It is very helpful that the meeting organisers have posted most of the slides for the oral presentations online, together with free access to the conference abstract book.

Abstracts and presentations are available at these links:

http://regist2.virology-education.com/abstractbook/2012_1.pdf

http://regist2.virology-education.com/2012/2ndHIV&Women/9_Jan.html

http://www.virology-education.com

Reports in this issue include:

• Similar efficacy and a few gender related differences in side effects with rilpivirine vs efavirenz at 96-weeks
• The effect of BMI on efficacy, safety and tolerability of lopinavir/r in women
• Hormonal contraception and higher risk of non-AIDS-defining events in Nashville cohort
• Poorer adherence and loss to follow up in Kenyan women who are pregnant when enrolled to ART programmes

Rilpivirine (RPV) did not show teratogenicity risk in pre-clinical studies and is therefore FDA pregnancy category B, nor does it interact with the oral contraceptives norethindrone and ethinyl estradiol. For these reasons, it could be a useful option for women of child bearing potential.

RPV was non-inferior to efavirenz (EFV) when combined with a nucleos(ti)de backbone in the pooled 96-week analysis of the phase 3 ECHO and THRIVE trials but only for baseline viral load strata <500,000 copies/mL. The primary endpoint was viral suppression to <50 copies/mL at week 48 by TLOVR analysis, with non inferiority defined by 95% CI compared to control not crossing the lower margin of -12%.

An investigation was conducted to look at safety and efficacy outcomes in women participating in these trials specifically and in comparison to men. This analysis included data from 236 (22%) women and 860 men, of these, 121 women and 429 men were randomised to RPV, and 115 women and 431 men to EFV. The women and men had similar median age of about 35 years, baseline CD4 counts of 243 and 258 cells/mm3 and viral loads of 4.9 and 5.0 log10 copies/mL respectively. Of the participants, a greater proportion of women than men (45% vs 18%) were black, and a smaller proportion (33% vs 70%) were white and Latina/o (16% vs 28%).

At 96 weeks, CD4 increases were similar in women and men in the RPV and EFV groups (approximately 225 cells/mm3).

Overall, 14% vs 6.1% of women failed virologically and/or discontinued treatment in the RPV and EFV arms respectively. The difference between the two arms was greater in the first year of treatment with 11.6% vs 3.5% failing compared to 2.5% vs 2.6% in the second year. These proportions were similar for men participating in the study: overall 14.2% vs 7%, year one 11.4% vs 4.4%, and year two 2.8% vs 2.6%, in the RPV and EFV arms respectively.

Stratification by baseline viral load showed similar rates of virological suppression for women and men with <100, 000 copies/mL receiving RPV or EFV (approximately 80%). Between >100,000 and 500,000 copies/mL, women in the RPV arm did slightly better than those receiving EFV, respectively 81% and 73% had viral loads <50 copies/mL at 96 weeks. The results for men in this viral load stratum were similar across the two arms, 72% and 73% for RPV and EFV. Above 500,000 copies/mL only 30% of women in the RPV arm had viral loads <50 copies/mL but this percentage relied on results for 3/10 women. For women receiving EFV the proportion was 57% (8/140). Of the men 67% (29/43) and 79% (46/58) in the RPV and EFV arms had viral loads <50 copies/mL at 96 weeks.

Of women who reported adherence >95%, both those receiving RPV (n=94) and EFV (n=92) had 78% rates of virological suppression <50 copies/mL. For those reporting <95% adherence suppression rates were lower, 67% and 64% for RPV (n=18) and EFV (n=14) respectively.

For men who reported >95% adherence, 96-week suppression rates with RPV (n=364) and EFV (n= 336) were 82% and 85%. Rates for those reporting <95% adherence were 52% with RPV (n=50) and 68% with EFV (n=59).

Resistance was analysed in a very small subset of women, RPV (n=15) and EFV (n=5). This revealed 20% of virologic failures with wild-type virus and 60% of with NNRTI resistance. There were more NRTI mutations in the women receiving RPV than EFV, 47% vs 0% and the most common were E138K (33%) and M184I (27%).

At week 96, rates of adverse events (AEs) leading to discontinuation of treatment were similar across treatment arms and genders. Incidence of grade 2 to 4 adverse events was significantly lower with RPV than EFV in women, 15.7% vs 34.8% and men, 17.5% vs 32.7%, both p<0.001.

Nausea occurred more frequently in women than men receiving both RPV and EFV, 19% vs 11.2%, 18.3% vs 9.7%, both p<0.05. But the incidence of treatment-related psychiatric adverse events was significantly lower in women than men receiving RPV, 9.1% vs 18.2%, p<0.05). Both these rates were lower than those in women and men receiving efavirenz, 16.5% vs 29.5%, p<0.05).

There were lower rates of abnormal dreams and nightmares in women than men receiving RPV 4.1% versus 11.4%, p<0.05. Women also experienced less of these events than men with EFV, 8.7% vs 17.4%, p<0.05. Rates of diarrhoea were similar in women and men receiving RPV, 13.2% versus 16.3%, but lower in women than men receiving efavirenz, 9.6% vs 18.6%, p< 0.05.

Women and men receiving RPV reported lower incidence of neurologic AEs compared to those receiving EFV, 15.7% vs 34.8%, p<0.05, and 17.5% vs 32.7%, p<0.001, for men and women respectively. There was also lower incidence of dizziness, 12.4% vs 27.8%, p<0.05 and 8.8% vs 28.8%, p<0.0001; and rash, 5.8% vs 16.5%, p<0.05 and 6.8% vs 12.5%, p<0.05.

Women and men receiving RPV had less grade 3 or 4 laboratory abnormalities 7.4% vs 11.5% and 10% vs 18.7% but this only reached statistical significance in men, p<0.05.

There were less grade 1 to 3 elevations in LDL cholesterol with RPV than EFV in women 19.9% vs 49.6%, p< 0.05, and men 19.6% vs 43.1%, p<0.001.

For all groups, there were significant increases from baseline in limb fat at week 96 with no statistical differences between treatment groups. Women receiving RPV appeared to have greater increase than the EFV group, median 1592g vs 641g. For men the two groups had similar median increases 828g vs 835g.

There was a trend towards greater BMD changes in women for both arms, but this was in a small sample size (n=30).

Reference:

Short W et al. Sustained efficacy and safety observed for RPV vs EFV plus FTC/TF and with a few gender differences in pooled 96-week ECHO and THRIVE analysis. 2nd International Workshop on HIV and Women. 9-10 January 2012, Bethesda, MD. Oral abstract O_14A.

Body mass index (BMI) can lead to alterations in pharmacokinetics and pharmacodynamics. Data describing the relationship between BMI and clinical outcomes of ART in women are limited.

Investigators from Abbott conducted a meta-analysis in women taking lopinavir/ritonavir (LPV/r)-based regimens in order to look at the effect of BMI on efficacy, safety, and tolerability. Ashwaq Hermes presented findings from this study.

All prospective randomised controlled trials (RCTs) in the company database in adults receiving LPV/r in regimens with two NRTIs, having BMI data, and baseline to week 48 efficacy, safety, and tolerability data were included.

Women were stratified by baseline BMI (kg/m2) into <18.5, ≥18.5-< 25, ≥25-<30 and ≥30 groups. As the number of women with BMI <18.5 was low (n=28), the investigators selected categories of <25 (normal), ≥25-<30 (overweight) and ≥30 (obese) for the analyses.

The meta-analysis included 485 women from seven RCTs, 258 with normal BMI, 130 were overweight women, and 97 categorised as obese. There were statistically significant differences (p<0.05) among the normal, overweight, and obese groups in baseline demographic characteristics: percentage of white women, 53.9%, 36.9% and 25.8% respectively; percentage of Latina women, 17.4%, 33.8%, and 20.6%, respectively and rate of hepatitis C co-infection, 17.2%, 10.8%, 6.2%, respectively.

There were also statistically significant differences in the three groups in baseline disease characteristics: mean viral load, 4.6, 4.4, and 4.3 log10 copies/mL, respectively, and mean CD4 counts 214, 244, and 278 cells/mm3, respectively.

Efficacy was similar across the groups at week 48. Similar proportions of women had viral load <50 copies/mL, 65.1%, 57.7% and 57.7%, respectively (ITT analysis). Mean increases in CD4 counts were also similar across the normal, overweight, and obese groups, 197, 158, and 172 cells/mm3, respectively.

Incidence of grade 3 and above adverse events (AEs) was also similar across the groups, 29.5%, 29.2%, and 41.2%, respectively, p=0.087. Differences were seen in the incidence of moderate/severe abdominal pain, 0.8%, 0%, 7.2%, respectively and diarrhea 9.3%, 10.8%, and 22.7%, respectively in the normal, overweight and obese groups, both p<0.05. These AEs were significantly higher, p<0.05, in the obese women compared with the other two groups. There was no significant difference in the incidence of nausea and vomiting among the three groups.

The investigators noted that increasing BMI is associated with a greater prevalence of diarrhea and abdominal pain, but not nausea or vomiting, in the general population. Also dietary differences among the BMI groups could be confounding, and this information was not collected or controlled for in the meta-analysis. Furthermore people with high BMI have elevated incidence of non-alcoholic fatty liver disease, which is associated with liver fibrosis and changes in drug metabolism.

They concluded that direct comparisons of dose safety and efficacy by BMI groups are needed to increase the understanding of obesity related changes and the impact on treatment.

comment

As reported in other studies, increased weight did not lead to higher rates of virological failure, suggesting that pharmockinetics for lopinavir do not have a direct association with higher weight, even though the PK data were not available or analysed.

However, although the results were not significantly different, it is unclear whether a formal test of equivalence was performed. There is >7% difference between the normal group and the other two groups and response rates appear lower in heavier groups. It would be interesting to see the confidence intervals for the differences which would have to be to conclude equivalence. The CD4 count increases also seem to be lower.

It would also have been interesting to see whether lopinavir/ritonavir has an impact on BMI in relation to baseline BMI.

Reference:

Hermes A et al. A meta-analysis of the effect of BMI on efficacy, safety, and tolerability of lopinavir/ritonavir in HIV-infected women in randomised clinical trials. 2nd International Workshop on HIV and Women. 9-10 January 2012, Bethesda, MD. Oral abstract O_15.

Studies evaluating the effect of hormonal contraceptives (HC) on HIV disease progression have shown conflicting results. Previous findings have been from resource limited settings (RLS) and have not looked at the effect of HC on non-AIDS defining events (non-ADE).

Mainly observational data from Africa and Asia has shown both higher and lower rates of HIV disease progression in women receiving HC. Observational data in HIV negative women has shown an association between HC and metabolic complications.

Vlada Melekhin presented findings from a retrospective cohort study of HIV-positive women attending the Comprehensive Care Center (Nashville, TN) between 1998-2008. The study investigated the association between HC (oral and injectable methods used >28 days) and AIDS-defining events (ADE), non-ADE (ie cardiovascular, renal, liver, and metabolic diseases and non-AIDS associated malignancies) and death.

Eligible women were <55 years old with no history of pulmonary or deep venous thromboembolism, breast cancer, hysterectomy, or bilateral tubal ligation and not pregnant at first clinic visit. Women with no HC were evaluated from their first clinic visit and those using HC at HC start.

Logistic regression analysis included age, race, baseline CD4 count, viral load, and haemoglobin, CD4 nadir, history of ADE, non-ADE, HCV, antiretroviral (ART and non-ART) use, smoking status, IV and non-IV drug use, year of study start, and year of HC start.

Of 467 HC-eligible women, 112 (24%) were on HC at any time during the follow up. At baseline women on HC were younger, median 28.6 vs 35.6 years. They had higher CD4 count 523 vs 364 cells/mm3 and nadir, 340 vs 280 cells/mm3, and lower median viral load, 3.1 vs 4.1 log10 copies/mL. They were less likely to be coinfected with HCV, 5% vs 15% or inject drugs, 16% vs 27%, both p<0.03.

There was no statistical difference in ART use between the HC and no HC groups, 30.4% vs 26.8%, respectively, nor in prior ADE or non-ADE.

Of the 112 women using HC, 51 used oral and the remaining 61 used injectable for a median duration of 7.6 and 13 months respectively, p=0.26.

HC users had longer follow-up compared to non HC users, median 2.8 vs 1.5 years for ADE, 2.8 vs 1.6 years for non-ADE and 3.8 vs 2.1 years for death.

The investigators reported a lower proportion of deaths in the HC group, 6% vs. 15%, p=0.01. But these women had more new cardiovascular non-ADEs, 12% vs. 5%, p=0.02.

In the adjusted analyses, HC use was associated with a statistically significantly higher risk of non-ADE HR 2.0, (95% CI 1.28, 3.1), p=0.02 and non-ADE/death HR 1.89, (95% CI 1.25, 2.87), p=0.03). Risks of ADE and ADE/death were also higher among HC users but did not reach statistical significance: HR 1.51 (95% CI 0.59, 3.85), p=0.39 and 1.49 (95% CI 0.72, 3.11), p=0.29, respectively. Women using injectable HC were at a higher risk of non-ADE and non-ADE/death, HR 2.0 and 1.9 respectively, both p=0.03 and those using oral HC only non-ADE, HR 1.9, p=0.02.

The investigators plan further analyses from this cohort including looking at the effect of ART and suggested as the number of women with HIV who are of child-bearing age increases, it is important to better understand any negative effect of HC on their health.

comment

Investigations into the use of hormonal contraceptive methods and its effect on disease progression in HIV positive women have led to conflicting results,

One randomised controlled trial conducted in Zambia showed risk of CD4 decline or death with hormonal contraception, compared to use of the copper IUD. [2] But the study was designed to look at the incidence of pregnancy and pelvic inflammatory disease in the IUD users and there was considerable discontinuation and switching between methods. Data from several observational studies do not confirm this effect.

The association with non-AIDS events found by Melekin are interesting but should be interpreted cautiously given that two large trials have reached very different conclusions. Observational data is vulnerable to unmeasured confounding and (as they are in the general population) lifestyles are very different between those that use hormonal contraception and those that do not. These differences could (feasibly) explain differences in incidence of some of these serious events. For example, even smoking and alcohol use may be different in the groups. Whille the researchers may have used propensity scores, these do nothing to tackle unmeasured confounding (and are arguably little better than standard multivariable logistic regression models).

The WHO recently held a stakeholders meeting to review the evidence on hormonal contraception and HIV, not only to consider the effect on disease progression but also female to male HIV transmission and HIV acquisition by negative women. The organisation and partners are producing three systematic reviews and there will be a statement from the consultation.

Currently the WHO medical eligibility criteria for contraceptive use defines hormonal contraceptives as category 1 – ie no restriction on the use of the methods for women with HIV (including AIDS).

References:

1. Melekin V et al. Hormonal contraceptive use is associated with a higher risk of non-AIDS-defining events in HIV-1-infected women. 2nd International Workshop on HIV and Women. 9-10 January 2012, Bethesda, MD. Oral abstract O_13.
2. Stringer EM et al. A randomised trial of the intrauterine contraceptive device vs hormonal contraception in women who are infected with the human immunodeficiency virus. Am J Obstet Gynecol 2007 August; 197 (2):144-148. Free full text:
   http://www.ajog.org/article/S0002-9378(07)00399-7/fulltext

There are concerns that women diagnosed with HIV during pregnancy may have greater difficulty with adherence to ART than those who are already aware of their status. This may lead to increased rates of vertical transmission and the development of drug resistance.

April Bell showed findings from a retrospective analysis of data collected from January 2006 to July 2011 by the United States Agency for International Development-Academic Model Providing Access to Healthcare (USAID-AMPATH) programme in Western Kenya.

The study compared adherence rates and pregnancy outcomes between women enrolled in the programme during pregnancy and those who became pregnant after they were already enrolled. Women from both groups were ART-naïve when their pregnancy was identified. Those meeting the eligibility criteria for treatment in Kenya at the time – CD4 <200 cells/mm3 – started ART immediately and those with CD4 >200 cells/mm3 started at 28 weeks gestation.

The women enrolled during pregnancy were younger, with a median age of 27 (IQR 23.2-31.7) years (n=8926), compared to 30.8 (IQR 26.6 – 35.1) years in the group already enrolled (n=5108). At enrollment a higher proportion were married, 69.6% compared to 52% and had a higher median CD4 count 371.5 (IQR 222 – 543) cells /mm3 compared to 282 (IQR 133-461) cells/mm3 for women who became pregnant when they were already enrolled in the programme. All comparisons, p<0.0001.

The women who were pregnant at enrollment were less adherent, 89.7% compared to 93.2% with perfect adherence, and were more likely to be lost to follow up before delivery, 29.6% compared to 3.4%, both p<0.0001.

Among the women who remained in the programme post-partum, there was no difference in the rate of mother-to-child transmission, 7% compared to 8.8%, p=0.0053, or early infant death, 3.2% compared to 4.2%, p=0.032, in those enrolled during pregnancy or became pregnant after enrollment respectively.

Although this study was limited by incomplete data, the investigators were able to conclude that women who are pregnant at enrollment into an HIV care programme are at higher risk for loss to follow up and poor adherence than those already enrolled in care at the time of pregnancy.

They suggested, “Interventions targeting women newly diagnosed with HIV infection during pregnancy are necessary to improve retention and adherence to therapy”.

Reference:

Bell A et al. Adherence and retention rates: a comparison of women enrolled in an ART programme during pregnancy and those who become pregnant after enrollment. 2nd International Workshop on HIV and women. 9—10 January 2012, Bethesda, MD. Oral abstract O_17.

Introduction

This meeting had a limited numbers of attendees and brought together an impressive group of leading researchers.

The abstract book and late breaker abstracts are available in PDF format from the conference website and links:

http://www.hiv-workshop.com/workshop-2011.htm

http://www.hiv-reservoir.net/index.php/the-news/189-abstract-book-2011-hiv-persistence-workshop.html

The site also contains daily rapid summaries of the workshop that will be followed in the next few weeks by more detailed reports.

As has been extensively documented, Brown’s apparent cure resulted from a debilitating odyssey of treatments required for the grim diagnosis of acute myelogenous leukemia, enhanced with a mix of insight and good fortune on the part of his doctor Gero Hutter, who was able to provide a stem cell transplant from a donor lacking the major HIV co-receptor CCR5.

The sea change wrought by this fortuitous ‘proof of concept’ was much in evidence at the 2011 persistence workshop this past December; the tentative forays into basic science that were once emblematic of the field are now mixed together with more ambitious plans for advancing ideas into the clinic. Perhaps most strikingly, two large pharmaceutical companies—Gilead and Janssen/Tibotec—described their use of industrial scale screening to search for compounds that are active against latent HIV; this represents an unprecedented expansion of efforts once confined to under-resourced academic labs.

A number of online resources are available with information on presentations at the 2011 persistence workshop: Lafeuillade runs a website called the Reference Portal on HIV Reservoirs & Eradication Strategies which includes an expanding number of reports, video interviews and commentary. [1]

David Margolis from the University of North Carolina has written a comprehensive report for Jules Levin’s National AIDS Treatment Advocacy Project (NATAP) website. [2] Jon Cohen also covered one the most notable presentations in the journal Science. [3]

This report and commentary represents my subjective take on events.

To try and briefly summarise the top-line stories that emerged from the 2011 meeting:

• A triumvirate of researchers – Courtney Fletcher, Mario Stevenson and Tim Schacker – presented data suggesting that sporadic, very limited rounds of HIV replication may occur in some individuals on ART due to poor penetration of certain drugs into the lymphoid tissues. However, preliminary data were only available from a small number of participants (~4-5) so the implications are still uncertain. According to the clinicaltrials.gov entry for the study, it is now expanding from the original enrollment target of 12 to 40 so additional information should soon be forthcoming. [4] Alain Lafeuillade has posted an interview with Mario Stevenson about the findings, and these presentations were the subject of Jon Cohen’s story in Science. [5]
• An Italian research group led by Andrea Savarino described a retrospective analysis involving 18 rhesus macaques infected with SIVmac251 that participated in various studies combining ART with drugs targeting the viral reservoir. The analysis found an association between the number of ‘anti-reservoir’ drugs animals received and the likelihood of controlling SIV to undetectable levels after ART was interrupted; however only three macaques controlled SIV to this degree so the findings should be considered very preliminary. The workshop organisers issued a press release about the data suggesting that for the first time they show that anti-reservoir drugs may be able to contribute to what is now frequently referred to as a ‘functional cure’ (control of viral load in the absence of ART). In an interview with Alain Lafeuillade, Savarino is careful to note that the findings require confirmation in human studies because they could relate to unknown factors specific to the three macaques that controlled SIV in the experiment. [6] This caveat is underscored by the fact that there are relatively few studies involving ART treatment of SIVmac251 in macaques to provide context, and in those that have been published there appear to be some examples of animals that spontaneously controlled viral load after ART interruption (both in control groups and in recipients of a DNA-based therapeutic SIV vaccine).
• David Margolis from the University of North Carolina presented the first data on the use of a histone deacetylase (HDAC) inhibitor named SAHA (aka vorinostat) in individuals with HIV. HDAC inhibitors are at the forefront of efforts to pharmaceutically urge HIV out of latency, so news from Margolis’s trial has been eagerly awaited. While very preliminary, and derived from just four participants, the results so far suggest that the approach is able to increase HIV expression by latently infected cells. It took Margolis many years to get the trial started due to concerns about the safety of HDAC inhibitors (which are used as cancer treatments and can cause serious toxicities) but no serious side effects have occurred to date. As Margolis stressed, much more work is needed before any conclusions can be drawn about the promise of the approach.
• The burgeoning involvement of the pharmaceutical industry in cure-related research – represented by presentations from Romas G from Gilead and Roger Sutmuller from Janssen/Tibotec – was important news because it promises to transform the drug discovery effort by increasing the number of compounds that are being screened by many orders of magnitude.

The workshop agenda was divided into discrete topic areas spread over three days. The first session addressed the subject of animal models, and was led off by Jeff Lifson from the National Cancer Institute (NCI) at Frederick who has nearly two decades of experience studying SIV infection in rhesus macaques. Lifson outlined some of the considerations in developing an appropriate model for cure-related studies, which include mimicking the degree of viral suppression achieved with ART in humans and developing tools to comprehensively assess the impact of additional interventions on SIV reservoirs.

The models currently in use include:

• Macaques infected with hybrid SIV/HIV viruses encoding HIV reverse transcriptase (SHIV-RT), treated with efavirenz, emtricitabine and tenofovir
• Macaques infected with SIVmac251 or SIVmac239 treated with multi-drug regimens (e.g. tenofovir, emtricitabine, raltegravir and ritonavir-boosted darunavir +/- maraviroc)
• Pigtailed macaques infected with SIV/17E-Fr and SIV/Delta B670 treated with tenofovir, integrase inhibitor, saquinavir, atazanavir (this model is primarily being used to assess issues relating to viral activity in the brain)

Lifson described a study conducted by his laboratory in which macaques were infected with the highly virulent challenge virus SIVmac239 and, after sixteen weeks, treated with a multi-drug antiretroviral regimen comprising an integrase inhibitor, tenofovir, emtricitabine, and ritonavir-boosted darunavir. Suppression of viral load to less than 30 copies/mL was eventually achieved, but Lifson noted that it took longer than is seen with HIV in humans. Like the vast majority of macaque studies, the experiment involved Indian rhesus macaques, and Lifson suggested that viral load suppression might be easier to achieve in Chinese rhesus macaques (this subspecies has been shown to control SIV somewhat better in the absence of ART). Lifson acknowledged that refinement of the SIV/macaque model for cure-related research is ongoing, and he cautioned against the premature adoption of any one approach as a standard. As an example of the pitfalls of premature standardisation, he cited the HIV vaccine field’s mistake in adopting a SHIV89.6p challenge model that turned out to have essentially no relevance to human HIV infection.

One potentially important new technology that Lifson highlighted is called digital PCR, which is vastly superior to traditional PCR for measuring small quantities of nucleic acid in samples. PCR amplifies nucleic acid sequences from a single sample by inducing rounds of copying of the original sequence, then back-calculating how many were originally present using a formula that takes into account the number of rounds of copying; however these calculations can be imprecise for a number of reasons. Digital PCR divides a sample into many discrete ‘microfluidic’ wells and then uses PCR to look for the nucleic acid sequence of interest in each well, providing a readout as to whether the sequence is absent (0) or present (1). The total amount of nucleic acid sequence that was present is then calculated based on the number of negative and positive wells, using an approach called a Poisson distribution. Digital PCR assays have only recently been commercialised and a number of laboratories are now busy using them to measure SIV and HIV in research studies.

The presentations following Lifson illustrated the diversity of animal models in use, and the uncertainties associated with them. Andrea Savarino from the Istituto Superiore di Sanità in Rome provided an update on experiments conducted by his group involving macaques infected with SIVmac251. In a paper published in AIDS last year, Savarino and colleagues reported that the gold-based rheumatoid arthritis drug auranofin reduced the reservoir of SIV-infected cells in animals treated with combination ART. [7]

At the workshop, Savarino presented results of a retrospective analysis of 18 macaques (including those included in the experiments reported in the paper) that have received various combinations of antiretrovirals and ‘anti-reservoir’ drugs including auranofin and buthionine sulfoximine (BSO). The breakdown of the antiretroviral regimens employed was as follows:

• ART: tenofovir, emtricabine, raltegravir
• Intensified ART (iART): tenofovir, emtricabine, raltegravir, ritonavir-boosted darunavir
• Mega-ART: tenofovir, emtricabine, raltegravir, ritonavir-boosted darunavir, maraviroc

Three of the 18 macaques have controlled SIVmac251 to undetectable (<40 copies/mL) levels after interruption of all treatment for several months, and Savarino reported that there was a significant correlation between the number of ‘anti-reservoir’ drugs received and this salutary outcome (for the purposes of this analysis, the CCR5 inhibitor maraviroc was counted as an anti-reservoir drug due to evidence that it reduced the amount of SIV DNA when added to intensified ART and preliminary results from a human study suggesting it may impact reservoirs). Some macaques also received the HDAC inhibitor SAHA, but an impact on the SIV reservoir could not be demonstrated.

The complicated sequence of treatments and outcomes in the three macaques that have controlled viral load off ART can be roughly summarised as follows:

• Macaque P252: ART, ART+auranofin, iART+auranofin, iART+SAHA, iART+auranofin, treatment interruption, viral load control to limit of detection, viral load rebound, Mega-ART, treatment interruption, viral load control, viral load rebound, viral load control, viral load rebound, Mega-ART, viral load control, viral load rebound, Mega-ART+BSO, viral load control (100+ days)
• Macaque P157: ART, iART, Mega-ART+auranofin+BSO, treatment interruption, viral load rebound, viral load control (~60 days), viral load blip, viral load control (~50+ days)
• Macaque P177: ART, iART, Mega-ART, Mega-ART+auranofin, treatment interruption, viral load rebound, Mega-ART, treatment interruption, viral load rebound, viral load control, viral load rebound, viral load control (~50+ days)

The data appear encouraging but there are some potential caveats:

• The model of SIVmac251 infection treated with combination ART (the drugs used in the study included tenofovir, emtricabine, raltegravir, ritonavir-boosted darunavir and maraviroc) is not well characterised, at least in terms of the published literature
• There were very few control animals, and the results are not from a single study but rather from multiple experiments, sometimes involving the same macaques being rolled over from prior experiments
• As can be seen from the sequence of events in the three controlling macaques, the treatments were complex and there was variability between animals in terms of exactly when different interventions were administered

As Savarino stresses in his video interview with Alain Lafeuillade, human trials are now required to ascertain if the macaque results can be translated to HIV.

Paul Luciw presented results of an experiment in which macaques infected with SHIV-RT had prostratin and valproic acid added to long-term ART (efavirenz, emtricitabine and tenofovir) prior to an interruption. Luciw showed evidence of reduced viral RNA and DNA in tissues but when treatment was interrupted there was no significant difference in viral load rebound compared to macaques treated with ART alone. Daria Hazuda from Merck has included several of Luciw’s slides in her recent presentations on cure research so the main findings can be viewed online, however note that prostratin is only referenced as a ‘protein kinase C activator’ and valproic acid as an ‘HDAC inhibitor’. [8]

Luciw also mentioned that he repeated the experiment adding raltegravir to the ART regimen and in that case there was no additional viral RNA and DNA reduction in tissues resulting from the anti-reservoir drugs, but he was running out of time and was unable to give any details; this finding is perhaps a reminder of how much uncertainty still surrounds macaque models for cure research.

Jerome Zack is trying to make drug-delivery nanoparticles out of weird cellular particles called ‘vaults’ made of three proteins and a bit of RNA. [9] Zack presented some preliminary evidence that they can be engineered to deliver potential latency activators prostratin and bryostatin, Zack is also working with Paul Wender at Stanford to develop better analogues of these drugs to use. The goal is to come up with some lead vault-delivered anti-latency compounds to test in the BLT humanised mouse model.

Shifting topics to the virological aspects of HIV persistence, Sarah Palmer from the Karolinska Institute reported results of an intensive evaluation of viral genetics pre-ART and on long-term ART (up to >12 yearrs) in 12 people (seven treated at acute infection, five during chronic infection) to look for evidence of viral evolution that would be indicative of ongoing replication. No evidence suggestive of HIV replication was found in various CD4 subsets and other cell types in blood, lymph tissue, bone marrow and gut. Palmer noted that no hematopoetic progenitor cells (HPCs) containing HIV DNA could be found; occasional positive signals from HPC samples turned out to be due to low-level contamination with CD4 cells. This finding was recently echoed in a paper from Bob Siliciano’s group at Johns Hopkins. [10]

Palmer drew attention to one case where a large amount of HIV DNA containing a huge deletion encompassing all of the protease gene was discovered. Since HIV can’t replicate without protease, this demonstrates that the division of CD4 T cells carrying integrated, non-functional proviral HIV DNA can contribute to what may appear to be an HIV reservoir by some measures (but really isn’t because the virus is defective). Mario Stevenson coined the term ‘junkyard DNA’ for these non-functional proviruses, and it was quickly adopted at the workshop.

Tae-Wook Chun from the National Institute of Allergy and Infectious Diseases (NIAID) offered some data suggesting HDAC inhibitors may not be all they’re cracked up to be in terms of reversing HIV latency, in the hands of his lab they didn’t induce a significant amount of viral RNA from latently infected cells compared to prostratin (which is a potent activator generally considered too toxic for human use). Chun also said that the latently infected cells induced to produce viral RNA don’t seem to die (“we haven’t seen any evidence of cell death’), suggesting that induction using HDACs might have little effect in the absence of an immune response capable of killing the infected cell.

Day two

Day two of the persistence workshop featured the presentations from industry, with Romas Geleziunas from Gilead and Roger Sutmuller from Janssen/Tibotec talking back-to-back about the ongoing work at their companies.

Gilead is looking at both virus activators and immune modulators, with Romas Geleziunas seemingly already having taken on board what Tae-Wook Chun had suggested the previous day: reactivating latent infection may not be enough to kill a cell, hence immune mechanisms may need to be induced to deliver the coup de grace. Geleziunas described Gilead’s high throughput primary cell screening assay, which is a modified version of an assay developed by Vincente Planelles and Alberto Bosque. [11]

So far they’ve identified three HDAC inhibitors from the Gilead drug library, imaginatively named 001, 002 and 003. 001 is 10-fold more potent than SAHA but inhibits all classes of HDACs (which I think is a bit of a worry from a toxicity perspective) while 002 is of interest because while less potent it doesn’t score positive on the AMES test (the standard test for assessing mutagenic potential). 001 and 003 were both AMES positive. Rats tolerated 3 weeks of 002 in a preliminary safety study. Romas noted that HDAC inhibitors only activate a fraction of the virus expression seen with pan-activating CD4 T cell stimulation using CD3 and CD28 antibodies, raising the question of whether the HDAC inhibitors are only activating a proportion of the latently infected CD4 cells, or rather causing less virus expression per cell. This question remains to be resolved.

High throughput screening of a Gilead library and a commercially available drug library produced a 1% hit rate, identifying 89 compounds that could be grouped into 15 clusters based on their structures. One was a calcium pump inhibitor named thapsigargin, possibly after a character from Lord of the Rings. It was a ‘robust activator’ of latency in cells from 6 out of 6 donors. Romas didn’t say anything more about it and Wikipedia offers an explanation as to why: “It is a tumor promoter in mammalian cells”. Another was a “broad spectrum nonspecific tyrosine kinase inhibitor” called tyrphostin A which worked on cells from 3/6 donors. Since they hadn’t expected to find kinase inhibitors, they then tried screening a library of those and got a 20% hit rate. Evidence of activity at low concentrations and dose responses were seen. Next steps are to confirm activity with more selective kinase inhibitors and explore the signaling pathways that are causing these compounds to work.

Switching to the topic of bolstering immunity, Romas said Gilead is looking at a TLR7 agonist it has in development for hepatitis B. It’s been tested in chimps and woodchucks, where it has shown antiviral activity and dose-dependent induction of alpha interferon production and T cell and B cell activation. In woodchucks, it led to induction of antibodies against the hepatitis B surface protein. A small phase I human study has been safely conducted, also showing evidence of some T cell and B cell activation. Next step is to study the impact on HIV-infected cells and potentially test it in animal models in combination with HDAC inhibitors.

Meanwhile the overarching goals of Gilead’s program continue to be:

• More high throughput screening
• Uncover novel mechanisms (e.g. as may happen as a result of the identification of kinase inhibitors)
• Discover new chemical entities (NCEs).

Roger Sutmuller from Janssen/Tibotec then described his company’s efforts which have not been discussed publically before. He outlined the basic goal of discovering safe and effective compounds to reactivate latent HIV i.e. those that cause little or no cell activation and ideally have the potential to be combined. Unlike Gilead, Tibotec starts with a Jurkat cell line assay to identify compounds, after which they have a preplanned set of steps involving evaluation of:

• Toxicity/immune stimulation
• Virus reactivation in primary T cell assays
• Virus reactivation in latently infected cells from HIV+ individuals ex vivo
• Medicinal chemistry selection of lead compounds
• Testing in a humanised mouse model developed by Roberto Speck
• Testing of the pathways involved in drug activity eg using microarrays, HIV mutants with various signaling elements disabled, short-interfering RNAs etc.

Using the Jurkat cell line assay, 35,000 compounds have been screened to date, and the next step is to screen 480,000 compounds from a Johnson & Johnson ‘diversity library.’ Of those screened to date, 800 HDAC inhibitors have popped out (a 20% hit rate), 25 protein kinase C agonists (a family prostratin belongs to) and 600 unknowns that can be grouped into 11 different ‘chemotypes.’

Sutmuller went on to describe their in-house primary T cell assay, which involves fresh cells expanded in the lab and infected with an HIV encoding green fluorescent protein (GFP). Cells are rested to create latency and then drug activity is measured based on the extent to which the cells light up green. They’re using this assay to screen medium sized libraries; it can handle about 2,000 compounds per week. He showed some data from one compound ‘229,’ which induced virus at about half the level of pan-stimulator PMA, and worked even better in combination with SAHA. The next step is to study these and other compounds in Roberto Speck’s humanised mouse model, which involves 3TC and TDF given in food pellets and a long-acting version of TMC 278 that is delivered by weekly injection. They have seen good viral suppression and can recover latently infected CD4 cells using this system.

Among the other highlights from day two, Una O’Doherty from the University of Pennsylvania showed that CD8 T cells from elite controllers can kill what appear to be latently infected CD4 cells because they express the HIV Gag protein, just with much slower kinetics than seen with activated CD4 cells (and without causing spreading infection). O’Doherty suggested that perhaps this means latently infected CD4 cells aren’t as invisible to the immune system as has been thought, which provoked some controversy because—as she happily acknowledged—it is not yet known whether the same holds true for latently infected CD4 cells from individuals on ART.

In an effort to hone in on which elements of the Berlin patient’s treatment were necessary to achieving the apparent cure of HIV infection, the ever-curmudgeonly John Mellors (University of Pittsburgh) presented an analysis of ten people who had undergone myeloablative chemotherapy and autologous stem cell transplants for lymphoma. None of these individuals were cured of HIV infection, leading Mellors to conclude that in the case of Timothy Brown, the CCR5-negative transplant was important, possibly along with the graft-versus-host disease Brown experienced. In the Q&A afterwards, workshop attendee Mike McCune from UCSF suggested that total body irradiation (TBI) might also have played a role.

Santiago Moreno (Hospital Ramon Y Cajal, Madrid, Spain) presented some preliminary evidence that the CCR5 inhibitor maraviroc may activate a protein complex named NF-kappaB when the drug binds to the CCR5 receptor. Because NF-kappaB activation can stimulate latent HIV, Moreno suggested that maraviroc might have anti-reservoir activity, as was previously suggested by a small uncontrolled pilot study conducted by Moreno’s laboratory and reported at a symposium prior to the 2010 International AIDS Conference in Vienna. However, results from a randomised trial of ART intensification with maraviroc were debuted at the persistence workshop by Maria Puertas, and this study was unable to document any additional declines in HIV reservoirs associated with receipt of the drug (HIV DNA levels fell by ~8-fold in both arms).

In a session on acute HIV infection, Marty Markowitz from Aaron Diamond AIDS Research Center presented 96-week results from a 3-drug vs. 5-drug treatment study, showing essentially no significant differences in a variety of reservoir and immunological measures in blood and gut. There was a slight reduction in cell-associated HIV RNA levels at week 96 in the 5-drug group but Markowitz felt this was unlikely to be meaningful. Jintanat Ananworanich (HIV Netherlands Australia Thailand Research Collaboration) described a study involving treatment of people with very, very early HIV infection, in which 60 people have so far been enrolled, with an average time from screening to enrollment of just 3 days. This would not seem like much time for someone to process the news that they have become HIV infected and make a decision to enter a trial involving a multiple treatments and sampling from the peripheral blood, CNS and GI tract, but Ananworanich said “acceptance rates are quite high.” Individuals were in what in the following Fiebig stages of seroconversion:

• 34% stage I: within 5 days of infection
• 9% stage II: within 10 days of infection
• 48% stage III: within 13 days of infection
• 9% stage IV: within 19 days of infection

24-week results on a subset of participants indicated significantly smaller reservoirs in blood and gut of stage I vs. III or IV, with total and integrated HIV DNA being undetectable in a proportion of the earliest-treated individuals.

The very last presentations of day two involved the tag team of Timothy Schacker (University of Minnesota), Courtney Fletcher (University of Nebraska) and Mario Stevenson (University of Miami) outlining very preliminary results from their small study of viral replication in anatomical and cellular reservoirs. A total of 12 individuals are enrolled, ART naive at baseline but then treated (mostly with TDF, FTC and ritonavir boosted atazanavir) and analysed regularly up to six months. Not all individuals have data available yet, and the number of individuals from whom data were reported varied between the different presenters. Courtney Fletcher looked at drug levels in nine people, finding that some drugs (particularly atazanavir, FTC and efavirenz) may not reach adequate levels in lymph nodes and gut. Mario Stevenson then showed that in some study participants, 2-LTR circles increased in lymph tissue after starting ART, in one case along with a rise in proviral DNA. In one other individual, levels of both 2-LTR circles and proviral DNA went down. Stevenson stated: “this does not necessarily denote ongoing replication” but proposed an alternative model in which a population of long-lived cells can generate virions that infect one more cell and that’s it – just one cycle of replication, in other words. He stated this would not lead to viral evolution but could replenish the latent reservoir. In the Q&A, John Coffin from the NCI got up to the microphone and noted that since latency is a rare event in infected cells, and since Stevenson was saying these were single-cycle rounds of infection, the number of times latency would be created is not known, and may well not be often enough replenish the reservoir.

Timothy Shacker closed out the talks with a description of his efforts to correlate Fletcher’s and Stevenson’s results with measurements of viral RNA on the follicular dendritic cell (FDC) network in lymph tissue (using in situ hybridisation). Schacker created 3D graphs for several participants that included 2-LTR circle levels, DNA levels, levels of viral RNA on FDCs and, lastly, drug levels. There appeared to be correlations between the various measures, but how many people had evidence of ongoing HIV replication cycles was unclear. Schacker noted that there was a significant inverse correlation between levels of FTC diphosphate in lymph tissue and viral RNA on FDCs. Additional results from the expanded version of this study are needed in order to understand if this is a broadly applicable phenomenon, and whether poor tissue penetration of antiretrovirals represents an under-appreciated obstacle to curing HIV infection.

Day three: Margolis reaches a milestone, the crowd thins for functional cures

The major news on day three of the workshop was the presentation by David Margolis (University of North Carolina) of very preliminary results from the phase I/II study of the HDAC inhibitor vorinostat (SAHA). The trial has a complicated schema, largely due to the safety concerns of the FDA regarding the drug, which scores positive on the AMES mutagenic test (a red flag for regulators even though the significance is not fully understood).

The first step of the protocol involved screening potential participants to assess whether vorinostat could reactivate latent HIV from their CD4 T cells ex vivo. Thirteen individuals had ~4 billion lymphocytes extracted by leukopheresis, then sorted into discrete pools of 1 million purified resting CD4 cells each (ending up with 24-36 pools per participant). These pools were exposed to either vorinostat or no drug, and a mean level of HIV RNA per million cells (and a standard deviation) was calculated for each person (the assay used can measure down to 10 copies per million cells). Margolis noted that the statistical approach used to calculate the mean RNA levels is robust but complicated, and a paper explaining it is currently in press at an unnamed statistics journal.

Four of the thirteen people screened showed a statistically significant upregulation of HIV RNA expression in this analysis and were therefore recruited into the next step of the trial. A 200mg dose of vorinostat was given first for safety, followed by a 400mg dose to study pharmacokinetics and for analyses of histone acetylation and acetylation of the p21 gene (in other words, analyses of the effects of the drug on cellular genetic machinery and not HIV). The pharmacokinetic data mirrored that reported in cancer studies and cellular acetylation (both total and p21 gene) was maximal by 8 hours then trended down by 24 hours.

A final 400 mg dose of vorinostat was then administered with leukopheresis performed 4-6 hours afterward based on the pharmacokinetic data indicating this would be around the time of maximum activity. No grade 1 or greater toxicities were seen, and HIV RNA expression increased compared to baseline in all four individuals by a mean of 4.4-fold (range: 3-6.6 fold). HIV RNA in peripheral blood was also assessed using a single copy assay but no change was detected, perhaps not surprisingly given that this was a single dose study.

Margolis was obviously very encouraged by the data and stated that they had successfully “demonstrated induction of full length HIV RNA expression within a window of time after a single vorinostat exposure.” He concluded that obstacles to HIV RNA expression can overcome “at least in some cells.” But he stressed that many questions remain, including:

• Is there an equal effect to multiple doses or does it become attenuated?
• How much exposure is needed?
• Should drug be administered continuously or pulsed?
• Will toxicities emerge?
• What number of cells is needed to measure relatively rare reactivation events?
• Does RNA expression lead to virion production or clearance of cell?
• Are additional inducers needed?
• Are additional interventions needed to clear the latently cells that have been induced to express HIV RNA?

The final session of the meeting was on functional cures. Dishearteningly, the crowd of attendees thinned noticeably but the first presenter, Paula Cannon, was undeterred. “This is the first time people are going to be talking about functional cures,” she opened sunnily. “I know you’re all very obsessed with the reservoirs but we don’t really care about the reservoir – if there’s a little bit of virus left in the body, so what?” Having stuck fear into the hearts of any remaining reservoir obsessives, she then outlined what she meant, highlighting three key goals for those in pursuit of a functional cure:

• Reducing the pool of HIV target cells and thereby reducing the harmful immune activation and inflammation that is central to pathogenesis.
• Creating HIV-resistant HIV-specific CD4 T cells.
• Taking advantage of HIV as a selection agent to drive the expansion of resistant cells.

Cannon went on to review the Sangamo zinc finger nuclease (ZFN) approach to deleting CCR5, the work conducted by her laboratory to adapt it to modify hematopoietic stem cells (HSCs), and the efficacy demonstrated in a published experiment in which humanised mice were engrafted with the CCR5-deleted stem cells and challenged with HIV. Work is now underway to advance the approach into HIV positive people who need stem cell transplants as treatment for lymphoma, in collaboration with John Zaia and David DeGusto from City of Hope who have previous experience of studying gene-modified HSCs in this setting. Cannon explained that preparation for the trial has involved switching from relatively easy-to-use HSCs obtained from fetal cord blood to rather more uncooperative adult stem cells. These cells are called mobilised peripheral blood stem precursor cells (mPSCs) and sampling involves giving G-CSF for four days then conducting apheresis to extract white blood cells, followed by ex vivo purification of CD34+ cells. This procedure has now been performed on 13 donors, obtaining 42 billion white blood cells of which around 0.5% were CD34+ cells; Cannon estimates that around 1% of the CD34+ cells are ‘true’ stem cells. These mPSCs are now being used in mouse studies to address a number of issues prior to human testing.

One such experiment assessed whether pre-existing immunity to adenovirus might be problematic, because an adenovirus vector is used to deliver the zinc finger nuclease into the mPSCs. Mice were given a high titer of anti-adenovirus antibodies prior to delivery of the mPSCs and, encouragingly, no difference was seen in the extent of engraftment compared to controls given phosphate buffered saline (PBS).

Next steps include large scale tumorigenicity studies in ‘NOD scid gamma’ (NSG) mice and evaluation of modified mPSC under ‘maximising’ conditions to test the upper limit of on and off target effects (there is some evidence that ZFNs can disrupt genes other than the CCR5 target, particularly a similar region of the CCR2 gene). Mice given the maximised mPSCs will be kept for many months and extensively analysed for safety.

Following Paula Cannon, Carl June gave an update on the use of the same technology to modify CD4 T cells that are extracted from individuals with HIV using apheresis, expanded and modified in the laboratory, and reinfused into the same individual. Previous presentations of data from these phase I trials has generated considerable excitement, because the proportion of modified CD4 T cells persisting in the blood and gut of participants far exceeds the extremely modest levels obtained with prior gene therapies delivered using the same approach. Significant CD4 T cell count increases have also been documented out to nine months of follow up. Unusually, CD4:CD8 ratios have also significantly improved from an average of 0.5 at baseline to 1.5 at last analysis; this type of improvement is rarely observed as a result of ART, and may have implications in terms of improving long-term health because inverted CD4:CD8 ratios are a well-documented risk factor for illness in the HIV-uninfected elderly.

Most intriguing, however, is a trial involving a 12-week analytical treatment interruption (ATI). Data is now available from six individuals who have undergone the ATI and while all experienced a viral load rebound, levels began falling prior to the reinitiation of ART, which June noted was not the case in a prior gene therapy study involving an ATI (an evaluation of a candidate named VRX496).

One notable individual controlled viral load to below the level of detection (<50 copies/mL) before ART was restarted. This person turned out to be heterozygous for the delta32 CCR5 deletion, which means that the ZFNs could work more efficiently because only one CCR5 gene in each cell had to be disrupted in order for CCR5 expression to be completely abrogated (instead of two as is normally the case). Importantly, June found a significant correlation between the proportion of modified CD4 T cells and viral load control during the ATI. This suggests that an antiretroviral effect is achievable with the approach, and that the potency of the effect may be boosted if the proportion of modified cells can be increased.

In the Q&A period, June was asked if he had assessed whether gene-modified HIV-specific CD4 T cells may have contributed the viral load results; he replied that HIV-specific CD4 T cell responses have not yet been analyzed in the ATI trial.

The last two talks in the final workshop session addressed the development of methods that attempt to specifically target latent HIV and excise it from the DNA of infected cells (or damage the provirus in order to render it non-functional). On paper, at least, these approaches sound very appealing but it was clear that significant hurdles remain. Jan van Lunzen (University Medical Centre Hamburg-Eppendorf) discussed the modification of an enzyme called Cre recombinase to target HIV DNA. The modified version, dubbed Tre recombinase, has successfully excised proviral DNA from cells in vitro and work is now underway to study how it might be delivered. Next steps involve studies in humanised mice using a lentiviral vector to deliver the Tre recombinase to CD34+ stem cells; the vector is designed to be ’self-inactivating’ in cells that do not contain HIV DNA. As an aside, Jan van Lunzen also mentioned a patient of his who started ART during early infection, was treated for five years, then stopped six years ago, had a small viral load blip and has been undetectable ever since. HIV RNA cannot be found in blood, gut or CNS. According to van Lunzen, the individual has a “very strong HIV-specific CD4 response,” and he highlighted the case as being similar to Christine Rouzioux’s report of five individuals treated very early who have controlled viral load to undetectable levels off ART for an average of around five years. [12] These case reports may bode well for prospects for a functional cure, van Lunzen suggested.

Keith Jerome from the Fred Hutchinson Cancer Research Center recounted the efforts of his group to employ different enzymes, endonucleases, to target latent HIV. The idea in this case is to induce mutations in the HIV provirus in order to render it non-functional. Some success has been achieved in vitro but considerable challenges remain in terms of improving the efficiency of targeting and developing delivery methods that might be able to get the endonucleases to where they are needed. Jerome’s work is now being supported by a Martin Delaney Collaboratory grant from NIH.

The last word at the 2011 persistence workshop was given to Nobel laureate Françoise Barré-Sinoussi, who outlined the International AIDS Society’s development of a Global Scientific Strategy ‘Towards an HIV Cure’ and encouraged audience members to attend an IAS symposium on the subject that will take place in Washington DC immediately ahead of the 2012 International AIDS Conference. Barré-Sinoussi also stressed the importance of the work and the need to continue the momentum which has placed curing HIV infection back at the top of the research agenda.

The 6th International Workshop on HIV Persistence, Reservoirs & Eradication Strategies is scheduled for 2013 in Miami.

References

1. Reference Portal on HIV Reservoirs and Eradication Strategies website.
   http://www.hiv-reservoir.net/
2. Margolis D. HIV Persistence during Therapy 5th International Workshop. natap.org, 2011.
   http://www.natap.org/2011/HIV/122111_01.htm
3. Cohen J. Tissue says blood Is misleading, confusing HIV cure efforts. Science 23 December 2011: Vol. 334 no. 6063 p. 1614.
   http://www.sciencemag.org/content/334/6063/1614.summary
4. Clinical Trial. Tissue drug levels of HIV medications.
   http://clinicaltrials.gov/ct2/show/NCT01490346
5. Mario Stevenson Interview. (9 December 2011).
   http://www.youtube.com/watch?v=_fhb95p__9g
6. Andrea Savarino Interview on HIV Cure. (10 December 2011).
   http://www.youtube.com/watch?v=xIqdDU_OEFU
7. Lewis MG et al. Gold drug auranofin restricts the viral reservoir in the monkey AIDS model and induces containment of viral load following ART suspension. AIDS. 25(11):1347-1356, July 17, 2011.
   http://journals.lww.com/aidsonline/toc/2011/07170
8. Hazuda D. Powerpoint can be downloaded at the bottom of this page, Luciw’s data is on slides 21-25):
   http://www.iasociety.org/Default.aspx?pageid=416#session5
9. TEDxUCLA – Leonard Rome – online video. (29 August 2011).
   http://tedxtalks.ted.com/video/TEDxUCLA-Leonard-Rome-Vaults-mo
10. HIV-1 DNA is detected in bone marrow populations containing CD4+ T cells but is not found in purified CD34+ hematopoietic progenitor cells in most patients on antiretroviral therapy. J Infect Dis. (2012). First published online: 24 January 2012.
    http://jid.oxfordjournals.org/content/early/2012/01/24/infdis.jir884.abstract
11. Bosque A et al. Studies of HIV-1 latency in an ex vivo model that uses primary central memory T cells. 2011 Jan;53(1):54-61. Epub 2010 Oct 21.
    http://www.ncbi.nlm.nih.gov/pubmed/20970502
12. Hocqueloux C et al. Long-term immunovirologic control following antiretroviral therapy interruption in patients treated at the time of primary HIV-1 infection. 19 June 2010 – Volume 24 – Issue 10 – p 1598-1601.
    http://journals.lww.com/aidsonline/Fulltext/2010/06190/Long_term_immunovirologic_control_following.27.aspx

Unfortunately abstracts are not yet available online, and although webcasts, podcasts and PowerPoint slides are available these require a login name and password (obtainable by email from the EACS secretariat).

The same login details can also be used to access training resources from a pre-meeting training for doctors and other resources. iPhone and iPad versions are accessible using the free Talks On The Go App.

http://www.europeanaidsclinicalsociety.org/

The following reports are included in this issue.

• European guidelines (EACS) – 2011 update
• Raltegravir achieves superiority over efavirenz after four years
• Higher plasma levels of tenofovir and darunavir but not efavirenz in older patients
• Ritonavir levels reduced with high fat meal (900 kcal)
• Transplacental transfer of raltegravir and delayed plasma clearance in preterm neonates

The launch of 2011 guidelines from the European AIDS Clinician Society (EACS), extensively revised and updated, was probably one of the scientific highlights of the conference.

The three guidelines, previously printed separately, have now been collated together in a slightly larger format. This makes the print edition now more comprehensive and also easier to use and reference.

Based on essential bulleted lists, treatment algorithms, and reference tables the guidelines are an excellent format for rapidly reviewing the current best standard of care in four key areas of HIV management:

1. Assessment and monitoring at initial and subsequent visits
2. ARV treatment in adults
3. Prevention and management of non-infectious co-morbidities
4. Clinical management and treatment of hepatitis B and C in HIV-positive patients

Although additional resources have been produced to compliment the guidelines in many important areas of HIV management, these had no yet been posted online when we went to press.

The guidelines (version 6.1) can be downloaded free as a PDF file and print copies can be order on the EACS website:
http://www.europeanaidsclinicalsociety.org/

Print versions can be ordered from EACS:
info@eacsparis.org

Four year results from a five year, double-blind, randomised, non-inferiority study comparing raltegravir to efavirenz (each with tenofovir plus FTC) in treatment-naïve patients were presented by Jurgen Rockstroh.

The study design, matched baseline characteristics and safety and efficacy results from earlier analyses have already been presented at earlier meetings. The new subgroup analyses (including baseline CD4 <200 copies/mm3, viral load >100,000 copies/mL, hepatitis and demographic responses) focused on virological efficacy with discontinuations related to viral failure included but discontinuations for other reasons excluded and using an observed failure approach.

From approximately 280 patients in each arm at baseline, 223 (79%) and 197 (70%) completed the 192 week analysis, in the raltegravir and efavirenz arms respectively. Discontinuations were all less frequent in the raltegravir arm: virological failure (n=5 vs 8); side effects (n=13 vs 26); and loss to follow-up (n=8 vs 17)

At 192 weeks, the primary analysis of viral suppression to <50 copies/mL (non-completer=failure) saw raltegravir achieve statistical superiority compared to efavirenz [76% vs 67% (difference = +9.0; 95%CI 1.6, 16.4, p < 0.001: with the lower limit for non-inferiority set at –12% and superiority being achieved when both confidence intervals became greater than 1.0].

CD4 increases were + 60 cells/mm3 higher in the raltegravir arm (95%CI 24, 95).

Overall clinical events (96% vs 98%, p = 0.16), discontinuations due to drug-related events (5% vs 8%, p = 0.173) and serious adverse events (18% in each arm, p = 0.91) were similar between the two study groups, raltegravir was associated with significantly fewer drug-related events (50% vs 80%, p < 0.001).

There were no statistically significant differences in response between groups by gender, age, race/ethnicity, viral load >100,000 c/mL, CD4 > 200 cells/mm3, hepatitis coinfection or HIV sub-type. Raltegravir showed a significantly stronger virological response in the <100,000 c/mL group (93% vs 81%; difference +12; 95% CI 3, 22). Interpretation of a difference in favour of raltegravir when baseline CD4 was 50-<200 cells/mm3 is complicated by a trend to favour efairenz when CD4 counts were <50 cells/mm3.

comment

These results support durability and safety of raltegravir. they also show that after week 192 raltegravir achieves superiority compared to efavirenz with the difference largely driven by efavirenz-related side effects.

The CD4 difference may also be important for patients with sub-optimal CD4 responses on other HAART combinations.

Reference:

Rockstroh JK et al. Long-term efficacy of raltegravir or efavirenz combined with TDF/FTC in treatment-naïve HIV-1-infected patients: week-192 subgroup analyses from STARTMRK. 13th EACS, 12–15 October 2011, Belgrade. Abstract PS 1/1.

Several studies looked at the association between older age and antiretroviral pharmacokinetics (PK).

Tenofovir

Muge Cevik from the Chelsea and Westminster Hospital London reported results from a PK study suggesting that tenofovir clearance is significantly reduced with increasing age and resulting in higher drug levels (AUC and Ctrough). [1]

This included steady-state plasma levels from 52 men and 2 women (12 of whom were on PI/r-based combinations). Median age was 54 years (range 40–81 years) with only two people younger than 50. Samples were drawn randomly and population pharmacokinetics applied to predict values.

Tenofovir median clearance (CL/F), AUC (24hr) and Ctrough (C24) were 110.0 L/r (27.4–248.3). 2.2 mg.hr/L (1.0–9.0), and 0.06 mg/L (0.01–0.3) respectively.

Increasing age was significantly associated with slower clearance (p=0.0012), higher AUC (p=0.0012) and higher Ctrough (p=0.0017). People older than 60 had significantly lower clearances (p=0.0447) and higher AUC (p=0.0457) than those younger than 60.

No PK differences were seen between PI and NNRTI based combinations (p=0.08).

Efavirenz and darunavir/ritonavir

A similar analysis was presented by Ahmed and colleagues from the same group at Chelsea and Westminster on the PK of efavirenz or darunavir/ritonavir used by older patients (median age was 54 years (range 27-77) and 56 years (28-76), respectively). [2]

In 70 men and 7 women taking efavirenz, no differences were seen in any PK parameter when comparisons were made between people older and younger than 50 (all p-values >0.05 for between age comparisons).

In 33 men and one woman taking darunavir/ritonavir (23 using once-daily) oral clearance was significantly lower in people over 50 years old (10.3 vs 13.0 L/h; p=0.027) with higher AUC (80.9 vs 61.6 mg.h/L; p=0.021) and Ctrough levels (1.9 vs 1.2 mg/L; p=0.008) than those younger than 50.

Once-daily vs twice-daily could not be assessed because of unequal age distribution between the two dosing regimens.

References:

1. Cevik M et al. Tenofovir (TFV) pharmacokinetics (PK) in HIV infected individuals over 40 years of age. 13th EACS, 12–15 October 2011, Belgrade. Abstract PS 6/1.
2. Ahmed A et al. Efavirenz and Darunavir Plasma Concentrations in HIV-infected Patients Aged 50 Years or over. 13th EACS, 12–15 October 2011, Belgrade. Abstract PE6.2/1.

Researchers at Makerere University, Kampala and the pharmocology group at Liverpool University reported a significant interaction between high fat meals and ritonavir as a booster in lopinavir/r (Kaletra).

Three meal conditions were studied in an open-label, three part, cross over study in 12 HIV positive people (6 men, 6 women) using lopinavir/r (2 x 400/100 mg tablets) as second-line therapy. Median (IQR) age and weight of patients was 48 (44 – 49) years and 62 (59-68) kgs.

Intensive PK sampling after a moderate (20 g fat) and high (36 g) fat meal (on Day 1 and 8 respectively) were compared to fasted state on Day 15.

Compared to the fasting, administration with a high fat meal resulted in 29% lower ritonavir AUC (geometric mean ratio 0.71; 90%CI 0.61-0.84) and 29% lower Cmax (GM 0.71; 90%CI 0.60-0.84) while C12 increased non-significantly by 12% (GM 1.12 (90%CI 0.94-1.33).

Reference:

Lamorde M et al. – Steady-state exposure of ritonavir is reduced by a high fat meal in Ugandan patients receiving lopinavir plus ritonavir co-formulated tablets. 13th EACS, 12–15 October 2011, Belgrade. Abstract PE6.6/1 (BPD1/1).

Preterm birth is common in infants born to HIV positive mothers and is associated with an increased risk of mother to child transmission. Oral drug absorption in infants is unpredictable due to the immaturity of the gastro intestinal tract at this age. Preloading the foetus with maternal nevirapine (NVP) is common in these cases.

Raltegravir (RAL) is pregnancy category C and data to guide its use in pregnancy are limited. However, it has been used to achieve a rapid reduction in viral load before delivery, for preloading the foetus where poor oral absorption is anticipated and in cases where there is resistance or intolerance to other antiretrovirals.

RAL is absorbed rapidly (with a T max of about three hours) it takes two days to reach steady state concentrations and has an elimination terminal half-life of 9 hours. It uses the UGT 1A1 metabolic pathway. About half of the oral dose is excreted unchanged in the stool and 30% in the urine (about a third of which is as unchanged RAL and the remainder as the metabolite). There is considerable inter patient variability in its metabolism.

In an oral presentation, Aseel Hegazi from St Georges University Hospital, London showed three maternal infant case studies in which pregnant mothers of preterm neonates received RAL as part of their prevention of mother to child transmission (PMTCT) regimens. [1] The investigators looked at transplacental transfer of the drug and plasma clearance in the infants.

The same group has previously described the use of RAL in PMTCT regimens in mothers of three term infants. In these cases they found good transplacental transfer with higher concentrations in the infants than the mothers approximately three hours after delivery. [2] They also reported persistence of neonatal concentrations at three days (although below the therapeutic range). They suggested that poor neonatal and foetal maturity of the UGT-dependent pathways could account for this. And that it is possible that increased activity of UGT1A1, associated with progesterone, observed in pregnant women contributed to the disparity.

In the three cases of RAL use in preterm delivery, paired blood samples were taken as close as possible to delivery and then post partum. Maternal and neonatal RAL plasma concentrations were measured using liquid chromatography and mass spectrometry. Table 1 summarises these cases.

Dr Hegazi concluded that therapeutic RAL plasma concentrations (> 15ng/mL) might be persistent for up to five days in preterm neonates. She noted that this is longer than that observed in term infants and is probably linked to immature UGT1A1 mediated gluronidation. She suggested that maternal RAL preloading might be a good alternative to NVP where oral absorption is unreliable (particularly with preterm infants) and maternal options are limited

comment

These case studies are interesting and this use of RAL could prove important. RAL used this way is likely to be mentioned in the next BHIVA guidelines (although data is very sparse so evidence will be weak).

IMPAACT 1097 is a washout (passive) PK and safety study designed to investigate this phenomenon in neontates. It is the first clinical trial of an investigational drug to look at neonatal PK. It is recruiting mothers already receiving RAL in pregnancy and the infants will be sampled at intervals up to 30 to 36 hours after dosing.

After a review of PK and safety data from this and IMPAACT1060 – which is investigating this drug in children in de-escalated age bands with those below two years, receiving a granule formulation, now being studied – the company is planning a study of infants born to HIV positive mothers from immediately after birth until their status has been confirmed.

References:

1. Hegazi A et al. Raltegravir in the prevention of mother to child transmission of HIV: effective transplacental transfer and delayed plasma clearance observed in preterm neonates. 13th EACS, 12–15 October 2011, Belgrade. Oral abstract PS 6/7.
2. Mckeown D A et al. High neonatal concentrations of raltegravir following transplacental transfer in HIV-1 positive pregnant women AIDS. 24 September 2010. Volume 24. Issue 15. p 2416–2418.

• Statin blocks negative impact of PIs on bone formation in vitro
• HIV linked to frailty in middle-aged IDUs, especially with poor HIV control

Ritonavir-boosted or unboosted atazanavir or lopinavir promoted stem cell changes that could lead to decreased bone formation, according to results of cell studies by Jacqueline Capeau and colleagues at Saint-Antoine Hospital in Paris. Exposing the cells to pravastatin blocked these protease inhibitor (PI)-induced changes. [1]

Bone density declines with HIV infection, and that decline can accelerate with antiretroviral therapy. Treatment with certain PIs or tenofovir heightened the risk of osteopenia and osteoporosis in longitudinal studies. SMART trial participants randomised to take antiretrovirals continuously had more bone loss than those randomised to CD4-based treatment interruptions. [2]

Indinavir and nelfinavir – two PIs rarely used today – resulted in poorly functioning osteoblasts, the cells responsible for new bone formation. Capeau and coworkers planned a series of cell studies to see if two currently prescribed antiretrovirals, lopinavir and atazanavir with or without ritonavir, affect cell properties that could promote bone loss in people with HIV.

The researchers used menenchymal bone marrow stem cells from young, healthy donors. They passaged these stem cells every 5 days to simulate ageing. For up to 40 days, they exposed the cells to doses of lopinavir, atazanavir, and ritonavir equivalent to maximum concentrations of those PIs typically attained in people taking them at prescribed doses.

Stem-cell numbers dropped sharply after 10 to 15 days of lopinavir (with or without ritonavir) and after 20 to 25 days of atazanavir (with or without ritonavir). The PIs had no effect on cell survival, a finding suggesting that decreased proliferative capacity accounted for these declines in stem cell number.

Assessing stem-cell senescence by measuring senescence-associated beta-galactosidase activity, the researchers found that both atazanavir and lopinavir significantly induced premature senescence. Increased reactive oxygen species (ROS) production in these cells suggested that oxidative stress may be responsible for cell ageing. Atazanavir and lopinavir also raised levels of superoxide dismutase, an antioxidant enzyme, in these cells. Both PIs increased expression of the cell-cycle inhibitors P16 and P21. Finally, the two PIs induced accumulation of prelamin A, a cell-ageing marker associated with cell senescence.

Mesenchymal stem cells normally differentiate into an even balance of osteoblasts and adipocytes (fat cells). With ageing, differentiation to adipocytes begins to outweigh differentiation to osteoblasts. Age-related bone loss is marked by increased bone marrow fat, which leads to decreased bone formation.

Stem cells pretreated with lopinavir or atazanavir lost their ability to differentiate into osteoblasts, a result Capeau suggested could mean these PIs irreversibly affect the menenchymal stem cell pool in bone marrow. Lopinavir-exposed stem cells also failed to differentiate into adipocytes, while atazanavir-exposed cells promoted increased differentiation into adipocytes. The investigators proposed that atazanavir could lower the number of osteoblasts in treated people by upsetting the balance between adipocytes and osteoblasts in bone marrow.

When Capeau and colleagues exposed stem cells to pravastatin and the study PIs, they found that this statin prevented PI-induced senescence, reduced oxidative stress, and restored the differentiation of stem cells to an even balance of osteoblasts and adipocytes.

The researchers concluded that their cell-study data “show that some PIs can alter osteoblast formation by a direct effect on osteoblast differentiation and also by inducing premature senescence of the bone marrow progenitors.”

References

1. Hernandez-Vallejo S et al. Some HIV protease inhibitors induce premature senescence and alter osteoblastic cell fate determination of human bone marrow mesenchymal stem cells. 2nd International Workshop on HIV and Aging. October 27-28, 2011. Baltimore, Maryland. Abstract: O_14.
2. Grund B et al for the INSIGHT SMART Body Composition Substudy Group. Continuous antiretroviral therapy decreases bone mineral density. AIDS. 2009;23:1519-1529.
   http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2748675/?tool=pubmed.

Comparing HIV-positive and negative injection drug users (IDUs) in a large Baltimore cohort, researchers determined that HIV infection independently raised the risk of objectively defined frailty and prefrailty. Frailty and prefrailty risks were highest in people with a CD4 count below 350 and a detectable viral load. [1]

Frailty boosts chances of hospital admission, disability, and death in older people without HIV.

In the Multicenter AIDS Cohort Study of HIV-positive and negative gay men. HIV infection raised the odds of earlier frailty [2], and frailty before combination antiretroviral therapy begins independently predicted AIDS or death [3]. But the impact of HIV on frailty risk and clinical outcomes is still in an early phase of study.

In this analysis Johns Hopkins University researchers focused on 1206 current or former IDUs with or without HIV infection seen from 2005 through 2009 in a prospective observational cohort. All cohort members were at least 18 years old and made twice-yearly visits for follow-up. The Hopkins team defined frailty (by the Fried system) as meeting 3 or more of 5 criteria: weakness determined by grip strength, slowed walking speed, weight loss, low physical activity, and exhaustion. They defined prefrailty as one or two of these criteria.

Of the 1206 IDUs assessed, 345 (29%) had HIV infection. Median age stood at 48 years, and one third in both the HIV-positive and negative groups were women. Higher proportions in the HIV group were African American (95.7% versus 87.6%), had less than a high school education (65.2% versus 57.0%), and were hepatitis C positive (93.3% versus 81.0%) (P < 0.05 for all comparisons). Lower proportions of HIV-positive cohort members were recent injectors (36.2% versus 47.4%), actively used alcohol (48.3% versus 56.8%), abused prescription drugs (6.4% versus 12.9%), or had a spouse or common-law partner (4.7% versus 8.8%) (P < 0.05 for all).

HIV-positive people had a median CD4 count of 290, a median CD4 nadir of 138, and a median viral load of 3.1 log (about 1250 copies). Half (51%) were taking combination antiretrovirals, and 21.7% had an AIDS diagnosis.

Overall frailty prevalence stood at 8.3%, with rates of 10.7% in the HIV group and 7.3% in the HIV-negative group; 59% of cohort members met prefrailty criteria. Through 4652 person-visits, both frailty and prefrailty proved more common in older IDUs, women, those with less than a high school education, people without a spouse or partner, those who abused prescription drugs, and those with depressive symptoms. African-American cohort members had a lower risk of prefrailty. Adjusting for all these factors, the researchers determined that HIV infection raised the prefrailty risk 28% (adjusted odds ratio [AOR] 1.28, 95% confidence interval [CI] 1.06 to 1.53), while raising the frailty risk 75% (AOR 1.75, 95% CI 1.27 to 2.39).

Compared with HIV-negative IDUs, HIV-positive cohort members had a higher risk of prefrailty or frailty with worse HIV disease status, as noted by the AORs (and 95% CIs) in Table 1.

The Hopkins teams evaluated frailty as a predictor of new hospital admissions in all 1206 cohort members from July 2005 through December 2009. During that time there were 374 hospital admissions, and the admission rate was significantly greater in frail than in nonfrail people (P = 0.006).

Compared with nonfrail cohort members, prefrail people did not have an independently higher risk of hospital admission, but frail people had a 60% higher risk (adjusted hazard ratio [AHR] 1.59, 95% CI 1.10 to 2.30). Female gender made hospital admission 66% more likely, homelessness raised the odds by 42%, active alcohol use by 32%, hepatitis C by 90%, and prescription drug use by 56%. Compared with HIV-negative people, HIV-positive people with a CD4 count under 350 and a detectable viral load had a doubled risk of hospital admission (AHR 2.12, 95% CI 1.61 to 2.79).

The Johns Hopkins investigators concluded that HIV infection boosts the risk of prefrailty and frailty in current and former IDUs. They proposed that “early identification of frail and prefrail IDUs may provide opportunities for arresting progression to adverse clinical states.”

References:

1. Piggott D et al. Frailty and incident hospitalization in a cohort of HIV-infected and uninfected injection drug users (IDUs). 2nd International Workshop on HIV and Aging. October 27-28, 2011. Baltimore, Maryland. Abstract O_06.
2. Desquilbet L et al. HIV-1 infection is associated with an earlier occurrence of a phenotype related to frailty. J Gerontol A Biol Sci Med Sci. 2007;62:1279-1286.
3. Desquilbet L et al. A frailty-related phenotype before HAART initiation as an independent risk factor for AIDS or death after HAART among HIV-infected men. J Gerontol A Biol Sci Med Sci. 2011;66:1030-1038.

Unfortunately the conference restricts public access to this research. Although abstracts available online (for a short time) the database to access abstracts is not very user friendly and the long URLs often change, making referencing problematic.

Also, few, if any studies are supported by webcasts or the option to view slides or full PDF posters.

The following studies are largely thanks to natap.org.

• Monitoring kidney function change with cobicistat
• Intracellular raltegravir concentrations better with twice-daily than once-daily dosing

Cobicistat is a pharmacokinetic (PK) booster currently in phase 3 studies that unlike ritonavir has no direct antiretroviral activity. This Gilead booster might facilitate a wider range of coformulated boosted medicines: with elvitegravir and Quad (boosted elvitegravir plus Truvada) and with products developed by other companies (darunavir and atazanavir).

An early caution is that cobicistat produces significant reductions in estimated glomerular filtration rate (eGFR). These do not indicate clinically significant changes but will pose a problem for interpretation of routine monitoring tests where clinical changes in eGFR are a concern.

If average actual GFR (aGFR) is used to monitor cobicistat, determined by iohexol clearance (a probe drug excreted almost exclusively by glomerular filtration), no changes are observed.

At ICAAC, Gilead researchers presented results from a placebo controlled study in 36 HIV negative participants with normal renal function (eGFR >80 mL/min) and 18 HV negative participants with mildly impaired renal function (eGFR 50-79 mL/min).

Participants with normal function were randomised (12 per group) to one of three groups: 150 mg cobicistat + placebo; 100 mg ritonavir + placebo; or double placebo for 7 days, with both eGFR and aGFR measured at baseline, day 7 and day 14 (following a 7 day washout). All participants with reduced renal function took cobicistat for seven days with similar monitoring.

Independent of baseline eGFR, volunteers taking cobicistat experienced significant average reductions in eGFR by day seven which resolved seven days after discontinuation, with but showed no significant changes in aGFR (see Table 1). Similar changes were seen using either Cockcroft-Gault or MDRD to calculate eGFR. Participants taking ritonavir or placebo showed no significant changes in either measure.

The researchers interpret these findings to show that true GFR is not affected by cobicistat which affects proximal tubular secretion of creatinine.

While these results are reassuring in terms of clinical impact of cobicistat it is unclear how patients using other medications that affect eGFR would be managed in order not to misinterpret a genuine impact on real GFR.

Source: Mascolini M. Kidney Function Change With Cobicistat Calculated in HIV-Negative Volunteers. NATAP.org
http://www.natap.org/2011/ICAAC/ICAAC_66.htm

Reference:

German P et al. Effect of cobicistat on glomerular filtration rate (GFR) in subjects with normal and impaired renal function. 51st ICAAC, 17-20 September 2011, Chicago. Abstract H2-804.

Intracellular concentrations of raltegravir stayed above the 95% effective concentration (EC95) in higher proportions of people taking this integrase inhibitor twice daily than in those taking it once daily, according to results of a 13-person study [1]. The average intracellular-to-plasma ratio was 0.37.

Raltegravir is licensed for adults at a dose of 400 mg twice daily with or without food. A randomised trial of twice- versus once-daily raltegravir for antiretroviral-naive people found that 318 of 382 (83%) in the once-daily group versus 343 of 386 (89%) in the twice-daily group had a viral load below 50 copies/mL after 48 weeks, a significant difference (-5.7%, 95% confidence interval -10.7 to -0.83, P = 0.044) [2]. The investigators concluded that “despite high response rates with both regimens, once-daily raltegravir cannot be recommended in place of twice-daily dosing.”

The study of plasma and intracellular raltegravir concentrations involved 12 people taking 400 mg of raltegravir twice daily and 1 taking 800 mg once daily for more than 1 week [1]. People on the twice-daily dose who had a viral load below 50 copies were offered a switch to once-daily dosing for at least 3 days so the investigators could assess raltegravir after once-daily dosing. Six people agreed.

In the twice-daily group, the researchers collected 26 paired samples of plasma and peripheral blood mononuclear cells (PBMCs) 2, 4 or 6, and 12 hours after dosing. In the once-daily group they collected 12 paired samples over the 24-hour dosing interval. Among people taking raltegravir twice daily, 3 had a detectable viral load; 2 of these were considered blips, and one load of 2649 copies came during the first 6 weeks of treatment.

No one taking raltegravir twice daily had a plasma trough concentration below the EC95 (14 ng/mL). Three of 12 had an intracellular trough below the EC95, at 7, 11.1, and 13.3 ng/mL.

Two of 6 people taking raltegravir once daily had a plasma trough below the EC95, at 7 and 13.8 ng/mL. Three of these 6 had an intracellular trough below the EC95, at 1.56, 4.06, and 6.56 ng/mL.

The mean ratio of intracellular-to-plasma concentrations was 0.37 and did not change over time. The ratio was higher than reported previously, probably because cell-wash steps in older methods flushed out some intracellular drug.

The researchers proposed that the high plasma and intracellular troughs with twice-daily raltegravir “may contribute to the efficacy observed with this regimen.”

UK researchers just published results of a 24-person study comparing plasma and intracellular raltegravir concentrations with once- and twice-daily dosing, with or without darunavir/ritonavir [3]. Study participants were taking 400 mg of raltegravir twice daily for at least 21 days. They added 800/100 mg of darunavir/ritonavir once daily for 14 days. During that 14-day period, people were randomised to continue twice-daily raltegravir (group 1) or to switch to 800 mg once daily (group 2).

Geometric mean ratios (and 90% confidence intervals) of raltegravir area under the concentration-time curve without and with darunavir/ritonavir for group 1 were 0.90 (0.73 to 1.44) in plasma and 1.02 (0.81 to 1.67) in cells and for group 2 were 1.21 (1.03 to 1.77) in plasma and 1.27 (1.07 to 1.94) in cells. These researchers concluded that “no remarkable interactions between darunavir/ritonavir and raltegravir in plasma or cells were seen.”

References

1. Sandkovsky US, Swindells S, Robbins BL, Nelson SR, Acosta EP, Fletcher CV. Measurement Of plasma and intracellular concentrations of raltegravir in patients with HIV infection. 51st Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC). September 17-20, 2011. Chicago. Abstract A1-1738b.
2. Eron JJ Jr, Rockstroh JK, Reynes J, et al. Raltegravir once daily or twice daily in previously untreated patients with HIV-1: a randomised, active-controlled, phase 3 non-inferiority trial. Lancet Infect Dis. 2011 Sep 19.
   http://www.thelancet.com/journals/laninf/article/PIIS1473-3099(11)70196-7/fulltext
3. Jackson A, Watson V, Back D, et al. Plasma and intracellular pharmacokinetics of darunavir/ritonavir once daily and raltegravir once and twice daily in HIV-infected individuals. J Acquir Immune Defic Syndr. 2011 Sep 15. Epub ahead of print.
   http://www.ncbi.nlm.nih.gov/pubmed/21926632

The conference has an open-access searchable abstract database online.
http://www.ias2011.org/

The ‘Programme at a glance’ can be searched for key words but requires a free software upgrade Silverlight which is quick and easy to do. Then from this page you can search abstracts or presentations.
http://pag.ias2011.org/

Sessions with PowerPoint slides or webcasts have relevant icons next to them. As with previous years, the PowerPoint links on the left under the session time are not active, so to download PowerPoint files scroll down to the bottom of the session page.

Reports in this issue of HTB include:

• Cure research and viral reservoirs
• Orange Farm circumcision results dispel concerns about risk compensation
• Randomised trial of ART in TB patients with high CD4 counts

Unless stated otherwise, all references are the Programme and Abstracts of the 16th IAS Conference on HIV Pathogenesis, Treatment and Prevention, 17–20 July 2011, Rome.

In addition to the prevention studies and the progress on pipeline drugs that made most headlines (see the previous issue of HTB), a third set of presentations through the meeting supported the IAS Conference Statement on the need for the cure. [1]

That ‘the Cure’ might again re seen as an achievable goal for research was resuscitated in keynote lectures from NIAID lead Anthony Fauci several years ago and several medical networks, including the IAS, already hold annual meetings to coordinate different approaches. US public funding now requires cure research as a key work stream for HIV treatment networks.

Whilst the scientific challenge of curing HIV has been the consitent focus for many committed researchers, the renewed level of funding is clearly driven by the financial challenge of providing lifelong global treatment. Even when the generic costs are reduced to less than $100 per person per year, current treatment programmes need to more than double and then be sustained for coverage to meet the existing need. Perhaps increasing the resources for cure research is therefore perhaps also the most ethical way to be able to withdraw from responsibility for funding global treatment.

However, these sessions in Rome were mostly held in the smaller rooms, filled to capacity. They were also frustratingly insular with few being available as webcasts or slides to download, including the plenary session by IAS President Elect, Françoise Barré-Sinoussi. [2]

Neither the IAS pre-conference workshop (New concepts in HIV Immunopathogenesis, Treatment and Vaccine Strategies) nor the rapid summary report from that workshop in the main IAS conference by Nicolas Chomont was webcast. However, slides are available from some of the workshop sessions and for the summary by Chomont. [3, 4]

A satellite meeting cosponsored by amfAR and IAS also was not webcast, although the slide presentations can be downloaded. [5] This meeting focused on whether:

• viral replication persists on HARRT;
• eradication research can progress in animal models or is dependent on human studies;
• eradication is most likely to come from targeting the viral reservoir or more recent approaches using gene therapy.

Several other presentations at the conferences looked at strategies to selectively reactivate the reservoir of latently infected resting CD4 cells, either at the pre- or post-integration step. This challenge is highlighted by the pool being estimated to be less than one in a million resting cells for someone on stable treatment with undetectable viral load.

Some researchers believe that success in this goal might eradicate HIV, though this is dependent on whether current treatment suppresses replication sufficiently to halt viral evolution. This might turn out to be possible, as it has been the conclusion from several intensification studies that have shown no further reduction on low level viraemia after increasing the potency of a three drug combination with a fourth drug, including an integrase inhibitor. [6] An oral presentation from Brunetta and colleagues reported no impact on CD4 reservoirs in gut-associated lymphoid tissue obtained from sigmoid colon biopsies at 48 weeks of follow up following intensification with raltegravir. [7]

A case reported by Chun and colleagues in an article in AIDS last year perhaps also supports this view. [8] This paper described one person – ‘the Toronto patient’ – who was enrolled and treated prior to seroconversion. Viral load was suppressed to <50 copies/mL on HAART for more than ten years, driving the pool of infected CD4 T cells down to less than one in 1.7 billion cells. Against advice, the person decided to stop treatment under research conditions. Viral rebound only occurred after 50 days with an increased to 1600 copies/mL followed by spontaneous suppression by day 95 back to undetectable. Subsequently, viral load steadily increased to approximately 8600 copies/mL on day 143 when treatment was restarted.

So one interpretation of this case could be to emphasise the difficulty of eradication – even with such an early, effective and sustained level of treatment. Another interpretation is that eradication might almost have been achieved. Perhaps another month, or year, or few years on treatment might have been sufficient to final exhaust the remaining pool on resting infected cells. This study is unlikely to be repeated.

Another more optimistic, but also unexplained, set of cases includes the 32 patients from the ANRS Visconti study reported at CROI this year. This group received antiretroviral therapy within ten weeks of seroconversion for a median of three years (1-7.5 years). Five of these people sustained virological control for a median of 6 years (range 4-10) after treatment discontinuation. [9] It is unclear why similar cohorts (Rosenberg, Walker et al.) have not had the same success.

However, over time, so long as treatment is maintained, the resting pool of infected cells might be able to be agitated to become active, most likely by using multiple approaches. This could reduce the time needed to eliminate this reservoir from decades down to years – with residual virus mopped up by antiretrovirals, allowing treatment to be stopped.

Importantly, research into activation of latently infected cells is already investigating a broad group of drugs that are already licensed. Studying HIV transcription at the molecular level is driving the understanding of differences between latent and productively infected CD4 cells including HDAC-1 and methylation sites in latent infection with the hope that these targets might switch cells away from latency.

A comprehensive review of potential molecules by Sharon Lewin and Christine Rouzioux in the 24 April edition of AIDS [10] was the basis of one of the presentations at the IAS cure workshop. [11]

These include histone deacetylase (HDAC) inhibitors (vorinostat, romidepsin, panabinostat, entinostat, belinostat, givinostat and at least nine others), a methylation inhibitor (5-azacytidine), cytokines (IL-7 – Eramune group, IL-15) and an antialcoholic (disulfiram). Immune modulators with similar potential incude antibotics (minocycline), antirheumatics (auranofin), anti-PD-1 (MDX-1106) and protein kinase C modulators (bryostatins and others). Many of these compounds are already being studied in HIV-positive people.

An oral presentation by Claire Vandergeeten from the Vaccine and Gene Therapy Institute reported results from in vitro studies that suggest that IL-15 therapy may be used as a strategy to deplete the latent HIV reservoir while IL-7 maintains the reservoir both in vitro and in patients on stable HAART. [12]

This research is important and exciting. Many of these compounds have been studied for several years and for other studies are ongoing. A combination therapy approach is therefore likely to have a greater chance of success, for example, valproic acid or vorinostat plus prostratin. [13]

However, other researchers believe that an as yet unidentified sanctuary site would prevent the latent reservoir from being a slowly diminishing pool that theoretically might wear itself out, with or without stimulation to do so. This includes Steven Deeks at UCSF who co-chairs the IAS working group on cure research and was heads a recent $4 million grant from the US NIH to develop a strategy to eradicate HIV. [14]

This raises the importance of finding out whether any compartments are actively replenishing the viral reservoir, currently untouched by the maximal suppression measured by plasma viral load.

If this is the case, then any strategy to activate latently infected cells would not be successful. In November 2010, Yuki and colleagues reported that ongoing replication (measured by unspliced HIV RNA in CD4 T cells) is present in higher levels in gut sites (duodenum, terminal illeum, right colon and rectum) compared to that in PBMCs in patients on HAART with viral load suppressed to <40 copies/mL. [15]

The same group then showed that intensification with raltegravir in this group of patients produced a reduction in levels of unspliced RNA in the terminal ileum and a trend towards reduced T cell activation in other gut sites. [16]

For these researchers, looking for the impact of intensification studies in plasma viral load is searching in the wrong place. If tissue compartments are a source of ongoing viral replication, the spillover pool found in plasma may have limited relevance.  Other cellular sites distinct from CD4 T cells contributing to the latent cellular reservoir include macrophages, hematopoetic stem cells, naive T cells, astocytes, thymocytes and others.

Also, Maria Buzon and colleagues in Nature Medicine in 2010 reported increases in episomes following raltegravir intensification (shown by an increase in 2LTR in PMBCs) as evidence of de novo infection and reduced levels of activation. The extremely low level or replication is used to account for the non-development of resistance even over years. Also perhaps indicating that the chronic source for new virus drives a limited number of rounds of infection. [17]

Finally, in one of the few late breaker presentations with slides posted online, Hiroyu Hatano working with Deeks’ group at UCSF reported that viral persistence was consistently associated with markers of immune activation and dysfunction (including PD-1 expressing cells) rather than plasma viral load. These measures were particularly elevated in people on treatment with low CD4 counts despite treatment (less than 350 cells/mm3 compared to higher), suggesting that patients below this cut-off present a more difficult and sobering challenge to any approach to a cure.

More optimistically, Hatano noted that the preferential expression of PD-1 by latently infected cells supports targeting this molecule as a strategy for depleting HIV reservoirs. The AIDS Clinical Trials Group (ACTG) in the US is planning an exploratory trial of Merck’s experimental PD-1 inhibitor for this purpose. [18]

References:

Unless stated otherwise, all references are to the abstracts and conference programme of the 6th IAS Conference on HIV Pathogenesis, Treatment and Prevention, 1720 July 2011, Rome.

1. The Rome Statement for an HIV Cure: Major HIV/AIDS Stakeholders Call for HIV Cure Research to be Accelerated. International AIDS Society, July 2011.
   http://www.iasociety.org/Default.aspx?pageId=583
2. Barré-Sinoussi F. Discussing past and future accomplishments of HIV research. Abstract MOSS0103.
   http://pag.ias2011.org/session.aspx?s=83
3. Towards an HIV Cure: Insight into Residual Viral Replication, Establishment of Reservoirs and Understanding Mechanisms of Persistence. Conference workshop WEWS03.
   http://pag.ias2011.org/session.aspx?s=70
4. Chomont N. New concept in HIV: HIV immunopathogenesis, treatment and vaccine strategies – report back from pre-conference. Symposium WESY01.
   http://pag.ias2011.org/session.aspx?s=15
5. Controversies in HIV Cure Research. Joint IAS and amfAR workshop MOSA02.
   http://pag.ias2011.org/session.aspx?s=39
6. McMahon D et al. Short-course raltegravir intensification does not reduce persistent low-level viremia in patients with HIV-1 suppression during receipt of combination antiretroviral therapy. Clin Infect Dis. 2010 March 15; 50(6): 912–919.
   http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2897152/
7. Kovacs C et al. Effect of intensification of long-term highly active antiretroviral therapy (HAART) with raltegravir on proviral HIV-1 DNA in gut associated lymphoid tissue (GALT): a randomized, placebo controlled trial. 6th IAS, Rome 2001. Oral abstract MOAA0103.
   http://pag.ias2011.org/Abstracts.aspx?SID=53&AID=1432
8. Chun T-W et al. Rebound of plasma viremia following cessation of antiretroviral therapy despite profoundly low levels of HIV reservoir: implications for eradication. AIDS: 27 November 2010 – Volume 24 – Issue 18 – p 2803–2808.
   http://journals.lww.com/aidsonline/Fulltext/2010/11270/Rebound_of_plasma_viremia_following_cessation_of.6.aspx
9. Saez-Cirion A et al. Long-term HIV-1 control after interruption of a treatment initiated at the time of primary infection is associated to low cell-associated HIV DNA levels: ANRS VISCONTI study. 18th CROI 2011, Abstract 515.
   http://www.retroconference.org/2011/Abstracts/41477.htm
10. Lewin SR, Rouzioux C. HIV cure and eradication: how will we get from the laboratory to effective clinical trials? AIDS: 24 April 2011 – Volume 25 – Issue 7 – p 885-897.
    http://journals.lww.com/aidsonline/pages/articleviewer.aspx?year=2011&issue=04240&article=00001&type=Abstract
11. Lewin S. Contribution of the immune system to HIV persistence. Workshop: Towards an HIV Cure: insight into residual viral replication, establishment of reservoirs and understanding mechanisms of persistence, July 2011, Rome.
12. Vandergeeten C et al. Differential impact of IL-7 and IL-15 on HIV reservoir persistence. 6th IAS, Rome 2011. Oral abstract MOAA0101.
    http://pag.ias2011.org/Abstracts.aspx?SID=53&AID=2604
13. Reuse S et al, Synergistic activation of HIV-1 expression by deacetylase inhibitors and prostratin: implications for treatment of latent infection. PLoS ONE 4(6): e6093.
    http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0006093
14. NIH supports new research strategy for finding a cure for HIV. (July 2011).
    http://www.ucsf.edu/news/2011/07/10201/nih-supports-new-research-strategy-finding-cure-hiv
15. Yukl SA et al. Differences in HIV burden and immune activation within the gut of HIV-positive patients receiving suppressive antiretroviral therapy. Journal of Infectious Diseases. Published online 12 October. 2010;202:000-000. DOI: 10.1086/656722.
    http://jid.oxfordjournals.org/content/202/10/1553.full
16. Yukl SA et al. Effect of raltegravir-containing intensification on HIV burden and T-cell activation in multiple gut sites of HIV-positive adults on suppressive antiretroviral therapy. AIDS. 2010 Oct 23;24(16):2451-60.
    http://journals.lww.com/aidsonline/Abstract/2010/10230/Effect_of_raltegravir_containing_intensification.4.aspx
17. Buzón M et al. HIV-1 replication and immune dynamics are affected by raltegravir intensification of HAART-suppressed subjects. Nature Medicine 16, 460–465 (2010).
    http://www.nature.com/nm/journal/v16/n4/full/nm.2111.html
18. Hatano H et al. Cell-based measures of viral persistence are associated with immune activation and PD-1+-expressing CD4+ T cells. 6th IAS Conference, Rome 2011. Oral late breaker WELBA01.
    http://pag.ias2011.org/abstracts.aspx?aid=4801

Amidst the excitement about HPTN 052 at the Rome IAS meeting, the results of the ANRS Orange Farm circumcision programme received little publicity, despite stunning data.

Orange Farm was the site of the first of three randomised control trials that showed that circumcision reduces the risk of men contracting HIV in a predominantly heterosexual population. Following the trial, the researchers implemented a scaled up circumcision programme in Orange Farm. Bertran Auvert presented a late-breaker describing the results of this programme. [1]

There are several important findings from this study:

• Post-trial uptake was large. Orange Farm has now carried out approximately 25,000 circumcisions.
• No deaths or permanent injuries have occurred due to circumcision. There have been ten hospitalisations and in all these cases the adverse events were resolved.
• The benefit of circumcision on HIV incidence is durable.
• There was no evidence that incidence was affected by risk compensation.

Orange Farm is a township of about 110,000 adults about 45kms from Johannesburg. Since January 2008, free voluntary medical male circumcision to all boys and men older than 15 has been offered by the ANRS-sponsored project. The intervention includes community mobilisation and outreach, counselling, condom distribution, STI treatment, HIV voluntary counselling and testing and ART if eligible.

A baseline cross-sectional survey was done in 2007. This was a random sample of just under 1,200 males aged 15 to 49 years. The response rate was 74%. Male circumcision status was determined by genital examination. A second cross-sectional survey was done in 2010. It was almost the same size and the response rate was 88%. This survey included a background and sexual behaviour questionaire. Again male circumcision status was determined by genital examination. Blood samples were tested for HIV, ARVs and for recent infection (within 6 months) using a population incidence detuned HIV test (Calypte EIA BED).

Uptake

Male circumcision prevalence changed from 15.6% (95%CI: 13.6%-17.8%) of 15-49 year-olds in 2007 to 49.4% (95%CI: 46.5%-52.3%) in 2010. Using this data, the researchers calculated uptake, which increased across all age groups in the 2008-2010 period. In 15-49 year-olds it was 40% (95%CI: 38.0% to 43.5%) and 49.1% in 20 to 24 year-olds (95%CI: 42.1% to 52.4%). This substantial increase led Auvert to comment, “We are changing the social norms.”

In a comparison of 590 circumcised versus 605 uncircumcised men, circumcised men were younger, more educated, less likely to be married and more often aware of their HIV status. No difference in sexual behaviour was detected. For example reported condom usage was consistent (adjusted OR: 0.84; 95%CI: 0.63-1.1; p=0.26).

HIV prevalence and incidence

Among 586 uncircumcised men in the survey, 117 were HIV-positive (20%; 95%CI: 16.7%-23.2%). Among circumcised men, 36 out of 582 men were HIV-positive (6.2%; 95%CI: 4.3%-8.2%). This is a 55% reduction (95%CI: 39% to 70%).

In the 15-34 age group, the BED assay indicated that incidence in uncircumcised men was 3.7 per 100 person-years (95%CI: 2.2-6.1) and 0.6 per 100 person-years in circumcised men (0.19-1.9). The adjusted relative risk was 0.24 (95%CI: 0-0.66). Interestingly, this is equivalent to a 76% reduction that is exactly what the as-treated effect of the Orange Farm randomised control trial was.

Because of problems with the BED assay, a modelling exercise was also done in which HIV incidence was calculated from HIV prevalence data to determine the effect of circumcision on incidence. In this separate analysis the reduction in incidence was 83% (95%CI: 64%-98%) in 15-34 year-olds, consistent with the BED-based estimate.

It was estimated that without male circumcision, HIV prevalence would have been 25.1% higher in 15-49 year-olds in Orange Farm (95%CI: 13.1%-39.1%) and HIV incidence would have been 57.9% higher (95%CI: 17.0%-131%).

comment

The key limitation to a study like this is that it is observational. But randomised controlled trials have already proven the efficacy of circumcision. This was the first prospective study to show the benefits of circumcision in a real-world operational setting.

A widely expressed concern about circumcision is that risk compensation would undo much of its benefit. The finding that the operational effect of circumcision matched the as-treated effect of the Orange Farm clinical trial addresses this concern. The lack of difference in reported condom usage also indicates that risk compensation is not a factor, but this must be discounted against the fact that survey participants give answers that they believe are consistent with societal expectations rather than what they actually do.

There should be no further objections to scaling up voluntary medical male circumcision in appropriately equipped facilities. The South African Department of Health has committed to scaling up circumcision and implementation is taking place in several provinces. PEPFAR and the Gates Foundation have both committed to funding circumcision programmes across sub-Saharan Africa. However South African guidelines have still not been published, albeit that a draft exists. These guidelines need to be finalised and published. An implementation plan also needs to be devised.

The Orange Farm researchers hope soon to be able to do an analysis of the effect of medical male circumcision on incidence in women.

Reference:

Auvert B. 2011. Effect of the Orange Farm (South Africa) male circumcision roll-out (ANRS-12126) on the spread of HIV. 6th IAS Conference on HIV Pathogenesis, Treatment and Prevention. 17-20 July 2011, Rome, Italy.
http://pag.ias2011.org/Abstracts.aspx?SID=43&AID=4792

Thank you to Bertran Auvert, Dino Rech and Dirk Taljaard for assisting my understanding of this study.

Declaration of interest: Nathan Geffen am a member of the Orange Farm circumcision scientific committee.

The benefits of initiating antiretroviral treatment (ART) in TB co-infected patients with CD4 counts below 350 cells/mm3 have been demonstrated in a number of recent studies. [1-2]

Now an open-label randomised controlled trial by Nanteza and colleagues has been published that looks at patients with higher CD4 counts. The trial, run in Uganda, compared 6 months of ART concurrent with TB treatment versus only TB treatment in patients with CD4 counts > 350 cells/mm3. The trial was small, but it provides some evidence that there is clinical benefit to placing patients with high CD4 counts on ART. [3]

From October 2004 through September 2008, 4,951 people were screened. Of these 250 patients co-infected with TB and HIV and with CD4 counts >350 cells/mm3 were enrolled. Of these 232 were randomised and 214 remained on the trial and eligible for analysis. The final control and intervention arms each had 107 patients. Patients in the intervention arm were given ART (abacavir, lamivudine and AZT) for six months. Participants were followed up for 24 months. The trial was designed and approved before several studies demonstrated that structured treatment interruptions are not a viable treatment option. The authors explained that the rationale of the study was that a punctuated course of antiretroviral therapy in patients with high CD4 cell counts would suppress viral replication during therapy for tuberculosis, block the effects of immune activation on T cells harboring HIV, slow the pace of HIV disease progression, and improve clinical outcomes.

The baseline characteristics of the patients were mostly well matched. There were slightly more men in the control arm (63% versus 52%). Nearly all patients had at least one TB symptom, especially cough. Median age was 31 years. Nearly 90% of patients were both smear and culture-positive. Interestingly 8% of patients were smear-positive and culture-negative, while only 3% were smear-negative and culture-positive. The intervention and control arms had similar median CD4 counts (between 500 and 550 cells/mm3) and median viral load (4.6 and 4.7 log10 copies/mL).

The primary endpoint was a composite of CD4 cell count <250 cells/mm3, clinical AIDS, or death. In the intervention arm, 17 people reached this endpoint versus 25 in the control arm (p=0.17). Most people reached the endpoint on the basis of CD4 count, 15 in the intervention arm and 18 in the control arm. They initiated lifelong ART. Although not statistically significant, there were consistently fewer endpoint events in the intervention arm throughout the trial. At 12 months of follow-up the difference between the arms reached significance (98% and 90%, respectively; p=0.02), but became non-significant by the end of the follow-up period.

There were two deaths in the ART arm and no clinical endpoints. There were three clinical endpoints and four deaths in the control arm. Despite the tiny numbers, this was significant (p=0.048).

In the intervention arm, 86% of participants achieved a viral load <400 copies/mL at three and six months. Viral load rebounded upon discontinuation of treatment to near baseline. The average viral load of the control group did not change significantly over the 24-month period.

There were 45 versus 28 adverse events in the control and intervention groups respectively. When considered individually, the risk of a grade 3 or 4 adverse event was 76% greater in the control arm than in the intervention arm (rate ratio, 1.76; 95% CI: 1.24–2.53). About half the adverse events took place during the six months treatment (ART or TB) stage of the study. Neutropenia was high and not significantly different in both arms (17 versus 25% in the intervention and control arms respectively). The authors therefore concluded that neutropenia is common in patients with tuberculosis, even when CD4 counts are >350 cells/mm3 and that treatment with concurrent antiretroviral therapy only partially mitigates the effect of HIV infection on bone marrow suppression. As would be expected in a trial of people with relatively high CD4 counts, no Immune Reconstitution Syndrome was detected.

No patients were culture-positive after six months of TB therapy. The average time to culture conversion was 37.5 days in the intervention group versus 29 in the control, but this was not significant (p=0.37).

The authors state that the trial provides limited further support for early initiation of treatment.

comment

Although small and despite the outdated and short treatment intervention, this study does provide limited support for initiating all HIV-positive patients with TB on ART, even at high CD4 counts.

The World Health Organisation (WHO) recommends that treatment be provided to all patients with TB irrespective of CD4 count. The South African government has recently announced that it will treat all patients with CD4 counts <350 cells/mm3. This study offers some evidence, albeit not compelling, that South Africa should go further and implement the WHO recommendation. It seems likely that South Africa’s new HIV and TB National Strategic Plan for 2012-2016 will provide for this.

Also interesting was that in this Ugandan setting 25% of the TB and HIV co-infected patients who screened for the trial had CD4 counts above 350 cells/mm3.

In Khayelitsha, Cape Town, nearly one in five HIV-positive patients presents with a CD4 count >500 cells/mm3 (communication with MSF). These statistics show that the question of determining the optimal starting point affects many people and is an important one.

We look forward to the results of the TEMPRANO and START trials that have been designed to answer this question.

References:

1. Abdool Karim SS et al. Timing of initiation of antiretroviral drugs during tuberculosis therapy. N Engl J Med 2010; 362:697–706.

http://www.nejm.org/doi/full/10.1056/NEJMoa0905848

2. Blanc FX et al. Significant enhancement in survival with early (2 weeks) vs. late (8 weeks) of highly active antiretroviral treatment (HAART) in severely immunosuppressed HIV-infected adults with newly diagnosed tuberculosis. Vienna: International AIDS Society, 2010:284–5.

http://pag.aids2010.org/Abstracts.aspx?AID=17091

3. Nanteza MW et al. 2011. A randomized trial of punctuated antiretroviral therapy in Ugandan HIV-seropositive adults with pulmonary tuberculosis and CD4+ T-cell counts >= 350 cells/uL. JID 2011:204 (15 September)

http://jid.oxfordjournals.org/content/204/6/884.abstract

Introduction

The biannual IAS Conference on HIV Pathogenesis, Treatment and Prevention is more scientifically focused than the World AIDS Conference held in alternate years and is considerably smaller. This makes both attending and reporting more manageable and concentrated.

This year the conference was held in Rome. While plenary lectures were held in the concert halls designed by Renzo Piano (the architect designing the Shard Tower in London), smaller meetings were often in rooms with a capacity of only 50 people and the poster and exhibition halls were less than ideal, being held in the venues garages.

Even prior to the conference, the frenzy to gain media coverage filled email boxes with press releases that made it clear that the meeting would be dominated by prevention studies.

The leading prevention reports, first and most importantly, involved the reduction in transmission from use of HIV treatment for HIV-positive people. The risk is not reduced to zero, but it is getting close, especially when condoms remain the mainstay of prevention work. It means that if a condom breaks, slips off, or is not used at all, an HIV positive person who has had an undetectable viral load for over six months would find it difficult to transmit HIV.

The shift in medical consensus is dramatic. While the Swiss Statement three years ago was met with anger publically from many prominent doctors, in private most also recognised that viral load was the driving factor behind transmission risk. It was good to hear Pietro Vernazza who authored the Swiss paper ask Myron Cohen after presenting the results of HPTN 052 on whether Cohen’s new results had prompted a change of heart.

IAS in Rome included four oral presentations from the HPTN 052 study. Together they showed that HIV-positive people in high incidence resource limited settings (predominantly Africa, Asia and Latin America) who started treatment at a CD4 count of 350-550 reduced the risk of transmitting HIV to their HIV-negative primary partner by 96% compared to people waiting until their CD4 count was 250. This was a study that intensely integrated other prevention strategies – condom provision and counselling reduced transmission too, but treatment extended this significantly further.

The second way that treatment reduces transmission was supported by new studies reporting the benefits of PEP/PrEP strategies. These involved HIV negative people taking a daily pill of tenofovir/FTC, or tenofovir alone, which led to reductions in their risk of catching HIV.

As with previous meetings, the conference has an open-access searchable abstract database online.

http://www.ias2011.org

However, many oral presentations are not included as webcasts or PowerPoint slides.

The ‘Programme at a glance’ can be searched for key words but requires a free software upgrade Silverlight which is quick and easy to do. Then from this page you can search abstracts or presentations.

http://pag.ias2011.org/

Sessions with PowerPoint slides or webcasts have relevant icons next to them. As with previous years, the PowerPoint links on the left under the session time are not active, so to download PowerPoint files scroll down to the bottom of the session page

We have also included reports from the 3rd International Workshop on HIV Pediatrics immediately preceding IAS 2011.  This small annual meeting is becoming quite established and although abstracts are often submitted to both meetings, in this one they may often get an oral presentation instead of just a poster. For those specialising in paediatrics this meeting is a welcome opportunity to present and discuss work in a dedicated forum. Abstracts and presentations are online. We have included references from both meetings in our paediatric reports.

http://regist2.virology-education.com/abstractbook/2011_8.pdf

http://regist2.virology-education.com/2011/3HIVped/15_July.html

Reports in this issue of HTB include:

• Webcasts for major research at IAS

• Treatment is prevention: ARV treatment in HPTN-052 reduces transmission by at least 96%: single transmission in treatment arm occurred prior to viral suppression

• Daily oral tenofovir/FTC PrEP reduces heterosexual transmission by 63% in the TDF2 study

• Tenofovir/FTC vs tenofovir as daily oral PrEP: preliminary results from Partners PrEP

• Elvitegravir vs raltegravir: 48 week results in treatment-experienced patients

• Dolutegravir: 48 week results from phase II study in treatment-naïve patients

• Lersivirine: 48 week results compared to efavirenz in phase 2 treatment-naïve study

• Maraviroc plus atazanavir/r without nukes versus standard of care: 48 week results

• SPARTAC trial: treatment in primary infection for 48 weeks shows small delay in disease progression

• Hearing loss not associated with HIV in MACS and WIHS cohorts

• Pharmacokinetics of darunavir and fosamprenavir in pregnancy

• Low birth weight and preterm delivery

• Hormonal contraception and HIV transmission risk

• No difference in AIDS-free survival in children starting ART with a CD4% between 15%–24% compared to deferring until less than 15% in the PREDICT trial

• Paediatric antiretroviral pipeline: update on etravirine and maraviroc

• Prematurity not associated with early mortality in infants on ART

• More metabolic abnormalities in children receiving a PI compared to NNRTI in NEVEREST study

• Free online resource for treatment decisions without access to genotype resistance tests

Unless stated otherwise, all references are the Programme and Abstracts of the 16th IAS Conference on HIV Pathogenesis, Treatment and Prevention, 17–20 July 2011, Rome.

Firstly, treatment as prevention – most notably in a study called HPTN-052 and PrEP studies. The Treatment Is Prevention session include links to abstracts, slides and webcasts from the HPTN-052 study.

http://pag.ias2011.org/session.aspx?s=98

Secondly, research relating to reservoirs, eradication and the cure was established throughout the programme. This included overviews of the different ways that this could be approached and preliminary results for early potential targets. Unfortunately very few of the lectures in these sessions are webcast. However, the overview by Anthony Fauci, is online and worth watching to understand the new strengthening steer from the US that dominates global research.

Tony Fauci: 30 years of HIV/AIDS: a scientific journey and a look to the future

http://pag.ias2011.org/flash.aspx?pid=409

Towards an HIV cure: insight into residual viral replication, establishment of reservoirs and understanding mechanisms of persistence

http://pag.ias2011.org/session.aspx?s=70

Towards an HIV cure: new strategies for an old challenge

http://pag.ias2011.org/session.aspx?s=15

Controversies in HIV cure research satellite meeting

http://pag.ias2011.org/session.aspx?s=39

Thirdly, there were important studies about New drugs and diagnostics and new strategies with oral presentations on new integrase inhibitors (elvitegravir and dolutegravir) and NNRTIs (lersivirine), using of HSV drugs (acyclovir/valacyclovir) and currently approved meds (maraviroc/atazanavir). Plus, also in the late breaker session, an exciting new rapid antibody test for cryptococcal meningitis developed in the UK that can be used at the point of care. These were presented mainly in the following two sessions.

New drugs and strategies

http://pag.ias2011.org/session.aspx?s=55

Late breaker Track B

http://pag.ias2011.org/session.aspx?s=44

Simon Collins, HIV i-Base

Four of the six oral presentations in the ‘Treatment is prevention: the proof is here’ session reported on the results from HPTN 052. [1] This study had been unblinded four years earlier than planned due to a review by the data and safety monitoring board (DSMB), with all patients now being offered active treatment.

In summary, HIV-positive people on treatment had a 96% reduction in sexual transmission to their HIV-negative partners if they started ARVs with a CD4 count of 350-550 cells/mm3 compared to waiting until it dropped below 250 cells/mm3. As with all prevention studies, condoms, testing and intensive counselling was included throughout the study.

The main study results were presented by Myron Cohen from University of North Carolina. [2]

HPTN 052 screened over 10,000 couples in order to randomise 1763 HIV-positive people with CD4 counts 350-550 to either immediate or delayed HIV treatment (CD4 confirmed <250 or an AIDS-defining illness). Screening failure was mostly due to CD4 or other criteria in the positive partner, but 300 couples were already both HIV-positive. This was an international study predominantly recruiting in Africa (Botswana, Kenya, South Africa and Zimbabwe, n=954), Asia (India and Thailand, n=531) and Latin America (Brazil, n=276). Men and women were equally distributed as the positive partners. Median baseline CD4 count was 436 cells/mm3 (IQR 365-522) and viral load was approximately 25,000 copies/mL (IQR 6,000-80,000) respectively.

This was generally a low risk population with only 6-8% reporting recent unprotected sex and only 16% aged 18-25 years (~ 60% were 25-40 years and ~20% > 40 years).

The primary transmission endpoint was the prevention of virologically linked transmissions with a primary clinical endpoint of WHO Stage 4 events (including pulmonary TB, severe bacterial infections and death).

Transmission events (n=39) occurred significantly less frequently in the immediate (n=4) compared to the deferred (n=35) treatment arms, p<0.0001. Of these, only 28/39 were linked transmissions (within the couple) with 1 case in the immediate arm vs 27 cases in the deferred arm, p=0.001 (see below for details). Eleven transmissions were either unlinked or undetermined. This translated to incidence rates of linked transmission of 0.1 (95%CI 0.00-0.04) vs 1.7 (95%CI 1.1-2.5) per 100 person years respectively over a median follow-up of 1.7 years.

The single transmission in the immediate treatment arm was detected at the first follow-up visit. However viral diversification analysis estimated that transmission occurred prior to the positive partner initiating treatment (baseline 87,000 viral load) or certainly prior to viral suppression to <400 copies/mL which was recorded at day 28.

Other transmission risk factors were similar between arms, including rates of STIs (low at <5% in both index and partner at baseline and during the study), sexual activity (approximately 70%) and condom use (>90% by all throughout).

Viral suppression (<400 copies/mL) was maintained by >90% of participants in the immediate arm. There was a slow increase in this percentage over time in the deferred arm as people started treatment (from <10% over the first year, 20% by month 24 and increasing to 50% at month 45, though with much fewer patients). The median viral load closest to the time of transmission in the deferred arm was considerable at 80,000 copies/mL but had a wide range from 600 to 630,000 copies/mL.

In multivariate analysis, treatment was the strongest protective effect [HR=0.04, 95%CI 0.01-0.28] compared to condom use [HR=0.33; 95%CI 0.12-0.91]. Factors associated with increased transmission included baseline viral load [per log increment: HR 2.84, 95%CI 1.51-5.41] and baseline CD4 count [per 100 count increment: HR 1.24 95%CI 1.00-1.54].

The second presentation by Susan Eshelman from Johns Hopkins University School of Medicine focused on the analysis of linked transmission. [3] This included a helpful introduction to the three types of phylogenetic analyses used: phylogenetic analysis of HIV pol sequences using population sequencing, and statistical analysis of genetic distances from pol sequence pairs for the clearest cases (n=26), and phylogenetic analysis of env sequences obtained by deep sequencing for more complex cases (n=12). Together these provided a high level of reliability for indentifying whether the source of new infections was the HIV-positive partner or whether this was from another partner.

Transmissions in previous serodifferent couple studies have been from outside the main relationship in 25-50% cases.

The deep sequencing (‘ultradeep pyrosequencing’) supported linked two further cases and confirmed non-linkage for seven others (4 in the immediate and 3 in the deferred arm). Three cases remained unidentified (all in the deferred arm). Transmission linkage was not associated with index partner gender or CD4 count, geographical regions or time on study but this was strongly associated with study group and number of sexual partners in the three months prior to new seroconversions.

Results on the clinical outcomes for the HIV-positive participants in HPTN 052 were presented by Mina Hosseinipour from the UNC Project, Malawi. [4]

Results comparing the two groups were presented as ITT analyses and included the approximate 20% (184/877) people randomised to the deferred arm who started treatment during follow-up.

Over two years, median CD4 counts increased from 442 to 662 cells/mm3 in the immediate group compared to reducing from 428 to 390 cells/mm3 in the deferred arm. These differences are blunted as the deferred arm includes the response for the 20% people who started treatment. Viral suppression was achieved and maintained <400 copies/mL by >90% of the immediate arm. Less than 5% of patients on immediate treatment experienced virological failure during follow-up with most (60%) of these switching to a second-line combination.

The decision to start treatment in the deferred arm was driven by CD4 declines in 75% of cases. This occurred at a median count of 225 cells/mm3 (IQR 199–247), with 25% over people not starting until their CD4 count was less than 200. Treatment in both arms was predominantly AZT/3TC/efavirenz (70%) with ~10% using AZT/3TC/atazanavir, and ~10% using tenofovir/3TC/efavirenz. CD4 responses in the deferred arm were similar to absolute increase in the immediate treatment arm but remained significantly lower at all timepoints, reflecting the lower counts when starting treatment. Although there are fewer patients with longer duration of follow-up in the deferred arm, other studies have reported that baseline CD4 correlates with CD4 response after treatment.

The analysis by geographical region reported that about 80% of both the linked and unlinked transmission events occurred in African sites, likely a reflection of the higher background population prevalence rates in those countries, although the researchers highlighted higher rates of unprotected sex in the last week (by 9% vs 4% of African vs non-African) and higher sexual activity (>3 acts). However, baseline CD4 count, viral load and adjusted time to initiation, median adherence (99%) and treatment responses were similar between African and Asian sites.

Further details on clinical outcomes were presented by Beatriz Grinsztejn from the Oswaldo Cruz Foundation, Rio de Janeiro. [5]

Primary clinical events occurred at least once in 105 participants over 3304 person-years (PY) of follow-up; 40 in the immediate arm (2.4/100PY) and 65 in the delayed arm (4.0/100PY), hazard ratio (HR) 0.59, 95% CI: (0.40, 0.88), p=0.01. Seventeen people experienced more than one event. Time to event was significantly shorter in the deferred arm (HR 0.6, 95%CI 0.4, 0.9, p=0.01)

CD4 counts were significantly higher in the immediate arm vs deferred arms for all clinical endpoints (TB 518 vs 316; bacterial infection (mainly pneumonia) 551 vs 337 and death 476 vs 372 cells/mm3 respectively).

The between-arm difference was driven by extrapulmonary tuberculosis with 3 cases in the immediate versus 17 cases in deferred arms (p< 0.002). These were peripheral lymph nodes (2 vs 4), abdominal (0 vs 8), pleural (1 vs 3), skeletal (0 vs 1) and meningeal (0 vs 1). Isoniazid prophylaxis was only being used by 4% of patients in each arm at baseline.

Of the 23 deaths observed, there was no difference between arms: 10 in the immediate arm and 13 in the delayed arm [HR 0.77, 95% CI: (0.34, 1.76), NS p>0.5]. Causes of death were similar, but with 3 vs 3 suicides; 0 vs 2 accidents; and 3 vs 6 unknown).

Adverse events potentially related to ART were reported in 24% of subjects in the immediate arm and 5% in the delayed arm, but severe or life-threatening events occurred equally in 14% of each group and grade 4 lab events were also similar (in <1-2% of participants).

Since the DSMB recommendation in April 2011, all participants in the deferred arm have been offered HAART based on the strength of the study findings. This study continues to monitor all participants and results will add to clinical data from use of earlier vs later treatment in people with CD4 counts >350 cells/mm3.

comment

These results add to research that not only correlates viral load with risk of sexual transmission but specifically demonstrates a protective impact with treatment. The two cases of transmission in the early treatment arm (a second was discussed during the presentation) were both detected at the beginning of the study prior to the positive person reaching suppressed viraemia <400 copies/mL.

The fewer clinical endpoints from earlier treatment for the HIV-positive partners in this study are important but were driven by extrapulmonary TB. This clinical difference has significance for people in geographical regions where this study was run, but this aspect of the results was unexpected and has yet to be explained. A more generalisable benefit to people in Western countries is probably the reduced CD4 response in the deferred arm and this needs to be supported by longer follow-up. The ongoing START study will report on whether clinical benefits result from earlier treatment in Western countries.

It would be interesting to model the potential number of transmissions that have already been prevented over the last ten years from the seven million people globally on HAART. Given the financial constraints of access to treatment the additional impact on prevention should be included in future cost: benefit analysis.

The results from HPTN 052 clearly support offering an option for treatment to HIV-positive people who have HIV negative partners. This has been included in UK (BHIVA) guidelines for many years.

When access to treatment is limited with a waiting list using CD4 upper cut-offs to access treatment, those with the most severe medical should clearly be prioritised. However, the majority of the nine million people currently identified by UNAIDS and WHO analyses as requiring but not yet able to access treatment are likely to be undiagnosed. Broadening the CD4 criteria for access to treatment as prevention at higher CD4 counts is unlikely to directly deny access to treatment for more advanced patients.

It was unfortunate that a WHO guideline due to be launched at the IAS meeting, that included the recommendation for treatment people with CD4 counts higher than 350 and who have HIV-negative partners, based on the HPTN 052 study was withdrawn at the last minute. [6]

Although printed for a launch at the conference there is concern that while the scientific evidence is clear – and this should be the focus for clinical guidelines – practical issues on implementation have stalled their release perhaps under pressure from prominent WHO funders. It is difficult to understand how such a useful document that included broad community consultation and approval to the stage of print would have been retracted at such a late stage. WHO say this is due to a need to make “small modifications” and “to review their modeling data they used to inform investment structures”. The timeline for these changes are 2-3 months.

This plausibility for intervention from outside the extensive WHO guidelines writing and advisory panels is supported by an article in Science magazine that names the Gates Foundation specifically related to their interest in the latest PrEP results also being included. [7]

References

Unless stated otherwise, all references are the Programme and Abstracts of the 16th IAS Conference on HIV Pathogenesis, Treatment and Prevention, 17–20 July 2011, Rome.

1. Treatment is prevention: the proof is here. Oral abstract session: Monday 4.30-6.00pm.
2. Cohen M et al. Antiretroviral treatment to prevent the sexual transmission of HIV-1: results from the HPTN 052 multinational randomized controlled trial. Oral abstract MOAX0102. Webcast
3. Eshleman S et al. Analysis of genetic linkage of HIV from couples enrolled in the HIV Prevention Trials Network (HPTN) 052 trial. Oral abstract MOAX0103.
4. Hosseinipour M et al. Immunologic and virologic disease progression and responses to ART across geographic regions: outcomes from HPTN 052 study. Oral abstract MOAX0104. Webcast
5. Grinsztejn B et al. Effects of early versus delayed initiation of antiretroviral therapy (ART) on HIV clinical outcomes: results from the HPTN 052 randomized clinical trial. Oral abstract MOAX0105. Webcast.
6. WHO. Couples HIV testing and counselling and antiretroviral therapy for treatment and prevention in serodiscordant couples: Recommendations for a public health approach. 2011. Final version approved but not yet released. (PDF file)
7. Cohen J. New prevention data leads WHO to delay guidelines for couples. Science Insider (25 July 2011).

Simon Collins, HIV i-Base

Results from the double-blind placebo-controlled TDF2 study presented in an oral presentation in Rome provided additional supportive data for the benefit of daily oral tenofovir/FTC (Truvada) to reduce sexual heterosexual transmission. [1]

While the iPrEX study first reported a strongly protective impact in high risk MSM, the lack of protection in the FemPrEP study earlier this year has still to be explained. [2, 3]

TDF2 randomised 1200 sexually active HIV-negative adults (age 18-49: approximately 90% were between 21-29 years) and followed them for a year. Over 90% of participants were single with only 3% having low educations (primary or less) with >70% having secondary and >20% having post-secondary education. HIV testing was monthly and as with all prevention studies, intensive counselling on safer sex and free condom distribution was provided throughout the study. An indication of the background risk in this population is that 16% of people failing screening (197/2533) were excluded due to already being HIV-positive and 20% due to not being sexually active.

A slightly higher percentage of people in the active vs placebo arm (34% vs 31%) did not complete the study due to loss to follow-up, withdrawal of consent, relocations or other reason. The study had a good gender balance with 45% women.

With 33 seroconversions, primary efficacy results reported a 63% reduced risk of transmission with Truvada based on 9 new infections in the active arm compared to 24 in placebo group (difference 62%: 95%CI 21.5 to 83.4, p=0.0133).

When restricting the analysis (post hoc?) to infections within 4 weeks of a study visit (ie where the monthly visit schedule was being followed and the participant was under a prescription period) the association became stronger. Out of 23 seroconversions, 4 occurred in the active arm and 19 in the placebo group with 78% protection efficacy (95% CI 41.2 to 93.6, p=0.0053).

Although it was emphasised that the study was underpowered to draw any conclusion by gender, in an ITT analysis (33 cases) the intervention appeared protective in men (p=0.026) but not women (p=0.107) and in the observed results (23 cases) the protection was seen on women (p=0.021) but not men (p=0.065). Whilst interesting to see if a gender effect can shed light on the results from FemPREP, this will need to come from larger study numbers.

Resistance developed in one person enrolled in the active arm whose acute HIV infection was undiagnosed with K65R, M184V and A62V conferring nucleoside cross resistance. The person has achieved viral suppression after starting treatment with AZT/3TC/lopinavir/ritonavir. One person in the placebo group had low-level K65R suggesting an infection with drug resistant HIV.

Side effects were commonly reported in both arms, usually mild, with nausea (19% vs 7%, p=0.0001) and vomiting (11% vs 7%, p=0.005) occurring more significantly in the active arm compared to the placebo arm, but resolving within the first month. There were no differences in laboratory abnormalities with one case of elevated creatinine in the active group that resolved when treatment was stopped.

References

1. Thigpen MC et al. Daily oral antiretroviral use for the prevention of HIV infection in heterosexually active young adults in Botswana: results from the TDF2 study. Oral abstract WELBC01. Webcast.
2. Grant RM et al. Preexposure chemoprophylaxis for HIV prevention in men who have sex with men. NEJM. 23 November 2010 (10.1056/NEJMoa1011205). Free full access.
3. FHI statement on the FEM-PrEP HIV prevention study: FHI to initiate orderly closure of FEM-PrEP. (18 April 2011).

Simon Collins, HIV i-Base

The final presentation in the Treatment is Prevention session was a summary of the first results from the Partners PrEP study that randomised the HIV-negative partner in 4758 HIV serodifferent heterosexual couples to daily tenofovir (TDF) vs tenofovir/FTC (TVD) vs placebo in a 1:1:1 ratio. [1]

The results presented were based on a DSMB analysis a week before the conference that recommended unblinding the placebo arm and switching those participants to active drugs. This was based on significantly reduced risks of transmission in both the active arms and was 18 months earlier than the planned study endpoint.

This study was run in nine sites in Kenya and Uganda with all participants receiving intensive healthcare and adherence counselling including free condoms. The negative partners were seen monthly for counselling with HIV and pregnancy testing and the positive partners were monitored for their HIV care every three months.

Baseline demographics were similar across the three arms and included age (of the negative partner) 33 years (IQR 28-40), with the positive partner diagnosed a median of 0.4 years (IQR 0.1-2.0 years), CD4 count 490 cells/mm3 (IQR 370-660) and viral load 3.9 log copies/mL (3.2-4.5 logs).

Nearly all couples were married (98%) with duration of relationship a median of 7 years (IQR 3-14). The positive partner was a woman in 40% of couples. Approximately 20% of positive partners started treatment during the study for their own health.

Study retention was high with fewer than 5% discontinuations over 7337 person years of follow up (median 12 months). Adherence was also estimated high at 97% based on pill count using returned bottles (98% of bottles were returned).

Up to May 2011, there were 90 new infections, 12 of which were HIV-positive at randomisation (3 TDF, 3 TVD, 6 placebo). Of the 78 transmissions that occurred as events for the primary endpoint, 18, 14 and 47 occurred in the TDF, TVD and placebo arms respectively. The was an incidence of 0.74, 0.53 and 1.92 per 100 patient years that produced protection rates of 62% (95%CI 34-78%, p=0.0003) in the tenofovir and 73% (95%CI 49-85%, p<0.0001) in the tenofovir/FTC arms compared to placebo.

The study reported of a similar response between the two active arms (p=0.18 for comparison, NS). However, protection was numerically greater with the dual therapy and the gender analysis reported wider confidence intervals for tenofovir monotherapy with lower levels that were lower.

For women, protection rates were 68% (29-85%) and 62% (19%-82%) in the TDF vs TVD arms; for men these were 55% (4-79%) vs 83% (49-94%). The plausibility for greater protection from dual therapy would be extended with either lower adherence or less-than-daily dosing, both of which might be key public health factors for considering use of PrEP outside of clinical trials.

Safety results were very similar between all three arms for serious events and lab abnormalities including creatinine increases (1%, mainly grade 1) and reduced phosphorus (9%, equal across arms). As with other PrEP studies, nausea and diarrhoea were significantly more common in the active arms, but generally only for the first month of treatment.

As with other PrEP research, results from the pharmacokinetic will be important to correlate drug levels with level of protection and partner viral load with risk of infection. Resistance results will also be analysed.

Reference

Baeten J et al. Antiretroviral Pre-Exposure Prophylaxis for HIV-1 prevention among heterosexual African men and women: the Partners PrEP Study. Oral abstract MOAX0106. Webcast.

Simon Collins, HIV i-Base

Elvitegravir is a once-daily integrase inhibitor being developed by Gilead. This was a double-blind, placebo-controlled study that randomised 702 treatment-experienced patients to compare elvitegravir (150 mg once-daily; reduced to 85 mg with atazanavir/r or lopinavir/r) to raltegravir (400 mg twice-daily), each with a background regimen of sensitive boosted-PI plus a third sensitive drug selected by phenotype (from NRTI, maraviroc, etravirine or T-20) and including the use of 3TC/FTC with the M184V mutation. The primary endpoint was proportion of patients with viral load <50 copies/mL at week 48 (TLOVR analysis, ITT), This was a non-inferiority study with the lower limit of the 95%CI set at –10%.

Baseline characteristics included mean age 45 years; 18% were women; mean CD4 count 260 cells/mm3 (45% cell <200), median viral load ~ 20,000 copies/mL (with 26% >100,000 copies/mL) and approximately 5% and 15% of patients were coinfected with HBV or HCV respectively. Approximately 63% patients had primary resistance to two or more classes (PI 33%, NRTI 72%, and NNRTI 61%), balanced between arms.

Choice of background PI was largely darunavir (58%), lopinavir/r (19%) or atazanavir (16%). The third drug was an NRTI in 80% of patients (tenofovir 59%, tenofovir/FTC 27%, abacavir 4%, 3TC 3%, other 7%) with 13% using etravirine and 6% using maraviroc.

At week 48 a similar virological response rate was reported in each arm: 59% vs 58% in the elvitegravir vs raltegravir arms respectively which was strongly significant for non-inferiority (difference 1.1%, 95%CI –6.0% to +8.2%; p= 0.001). Approximately 20% of patients in each arm were reported as discontinuing due to virological failure: due to viral rebound (11% vs 16%) or never suppressing (8% vs 5%) in the elvitegravir vs raltegravir arms respectively with 1% or patients in each arm failing due to a switch of background drugs. A summary of virological and safety results is included in Table 1.

However, discontinuations in a following slide were reported at 24% in each arm, mainly due to non-adherence, loss to follow-up or withdrawn consent and are detailed in Table 2, with virological failure reported in only 9 patients in each arm.

When looking at drug resistance in the patients with virological failure, this included 61 and 75 people in the elvitegravir and raltegravir arms respectively. In this dataset, failure with integrase-associated mutations was reported at a comparable number though with increased frequency (16/60; 27% vs 15/72; 21%) in the elvitegravir vs the raltegravir patients. Development of new PI- or NRTI-associated resistance was generally low and similar between the two groups.

Less than 5% of participants discontinued due to side effects. The only difference between arms in terms of adverse events was a higher rate of diarrhoea with elvitegravir (12% vs 7%), not associated with discontinuation. This was similar for laboratory abnormalities, with a slightly higher percentage of patients reporting grade 3/4 ALT/AST elevations with raltegravir (~1-2% vs 5%).

This study concluded that this demonstrated that once daily elvitegravir was non-inferior to twice-daily raltegravir in treatment-experienced HIV-positive patients.

comment

These are impressive results in treatment-experienced patients. The rate of 20% patients failing with integrase-associated mutations was considered low by the investigators given the low barrier to integrase mutations. This was partially explained by the low barrier to virological failure in the study design (<1 log by week 8).

Causes of the seven deaths were not apparently drug related. This included one intestinal perforation in the elvitegravir arm, and one lymphoma and two cardiovascular events in the raltegravir arm.

References

Molina J-F et al. Elvitegravir once-daily is non inferior to raltegravir twice-daily in treatment experienced patients: 48 week results from a phase 3 multicenter, randomized, double blind study. Oral late breaker abstract WELBB05. Webcast.

Simon Collins, HIV i-Base

Dolutegravir is an integrase inhibitor in development with ViiV/Shionogi that in earlier development was referred to as GSK-572. Results from the first monotherapy studies were presented only two years ago and the rapid development programme now includes phase III studies in naïve patients (using a 50 mg once-daily dose) with results already reported from phase II studies in experienced patients (using a higher 50 mg twice-daily dose).

Results from week 24 of a Phase IIb dose-ranging study treatment naïve study were presented in Glasgow last year with 90-96% of patients in the dolutegravir arms reducing viral load to <50 copies/mL compared to 78% patients in the efavirenz arm. These were updated to week 48 at in an oral presentation in Rome. The initial steep viral decline seen with integrase inhibitors as a class was probably a factor in choosing a primary endpoint at week 16 but this extended data is more crucial to understand sustainability.  [1]

The study randomised 205 treatment-naïve patients 1:1:1:1 to 10 mg, 25 mg or 50 mg of dolutegravir or efavirenz 600mg once-daily, plus either investigator choice of either tenofovir/FTC (used by two-thirds of patients) or abacavir/3TC.

Baseline viral load was originally low (approximately 30,000 copies/mL) with only 26% participants >100,000 copies/mL. This would increase effectiveness for the a viral suppression (requiring less than a 3 log drop to achieve undetectable levels. Participants were largely male (86%) and white (80%), with mean CD4 count of 324 cells/mm3 (63% were <350). Median age was 32 (range 20-79 years).

By week 48, suppression in the dolutegravir arms had dropped slightly to 88-91% compared to 82% in the efavirenz arms. No between group analyses were presented but the confidence intervals for all groups broadly overlapped. Virologic non-response/rebound rates were low and similar between arms (8%, 6%, 4% vs 6% in the efavirenz arm) with the lower response in the efavirenz arm driven by discontinuations related to side effects (0, 2%, 0 vs 8% respectively). In the small percentage of patients failing with viral rebound 0/3 (none in the 50 mg arm) showed evidence of integrase-related mutations, though patients were proactively switched early due to this potential concern.

Virological results were also presented using a <2 copies/mL viral load test – the first time perhaps for an ongoing Phase II study. At the 50 mg dose approximate suppression to <2 copies at weeks 16, 24 and 48 was 48% 65% and 52% respectively compared to 35%, 45% and 58% in the efavirenz arm. These are small study numbers and neither confidence intervals nor statistical comparisons were provided but these are unlikely to show significant differences. It may be notable that the <2 copies/mL results for the lower doses of dolutegravir were not presented. Given the increased research focus on greater suppression at levels below < 50 copies/mL and the conflicting results from some of the intensification studies with raltegravir (broadly finding no impact in blood but perhaps in some tissue sites) this class potential is likely to inform future studies.

CD4 increases were similar by week 48 with +231 cells/mm3 in the combined dolutegravir vs +174 in the efavirenz arm vs EFV (p=0.076), reducing a difference that was significantly higher at week 24.

No new serious adverse events were reported out to week 48. The two discontinuations from the dolutegravir arms were due to dyspepsia (25 mg arm) and Burkitt’s lymphoma.

Grade 2-4 side effects were similar between arms, except for rash and CNS-related side effects occurring only in the efavirenz group. Nausea (11%), diarrhoea (8%) and headache (5%) were most frequently reported with dolutegravir compared to dizziness (18%), fatigue, insomnia and rash (8% each) with efavirenz. Grade 3/4 laboratory abnormalities occurred in 12% vs 14% of the combined dolutegravir vs efavirenz arm. Dolutegravir was associated with mean increases in creatinine (6.4-11.9 mmol/L) at week 1 that were stable to week 20 and decreased by week 48. In vitro data have shown that dolutegravir causes a benign inhibition of creatinine secretion. These were detailed in a separate poster presentation. [2]

Lipid changes were generally greater in the efavirenz arm but there were no differences either from baseline or between drugs in the TC/HDL ratio at week 48 (due to the impact of efavirenz in increasing HDL).

The pharmacokinetic summary slide showed an impact of dose on drug levels over 24 hours, but these had low inter-patient variability and at all doses remained above the IC90 (0.064 ug/mL) with Cmin levels [geometric mean (CV%)] of 0.30 (71), 0.54 (67) and 1.20 (62) and an inhibitory quotient of 4.7, 8.4 and 19-fold for the 10 mg, 25 mg and 50 mg doses respectively.

References

1. Van Lunzen J et al. Rapid, robust and sustained antiviral response with once-daily (QD) dolutegravir (DTG, S/GSK1349572), a next generation integrase inhibitor (INI) in combination therapy in antiretroviral-naïve adults: 48 week results from SPRING-1 (ING112276). Oral abstract TUAB0102. Webcast.
2. Min S et al. Safety profile of dolutegravir (DTG, S/GSK1349572), a next generation integrase inhibitor (INI) in combination therapy in antiretroviral (ART)-naïve and ART-experienced adults from phase 2b studies. IAS. 17-20 July 2011. Rome. Abstract TUPE238.

Simon Collins, HIV i-Base

Lersivirine is a once-daily NNRTI from ViiV that was originally developed by Pfizer and previously called UK-453061 and that is promising due to a resistance pathway at V108I that appears distinct from the K103N or Y181C pathways associated with first-generation non-nukes.

This double-blind, placebo-controlled study randomised 193 patients (1:1:1) to either 500 mg or 750 mg of lersivirine or to standard dose efavirenz, each with once-daily tenofovir/FTC. The primary endpoint was the percentage of patients with viral load reduced to <50 copies/mL at 48 weeks with follow up out to 96 weeks (by ITT missing = failure analysis).

Although enrollment criteria for the study included a CD4 count >200 cells/mm3, at baseline this ranged from 122 to 955 (median 310) suggesting that a few more advanced patients were included on an experimental combination than the European regulatory guidelines recommend for Phase II studies. Baseline viral loads ranged from 1,500 to 1,600,000 (mean: 50,000 copies/mL). Approximately 35% of patients had baseline viral load >100,000 copies/mL and this was reflected in pre-specified analysis of the results.

Other baseline characteristics included: mean age 36 years (range 21-62); 27% were women; race: 60% white, 30% black, 10% other. While the majority of people had sub-type B, ~30% of people were sub-type C which was reflected in ~ 30% enrolled in sites in South Africa.

At week 48, the percentage of patients with viral load <50 copies/mL was 79%, 79% and 86% in the 500 mg, 750 mg and efavirenz groups respectively. Although the study was not powered to detect a difference in efficacy between arms, the lersivirine arms suggested a poorer response compared to efavirenz (500 mg: –9% difference; 80%CI –18.1, 0.8 and 750 mg: –8% difference; 80%CI –17.0, 1.2).

Results stratified by baseline viral load (which was lower in the >100K group) or geographical region (which was lower for sites in South Africa) did not contradict this finding, see Table 1.

A mean CD4 count increased of approximately +190 cells/mm3 from baseline was similar between arms.

Virological failure occurred in 4, 5 and 3 patients in the 500 mg, 750 mg and efavirenz groups respectively, with people on lersivirine generally failing with M184V plus NNRTI mutations when resistance was isolated. The one person with identifiable mutations in the efavirenz arm failed with K103N alone.

Overall, the combined safety analysis reported a similar incidence of side effects in each group but fewer grade 3/4 events in the lersivirine groups (n= 2 and 3) compared to efavirenz (n=8) see Table 2. Laboratory abnormalities were infrequent and evenly distributed between arms. Lipids were broadly stable for lersivirine compared to increases in TC, LDL, HDL and TG for efavirenz, but this resulted in little difference between the LSV and EFV groups (+0.24 and -0.06 vs -0.3) in the change in the TC:HDL ratio used to evaluate cardiovascular risk.

However, the study concluded that both lersivirine doses showed similar efficacy to efavirenz over 48 weeks in treatment-naïve patients and had different side effect profiles compared with efavirenz.

* EU, Latin America, Australia, Canada

comment

There is still a role for new NNRTI with activity against nevirapine and efavirenz associated resistance with an improved safety profile to efavirenz.

The higher reports of nausea and headache appeared to be low grade but limited data was available on duration and severity of these events.

Reference

Pozniak A et al. Efficacy and safety of lersivirine (UK-453,061) vs. efavirenz in antiretroviral treatment-naïve HIV-1-infected patients: week 48 primary analysis results from an ongoing, multicentre, randomised, double-blind, phase IIb trial (study A5271015). Oral abstract TUAB0101. Webcast.

Simon Collins, HIV i-Base

Updated results from a Pfizer-sponsored study of dual therapy maraviroc plus atazanavir/r compared to standard of care atazanavir/r plus tenofovir/FTC were presented at the meeting.

This is controversial due to the lower percentage of patients in the maraviroc arm reaching undetectable viral load <50 copies/mL at week 48 (75% vs 84%, no between arm statistical data presented due to lack of power in the study size), higher rates of toxicity and the decision to enroll a larger phase 3 study with the same design. These differences were also seen at week 48 when stratified by baseline viral <100,000 (77% vs 87%) and >100,000 (69% vs 77%).

These trends were apparent in the interim 24-week results presented at the IAS 2010 in Vienna: viral suppression then was 80 vs 89% together with increased side effects (ie 33% vs 23% grade 3/4 including 26% vs 13% hyperbilirubinaemia) in the maraviroc arm. [2]

A health economic interest in this study comes from the pharmacokinetic data supporting the use of half-dose (and therefore half-cost) maraviroc (patients are dosed at 150 mg daily when using atazanavir/r) and that in this combination maraviroc is only taken once daily.

Mean change from baseline in CD4 count at week 48 was similar with +215 vs +226 cells/mm3 in the maraviroc vs tenofovir/FTC arms respectively.

Grade 3/4 side effects were more frequent with maraviroc than tenofovir/FTC (18 vs 11 patients) and these were mostly due to hyperbilirubinemia. Creatinine clearance was stable with maraviroc but decreased by a median –12 mL/min with tenofovir/FTC. Serious adverse events were similar (10 vs 11 patients) with none related to assigned study drug.

A second presentation included an analysis of the results from patients who used 150 mg maraviroc with boosted PI, supporting the reduced dose compared to those using 300 mg twice-daily with either tipranavir or fosamprenavir in the maraviroc registrational trials. [3]

In summary, suppression to <50 copies/mL was reported in 45% vs 47% in the 150 mg/boosted PI vs 300 mg groups (vs 16% for the placebo group) with a similar close relationship between doses for people starting in advanced disease with baseline viral load >100,000 copies/mL (38% vs 39%) or CD4 counts <50 cells/mm3 (17% vs 18%).

comment

The limitations from low study numbers in phase 2 studies are important to remember when reviewing these results but it will be important to follow the phase 3 study of this dual therapy arm carefully. Some combinations that are less virologically effective perform better as switch options once people are stable on treatment.

A switch strategy, given the potential cost savings from the reduced dose of maraviroc might warrant a separate study.

References

1. 1. Mills A et al. 48-week results of a dual-therapy regimen of once-daily maraviroc (MVC) 150 mg in combination with ritonavir-boosted atazanavir (ATV/r) compared to emtricitabine/tenofovir (FTC/TDF) + ATV/r in treatment-naïve (TN) patients infected with CCR5-tropic HIV-1 (Study A4001078). 6th IAS Conference, 17–20 July 2011, Rome. Abstract TUAB0103. Webcast.
2. Portsmouth S et al. Safety and immunovirological activity of once daily maraviroc (MVC) in combination with ritonavir-boosted atazanavir (ATV/r) compared to emtricitabine 200mg/tenofovir 300mg QD (TDF/FTC) + ATV/r in treatment-naïve patients infected with CCR5-tropic HIV-1 (Study A4001078): A week 24 planned interim analysis. 18th IAS Conference, 18–23 July 2010, Vienna. Late breaker abstract THLBB203.
3. Taylor S et al. Efficacy of maraviroc (MVC) administered once daily (QD) or twice daily (BID) with boosted protease inhibitors (bPIs) to treatment-experienced patients. 6th IAS Conference, 17–20 July 2011, Rome. Abstract TUAB0106.

Polly Clayden, HIV i-Base

The main objective in the SPARTAC trial was to look at the impact on disease progression from two different short courses of antiretroviral treatment (ART) initiated during primary HIV infection compared to no immediate ART. Sarah Fidler from Imperial College London presented results from SPARTAC in an oral late breaker at IAS 2011.

In this study, adults with primary infection who were within 6 months of seroconversion were randomised to receive ART for 48 weeks, 12 weeks, or no therapy (standard of care, SOC). The primary endpoint was time from randomisation to either CD4 <350 cells/mm3 or initiation of continuous ART.

A sample size of 360 was calculated (using data from CASCADE) to provide 90% power to detect relative reduction in risk of time to primary endpoint of 50% – 25% in each of the ART arms – compared to SOC over four years of follow up.

A total of 366 participants were randomised from 35 sites in Australia, Brazil, Europe and Africa; 40% were from the UK and 35% South Africa. Of these, 60% were men (90% MSM) and 40% African women, with a median age overall of 31 years.

As would be expected, the median baseline CD4 was high at 543 cells/mm3 and viral load was 4.7 logs (~50,000 copies/mL). Participants were followed for a median of 4.2 years and 19% were lost to follow up. The majority (92%) of participants received lopinavir/r plus AZT and 3TC.

The investigators found no difference in time to primary endpoint in participants receiving 12 weeks of ART compared to SOC (HR 0.93: 95%CI 0.67-1.29, p=0.67). However, 48 weeks of ART conferred a statistically significant delay (HR 0.63: 95%CI 0.45-0.90, p=0.01). The median time to primary endpoint was 157, 184 and 222 weeks for the SOC, 12 week and 48 week arms respectively. Although, Dr Fidler noted that the 65 week (95%CI 17-114) delay in the 48-week arm was not significantly greater than the time spent on treatment.

A post-hoc analysis revealed two findings. There was a significantly more rapid rate of disease progression among participants identified within 12 weeks of acquiring infection in the SOC arm. Secondly, the delay to primary endpoint observed previously with 48 weeks of treatment compared to SOC was greater in participants who initiated ART initiated within 12 weeks of infection (HR 0.48: 95%CI 0.3-0.78). Overall, the investigators reported a non-significant trend to greater delay to primary endpoint the closer ART was initiated to the estimated time of seroconversion (p=0.09, NS).

There was a reduction in viral load of approximately half a log after interrupting ART in the 48-week arm compared to SOC, which was sustained until 60 weeks after stopping treatment. The mean CD4 count over the entire study period was 138 cells/mm3 higher in the 48-week arm than standard of care.

There were no significant differences between arms in AIDS, death or serious adverse events. In contrast to SMART there was no rebound in IL-6 and a drop in d-Dimer, compared to baseline, four weeks after stopping ART.

comment

By the time this study was completed the treatment landscape had changed considerably from when it was initially designed. So the big question now is “what would have happened with a continuous treatment arm?”.

As far as the implication for clinical practice is concerned, perhaps if someone is aware of their status and he or she wants to start treatment in primary infection there may be a slender argument to do so.  But if they do start and are doing well, given the modest time off treatment until starting again, why stop?

Reference

Fidler S et al. The effect of short-course antiretroviral therapy in primary HIV infection: final results from an international randomised controlled trial; SPARTAC. 6th IAS Conference on HIV pathogenesis, treatment and prevention. 17-20 July 2011. Rome, Italy.  Oral abstract WELBX06.

Simon Collins, HIV i-Base

Hearing loss has been associated as a complication in HIV-positive people but it is unclear whether HIV is a direct factor or whether symptoms are more strongly correlated to risk factors reported in the general population. This will be increasingly important as the HIV population ages.

Researchers from Washington DC measured cochlear function in 334 men and 178 women from two of the earliest population cohorts established to look at differences between HIV-positive and HIV-negative patients (MACS and WIHS respectively), and related to this to social factors including noise exposure and HIV and treatment history.

The mean age was 54 years for the men (46% were HIV-positive), and 45 years for the women (77% were HIV-positive). People were excluded if they had hearing-impaired clinical symptoms or recent use of orotoxic medication. Approximately 20% of people in each of the HIV-positive and HIV negative groups self-reported exposure to occupational noise.

Cochlear function was measured by distortion product otoacoustic emission (DPOAE) testing which is a non-invasive procedure using two separate tones to stimulate the cochlea. Each ear was measured twice, with a third test if results were inconsistent and the number of non-responses added as an outcome variable (0-4).

In multivariate analyses, a 10-year increase in age [OR 2.78; 95%CI 2.07, 3.73], being male [OR 5.60; 95%CI 2.98, 10.49], and being non-black [OR 2.75; 95%CI 1.57, 4.83] were significantly associated with a higher number of non-responses (all p<0.001), but not HIV status [OR 1.20; 95%CI 0.7, 2.02; p =0.52 NS]. However, neither occupational or non-occupational noise exposure was associated with reduced function (p=0.33 and p=0.93, respectively).

Age, race, and gender remained significant risk factors for increasing non-responses in the HIV-positive model. However, none of the HIV-related factors including use of monotherapy, combination therapy, HAART use, 100-cell increase in peak CD8, HIV viral load, and 100-cell increase in nadir CD4 count came near approaching statistical significance (with p-values ranging from 0.2 to 0.7).

The researchers concluded that HIV status, combination therapy, nadir CD4 count, peak CD8 count, and HIV viral load did not significantly predict decreased cochlear function in this patient group.

Reference

Torre P et al. Cochlear function among Multicenter AIDS Cohort Study (MACS) and Women’s Interagency HIV Study (WIHS) participants. 16th IAS Conference on HIV Pathogenesis, Treatment and Prevention, 17–20 July 2011, Rome. Poster abstract TUPE138. Poster. (PDF)

Polly Clayden, HIV i-Base

Physiological changes in pregnancy can affect drug disposition. Plasma concentrations of several PIs – including lopinavir, atazanavir and saquinavir – currently prescribed to HIV-positive pregnant women, are decreased during this period. Pharmacokinetcs (PK) for darunavir (DRV) and fosamprenavir (FPV) in pregnancy are not well characterised. Two posters presented at the paediatric workshop and IAS 2011 showed data from PK studies of these antiretrovirals in pregnant women. [1, 2]

Darunavir

Edmund Capparelli and colleagues from the IMPAACT P1026s study group presented PK and safety data of DRV dosed twice-daily (BID) and once-daily (QD) during the third trimester of pregnancy, at delivery and post partum. These data were shown at the paediatric workshop in Rome.

IMPAACT P1026s is an on-going, prospective, non-blinded study of antiretroviral PK in pregnancy. It comprises of two groups of women receiving ritonavir-boosted DRV either as 600/100mg BID, or 800/100 mg, QD, as part of an ART regimen during pregnancy and 6-12 weeks postpartum (PP).

All women had received at least two weeks of ART at the time of the evaluation. Intensive steady-state 12 or 24-hour PK profiles were performed during the 3rd trimester and PP. Cord blood and maternal samples were taken at delivery when possible. DRV concentrations were measured by HPLC (limit of detection 0.09 mcg/mL). The minimum exposure targets were DRV AUC0-12 or 24 of 43.6 or 56.5 mcg*hr/mL, for BID or QD, respectively. This represents >70% median for non-pregnant adults.
PK data were available for 31 women (19 BID, 12 QD). Two PP PK evaluations (1 BID and 1 QD) were excluded for non-adherence with no detectable DRV concentrations. Geometric mean 3rd trimester/PP ratios were 0.74 (90% CI 0.54-0.92) and 0.76 (90% CI 0.64-0.91) for AUC and 1.42 (90% CI 1.09-1.84) and 1.31 (90% CI1.10-1.55) for CL/Fs with BID and QD dosing respectively.

For the PK parameters presented below for 3rd trimester and PP the investigators indicated values with p<0.05 compared to PP with an asterisk (*). They found, AUC0-12 were median 50.7 (range 23.8-102)* mcg*hr/mL for 3rd trimester and 70.0 (range 40.3-175.5) mcg*hr/mL PP for women who received DRV/r 600/100mg BID. Of those with PK parameters available, 13/19 (68%) and 11/13 (85%) met the AUC-12 target. CL/F was 11.82 (range 7.58-26.4)* L/h and 8.57 (range 3.42-14.89) L/hr. C12h was 3.13 (range 0.78-8.85) mcg/mL and 2.81 (range 1.61-5.50) mcg/mL.

AUC0-24 were 67.7 (range 30.3-105.5) mcg*hr/mL and 87.9 (77.5- 150.2) mcg*hr/mL, for the women who received DRV/r 800/100mg QD. Of these 8/12 and 7/7 met the AUC0-24 target. CL/F was 11.82 (7.58-26.4) L/h and 9.10 (5.33-10.32) L/hr. C24h was 1.37 (0.15-3.49) mcg/mL and 2.59 (<0.09-3.96) mcg/mL.
A total of 20 paired samples of maternal delivery and cord blood were collected. Of these, 6 pairs had concentrations below the limit of detection. For the remainder (n=14) median cord blood DRV concentrations were 0.19 (<0.09-1.1) mcg/mL. Maternal delivery plasma DRV concentrations were 1.42 (<0.09-5.62) mcg/mL. The median ratio of cord blood/maternal delivery plasma concentrations was 0.24 (0.062-0.58) indicating limited transplacental transport of DRV.

The investigators concluded that lower troughs and AUC with QD compared to BID dosing combined with pregnancy lowering DRV exposure suggests BID dosing should be used in pregnancy and higher doses may be required.

Of note, not all women achieved viral suppression in both dosing groups (at delivery overall, 57% and 79% <50 and <400 copies/mL respectively), and there was at least one vertical transmission among 24 (77%) infants with data available at the time of this analysis.

Fosamprenavir

There are limited data describing safety and outcomes of FPV in pregnancy or plasma concentrations of FPV’s active metabolite, amprenavir (APV), during pregnancy, PP and in cord blood.

Michelle Cespides and colleagues from New York University School of Medicine showed findings from a phase I, open-label, single-centre study to evaluate APV PK following dosing of ritonavir boosted FPV 700/100mg BID in pregnant women. The investigators evaluated steady-state PK in the second and/or third trimesters and 4-12 weeks PP. Maternal plasma and cord blood samples were taken at the time of delivery. APV concentrations were measured by LC-MS/MS, and PK were determined using WinNonlin. This study was presented at IAS 2011.

The study evaluated 10 women receiving DRV/r based regimens. Cord blood samples were available from six deliveries. The median ratio of cord blood/maternal APV concentrations was 0.27, again, indicating limited transplacental transfer of this PI. Individual APV AUC was 22-34% lower, Cmax 9-41% lower and C12 27-28% lower in pregnancy than PP. See Table 1: Amprenanvir concentrations during pregnancy.

The investigators noted that although APV C12 was 27-28% lower in pregnancy, HIV was well suppressed for all subjects at delivery. Maternal and cord blood concentrations were above mean protein binding-adjusted IC50 (0.146 ug/mL) for wild-type virus.

Safety and outcomes data showed that FPV was well tolerated in this small study with no hepatic, renal, or adverse events attributed to ART.

At delivery, all women had viral loads < 400 copies/mL and nine women had <50 copies/mL. All infants were HIV PCR negative.

comment

The recommendation from the first study that higher doses of DRV may be required is consistant with US recomendations with other PIs such as lopinavir and atazanavir.

BHIVA guidelines do not recommended a dose increase.

References

1. Capparelli E et al. Pharmacokinetics of Darunavir Once or Twice Daily During and After Pregnancy. 3rd International Workshop on HIV Pediatrics. 15-16 July, 2011. Rome, Italy. Poster abstract P_72.
2. Cespedes M et al. Pharmacokinetics, cord blood concentrations, and tolerability of boosted fosamprenavir (FPV) in pregnancy. 6th IAS Conference on HIV pathogenesis, treatment and prevention. 17-20 July 2011. Rome, Italy. Poster abstract TUPE278.

Polly Clayden, HIV i-Base

Data describing the risk of low birth weight (LBW) and preterm delivery (PTD) associated with maternal HIV and antiretroviral exposure are conflicting and international consensus has not been reached. Two posters from the Antiretroviral Pregnancy Registry (APR) and the Perinatal HIV Research Unit (PHRU), Soweto, South Africa, presented at IAS 2011, showed findings from their analyses of LBW and PTD in their respective cohorts. [1, 2]

APR is a prospective exposure-driven birth registry to monitor for potential increased birth defects following ART exposure in pregnancy.

In this study, Karen Beckerman and colleagues analysed reports to the APR cohort from 1989-2010. They restricted outcomes to singleton live births without birth defects. After establishing overall LBW(<2500 g)/PTD(< 37 weeks) prevalence by type of ART (2 or more drug regimens with and without PI), the data were stratified for maternal age, race/ethnicity and CD4 count. Stratified analysis is based on the 2×2 chi-square test and Cochrane-Mantle-Haenzel.

The investigators found, among the 10082 live births with birth weight data available, 16% were < 2500 g. Of those with reported estimated gestational age, 12.8% were <37 weeks. There were significantly higher LBW/PTD women receiving PI-containing regimens vs regimens without PI (LBW RR=1.22, p<0.001; PTD RR=1.27, p<0.001). But after controlling for maternal age, race/ethnicity and CD4 count they found no significant increase in incidence of LBW/PTD associated with PI exposure among groups with lower pre-existing risk. See Table 1: Low birth weight, preterm delivery and PI exposure in APR.

The investigators concluded that among prospective reports to APR, increases in LBW/PTD were not associated with PI exposure in women with low background risk for these complications.

In a related study, Fatima Laher and colleagues from PHRU investigated risk factors for PTD in their cohort. They noted that prevalence for PTD is 17.5 in Southern Africa.

This study was a retrospective review of all HIV-positive pregnant women who received triple-combination ART between August 2004 and September 2010. Obstetric history, ART history, maternal CD4 count and viral load during pregnancy were recorded for all live births. Univariate analysis included variables associated with preterm delivery.

The investigators found, out of a total 223 pregnancies, 58 were electively terminated (26%), 19 were spontaneous miscarriages (8%), 16 did not yet have recorded outcomes (7%), and 4 were stillbirths (2%). There were 126 (57%) live births, and 62/126 (49%) were PTDs with median gestational age 34.7 weeks (IQR 33.0-35.7). Mothers of preterm and term infants were similar in age, median 31.7 and 30.9 years respectively. Virological suppression <50 copies/mL during pregnancy was similar in both groups, 84% and 80% respectively.

The majority of women, 111/126 (88%) initiated ART was before conception. Maternal CD4 count during pregnancy below 200 cells/mm3 [OR 1.2; 95%CI 0.5-2.8, p= 0.76], 350 cells/mm3 [OR 1.4; 95%CI 0.7-2.8, p= 0.37], or 500 cells/mm3 [OR 1.1; 95%CI 0.4-2.8, p=0.87], were not risk factors for PTD.

Final-trimester maternal use of EFV-based regimens [OR 2.5; 95%CI 0.9-6.9, p=0.09], or PI-based regimens [OR 1.4; 95%CI 0.6-3.0, p=0.4], were not predictive of PTD compared to NVP-based regimens.

The investigators concluded that preterm delivery is common among pregnant ART-recipients in Soweto. Maternal CD4 count and final-trimester ART type seem not to predict preterm delivery. They noted that their small sample size in this study is a limitation.

comment

The APR data are unsurprising as 85% of the pregnancies enrolled are from the US and the association with PTD and PIs is largely not observed in American cohorts. The Soweto data may reflect a high background PTD rate and, as the investigators note, a small sample size.

Recently published data from Botswana does show an increased risk of PTD in women reciving PIs in pregnancy. [3]

References

1. Beckerman K et al. Exposure to combination antiretroviral (cARV) regimens containing protease inhibitors (PI) during pregnancy and prevalence of low birth weight/preterm delivery (LBW/PTD) among women with low pre-existing risk for LBW/PTD: a stratified analysis of 10,082 p. 6th IAS Conference on HIV pathogenesis, treatment and prevention. 17-20 July 2011. Rome, Italy.  Poster abstract TULBPE018.
2. Laher F et al. Maternal CD4 and HAART type are not risk factors for preterm delivery amongst HIV-infected pregnant women receiving HAART in Soweto, South Africa. 6th IAS Conference on HIV pathogenesis, treatment and prevention. 17-20 July 2011. Rome, Italy.  Poster abstract TUPE319.
3. Powis KM et al. Increased risk of preterm delivery Among HIV-infected women randomised to protease versus nucleoside reverse transcriptase inhibitor-based HAART during pregnancy. Journal of Infectious Diseases. Volume204, Issue4. Pp. 506-514.

Polly Clayden, HIV i-Base

Some epidemiological and laboratory studies have suggested that hormonal contraception can increase HIV transmission risk in women. There has been little research into the risk of transmission from women to men. To date findings have been inconsistent and WHO has called for high quality studies to look at potential interactions between hormonal contraception and HIV transmission.

Investigators from The Partners in Prevention HSV/HIV Transmission Study compared HIV incidence among women using hormonal contraception to those who were not. This analysis evaluated both HIV acquisition among women and transmission from women to men. Renee Heffron presented findings from this study in an oral presentation at IAS 2011. [1]

This was a prospective cohort study of 3790 serodiscordant couples from seven countries in East and southern Africa. The couples were enrolled from two studies conducted between 2004 and 2010, a randomised trial of acyclovir to reduce HIV transmission (n=3321) and a prospective cohort study of immune correlates of HIV protection (n=469).

Study participants were 18 years of age or older, and at enrollment the HIV-positive partners were not eligible for ART according to national guidelines. HIV-negative partners were tested quarterly and HIV-positive partners had CD4 measurements every six months and plasma and genital viral load at enrollment and six months later. The investigators used standardised questionnaires to measure sexual behaviour and contraceptive use.

They compared rates of HIV acquisition in women and HIV transmission from women to men using multivariate Cox proportional hazards regression and marginal structural modeling. The analyses were limited to infections acquired from the study partner (evaluated by viral genetic sequencing).

The negative partners were women in about a third (n=1314) of the couples evaluated, and two thirds (n=2476) were men.

Among the negative women, 21.2% overall used hormonal contraception at least once during follow up, of which 16% used injectable contraception at least once and 6.7% oral contraception. The HIV acquisition rates were 6.61 and 3.78 per 100 person-years in women currently using and not using hormonal contraception [AHR 1.98; 95%CI 1.06-3.68. p=0.03]. For injectable contraception the incidence rate was 6.85 per 100 person-years [AHR 2.05; 95%CI 1.06-3.68, p=0.04] and for oral contraception it was 5.94 per 100 person-years, [AHR 1.8 95%CI 0.55-5.82, p=0.33].

Overall, 33.3% of HIV-positive partners of negative men used hormonal contraception, 26.8% injectable and 8.9% oral. In these couples, HIV transmission rates from women to men were 2.61 and 1.51 per 100 person-years in those whose partners used and did not use hormonal contraception [AHR 1.91, 95%CI 1.12-3.45, p=0.02]. For injectable contraception the incidence rate was 2.64 per 100 patient years [AHR 1.95; 95%CI 1.06-3.58, p=0.03]. The incidence also increased in the group using oral contraception, 2.50 per 100 patient years, but as with HIV acquisition in negative women this did not reach statistical significance in this subgroup [AHR 2.09; 95%CI 0.75-5.84, p=0.16].

Results from marginal structural model analyses were consistent with those shown from the Cox proportional hazards regression.

When the investigators looked at this a possible explanation, there were significantly higher genital viral load concentrations overall in women using hormonal contraception [OR 1.51; 95%CI 1.13-2.01, p=0.01]. For injectable contraception these were significantly higher [OR 1.67; 95%CI 1.21-2.31, p=0.02]. But not for oral contraception [OR 1.06; 95%CI 0.62-1.84, p=0.49].

Dr Heffron noted that this was the first study to demonstrate that hormonal contraception increased an HIV-positive woman’s risk of transmitting HIV to her partner.

She added that the benefits of effective hormonal contraception are unequivocal and must be balanced with the increase in risk of HIV infection. These possible risks should be discussed with women and couples alongside the importance of HIV prevention.

Strategies to improve access to and usage of lower dose and non-hormonal methods – IUDs, implants, patches or combination injectables are warranted – she concluded.

comment

These findings understandably caused quite a stir and urgently need more investigation. This was followed by the report from Partners in Prevention that pregnancy doubles the risk of transmission from HIV-positive women to her male partner (to be reviewed in the next issue of HTB). [2]

References

1. Heffron R et al. Hormonal contraceptive use and risk of HIV-1 transmission: a prospective cohort analysis. 6th IAS Conference on HIV pathogenesis, treatment and prevention. 17-20 July 2011. Rome, Italy. Oral abstract WEAX0206. Webcast.
2. Mulago NR et al. Increased risk of HIV-1 transmission in pregnancy: a prospective study among African HIV-1 serodiscordant couples. Advance online edition of AIDS 25, doi: 10.1097/QAD.0b013e32834a9338, 2011.

Polly Clayden, HIV i-Base

Information to guide initiation of treatment in children older than one year of age is scarce.

Results from the PREDICT trial – presented as late breakers at both IAS 2011 and the preceding pediatric workshop – found that deferring ART until CD4 count fell below 15% or the occurrence of CDC category C events did not affect AIDS-free survival in children compared to starting ART at a CD4 count between 15% and 24%. [1]

PREDICT was conducted in 299 children from nine sites in Thailand and Cambodia between April 2006 and September 2008. Children were randomised to receive immediate ART or defer until their CD4 reached less than 15%. The children’s baseline characteristics are shown in table 1.

The primary endpoints were AIDS free survival at week 144 and neurodevelopmental outcome by Beery visual motor interrogation test.

Age, CD4%, HIV RNA, weight-for-age z-score and height-for-age z-score are mean values.

Retention was high in this study (96%). At week 144, 69 (46%) children had started ART with a mean CD4 at initiation of 13.8% (SD+2.8%). Of these, 17 children were <5 years and had a mean CD4 count of 591 cells/mm3 (SD+508) and 52 children were >5 years and had a mean CD4 count of 309 cells/mm3 (SD+141).

AIDS-free survival was 97.9% (95% CI, 93.7 -99.3) in the immediate arm and 98.7% (95%CI 94.7-99.7) in the deferred arm. The incidence of CDC C events or death per 1000 person-years was 7.6 (95%CI 2.5-23.6) in the immediate arm and 4.9 (95%CI 1.2-19.7) in the deferred arm.

The incidence of CDC category B events per 1000 person-years was broadly similar in both arms, 88 (95%CI 61-123) in the immediate arm compared to 110 (95%CI 80-147) in the deferred arm. But there were more episodes of herpes zoster (2 vs 13) and thrombocytopenia (1 vs 10) in the immediate and deferred arms respectively. There were only two episodes of TB, one in each arm.

Weight for age z-score was similar, deferred vs immediate -0.12 (95%CI -0.25 to 0.01), p=0.074. But children grew at a slower rate in the deferred arm, height for age z-score, deferred vs immediate -0.23 (95%CI -0.38 to 0.08), p=0.003.

And at 144 weeks of follow up there was no significant difference by Beery visual motor test between the two arms; Beery score deferred vs immediate, 84.7 vs 86.8, p=0.5.

The investigators noted that at approximately three years of follow up, the rate of progression to AIDS is extremely low in both the immediate and deferred arms. The finding reflects a slow disease progression among HIV-infected children who survive the first year of life without treatment.

comment

This study is important and a bit of a surprise to many as it appears to contradict both adult data and that for young infants. But the median age in the study reflects a population that have survived without treatment for the first few years and therefore selects a group of healthier children without rapid disease progression.

References

Puthanakit T et al. Randomised clinical trial of immediate versus deferred antiretroviral therapy initiation in children older than one year with moderate immunodeficiency: the PREDICT Study (NCT00234091). 3rd International workshop on HIV paediatrics. 15-16 July 2011. Rome, Italy. Oral abstract LB 01.

Also presented at the 6th IAS Conference on HIV pathogenesis, treatment and prevention. 17-20 July 2011. Rome, Italy. Poster abstract TULBPE023.

Polly Clayden, HIV i-Base

Data were presented at the paediatric workshop and IAS 2011describing recent developments in the paediatric pipeline.

Etravirine

Thomas Kakuda from Tibotec showed pharmacokinetic (PK) data of the NNRTI etravirine (ETV) in treatment experienced children and adolescents aged 6 to <18 years. [1, 2]

These 24-week results are from PIANO (Pediatric trial with Intelence as an Active NNRTI Option). PIANO is an ongoing Phase II, open label trial looking at the safety, efficacy and PK of ETV 5.2mg/kg bid (maximum dose 200mg bid).

In this study, 101 children (6 to <12 years, n=41) and adolescents (12 to <18 years, n=60) received ETV plus background regimen of a boosted protease inhibitor plus nucleoside/nucleotide inhibitors with optional enfuvirtide and/or raltegravir for 48 weeks. The trial participants received 25mg and 100mg tablets of ETV.

Sparse samples for population PK were taken at weeks 4, 8, 12, 24 and 48. At week 24 two samples were collected, a trough and one at least an hour after ETV dose. ETV plasma concentrations were measured using a validated high performance liquid chromatography-mass spectrometry/mass spectrometry assay.

The investigators developed a paediatric population PK model based on previous adult modelling and supplemented with rich and sparsely sampled PK data from TMC125-C126 [HTB ref] and PIANO respectively. They used the model to determine ETV AUC12h and C0h for all participants enrolled in PIANO up to 24 weeks.

There were 476 plasma concentration time samples available from 101 participants completing 24 weeks. There was an overall mean (SD) AUC12h and C0h of 5236 (+4314) ng*h/mL and 347 (+342) ng/mL respectively. In children in the younger age group these values were 5764 (+4044) ng*h/mL and 381 (+321) ng/mL. In adolescents they were 4834 (+4483) ng*h/mL and 323 (+357) ng/mL respectively. Adult reference values from the DUET trial were 5506 (+4710) ng*h/mL and 393 (+391) ng/mL for AUC12 and C0h respectively.

The investigators observed slightly lower exposures in the adolescents compared to the adults despite the majority (93%) of adolescents receiving the adult ETV dose of 200mg bid.

A dose of 5.2 mg/kg ETV is expected to be recommended for this population.

A related poster authored by Gareth Tudor Williams and colleagues described safety and efficacy from the same study. [3] The incidence of serious adverse events (AEs, grade 3 or 4) was low. A total of eight participants discontinued the trail due to AEs, this occurred more frequently in the older (n=6) than younger (n=2) age group. The most common AEs were upper respiratory tract infection (n=27) and rash (n=23).

Approximately half (n=51) of participants achieved a viral load <50 copies/mL. Response rates were higher in children than adolescents, with 24/41 (59%) achieving an undetectable viral load compared to 28/60 (47%). Response was similar in participants in both age groups considered adherent (measured by pill count and questionnaire) compared to non-adherent, respectively 48% (<95% adherent) compared to 53% (>95% adherent).

Of 28 participants with available genotype results at the time of virological failure, 54% developed NNRTI resistance mutations, mainly Y181C, E138A and V901.

Maraviroc

Carlo Giaquinto and colleagues presented preliminary PK data for the CCR5 antagonist maraviroc (MVC) in children and adolescents aged 2 to <18 years. [4, 5]

Data are from Study A4001031 – an ongoing open-label, non-comparative, multi-centre study in two stages (1: dose finding; 2: safety/efficacy) in treatment-experienced children, infected with CCR5-tropic HIV-1, receiving MVC 40-450 mg BID with optimised background therapy (OBT).

MVC PK were determined at Week 2. Participants (n=31) were stratified into four age cohorts. They were dosed twice daily. The initial dosing was calculated according to body surface area (BSA) with adjustments to take into account interactions between MVC and OBT (adult-recommended doses with/without CYP3A4 inhibitors/inducers).

Doses were adjusted and PK reevaluated if average concentrations (Cavg) at week 2 were <100 ng/mL. Cavg was estimated from AUC (AUC12h) calculated from seven samples taken over 12 hours.

The investigators reported, out of 22 participants receiving MVC with a potent CYP3A4 inhibitor (protease inhibitor based regimens). Only one failed to meet the PK target with the initial dose (this was due to poor adherence). But all five participants who did not receive a protease inhibitor (two nevirapine based regimens; two raltegravir based regimens; one NRTI based regimen) needed at least twice the initial MVC dose.

At the time of enrolment into stage 2, one participant did not meet the target after two dose adjustments but responded well clinically so was therefore included in the PK analysis. See Table 1: Preliminary PK results for maraviroc in children and adolescents aged 2 to <18 years.

The authors concluded that these preliminary data show that BSA-based dosing of MVC with CYP3A4 inhibitors provides MVC exposures associated with near- maximal efficacy (Cavg>100 ng/mL) in all age groups studied. But they noted that additional PK analyses are required to evaluate appropriate dosing when MVC is administered without CYP3A4 inhibitors in children.

A second poster from the same group showed safety and efficacy from the same study. [6]

At the time of analysis 35 children had been randomised (n=2, n=12, n=6 and n=15 in cohorts 1 to 4 respectively) and had received at least one dose of MVC. The median duration of treatment was 396, 493, 435 and 211 days in cohorts 1 to 4 respectively. The investigators observed 101 non-serious AEs in 21 patients; they considered 17 of these in 8 patients to be treatment related. Of those with elevated liver function test results, none were of grade 3 or higher. There were 8 serious adverse events of which none were judged to be treatment related and all resolved. There were no deaths.

Viral load <50 copies was achieved by 17/24 (71%) and 11/17 (65%) of participants at weeks 24 and 48 respectively. Five participants had virological failure; in four, this was due to poor adherence. The fifth had emergence of dual-mixed virus and developed 3TC resistance.

Enrollment in this study is continuing and long-term data will be collected and analysed.

References

1. Kakuda TN et al. Population pharmacokinetics of etravirine in HIV-1 infected treatment experienced children and adolescents (6 to <18 years). 3rd International workshop on HIV paediatrics. 15-16 July 2011. Rome, Italy. Oral abstract PP_1.(PDF)
2. Kakuda TN et al. Population pharmacokinetics of etravirine in HIV-1 infected treatment experienced children and adolescents (6 to <18 years). 6th IAS Conference on HIV pathogenesis, treatment and prevention. 17-20 July 2011. Rome, Italy. Poster abstract TULBPE026.
3. Tudor-Willaims G et al. Safety and efficacy of etravirine in HIV-1-infected, treatment-experienced children and adolescents (6-17 years): week 24 primary analysis of the phase II PIANO study. 6th IAS Conference on HIV pathogenesis, treatment and prevention. 17-20 July 2011. Rome, Italy. Poster abstract TULBPE027.
4. Vourvahis M et al. Maraviroc (MVC) pharmacokinetics (PK) in CCR5-tropic HIV-1-infected children aged 2 to < 18 years: preliminary results from study A4001031. 3rd International workshop on HIV paediatrics. 15-16 July 2011. Rome, Italy. Oral abstract PP_4. (PDF)
5. Vourvahis M et al. Maraviroc (MVC) pharmacokinetics (PK) in CCR5-tropic HIV-1-infected children aged 2-< 18 years: preliminary results from study A4001031. 6th IAS Conference on HIV pathogenesis, treatment and prevention. 17-20 July 2011. Rome, Italy. Poster abstract MOPE232.
6. Giaquinto et al. Safety and efficacy of Maraviroc (MVC) in CCR5-tropic HIV-1 infected children aged 2 to <18 years. 3rd International workshop on HIV paediatrics. 15-16 July 2011. Rome, Italy. Poster abstract P_51. Also presented at the 6th IAS Conference on HIV pathogenesis, treatment and prevention. 17-20 July 2011. Rome, Italy. Poster abstract MOPE237.

Polly Clayden, HIV i-Base

Prematurity is a known risk for infant mortality. Other risks include maternal immunosuppression, delayed initiation of ART and low baseline CD4 percentage.

Investigators from the Perinatal HIV Research Unit (PHRU) in Soweto, South Africa showed findings at the 2011 paediatric workshop from a cohort study designed to investigate prematurity among children born in 2009 and initiated on ART before one year of age. The study was a database and record review.

The background characteristics of the infants at time ART initiation are shown in Table 1.

The investigators reported no difference in mortality between preterm and term infants, respectively, 3% vs 4% (OR 1.9; 95%CI 0.5-6.7). Lost to follow up was 8% overall.

Univariate analysis revealed non-significant p-values for all variables ie preterm vs term, baseline CD4%, baseline viral load, breast vs formula feeding and maternal PMTCT. The investigators noted the small sample size and that the mortality rate was low in this study.

They concluded that although HIV-infected preterm infants have significantly lower CD4% than term infants, with early ART initiation they are not at increased risk of mortality.

Reference

Lazarus E et al. Prematurity is not a risk factor for early mortality in HIV-infected infants on antiretroviral therapy. 3rd International workshop on HIV paediatrics. 15-16 July 2011. Rome, Italy. Poster abstract P_18.

Polly Clayden, HIV i-Base

NEVEREST was a study in which young children who were exposed to nevirapine as PMTCT and initiated on PI-based HAART were randomised to continue on this regimen or switch to a nevirapine based regimen (we report the final results from NEVEREST later in this issue of HTB).

NEVEREST investigators evaluated body composition and metabolic abnormalities in 156 children exiting the trial. The objectives were to compare lipid profiles, markers of inflammation and regional fat distribution in children receiving a PI-based regimen of LPV/r plus 3TC plus d4T to those switched to an NVP-based regimen. [1]

The children’s weight (kg) and height (cm) was measured and weight-for-age, height-for-age and BMI-for-age z-scores (WAZ, HAZ, BAZ) calculated. Fasting total cholesterol (TC), high density lipoprotein (HDL), low density lipoprotein (LDL), triglycerides (TG), C-reactive protein (CRP), viral load, absolute CD4 and CD4 percentage were obtained. Circumferences and skinfolds were also measured; waist to hip ratio (MWC:MHC) and skinfold sum (SFS) were calculated. Upper arm and thigh fat estimates (UFE, UTFE) were calculated by Rolland Cachera. Analyses were intent to treat.

At the time of analyses, children were a mean age of 5.1 (range 3.6 – 6.9) years and approximately half were boys; 85 (42 boys) were randomised to the PI arm and 71 (40 boys) to the NNRTI arm. There were no differences between the two groups in sex, age, total time on ART, time since randomisation, WAZ, HAZ or BAZ or proportion with viral load <50 copies/mL. But children in the NNRTI group had a higher CD4 count, 1480 cells/mm3 compared to 1356 cells/mm3, p=0.049.

The investigators found differences in metabolic measurements. Mean TC was greater in the PI group, 171 (SD+39) mg/dL vs 161 (SD+31) mg/dL, p=0.05 as was the proportion of children with hypercholesterolemia (TC >200 mg/dL), 18.8% vs 8.5%, p=0.03. They also observed lower mean HDL levels, 51 (SD+14) mg/dL vs 59 (SD+16) mg/dL, p=0.006 and higher mean LDL levels, 100 (SD 34) mg/dL vs 88 (SD+27) mg/dL, p=0.018, in the PI group. The mean TG level was also greater in the PI group, 94 (SD+39) mg/dL vs 72 (SD+29) mg/dL, p<0.001 as was the proportion with hypertriglceridemia (TG >150 mg/dL), 12.9% vs 2.8%, p=0.038.

The children in the PI group had significantly greater amount of total body fat compared to those receiving an NNRTI, with a mean SFS of 43 (SD+11.1) mm vs 39 (SD+10.1) mm, p=0.029 and % body fat by BIA (Horlick Equation) of 0.17 (SD+0.7) vs 0.14 (SD+0.08), p=0.042.

The percentage of fat in the upper arm did not differ between groups but the percentage of fat in the upper thigh was greater in the PI group, p=0.021. Also the PI group had a smaller ratio of trunk fat relative to thigh fat, p=0.03.

The investigators wrote: “These unfavourable alterations in lipids and lipoproteins are of great concern with respect to potential increase in long term CVD risk and should be considered in treatment strategies, such as the reuse of NNRTIs for NNRTI-exposed infants”.

References

Shiau S et al. Body composition and metabolic abnormalities of perinatally HIV-infected children in South Africa on long-term ARV treatment. 3rd International workshop on HIV paediatrics. 15-16 July 2011. Rome, Italy. Oral abstract O_2. Also presented at the 6th IAS Conference on HIV pathogenesis, treatment and prevention. 17-20 July 2011. Rome, Italy. Poster abstract 252.

Simon Collins, HIV i-Base

Results from a new online resource developed to predict treatment outcomes for settings without access to genotypic resistance tests were presented in a poster at the conference. [1]

The system was developed by training computer models to predict virological response to therapy using data from approximately 15,000 treatment changes drawn from over 15 countries. The models use CD4, viral load, treatment history and the drugs in the new regimen in making their predictions and can generate predictions of response at selected time points out to 48 weeks for all available combinations or for a selected combination.  The system includes the option to select drugs that are available in each country and to exclude drugs that are contraindicated.

The accuracy of the models was assessed with an independent test set of 800 cases. Two further test sets from Romania (n=39) and South Africa (n=56) were also reported together with subset of 57 cases from the 800 test set that had genotypes available.

The mean area under the curve and overall accuracy were 0.77 and 71% with the 800 test dataset (with similar results during cross validation). The comparable results were 0.68 and 67% for the Romanian and 0.69 and 68% for the South African test sets respectively. When the 57 case test set was used to compare the performance of the models with and without genotype information the results were 0.77 and 74% using the genotype, compared to 0.76 and 68% for the ‘no-genotype’ models.

The models are now available via the RDI’s online treatment selection tool HIV-TRePS. Importantly, the resource includes the option to include, with permission, anonymised information on treatment decisions and outcomes to be collected to help further development of the system. [2]

The resource has been developed by researchers at RDI who were involved in much of the original pioneering work into HIV drug resistance technology and more recently have been developing prediction tools to interpret genotype results using computer-developed neural networks.

Future reports on how this resource is used in practice will be important given the extremely restricted access to resistance testing in most resource-limited countries and that this is unlikely to change in the near future.

References

1. Larder BA et al. Predicting response to antiretroviral therapy without a genotype: a treatment tool for resource-limited settings. 16th IAS Conference on HIV Pathogenesis, Treatment and Prevention, 17–20 July 2011, Rome. Poster MOPE146.
2. The resource can be accessed free online after one-time free registration.

http://www.hivrdi.org

This year the annual BHIVA meeting was held in Bournemouth and most reports were in the previous issue of HTB.

The abstract book from the conference, published as a supplement to HIV Medicine is available to download free as a PDF file from the BHIVA website.

http://www.bhiva.org/ConferenceAbstracts.aspx

The report in this issue is:

• Treatment in seroconversion maintains HIV specific immune responses similar to long term slow progressors

Recent studies by Hocqueloux et al suggest that long-term control of viraemia is possible after discontinuation of prolonged ART initiated at seroconversion. [1] This study compared a cohort of 15 long-term non-progressors (LTNPs) with spontaneously controlled viraemia with a cohort of 20 long-term treated HIV-1 seroconverters (LTTS), all of whom started ART at the time of seroconversion resulting in ART-induced controlled viraemia. [2]

LTNPs were defined as having an absence of clinical progression with no CD4 T cell and without using treatment. They have controlled viraemia (are ‘elite controllers’) and low viral reservoirs.

Immunovirological parameters defined for this study included:

• The size of viral reservoirs and residual replication (cell associated HIV-1 DNA and RNA respectively)
• Cellular immunity (HIV-1 specific CD4 and CD8 T cells)
• The role of HIV-1 specific CD8+ T cells in viraemic control
• Polyfunctionality associated with virological control
• Slow improvement of HIV-1 specific CD8 T cell function in LTTS
• HIV-1 specific CD8 T-cells in LTNPs are functional in relation to cytokine production, proliferation and cytotoxic capacity

Inclusion criteria of the two study groups included:

• LTNPs: =>7 years with <1000 HIV-1 copies/mL, CD4 >500 cells/mm3 in absence of ART, clinically healthy and with no history of OIs
• LTTS: HIV-1-positive on ART since seroconversion, ART =>4 years and long-term viral suppression (<50 copies/mL)

Study participants were matched in terms of gender, age, ethnicity, transmission route, CD4 count, viral load and cell-associated DNA and RNA. The only significant difference (p=0.06) was between CD4/CD8 T-cell ratios.

Results of the study suggest comparable levels of highly polyfunctional HIV-1 specific CD4+ and CD8+ T cells in both LTTS and LTNPs. Polyfunctional T-cell profiles and low viraemia in the presence/absence of ART were seen in both groups. There was a trend towards a higher magnitude and breadth of HIV-1 specific CD8+ T cells in LTNPs compared to LTTS which is thought to have been driven by a response against the GAG proteins.

The study concluded that prolonged ART initiated at the point of HIV seroconversion is associated with immuno-virological features which resemble those of HIV-1 LTNPs.

References

1. Hicqueloux L et al. Long-term immunovirologic control following antiretroviral therapy interruption in patients treated at the time of primary HIV-1 infection. AIDS. 2010 Jun 19;24(10):1598-601.
2. Kinloch-de Loes S et al. HIV-1 specific T cells during prolonged antiretroviral treatment in HIV-1 seroconverters. 17th Annual BHIVA Conference, 6–8 April 2011, Bournemouth. Abstract 029.

Our final reports from this important conference are:

• Monitoring treatment in resource limited settings: results from PHPT-3 and Stratall ANRS12110/ESTER trials

• DART: high rates of viral suppression after five years and a single CD4 test with a threshold of 250 cells/mm3 could reduce unnecessary switching

• Lopinavir/r monotherapy used as second-line therapy in resource-limited settings

• Treating children previously exposed to single dose nevirapine: update on IMPAACT P1060 and NEVEREST

• Lopinavir/ritonavir oral solution toxicity in neonates

• Paediatric antiretroviral pipeline: darunavir and raltegravir

• Pharmacokinetics of different rifabutin dosing strategies with lopinavir/ritonavir-based ART

• Initiation of ART during breastfeeding can induce multidrug resistance in infants

Unless mentioned otherwise, all references are to the Programme and Abstracts of the 18th Conference on Retroviruses and Opportunistic Infections, 28 February–2 March 2011, Boston.

http://www.retroconference.org/AbstractSearch/

Webcasts are available at the following link:

http://www.retroconference.org/2011/data/files/webcast_2011.htm

In resource-limited settings, optimal monitoring and switching criteria from first-line to second-line therapy is unclear. Results from two trials were shown as oral presentations that suggest that monitoring viral load is not essential for switch to second line. [1, 2]

Marc Lallemant showed data from PHPT-3, which was conducted in Thailand. This was a randomised double-blind (until first switch) non-inferiority trial. Participants were randomised to CD4 or viral load monitoring, which was conducted every three months.

Dr Lallemant explained that the trial was designed for a setting with only two lines of treatment and where second line is far more expensive than first line. The investigators wanted to test whether monitoring and switching people without viral load compromised their health or their future options.

PHPT-3 enrolled HIV-positive adults (CD4 count 50 to 250 cells/mm3, not hepatitis B or C co-infected), starting NNRTI-based HAART.

In the CD4 monitoring arm, patients switched to second-line protease inhibitor (PI) -based therapy when they had confirmed CD4 decline of 30% or more from peak, and in viral load monitoring they switched when they had confirmed viral load >400 copies/mL.

The primary endpoint was death, new AIDS-defining event or clinical failure – defined as CD4 <50 cells/mm3 – at 3 years. Secondary endpoints included proportions switching to second line, time to switch, resistance mutations at failure and future treatment options.

The trial enrolled 716 patients of which 60% were women. Their median CD4 count at baseline was 144 cells/mm3 (range 90 to 200 cells/mm3).

Regimens were 65% efavirenz-based regimen and 66% of participants received tenofovir/FTC. Other study drugs were nevirapine and AZT/3TC. At 3 years of follow up 93.3% of patients were evaluable. Ten percent stopped treatment for toxicity across both groups.

There were 58 clinical failures overall, 28 and 30 in the CD4 and viral load groups respectively. The respective rates of clinical failure per patient years were 2.3 vs 2.5 and of death 1.1 vs 1.4.

In multivariate analysis, anaemia, adjusted HR 2.7 (95% CI 1.5-4.8), p=0.001; CD4 <150 cells/mm3, AHR 2.3 (95% CI 1.2-4.2), p=0.009 and viral load >5 log, AHR 1.8 (95% CI 1.0-3.0), p=0.04, were predictive of clinical failure at 3 years.

The probability of switch to second-line (excluding toxicity/intolerance) was 5.2% (95% CI 3.2-8.4%) vs  7.5% (95% CI 5.0 -11.1%) in the CD4 and viral load groups respectively, p= 0.10.

The respective median times to switch were 11.7 months (95% CI 7.7-19.4) vs 24.7 (15.9-35.0), p=0.001. And the median duration of viraemia >400 copies/mL was 7.2 months (IQR 5.8 to 8.0) vs 15.8 months (8.5 to 20.4), p= 0.002. But the median CD4 counts were 426 cells/mm3 vs 420 cells/mm3, respectively.

Dr Lallemant noted that 15/31 patients in the CD4 monitoring arm who switched to second-line had viral load <50 copies/mL at the time of switching.

Viral load was <50 copies/mL in 99% of patients at 3 years follow-up and patients with CD4 monitoring did not have fewer future treatment options, with the exception of one patient with multiple thymidine analogue mutations (D67N/M41L/L210W/T215Y).

Dr Lallemant concluded that, after 3 years, the rate of clinical failure was very low and did not differ between the two strategies. Most mutations had been selected at the time of virological failure. The additional time spent on failing treatment in the CD4 arm did not result in reduced future treatment options.

He noted that the conclusions from PHPT-3 are similar to those from DART and HBAC in adults and PENPACT-1 in children. He added that the need for viral load monitoring may be less important than close and regular safety, tolerability, adherence, and immunological monitoring. He remarked that the nurse/patient team with expert assistance from doctors, biologists and patient networks “maximizes efficacy and durability.”

This was followed by a related presentation of data from the Stratall ANRS12110/ESTER trial.

Charles Kouanfack showed findings from a trial designed to compare clinical monitoring alone with laboratory and clinical monitoring. This trial was conducted in 9 rural district hospitals in Yaounde, Cameroon.

Dr Kouanfack explained, in Cameroon, the national programme followed WHO guidance for a public health approach based on decentralised, integrated HIV care delivery in facilities where laboratory monitoring is generally unavailable. He noted that the 2010 guidelines also state that using viral load monitoring to detect treatment failure and switch is recommended but has “low quality evidence”.

Stratall ANRS12110/ESTER was a randomised non-inferiority trial enrolling HAART-naïve, HIV-positive adults with a WHO stage 3-4 disease or stage 2 and total lymphocyte count <1200 cells/mm3, who were followed for 2 years. Management was by the health workers in charge of routine activities.

The primary endpoint was mean increase in CD4. The increase in the clinical monitoring arm was judged to be non-inferior to that in the laboratory monitoring arm if the difference was less than or equal to 25%.

Secondary endpoints included: viral suppression, death, new stage 3 or 4 events, resistance, loss to follow up, adherence, treatment changes and toxicity.

Participants were monitored clinically 3 monthly in both arms and those in the laboratory monitoring arm also had CD4 and viral load measured every 6 months.

Switching to second line was indicated by grade 3 or 4 events in the clinical monitoring arm and persistent viral load >5000 copies/mL in the laboratory monitoring arm.

Of a total of 493 patients, 256 were assigned to clinical and 237 to laboratory monitoring. Of these, 93% were followed and included in the analysis. Patients were similar at baseline with CD4 counts of 179 cells/mm3 and 182 cells/mm3 in the clinical and laboratory monitored arms respectively. Both arms had high baseline viral loads of 5.6 log₁₀ copies/mL. Overall 70% were women. About 65% started treatment with d4T + 3TC + NVP.

The trial failed to demonstrate non-inferiority of clinical monitoring:  the mean increase in the CD4 count was 175 cells/mm3 (95%CI 151-200) vs 206 cells/mm3 (95% CI 181-231) in the clinical and laboratory arms respectively. This gave a difference –31 (–63 to +2), the non-inferiority margin was –52 (–58 to –45). The analysis was last observation carried forward.

The analysis also revealed that 13 (6%) laboratory-monitored participants switched to second-line regimens because of treatment failure, compared to none of the clinically monitored participants, p<0.001. But, viral suppression (49 vs 52%), resistance (both 10%), mortality (18 vs 14%), disease progression (36 vs 29%), adherence (both 64%), loss to follow-up (9 vs 8%), and toxicity (19 vs 25%) were similar between the two groups.

Dr Kouanfack concluded that failure to demonstrate non-inferiority of immunological recovery and the need to switch to second line in this trial supports the WHO recommendation of laboratory monitoring of HAART where possible.

He also concluded that the difference between the two strategies suggest that clinical monitoring alone can be used for at least the first two years of treatment in order to expand scale up and to take into account financial and infrastructural constraints in resource limited settings.

References

1. Jourdain G et al. PHPT-3: A randomised clinical trial comparing CD4 vs viral load ART monitoring/switching strategies in Thailand. 18th CROI, 27 February–3 March 2011, Boston. Oral abstract 44.
2. Kouanfack C et al. HIV viral load, CD4 cell count, and clinical monitoring vs clinical monitoring alone for ART in rural hospitals in Cameroon: Stratall ANRS 12110/ESTHER trial, a randomised non-inferiority trial. 18th CROI, 27 February–3 March 2011, Boston. Oral abstract 45LB.

Both webcasts: Research on Delivery of Care in Developing Countries. Monday 28 February 11 am.

DART was a randomised trial comparing clinically driven monitoring (CDM) to laboratory (CD4, haematology, biochemistry) plus clinical monitoring (LCM) of 3316 HAART-naïve adults conducted in Uganda and Zimbabwe. People in both monitoring arms showed high and similar 5-year survival rate – 90% vs 87% in the LCM and CDM arms respectively – differing by a small percentage that only occurred after two years of follow up. This compared to an historical 5-year survival rate prior to HAART of only 8% in the Uganda cohort. [1]

First line HAART in this trial was AZT/3TC plus either TDF (74%), ABC (9%) or NVP (16%). Participants needing to switch to second line received LPV/r plus NRTI/s and/or NNRTI. Neither the CDM nor LCM group had real time viral load monitoring.

Ugandan patients who did not participate in one of two, nested second line RCTs had a viral load test when they left the trial and joined the national programme.

Further findings from the DART trial were presented at CROI 2011.

Cissy Kityo and colleagues showed high rates of virological suppression at 5 years after HAART initiation among the Ugandan participants alive and in follow up. [2]

Both monitoring groups switched to second line therapy following WHO stage 4 or multiple stage 3 events; the LCM group also switched at CD4 <100 cells/mm3.

A viral load measurement was available the end of the trial for the majority of eligible participants: 1207 (80%) and 187 (70%) respectively receiving first and second line at exit. The viral load sample was taken at a median of 5.2 years after initiation of HAART and 2.7 years after start of second line for those who had switched.

Of the participants who remained on first line, 81.9% (95%CI 78.5-84.9%) in LCM and 74.2% (95%CI 70.6-77.6%) in CDM had viral load <200 copies mL, p=0.001. In the LCM group 5.6% (95% CI, 3.9-7.8% had viral loads <10,000 copies, which was lower than the 10.4% (95%CI 8.1-13.1%) of participants in CDM.

Of those who switched, viral loads were similar across the two monitoring groups, p=0.6. Viral load <200 copies/mL was achieved in 88.8% (95%CI 83.3-92.9%) of participants receiving second line.

When the investigators examined the CD4 count nearest to the exit viral load measurement (taken at a maximum of 6 months apart), they found a negative association, r=0.4, as would be expected.

Of 283 (20%) participants with viral load >200 copies/mL, 29% in the LCM group and 42.2% in CDM had CD4 <200 cells/mm3.

The investigators noted that CD4 counts <100 cells/mm3 were rare in either arm; only 2 people in LCM and 7 in CDM.

A related study showed a single CD4 test with a threshold of >250 cells/mm3 could reduce inappropriate switching in clinically monitored patients. [3]

Charles Gilks and colleagues investigated the relationship between CD4 count at switch and the reason for doing so in all 675 (361 LCM and 314 CDM) DART participants switching to second line.

In the CDM arm, 206 (66%) switched due to WHO stage 4 events and 76 (24%)/32 (10%) participants single or multiple WHO stage 3 events, respectively. In LCM 265 (73%) participants switched because their CD4 count fell below 100 cells/mm3, 43 (12%) for other CD4 reasons, 37 (10%) due to WHO 4 events and 6 (23%)/10 (3%) single or multiple WHO stage 3 events.

In the LCM arm, clinical failure provoked switching in 7 (2%) of patients with CD4 >250 cells/mm3; 3 due to WHO stage 4 events, 1 single WHO stage 3 event and 3 for other CD4 reasons. This compared to 64 (20%) of participants who switched with CD4 > in the CDM arm, p=0.001. The investigators noted, however, that deaths within one year of switching were similar in CDM whether participants switched above or below 250 cells/mm3, 11/64 (17%) vs 33/250 (13%) respectively.

In the CDM group, switching due to a single WHO grade 3 event was significantly more frequent with a CD4 count of >250 cells/mm3 (27/76, 36%) compared to multiple WHO stage 3 events (4/32, 12%) or WHO stage 4 events (33/206, 16%), p=0.001.

Viral load measurements at switch were available for 108 and 113 participants in the LCM and CDM groups respectively.  Of these, 15 (14%) vs 32 (28%) respectively were <400 copies/mL, p=0.009.

In the CDM group, 25/31 (81%) with clinical failure and CD4 > 250 cells/mm3 had viral load <400 copies/mL vs 7/82 (9%) with CD4 <250 cells/mm3, p<0.001.

The investigators noted a trend to switching for single WHO stage 3 events compared to multiple WHO stage 3 or stage 4, but this was not significant, p=0.22.

They concluded that among clinically monitored patients, a single CD4 test with a threshold of 250 cells/mm3 could identify up to 80% with viral load <400 copies/mL who are unlikely to benefit from second line therapy. In DART, nearly 40% of participants who failed clinically with a single WHO stage 3 event had CD4 >250 cells/mm3. They wrote: “Targeting this group would be particularly likely to avoid premature, costly switching to second line.”

References

1. DART trial team. Routine versus clinically driven laboratory monitoring of HIV antiretroviral therapy in Africa (DART): a randomised non-inferiority trial. The Lancet, Volume 375, Issue 9709. 9 January 2010.
2. Kityo C et al. High rates of virologic suppression among patients not receiving routine virologic monitoring after 5 years of first-line ART. 18th CROI, 27 February–2 March 2011, Boston. Poster abstract 677.
3. Gilks C et al. A single CD4 Test with threshold >250 cells/mm3 can markedly reduce switching to second-line ART in African patients managed without CD4 or viral monitoring. 18th CROI, 27 February–2 March 2011, Boston.  Poster abstract 676.

WHO guidelines recommend the use of boosted protease inhibitors second line in resource limited settings. Findings from strategies looking at using lopinavir/ritonavir (LPV/r) have been uncertain to date, both in limited and richer resourced settings.

Two posters at CROI 2011 presented data from studies evaluating LPV/r monotherapy, with showed further conflicting results.

ACTG 5230 evaluated lopinavir/ritonavir (LPV/r) monotherapy in a pilot study. It was a single arm multinational trial with sites in Malawi, Tanzania, South Africa, Thailand and India.

Participants had previously received first line NNRTI-containing regimens for at least six months and had detectable viral load 1,000–200,000 copies/mL. All participants received LPV/r monotherapy BID. The primary endpoint was remaining on monotherapy without virological failure at 24 weeks. This was defined as: failure to suppress viral load to <400 copies/mL by week 24, or confirmed rebound to >400 copies/mL at or after week 16 following confirmed suppression.

People with virologic failure received intensification with emtricitabine (FTC) 200 mg/tenofovir (TDF) 300 mg.

There were 123 participants enrolled in this trial. About 60% were women and they were a median of 39 years of age, with a median CD4 of 164 cells/mm3 and viral load of 4.34 log₁₀ copies/mL (17% were >100,000 copies/mL).

Other baseline characteristics included: 93% with >1 year HAART, 98% with >1 NNRTI mutation and 95% with >1 NRTI mutation (87% M184V, 84% TAM, 11% K65R, 4% Q151M/L).

The majority, of participants completed 24 weeks of follow-up with the exception of one death at week 20 with a viral load of <400 copies/mL.

The investigators reported, at week 24, 107 (87%; 95% CI 80-92%) of participants remained on LPV/r monotherapy without virologic failure.

Of the remaining, 15 met the criteria for virologic failure and one added FTC/TDF before failure. Of 13 participants with data after intensification, 11 (85%) suppressed viral load to <400 copies/mL.

At virologic failure, 2/11 participants who were successfully sequenced had selected new resistance mutations (both had A71T and V82F). The overall mean CD4 count increase from baseline to week 24 was 107 cells/mm3. Overall 31 (25%) of participants experienced grade 3 or 4 toxicities. The most commonly reported grade 3 or 4 toxicities (9% of participants) were metabolic (mostly elevated lipids). Self reported adherence was high; at week 24, 83% of participants reported no missed doses.

The investigators concluded that LPV/r monotherapy showed promising preliminary activity as second-line HAART following failure of first-line NNRTI-containing regimens at 24 weeks. The lower bound of the 90% CI (81-92%) of the observed success rate (87%) was above 65%.

Torsak Bunupuradah and colleagues from the HIV STAR Study in Thailand looked at LPV/r monotherapy as second line but they also evaluated viral suppression to <50 copies/mL and included a comparison arm with triple therapy.

The STAR investigators enrolled 200 participants with viral load >1000 copies/mL on NNRTI-containing first line therapy. Participants were randomised to receive either LPV/r monotherapy ot LPV/r + TDF + 3TC.

Treatment failure was defined as viral load >400 copies/mL at >24 weeks. Participants meeting these criteria in the monotherapy arm received intensification with TDF + 3TC.

Participants in this study were about 60% men with a median age of 37 years, CD4 of 188 cells/mm3, and viral load of  4.1 log10 copies/mL.

Prior to switching, 92% of participants were receiving 3TC, 63% d4T, 23% AZT and 5% TDF. Nevirapine and efavirenz were received by 86% and 14% participants, respectively. Without significant differences between arms, 15% of participants had ≥3 TAMS, 82% had M184V/I, 6% had Q151M, and 7% had K65R.

By intent-to-treat analyses at 48 weeks, the proportion of patients with viral load <400 copies/mL the LPV/r monotherapy arm was 75% vs 86% in the TDF/3TC/LPV/r arm, p=0.53. But, only 61% of the LPV/r monotherapy arm vs 83% in TDF/3TC/LPV/r arm had a viral load <50 copies/mL, p<0.01.

Major PI mutations were detected in 1 of 2 LPV/r monotherapy and 0 of 3 TDF/3TC/LPV/r treated participants with genotype results following treatment failure. There was no significant difference in CD4 count increase between arms: 114 vs 137 cells/mm3 in the LPV/r monotherapy and TDF/3TC/LPV/r arms respectively. One death (unrelated to study drugs) was reported in each arm. Serious adverse events were reported in two patients in the LPV/r monotherapy arm and seven patients in the TDF/3TC/LPV/r arm.

The investigators concluded that LPV/r monotherapy should be used with caution as a second-line option, particularly in settings where close viral load monitoring is not available.

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The ongoing EARNEST Trial (NCT00988039) will answer the question whether or not lopinavir/r monotherapy is a sufficiently potent regimen compared to lopinavir/r combined with two NRTIs or raltegravir.

Results from this trial are expected in 2013.

References

1. Bartlett J et al. A pilot study of LPV/r monotherapy following virologic failure of first-line NNRTI-containing regimens in resource-limited settings: The Week-24 primary analysis of ACTG 5230. 18th CROI, 27 February–2 March 2011, Boston. Poster abstract 583.
2. Bunupuradah T et al. Second-line LPV/r monotherapy was inferior to TDF/3TC/LPV/r in patients who failed NNRTI regimen: HIV STAR Study. 18th CROI, 27 February–2 March 2011, Boston. Poster abstract 584.

Two oral presentations at CROI 2011 showed further findings from studies looking at treatment in children previously exposed or unexposed to maternal/infant single dose nevirapine (NVP) in prevention of mother to child transmission (PMTCT) programmes.

IMPAACT 1060

IMPAACT P1060 was a randomised trial to determine whether NVP- or lopinavir/ritonavir (LPV/r)-based treatment performed better in young children exposed and unexposed to single dose NVP. All children received AZT plus 3TC. The trial comprised of Cohort 1 (exposed children) and Cohort 2 (unexposed children). Data from Cohort 1 have previously been reported and this part of the study was stopped early after a scheduled Data Safety Monitoring Board (DSMB) review, as there was an unsurprising trend towards more failure in the children receiving NVP- compared to LPV/r-based treatment.

Peter Palumbo presented results from Cohort 2. This cohort enrolled children aged 2 to 36 months, who met WHO criteria for treatment and were unexposed to single dose NVP. Children were stratified by age < or ≥ 12 months. Children with TB were excluded from the trial.

The study had a composite primary endpoint of treatment failure, which comprised viral failure (<1 log10 decline from baseline to after 12 to 24 weeks or >400 copies/mL at week 24), or permanent discontinuation of NVP or LPV/r, including death by 24 weeks. Rates were calculated from Kaplan-Meier curves for each treatment group and age group.

Secondary endpoints included time to virological failure by 24 weeks, time to treatment failure throughout follow up and time to virological failure or death throughout follow up.

P1060 Cohort 2 was fully enrolled with 288 children by March 2010 and had 48-week planned follow-up to March 2011.  In October 2010, the DSMB recommended that the study was unblinded. All children had completed 24 weeks of follow up.

Dr Palumbo reported that the children’s median age at enrollment was 1.7 years (73% >12 months) and their median baseline viral load and CD4 percentage were 535,632 copies/mL and 15% respectively. The majority (79%) of children were subtype C.  The median follow-up was 72 weeks.

At week 24, 87 children had reached an endpoint; 60 in the NVP and 27 in the LPV/r arms. The overall difference in failure rate was 21.5% (95% CI, 11.2-31.8) in favour of LPV/r, p<0.001. This was similar in both age groups: 22.0% (<12 months) and 21.3% (>12 months).

There was also a significant difference in time to off study drug, over the full length of the trial, p<0.001. There were 10 vs 3 deaths in the NVP vs LPV/r arms during the entire follow-up

period (none judged related to study drugs), but this did not reach statistical significance, p=0.63.

There was a notable amendment during the course of the trial. In 2007 the recommended NVP dose in WHO guidelines increased from the FDA recommended dose of 7mg/kg to 160-200mg/m2 (max 200mg). Only 32 children were enrolled under the lower dose compared to 115 at the higher one but the investigators saw no effect associated with this change.

Dr Palumbo noted that the main reasons for off study were more virological failure, toxicity and death in the NVP arm.

As both the NEVEREST and P1060 Cohort 1 data had suggested poorer weight and CD4 improvement in children receiving LPV/r compared to NVP, the investigators also looked at this in Cohort 2. They did not find a statistically significant difference in CD4 improvement between the two arms but there was a difference in weight z-score favouring NVP at 24 and 48 weeks, respectively p=0.007 and p=0.009.

When the investigators looked at NVP resistance in samples from subsets of children at baseline and time of virological failure, they found 2.4% (5/206) with resistance at baseline compared to 56% (10/18) at time of virological failure.

These results were different to those in the sister study, OCTANE P1060, in which maternal data demonstrated non-inferiority of NVP- to LPV/r-based treatment, by the study definition, for NVP- unexposed women.

This highlighted the “unique and challenging situation of early paediatric HIV infection”, Dr Palumbo said, including very high baseline viral load and the unforgiving nature of NVP resistance. LPV/r is already recommended for NVP-exposed children and discussions are ongoing as to whether this recommendation should expand to all young children, possibly up to three years of age.

These data once again point to the importance of developing new first and second line options for use in this age group.

NEVEREST

Louise Kuhn presented data from NEVEREST, a study designed to evaluate a treatment switch strategy from LPV/r to NVP in NVP-exposed children.

In this study, 323 children aged 6 weeks to 2 years and eligible for treatment were initiated on LPV/r plus 3TC plus d4T. After achieving a viral load <400 copies/mL and maintaining it for > 3 months, children were randomised (n=195) to either remain on LPV/r (n=99) or switch to NVP (n=96). Time to any viral load >50 copies/mL or confirmed >1000 copies/mL was compared using Kaplan-Meier methods and log-rank tests.

Fifty-two week data post switch from this study has been reported previously. These data revealed a higher proportion of children suppressed to <50 copies/mL (the primary endpoint) in the NVP arm but also a higher proportion in that group with confirmed >1000 copies/mL.

Dr Kuhn showed longer term results from this study with follow up of 18-53 months.

There were three deaths in each group. At 36 months post randomisation, as with the earlier analysis, more children in the NVP group (40.5%) maintained viral load <50 copies/mL than those in the LPV/r group, p=0.01. Again, more in the NVP (23.9%) than in the LPV/r (11.1%) had confirmed >1000 copies/mL, p=0.01.

This difference persisted at 48 months, for <50 copies/mL and >1000 copies/mL, respectively p=0.02 and p=0.08.

At 6 months 59.1% of the failures in the NVP group had occurred vs 10% in the LPV/r group. By 12 months these proportions were 100% in the NVP group and 50% in the LPV/r group. Dr Kuhn noted that among children in the LPV/r group, 6% of failures occurred between 12 and 48 months.

Treatment failure >1000 copies/mL was associated with the presence of pre-treatment NVP mutations, p=0.02. There was no difference in response between children in the NVP and LPV/r groups in children who had no pre-treatment NVP resistance. Half the children with detectable NVP mutations failed when re-challenged with NVP.

Dr Kuhn concluded that viral load testing can identify all switch failures and that switching can be accomplished safely if viral load testing is available. Also that pre-treatment screening for resistance can be used to identify the children who could benefit from this strategy.

References

1. Palumbo P et al. NVP- vs LPV/r-based ART among HIV⁺ infants in resource-limited settings: the IMPAACT P1060 Trial. 18th CROI, 27 February–2 March 2011, Boston. Oral abstract 129LB.
2. Kuhn L et al. Long-term outcomes of switching children to NVP-based therapy after initial suppression with a PI based regimen. 18th CROI, 27 February–2 March 2011, Boston. Oral abstract 128.

Lopinavir/ritonavir (LPV/r, Kaletra) oral solution is approved by the FDA for infants 14 days of age and older. US guidelines do not recommend its use in preterm infants.

LPV/r oral solution has particular pharmacokinetic properties that make its use complicated in neonates. It contains high volumes of both ethanol (356.3 mg/mL, 42% volume solute/volume solution (v/v) and propylene glycol (152.7 mg/mL, 15.3% v/v).

Neonates have reduced alcohol dehydrogenase and CYP3A4 activity and immature renal function. Ethanol is 95% and propylene glycol is 55-75% metabolised in the liver by alcohol dehydrogenase. Ethanol inhibits the metabolism of propylene glycol by alcohol dehydrogenase leading to elevated concentrations. LPV is metabolised by CYP3A.

Reduced hepatic metabolism and renal clearance in neonates, particularly in preterm infants, can lead to accumulation of all three ingredients to toxic levels.

Acute ethanol toxicity is linked to central nervous system (CNS) and respiratory depression, and gastritis. Propylene glycol is also associated with CNS and respiratory depression, as well as renal failure and metabolic acidosis. LPV has been shown to cause PR and QT interval prolongation and AV block in adults with very high levels of the drug.

Cases of toxicity in neonates – particularly preterm – have been reported to the FDA Adverse Event Reporting System (AERS).

A poster authored by Debra Boxwell and colleagues from the FDA showed data from case studies from a search of the AERS database for all reports of toxicity in children 2 years of age or under following dosing with LPV/r oral solution.

The search revealed 10 unduplicated cases in neonates of whom 8 were preterm. Of the preterm infants, 3 were born at 28 weeks gestation, 1 at 30 weeks, 2 at 32 weeks and 2 at 34 weeks.

The documented adverse events included cardiac toxicity (bradycardia, complete AV block, bundle branch block, or cardiac failure; (n=7), acute renal failure (n=5), increased serum creatinine (n=1), elevated serum lactate level (n=2), hyperkalemia (n=4), respiratory failure (n=2), hypotonia (n=1), abnormal EEG (n=1), and CNS depression (n=1).

Outcomes included 1 death, 2 life threatening and 4 hospitalisations. Therapy was initiated on the day of birth in 7 neonates, day after birth in 1, day 34 in 1, and unknown in 1.

Onset the first adverse event occurred within 1 to 6 days (n = 8). Discontinuation of Kaletra (n=9) resulted in recovery within 1 day in 1, 2 days in 2, 3 days in 2, 6 days in 3, 20 days in 1 and was unknown in 1.

WHO set 25mg/kg as a maximum acceptable daily intake of propylene gel when it is used as a food additive. The European Medicines Agency (EMA) recommends that a 12.5mg/dL blood concentration of ethanol after a dose of any medication should not be exceeded. In IMPAACT P1030 – a PK sub-study in full-term infants 6 weeks of age – the mean steady state of LPV was 5.2+1.8ug/m2 twice daily. When the FDA investigators looked at neonatal exposure to the three ingredients in the cases for which data were available, the results were far in excess of these recommendations. See Table 1: Neonatal exposure to lopinavir, ethanol and propylene glycol.

The investigators concluded that the ten cases to the AERS suggest that neonates, especially those born preterm, who received LPV/r oral solution, were at increased risk of toxicities from drug accumulation. They added that the improvement of symptoms when the drug was stopped support this association.

There are limitations to the AERS however. Because reporting is voluntary, the quality of reporting is very variable. The database is subject to under reporting as well as reporting bias and both the numerator and the denominator are unknown for any event reviewed. Therefore the incidence or estimated risk cannot be calculated.

comment

This analysis provoked a FDA label change and the lopinavir/r oral solution is not recommended for neonates particularly preterm.

Reference

Boxwell D et al. Neonatal toxicity of Kaletra oral solution—LPV, ethanol or propylene glycol? 18th CROI, 27 February–2 March 2011, Boston. Poster abstract 708.

Two posters at CROI 2011 presented pharmacokinetic (PK), efficacy and safety data of paediatric formulations of antiretroviral drugs. [1, 2]

Darunavir

ARIEL (TMC114-C228) is a 48-week, open-label, single-arm, phase II trial evaluating PK, safety and efficacy of darunavir/ritonavir (DRV/r) plus an optimised background regimen (OBR) in HIV-positive treatment-experienced children. Avy Violari and colleagues reported interim (24 week) data from ARIEL.

Children aged 3 to <6 years, weighing 10 to <20kg, with viral load >1000 copies/mL and <3 DRV resistance-associated mutations (RAM) at screening, received DRV. The formulation used in this study is a high concentrate oral suspension (100 mg/mL) – initially dosed at 20 mg/kg BID plus ritonavir (RTV) 2.6 to 3.2mg/kg BID with an OBR (>2 active NRTI) – over 48 weeks.

After a PK analysis at week 2, the DRV dose was amended to 25mg/kg BID children weighing 10 to 15kg and 375mg BID fixed for those weighing 15 to <20 kg (following Data Safety Monitoring Board recommendations).

A total of 27 patients – 55.6% male and mean age 4.6 years at screening – with DRV/r + an OBR. At baseline, the children’s median viral load was 4.51 log copies/mL, median CD4 count was 927 cells/mm3, and median CD4 percentage was 27.7% cells/mm3. The children had a median of 0 primary PI mutations at baseline and 4 PI RAM, 1 NRTI RAM, and 1 NNRTI RAM.

The majority of children, 23 (85.2%) experienced at least one adverse event (AE). One child discontinued treatment (due to grade 2 vomiting, believed to be associated with ritonavir). Most side effectss were grade 1-2. Grade 3-4 and serious side effects were reported in 18.5% and 11.1% of patients, respectively but none was considered treatment-related. Most commonly reported adverse events (occurring in over 10% of patients) were diarrhea, vomiting, pyrexia, nasopharyngitis, rhinitis, upper respiratory tract infection, hypokalemia, cough, acidosis, and alkalosis.

One child had a grade 3 laboratory abnormality – neutropenia – but this was present since baseline and not considered to be related to treatment.

There was a steady increase in response from week 2 to 24. By week 24, 55.6% of the children met the primary efficacy endpoint of viral load <50 copies/mL (ITT-TLOVR). The mean increase in CD4 at week 24 was 109 cells/mm3.

Two children had DRV RAMs at baseline but both were <50 cells/mL at week 24. Eleven children (40.7%) were considered virological failures. None of the six children with paired baseline/endpoint genotype samples developed PI or NRTI RAMS.

Raltegravir

P1066 is an open-label study of raltegravir (RAL) in treatment experienced HIV-positive children and adolescents. Sharon Nachman and colleagues reported PK, and week 12 and 24 efficacy and safety data for treatment-experienced children aged 2 to 5 years receiving the RAL chewable tablet formulation.

In this dose finding study, intensive PK was initially performed on 4 children and once PK targets were met, 8 more were enrolled. Inclusion criteria included viral load >1000 copies/mL, prior ART experience but naïve to integrase inhibitors. A RAL chewable tablet 6 mg/kg twice daily was added to the existing  regimen, intensive PK samples were taken between days 5 and 12. Once the dose was selected, an additional 9 children were enrolled to assess longer-term safety and efficacy.

PK parameters were evaluated and a dose was selected using an AUC12h target (range 14 to 25uM*h) based on available PK data with a C12h target to exceed the protein-adjusted IC95 of RAL against wild type virus. The investigators compared PK parameters to existing data from 6 to 18 year old children and adolescents receiving the adult formulation and 6 to 11 year old children receiving RAL chewable tablet. Of the 12 children, 67% were female, they were a mean, age of 3 years old, viral load 4.14 log10 copies/mL, CD4%, 33% cells/mm3, CD4 count, 1505 cells/mm3, and weight, 14.3 kg. They received a mean RAL dose of 6.24 mg/kg (0.67).

The geometric mean AUC12 was 8.8hr*mg/L, 18uM*h; C12h 32ng/mL, 71nM; Cmax 4329ng/mL, 9.7uM; CL/F 10.5L/hr and %CV 77%.A 6mg/kg BID dose (maximum 300mg) was selected.

At week 24, by ITT analysis, 62% (95% CI, 53-92) of children (n=21) were <400 copies/mL and 52% (95% CI, 30-74) <50 copies/mL. CD4 gain from baseline was a median of 4.1% (95% CI 2.0-9.9) and 218 (95% CI 39-290) cells/mm3.

No child discontinued RAL due to AEs in this study. One child had grade 3 ALT (2 events), grade >3 AST and ungraded elevated GGT (5 events), considered possible treatment related. Three children had grade >3 neutropenia (7 events) but this was not judged to be treatment related. Other non-treatment related events were: grade 3 bronchopneumonia, grade 3 hydrogen ion concentration, ungraded lactic acidosis, decreased blood glucose, acute gastro enteritis and impetigo.

References

1. Violari A et al. ARIEL: 24-week safety and efficacy of DRV/r in treatment-experienced 3- to <6-year-old patients. 18th CROI, 27 February–2 March 2011, Boston. Poster abstract 713.

2. Nachman S et al. Interim results from IMPAACT P1066: RAL oral chewable tablet formulation for 2- to 5-year-olds. 18th CROI, 27 February–2 March 2011, Boston. Poster abstract 715.

Interactions between rifampicin and protease inhibitors makes treating patients coinfected with HIV and TB more complicated.

Rifabutin is an alternative rifamycin, which can be used in patients receiving a protease inhibitor. Recent findings suggest that the current recommended dose of lopinavir/r (LPV/r) is suboptimal. There are limited data regarding the newer formulation of LPV/r.

Investigators from University of Cape Town, International Union Against Tuberculosis and Lung Disease and WHO evaluated the pharmacokinetics (PK) of rifabutin in co -infected patients on a first line TB regimen before and after the initiation of LPV/r-based ART.

Suhashni Naiker and colleagues showed findings from this study in a poster at CROI 2011.

A group of 16 patients on stable rifabutin-containing TB regimens were initiated on LPV/r-containing HAART. At HAART initiation they were randomised to receive either: rifabutin 150 mg daily for 1 month followed by 150 mg 3 times weekly, or 3 times weekly doses followed by daily doses.

The investigators measured serial rifabutin and 25-O-desacetyl rifabutin concentrations during a dose interval after 4 weeks of rifabutin 300 mg daily, after 4 weeks of 150 mg rifabutin daily with LPV/r-based HAART, and after 4 weeks of rifabutin 150 mg 3 times a week with LPV/r-based ART.

At baseline the participants were a mean (SD) of 31.6 (5.5) years, 59.0 (9.4) kg, 160.1 (7.1) cm and 147 (43) CD4 cells/mm3. Ten were men. Two were not included in the analysis due to poor adherence.

The investigators reported median AUC0-24 and Cmax, for participants receiving 300 mg rifabutin daily, 150 mg rifabutin three times a week, and 150 mg rifabutin daily, respectively, of 3026 ng/mL.h and 297ng/mL, 2307 ng/mL.h and 168 ng/mL, and 5010 ng/mL.h and 311ng/mL.

They found that rifabutin was well tolerated at all dosing strategies. There was one case of uveitis that occurred before  LPV/r was initiated, and one grade 2 transaminitis and one grade 2 neutropenia were also reported.

They concluded that rifabutin 150 mg daily used with LPV/r produces Cmax concentrations within the recommended target range of 300 to 900 ng/mL.

Reference

Naiker S et al. Pharmacokinetic evaluation of different rifabutin dosing strategies in African TB patients on lopinavir/ritonavir-based ART. 18th CROI, 27 February–2 March 2011, Boston. Poster abstract 650.

In resource-limited settings some HIV-positive women initiate ART during breastfeeding. This exposes infected infants to the risk emergence of resistance to the antiretrovirals in their mothers’ regimen.

Investigators from the Post Exposure Prophylaxis of Infants (PEPI)-Malawi trial – in which infants were received up to 14 weeks of extended nevirapine (NVP) or extended NVP plus AZT – evaluated resistance in infants whose mothers began ART post-partum.

Interim data from this analysis was first presented as a poster at CROI. [1] Further findings were reported in a subsequent article in the April 24 2011 edition of AIDS. [2]

Infant plasma samples were collected at 14 weeks of age and tested using the ViroSeq HIV Genotyping System and LigAmp – a sensitive point mutation assay – to detect K103N (limit of detection 0.5%) and Y181C (limit of detection 1%). Later samples collected at 6 and 12 months of age were also analysed using LigAmp.

The investigators found that at 14 weeks 82/108 (75.9%) of infants evaluated had detectable NVP resistance using the Viroseq assay. The proportion of infants with K103N and/or Y181C detected by LigAmp was similar, 78/108 (72.2%), p=0.45. There were no significant differences between rates of resistance among infants receiving extended NVP or NVP plus AZT measured by either assay. Nor were: duration of prophylaxis received prior to infant diagnosis, maternal CD4 count, maternal single dose NVP use, or in utero infection, significantly associated with NVP resistance.

At 6 months, 38 out of 46 (82.6%) samples analysed still had K103N and/or Y181C. Again, results were similar across study arms, p=1.0. And at 12 months 19 out of 29 (66.5%) evaluable infants had these mutations in similar proportions across arms, p=0.43.

Although the data was not presented, the investigators noted that there was no significant difference in the percentage of the total viral population of either K103N or Y181C in infants in the two groups with these mutations at 6 and 12 months of age.

The investigators concluded that the frequent persistence of the K103N and Y181C mutations in infants after exposure to extended NVP prophylaxis, with or without AZT, may compromise the infants’ subsequent response to NNRTI-based treatment.

References

1. Fogel J et al. Initiation of ART in women after delivery can induce multi-class drug resistance in breastfeeding HIV-infected infants: PEPI-Malawi Trial. 18th CROI, 27 February–2 March 2011, Boston. Poster abstract 761.
2. Fogel J et al. Analysis of nevirapine (NVP) resistance in HIV-infected infants who receive extended NVP or NVP/zidovudine prophylaxis. AIDS 2011 25 911-917.24 April 2011.

Introduction

An international workshop on nanomedicine for infectious diseases related to poverty was held in Magaliesberg, South Africa from 27–31 March 2011. The meeting was organised by Dr Hulda Swai, chair of the nanotechnology programme at the Council for Scientific and Industrial Research (CISR), a multidisciplinary science and research institute established in 1945 and funded by the South Africa Department of Science and Technology and Economic Commission for Africa (ECA). CSIR is one of two SA government-funded centres with nanomedicine programmes (the other is MINTEC). About 70 delegates from 20 countries attended the workshop.

Nanotechnology is being used in many different aspects of medical care including therapeutics, new drug delivery systems, diagnostics, imaging and surgical procedures. This article principally focuses on new formulations of drugs and drug delivery systems. Nanomedicine – the application of nanotechnology to medicine – has a relevance for HIV and related infections that rarely gets much attention.

Most of the focus on pipeline drugs for HIV and TB is on new compounds or new oral formulations of already approved drugs. However, for the last fifteen years various laboratories have been working with nanoformulations of antriretrovirals, though none have yet resulted in new medicines.

Size and scale

The simplest explanation of nanomedicine is based on a size ranging from 10-100 nm, though the EU definition has an upper range of 1000 nm. One nanometer is one-billionth of a meter (the width of about five atoms). See Table 1.

At this scale particles have different physical properties relating to surface to volume ratio, surface tension, surface charge and quantum dot effect and this can enhance drug bioavailability and solubility. Engineering molecules to target specific cellular and tissue targets has the potential to overcome barriers to sanctuary sites including the blood-brain barrier. Although nanotechnology is generally associated with the concept of the smaller particles, nanomedicine is actually based on drug formulations that are larger that pure drug molecules.

Note: relative size from 10 (0) to 10 (-10) is similar to comparing the size of the world to a golf-ball.

Attaching compounds to larger molecules or encapsulating them inside other molecules can deliver a drug to the target site more accurately. This can overcome one of the main limitations of current oral formulations, where over 90% of medicines are excreted unused.

These improvements include:

• Better bioavailability, as an example this could be achieved by designing formulations that overcome hydrophobic or hydrophilic properties of individual molecules.
• Reducing drug wastage by overcoming protein binding and during oral absorption, where >90% of the active compound of antiretroviral drugs are cleared by blood filtration through the liver or kidneys before it is able to act on HIV.
• More targeted delivery should reduce the quantity of raw materials needed. This, is turn, has the potential to have the biggest impact on drugs used in resource-limited settings. Even though the drugs are much cheaper in poorer countries a much higher percentage of the costs is related to the active pharmaceutical ingredients (API).
• Reducing toxicities related to the metabolism of current oral formulations. For example, if a nanoformulation is designed to increase active drug levels inside cells while keeping blood levels low this has the potential to reduce toxicities related to systemic drug levels.
• Sanctuary site penetration by developing formulations that target immune cells that can cross the blood-brain barrier. In a similar way molecules may be designed to use cells to evade drug transporters such as P-gp that limit penetration of other sites.

However, while the potential benefits are promising, they also bring significant challenges to safety and regulatory approval.

Review of nanomedicine

The conference opened with a plenary lecture from Professor Ruth Duncan from Cardiff University, who has been a leading researcher for over 35 years in community-based research (Cancer UK), industry and academia. This talk emphasised the diversity of expertise needed from different fields including pharmacology, chemistry, medicine, ethics, health policy and politics.

Material science has been reducing the size of everything. Nanotechnology has been a rapidly evolving field that includes a top-down approach (where compounds are reduced) and a bottom-up approach (assembling polymer materials from the bottom up). Advanced drug delivery systems have been the focus of research for over 40 years. Nanomedicines can prolong action using new control/release technology, can target specific organs, cells, or organ space in an organelle, and improve bioavailability, including penetration of the blood-brain barrier.

Systemic distribution of most drugs involves a high level of dilution throughout the body and low concentrations at the active target. Nano-sized particles have different pharmacokinetics, delivering drugs by endocytosis and phagocytosis (cell engulfment) rather than passing directly through a cell wall. In addition to advancing drug delivery some nanoparticles are active in their own right. There are multiple targets through the lifecycle of infection and progression, including latent infection, each using different strategies.

The definition of nanomedicine incorporates engineering tools used outside the patient, biomedical and medical materials, imaging, and drug delivery and formulation. Designing nanomedicine should be easy. Starting with a target disease and product profile it should be possible to choose technologies and benchmark these against current treatment, with a clear stop-go development. Good lead candidates then require five years preclinical trials before getting to human studies. These drugs need a constant awareness of good laboratory practice (GLP), good manufacturing practice (GMP), good clinical practice (GCP) and ethics.

While nanomedicine has an unprecedented opportunity for invention, the lack of current knowledge across the field risks newer researchers repeating mistakes that earlier research has overcome. Against this is a caution that over exaggeration of the benefits will raise unrealistic expectations. Professor Gordon highlighted a limitation from the commercial focus on the target, using genomics and low molecular weight compounds, where 95% of compounds fail, probably because pharmacokinetics is not sufficiently important earlier in the development phase.

The first nanomedicines developed in the1990s based on liposome formulations are at the end of their patent life and are now coming through as generics. However each nanomedicine construct has a different design and route of delivery and every drug must be reviewed separately due to the specific challenges of manufacturing and constructing lipid-based nanoformulations. This field is more complex than generic antiretrovirals for example, where reproducing a compound that has similar pharmocokinetic properties is closely associated to similar safety and activity. From a safety perspective, producing new molecules, each piece (polymer, linker, etc) needs to be designed, requiring preclinical research before human studies. Many polymers for example are rejected for safety. Non-biodegradable polymers can accumulate, especially with long exposure (potentially lifelong) treatment. Pegylation itself covers a wide range of polymers. Currently the EMA opinion on generic formulations of Doxil etc is that bioequivilence does not exist for a liposome, only bio-similar properties and that pegylation on surface of liposome cannot just be duplicated as generic.

This complexity involves molecules travelling in the bloodstream at hundreds of miles an hour and the challenge is how to “stick your arm out to catch them at the right site”. Shape and density are essential aspects of design including ionisation of polyelectrolytes and changes in cellular behaviours. Linkers need to be stable and release drug at the right time. Activating compounds can behave differently, including by sex. All of parts of the system will go somewhere and these need to be accounted for to ensure patient safety.

The importance of products used in clinical trials and subsequently approved to have passed good GLP and GMP was introduced as a theme that would be frequently revisited throughout the workshop.

Historical perspective of nanomedicines in cancer treatment

Dr Theresa Allen from the University of Alberta, Canada, and Alberto Gabezon from the Shaare Zedek Medical Center, Jerusalem gave lectures on the historical perspective of nanotechnology drawing on the impact that it has had on cancer therapy.

Although molecules on a nanoscale have been known since 1900 the term is associated with a lecture given by the Nobel physicist Richard Feynman in 1959 setting two challenges based on the theory that “atoms on a small scale behave like nothing on a large scale”.

Nanotechnology based products already in common use include sunscreens (nanomolecules of titanium dioxide and zinc oxide are translucent, not white), self cleaning windows, pharmaceutical inks printing on medicines, and fabrics and coatings treated to variously repel dirt, water, bacteria, fungus or creasing.

In medicine, applications include imaging, diagnostics, drug delivery and therapeutic agents able to cross biological barriers. Nanomedicines deliver proteins, plasmid DNA, siRNA and antisense, enhanced permeability and retention (EPR) effect for solid tumours (where molecules target tissues with increased permeability and are only released in inflamed tissue and tumour damaged sites).

Nanomedicines can improve properties of existing drugs, reducing toxicity and improving bioavailability (poor bioavailability currently wastes an estimated 68 billion dollars annually).

Liposome and other lipid-based systems are the first and most common lipid medicines. They are simple, self-assembling, with proven safety, with flexibility in terms of particle size, release rates and bio distribution. A principal behind nanoformulations is that a drug changes its biological pharmacokinetics and distribution properties, generally being protected from degradation and metabolism, and only behaving as a regular molecule when released as free drug.

Examples of widely used nanomedicines include Doxil (a pegylated liposome encapsulation formulation of doxorubicin used to treat Kaposi’s sarcoma [KS] and ovarian cancer), the antifungal AmBisome (a liposomal formulation of amphotericin-B) and pegylated interferon (used with ribavirin to treat hepatitis C), Abraxone (a nanoformulation of paclitaxil used to treat metastatic breast cancer), Rapamune (formulation of sirolimus that is milled down and stabilised to become soluble) and Genoxol PM (a micelle formulation of paclitaxil). See Figure 2.

The first oncology nanomedicine was Doxil, approved in 1995 as a treatment for AIDS-related KS under fast track orphan drug designation with development time of less than ten years. Subsequent approvals for relapsed ovarian cancer, metastatic breast cancer and as a substitute for doxorubicin resulted in Doxil becoming a commercial ‘blockbuster’ drug.

Doxil is a 100 nm diameter surface-graft pegylated long-circulating formulation where the active drug stays in a liposome, increases the concentration in sold tumours and slow releases in tumour cells. If ligands are added to particles, the resulting package can enter a cell in larger quantities and where it is then released.

Compared to the original doxorubicin, Doxil dramatically reduced clearance by 1000-fold (extending the half-life to 50-70 hours) and increased drug accumulation in tumour tissue (approximately 80 ug vs 0.5 ug in skin a few millimetres away). This enabled a 4-fold lower dose of Doxil to have a 30% higher cure rate and reduction tumour volume. Unfortunately, in this early example, side effects were not always reduced and a higher rate of palmar-plantar erythema rash occured in some patients.

Nanomedicine can also incorporate combination chemotherapy in single constructs using drugs that use different mechanisms and that have non-overlapping side effects. It is possible to mix two liposomes or to add multiple targets on each (for example to target to nothing, CD20, CD19 or both and produce additive effects). In oncology, combination approaches to target and kill blood vessels in tumour tissue starve the tumour of oxygen and nutrients (though peripheral tumour cells survive). New blood vessels in a tumour are damaged, disorganised and with incomplete endothelial lining, allowing particles up to 400 nm to penetrate. An early paper in 1982 showed the potential of liposomes to carry adriamycin in mice. Conventional liposomes reduced cardiovascular risk but did not target liver tumours and were leaking drug in blood. Changing the lipid concentrations in new formulations changed pharmacokinetics with a 20-fold increase in concentrations in the tumour and 4-fold reduction in spleen, highlighting the importance of liposome design.

Targeted siRNA or antisense molecules can be effective but the high charge restricts transfer across cell membranes. Adding ligands enables a Trojan horse effect so a drug can cross cell membranes. For example, antibody-targeted coated cationic liposomes can be used to inhibit anaplastic lymphoma kinase (ALK) in neuroblastima-bearing mice with anti-GD2-targetted CCLs entrapping ALK-siRNA. Free siRNA cannot cross cells but can in a nanoformulation.

A brief history (and reality check) of the time it has taken to develop nanomedicines starts with the first description of liposomes in the early 1960s, which became the foundation of many formulations. Liposomes are 100 nm nanoparticle spheres (visicles) consisting of a phospholipid shell where one end is hydrophobic (usually the outer surface) and one is hydrophilic, enclosing an internal water phase. Drug molecules can be either encapsulated in the water phase or entrapped in the lipid shell. See Figure 3.

Figure 3: Cross section of liposome used for drug delivery

Source: http://en.wikipedia.org/wiki/Liposome

Further research led to the development of “stealth liposomes” in 1987 that used polyethylene glycol (PEG) studding on the outer coating to evade the mononuclear phagocyte immune system. AmBisome was approved in 1990, SMANCS (styrene maleic acid neocarzinostatin) in 1993 and Doxil in 1995. Nano-based drug delivery systems began entering mainstream medicine after 2005 and by 2011 there are over 38 nano products on market (worth $6.8 billion annually) with generic systems on the horizon.

The challenge for nanoformulations is to get an appropriate drug using appropriate delivery system that is the right size (not damaging other tissue) with low toxicit, for sufficient duration to solve an unmet medical need.

Crucially this needs to be at a sufficiently low cost to make it usable in a way that is safer and more effective than current treatments. Involvement of Indian companies has successfully radically reduced costs in many areas. In addition to reducing antiretroviral treatment from $10,000 to under $100 per year, the cost of hepatitis vaccinations have been reduced from $20 to $1, psoriasis treatment has been reduced from $20,000 to $100 and cataract surgery is carried out at 1% of the UK cost.

While the political aspects of new technology for infectious diseases have a high level of collaboration between countries at end stage distribution, the level is low for the early stages of research and development. The research presented at this workshop is going some way to address this.

A nanoformulation of paediatric efavirenz

Dr Alejandro Sosnik from the University of Buenos Aires discussed the potential applications of nanotechnology for paediatric formulations of HIV medicine. [4]

HIV in rich countries has become a largely manageable adult disease with more than 25 antiretroviral drugs and early diagnosis and access to HAART has reduced mother to child transmission to rates less than 1%. However, in most poorer countries HIV remains an acute disease with an estimated 1000 new paediatric infections globally each day, mostly in sub-Saharan Africa. Only 10% of children with HIV currently have access to treatment, and this drops to 2% in some regions.

Paediatric treatment in all countries is limited, with only 12 approved paediatric formulations. In particular, the lack of appropriate formulations, difficulties of dose adjustment, pill swallowing, and poor taste add to the challenges of enabling children to achieve the similar benefits from the advances seen in adult HIV care.

Dr Sosnik presented an example of a potential nanoformulation for efavirenz from his research group. Although a paediatric liquid formulation of efavirenz is produced by BMS this is not available globally and the indication is for children older than 3 years (and greater than 13 kg). The current oral formulation has lower bioavailability (by 40-45%) than capsule formulations and “tastes like liquid Vaseline” confirmed by associated weight-loss and diarrhoea. Interpatient and intrapatient bioavailability varies by 55-58% and 19-24% respectively.

The research goal and important unmet medical need is to develop a highly concentrated aqueous formulation with higher bioavailability, working with existing polymers like PEO-PPO polymeric micelles (poloxamers andoloamines) that are already commercially available in a broad spectrum of molecular weights and that already have a proven safety record (ie ethylendiamine).

So far, efavirenz-loaded micelles using surface aptamers to target CD4 cells have been produced in an oral solution and animal bioavailability data has been published. [5 - 9]. A micelle is an aggregate of compounds that lower the surface tension of a liquid and that are evenly dispersed.

In male rats the efavirenz-loaded micellar formulation was compared to that of a suspension prepared with the content of efavirenz capsules in 1.5% carboxymethylcellulose PBS solution (pH 5.0), and an EFV solution in a medium-chain triglyceride (Miglyol 812).

This formulation showed that the encapsulation of efavirenz, which is otherwise poorly water soluble, into polymeric micelles of different poly(ethylene oxide)–poly(propylene oxide) block copolymers significantly improves oral bioavailability and reduces the interindividual variability.

The solution has an improved taste, using only excipients approved by the FDA, and requiring less API has the potential to lower production costs.

A protocol has already been approved by an ethics committee in Buenos Aires for first in-human pharmacokinetic studies in HIV-negative volunteers, which if successful will progress to studies in HIV-positive adults and then HIV-positive children.

One of the discussion points after the presentation included the use of the formulation first in HIV-positive adults. This has been by the research group and not being linked to clinical HIV centres in Argentina. Expanding on this, Dr Soznik explained, “funding is difficult, industry studies are dominant. We need to now see if human exposure is similar to rats. Further animal studies might help but these are too expensive”.

Other antiretroviral nanoformulations

Two other research groups at the workshop also presented data on formulations of antiretrovirals that are already approved, though none have yet been used in human studies.

Dr Lebogang Katata from CSIR in South Africa presented a poster on spray-dried efavirenz nanoparticles that was previously shown at the IAS conference in Vienna last year. [10, 11]

In this process, efavirenz is encapsulated in a polycaprolactone (PCL) polymer by a double emulsion spray drying technique using two organic solvents. The nanoparticles have an average size of 220.6 ± 0.950 nm when using ethyl acetate and 372.1 ± 19.96 nm using dichloromethane. This formulation also overcomes the hydrophobic nature of efavirenz to improve bioavailability and it has met other manufacturing standards including encapsulation efficiency and a smooth particle surface. It also results in prolonged release compared to formulations of free drug (suggesting weekly dosing might be possible). The group have also produced formulations of other antiretrovirals including AZT and d4T and plan combination formulations. But the timeline for human studies that were due to start this year is likely to be several years away.

A poster from Dr Helanie van der Merwe and colleagues at the North-West University, Potchefstroom, South Africa presented results from a third technology for producing nanoparticles of antiretrovirals. [12]

This research group has encapsulated abacavir and lamivudine (3TC) in visicles using a technology called pheroid, that resulted in higher bioavailability in in vitro studies.

Professor Anne Grobler from North-West University explained pheroid technology in an oral presentation. [13]

This talk started with an example of South African drug development of Exorex. This is a coal tar preparation for psoriasis that was initially developed by an American individual for his personal treatment and was then developed by the University into a global treatment, with the advantage of greater efficacy from using a much lower percentage of coal tar (1% vs 5%). [14]

Pheroids use a form of colloidal transport (usually at 100 nm size but sometimes larger) using long chain fatty acids, modified in different ways, ie with pegylation to have with hydrophilic tails. The technology is apparently easy, and inexpensive but it was originally difficult to predict the type of pheroid because it was not designed working with the active compound. All fatty acids are currently being taken safely by humans and the technology allows packaging of more than one drug in each vesicle.

In addition to abacavir and lamivudine the group have produced Pheroid formulations of antimalarial [15] and antituberculousis drugs [16, 17] that in preclinical development consisted of comparative in vitro and animal efficacy, bioavailability and pharmacokinetic studies and limited toxicology studies.

These studies reported enhanced rates of in vitro efficacy and in vivo bioavailability of first and second-line antimalarial compounds (including chloroquine, mefloquine, artemether and artesunate) and extended in vivo pharmackinetics and efficacy studies with formulation of tuberculosis drugs in mice.

Formulations of anti-TB medications

The CSIR research programme for formulations of TB medications is more advanced than that for HIV. Dr Rose Hayeshi, expanded on this programme. [18]

The group have encapsulated four first line anti-TB drugs (isoniazid, rifampicin, ethambutol and pyrazinamide) with an encapsulation efficiency varying from 50-65% in particles of 250-400nm, using a multiple emulsion spray-drying technique.

The polymer used is poly(lactide-co- glycolide), (PLGA). The particles are taken up by macrophages in vitro indicating feasibility of intracellular drug delivery. Studies in mice using fluorescently-labelled PLGA nanoparticles indicated distribution to a broad diversity of tissues including macrophages of the peritoneum cell exudates that cross the blood brain barrier. Safety in mice after up to 10 days exposure was supported by histopathology on all major tissues including the spleen, lungs, kidney, liver, spleen, heart and the brain, which found no evidence of lesions.

Pharmocokinetic studies in mice showed that the drugs were released over a period of six days and the minimum inhibitory concentration for rifamicin and izoniazid was maintained over this period. Pharmacokinetic curves are similar to those of free drug, with an initial increase from drug on the outside of the molecule followed by slow release compared to free drug.

A study in six TB-infected mice dosed daily for four weeks showed a reduction in colony forming units in spleen, liver although there is a need to target actively to lungs. When repeated over nine weeks an improved pathology was observed in the lungs. The nanoformulation is a weekly dose compared to current requirement for strict daily dosing.

The research group are now looking at scale-up plans for all formulations, improved preclinical, dose formulation and design and then first clinical studies. Currently single formulations are used but combinations are planned and the group is working with compounds held by the TB Alliance.

The discussion after this presentation focused on the need to clarify the mechanism and route of administration for TB drugs. This included whether the controlled release is in blood, tissues, or the cells. Oral formulations are difficult to target to cells, and compounds often need IV administration, which is not practical in resource-limited settings.

Aptamers in nanomedicine

Dr Makobetsa Khati, also based at the CSIR explained the utility of nanoaptamer biconjugates against infectious diseases with a focus on HIV. [19]

DNA or RNA aptamers were first developed in 1990 and are usually short strands of artifical oligonucleotides that are selected in vitro using SELEX (systematic evolution of ligands by experimental enrichment).

At a size that is less than 10 nm radius they can increase tissue specificity with minimal impact on the size of the formulation. Current uses include delivery of drugs to treat age related macula degeneration. Aptamers have the molecular recognition properties of monoclonal antibodies in terms of their high affinity and specificity. They are chemically simple, easy to make, have high target specificity, are non-toxic and non-immunogenic. Aptamers are used in many of the formulations already discussed in order to identify target cells for nanoformulations.

As aptamers can block entry this group has worked with oligonucleotides that are active against gp120. [15] The aptamer doesn’t affect cell metabolism with toxicity, immune of cardiovascular cells. Over 80% of cells were sensitive with IC50 <1 nm and non-toxic at concentrations over 1000 nm.

In humanised mice Neff and colleagues showed reduction in viral load and a protective effect on CD4 counts with either the anti-gp120 aptamer or an aptamer-siRNA combination (that provided more extensive inhibition, resulting in a significantly longer antiviral effect that extended several weeks beyond the last injected dose). [20]

In the discussion after the presentation it was noted that the loss of the patent for the one aptamer on the market has dramatically reduced the cost (from $100,000 down to $25 per mg. However, as they are highly negatively charged this raised the issue of pharmacokinetics and side effects (clotting, platelets and immune effects).

Other subjects covered by the workshop

The workshop explored a wide range of other potential indications for nanomedicines that are not covered in this report including pulmonary fungal and parasite infections (including paracoccidioides brasiliensis), tuberculosis, malaria and prostate cancer.

It also included sessions on regulatory issues (with reference to the recent first meeting of the EMA on nanotechnology) and practical aspects of intellectual property, patents, technology transfer and international research collaborations.

Meeting summary

Many aspects of these new technologies are still in early development, even though some nanoparticle medicines have been used so extensively used that they are now off-patent. Each molecule has specific efficacy and safety issues dependent on the particular manufacturing process and cellular target.

Antiretroviral compounds show exciting pharmacological properties in in vitro studies. Particularly greater potency and that they require lower quantities of the API. The benefits from suspension formulations in development from researchers on nevirapine in South Africa and efavirenz in Argentina could have global impact as paediatric formulations. However, they still need to show proof of concept for overcoming obstacle associated with lymphocyte target cells.

The potential for TB nanoformulations may be closer than those active against HIV. The CSIR researchers have nanoparticle formulations of the four main first-line TB drugs and are working with the TB Alliance on newer compounds including TMC 207.

Some issues from a regulatory safety perspective are still not resolved . The FDA is more advanced than the EMA. Nearly all research is preclinical, with animal or in vitro data to support advantages of nanformulations. Particles based on molecules with established safety data should be easier to assess than totally new constructs. But one of the discussions on regulation of generic formulations of Doxil indicated that the complexity of the manufacturing process is likely to require in vivo safety and efficacy data from new studies in every molecule rather than just bioequivalence studies that enabled easier widespread use of oral antiretrovirals.

It is also important to balance expectations with likely realities for this research. Nanoformulations look as if they will offer exciting solutions to some specific currently unmet challenges rather than the potential to revolutionise treatment for every disease indication. Specific targeting of malignant tissue and its supportive vascular structures is revolutionising approaches to treatment of some cancers.

The political aspect of the workshop included the importance of technology being developed in countries where the demand for final medicines exists. With broadly 10% of global research focused on the 90% of global medical need. Less than 2% of patents are held in Africa and intellectual property and patent legislation are determined to benefit companies based in the developed world.

Of note, several recent reviews have summarised research over the last 15 years into nanoformualations of antiretrovirals. [21, 22]. See Figure 4.

This research is generally conducted by small independent groups working without sufficient funding to discover whether the potential of this technology can be realised for HIV care.

Given the potential advantage of providing reduced toxicity, more durable formulations with improved pharmacokinetics (ie targeting macrophages to increase concentrations in lymph tissue and dramatically extending half-life and dosing intervals) and at a reduced cost makes it frustrating that so far none of these formulations has yet progressed to human studies.

Compiled from Malipeddi and Rohan [21], Govender [23] and others.

Further reading

The following links are included for further reading.

US NIH nanotechnology research. Links to an extensive NIH funded research programme established in 2005.

http://www.nibib.nih.gov/Research/NIHNano

http://www.nih.gov/science/nanotechnology

EMA’s First Scientific Workshop on Nanomedicine. September 2010.

Little information is online but a few large video files from some sessions are available to download.

http://vod.ema.europa.eu/100902

Griffiths G et al. Nanobead-based interventions for the treatment and prevention of tuberculosis. Nature Reviews Microbiology 2010 Nov; 8(11): 827-34.

http://www.ncbi.nlm.nih.gov/pubmed/20938454

Gupta U and Jain NK. Non-polymeric nano-carriers in HIV/AIDS drug delivery and targeting. Advanced Drug Delivery Reviews 62 (2010) 478–490.

http://www.ncbi.nlm.nih.gov/pubmed/19913579

Stockley P and Bunka DHJ. Aptamers come of age at last. Nature Reviews, August 2006, (PDF download)

http://www.cs.duke.edu/bioComp/references/hanyingAptamer/aptRev2.pdf

Wong HL et al. Nanotechnology applications for improved delivery of antiretroviral drugs to the brain. Advanced Drug Delivery Reviews 62 (2010) 503–517.

http://linkinghub.elsevier.com/retrieve/pii/S0169409X09003615

References

Unless stated otherwise, all references are to the programme and abstracts of the First Workshop on Nanomedicine for Infectious Diseases of Poverty, 27–31 March  2011, Magaliesberg, South Africa. The programme and abstract book can be downloaded free from the workshop website.

http://www.csir.co.za/msm/nano_workshop2011/

1. Duncan R. Nanomedicine : from research to health care. Plenary lecture.
2. Allen T. Nanomedicines: opportunities and challenges.
3. Gabizon A. Liposome drug delivery in cancer therapy: from vision to reality.
4. Soznik A. The pros and contras of innovation: issues to take advantage of novel technologies in neglected diseases, the HIV case. Programme page 19.
5. Chiappetta​‌ DA et al. Efavirenz-loaded polymeric micelles for pediatric anti-HIV pharmacotherapy with significantly higher oral bioavailability. Nanomedicine. 2010;5(1):11-23.
   http://www.futuremedicine.com/doi/abs/10.2217/nnm.09.90
   Full text online at:
   http://www.medscape.com/viewarticle/715261
6. Chiappetta DA et al. A highly concentrated and taste-improved aqueous formulation of efavirenz for a more appropriate pediatric management of the anti-HIV therapy. Current HIV Research, 2010.
7. Chiappetta DA et al. Oral pharmacokinetics of the anti-HIV efavirenz encapsulated within polymeric micelles, Biomaterials 32 (2011) 2379-2387.
   http://linkinghub.elsevier.com/retrieve/pii/S0142961210015371
8. Soznik A. Nanotechnology contributions to the pharmacotherapy of pediatric HIV: a dual scientific and ethical challenge and a still pending agenda. Editorial. Nanomedicine (2010) 5(6), 833–837.
9. Chiappetta DA et al. Synergistic encapsulation of the anti-HIV agent efavirenz within mixed poloxamine/poloxamer polymeric micelles. Nanomedicine: Nanotechnology, Biology and Medicine (in press). doi:10.1016/j.nano.2011.01.017.
   http://linkinghub.elsevier.com/retrieve/pii/S1549963411000219
10. Katata L et al. Spray dried PCL-Efavirenz nanoparticles for improving the current HIV/AIDS treatment. Abstract 9; page 53.
11. Katata L et al. Spray dried PCL-efavirenz nanoparticles for improving the current HIV/AIDS treatment. Poster abstract MOPE0031. Link includes ePoster.
    http://pag.aids2010.org/Abstracts.aspx?AID=13785
12. van der Merwe H et al. Enhanced in vitro delivery of abacavir and lamivudine entrapped in a Pheroid formulation. Poster 23, page 70.
13. Grobler A. The Comparative efficacy and pharmacokinetics of selected anti- infective agents in rodents and primates with and without entrapment in Pheroid technology. Oral presentation, abstract page 22.
14. Kotze AF et al. Assessment of the in vitro efficacy of selected artemisinin derivatives in combination with the Pheroid drug delivery system. Poster page 50.
15. Wiid I et al Increased bioavailability and in vitro efficacy of anti-tuberculosis drugs by the Pheroid delivery system. Poster 4; page 46.
16. Nieuwoudt L-M et al. PheroidTM technology enhance bioavailability of antituberculosis drugs in mice. Poster 18; page 68.
17. Hayeshi R et al. Evaluation of polymeric nano drug delivery systems for the treatment of TB. Abstract 19, page 67.
18. Khati M. The utility of nano-aptamer bioconjugates against infectious and other diseases common in Africa. Abstract page 18.
19. Dey AK et al. An aptamer that neutralizes R5 strains of Human Immunodeficiency Virus type 1 blocks gp120-CCR5 Interaction. J. Virol. November 2005 79: 13806-13810.
    http://jvi.asm.org/cgi/content/abstract/79/21/13806
20. Neff CP et al. An Aptamer-siRNA chimera suppresses HIV-1 viral loads and protects from helper CD4+ T cell decline in humanized mice. Sci Transl Med 19 January 2011: Vol. 3, Issue 66, p. 66ra6. DOI: 10.1126/scitranslmed.3001581.
    http://stm.sciencemag.org/content/3/66/66ra6.abstract
21. Mallipeddi R and Rowan LC. Progress in antiretroviral drug delivery using nanotechnology. International Journal of Nanomedicine 2010:5 533–547. Online open access.
    http://www.dovepress.com/progress-in-antiretroviral-drug-delivery-using-nanotechnology-a4896
22. Mamo T et al. Emerging nanotechnology approaches for HIV/AIDS treatment and prevention. Nanomedicine (London). 2010 February; 5(2): 269–285. doi: 10.2217/nnm.10.1.
    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2861897/
23. Govender T et al. Polymeric Nanoparticles for Enhancing Antiretroviral Drug Therapy. Drug Delivery, 15:493–501, 2008 DOI: 10.1080/10717540802321776.
    http://www.ncbi.nlm.nih.gov/pubmed/18720133

The abstract book from the conference, published as a supplement to HIV Medicine is available to download free as a PDF file from the BHIVA website.

http://www.bhiva.org/ConferenceAbstracts.aspx

Reports in this issue include:

• Atazanavir in pregnancy: 155 cases in London clinics
• Co-morbidity and late presentation – findings from an over 50s cohort
• Health outcomes for young adults with perinatally acquired HIV infection following transfer to adult services
• The use of calcaneal stiffness index to screen for osteoporosis in HIV-infected individuals
• Frequency and characteristics of long-term non-progressors and HIV controllers in the Chelsea and Westminster cohort
• Cerebral function in perinatally HIV infected young people and HIV uninfected sibling controls
• An audit of HIV care provision for Immigration Removal Centre patients

Atazanavir is increasingly used in pregnancy. There are limited data to guide this use.

In an oral presentation at the spring 2011 BHIVA meeting Miriam Samuel presented data from a retrospective case note review of atazanavir-exposed pregnancies at 12 London sites.

The review included 155 pregnancies in 145 women since December 2004.

The majority (118, 71.6%) of women were Black African and a small proportion were IDUs (5, 3.2%). Their median age was 32 years (15–47), 6 were Hepatitis B and 2 Hepatitis C co-infected. Only 15 (9.7%) were diagnosed this pregnancy and 105 (68%) were receiving HAART when they conceived. Their median CD4 was 401 cells/mm3 (range15–1161 cells/mm3).

Over half (93, 60%) conceived while receiving ATV; 20% of the remainder changed from another drug and 20% started with ATV first line in pregnancy. Only 9% of women received AZT and 16% abacavir but 72% received tenofovir as part of their nucleoside backbone.

Overall, atazanavir was well tolerated with 98/155 (63.2%) reporting no side effects. The commonest side effect was nausea, which was reported by 53/155 (34.2%) of women. Only 3/155 (1.9%) of women discontinued ATV due to side effects.

Of 21 women who switched to atazanavir due to GI side effects from a previous regimen (20 PI based), symptoms improved in 19. One woman stopped atazanavir due to persistent nausea.

There was a low overall incidence of hepatotoxicity; 9 (5.8%) women developed G1-4 raised transaminase. Of these, 5 women switched to atazanavir with pre-existing hepatotoxicity,1 from nevirapine and 4 from lopinavir/r. LFTs resolved in 3 women and 2 had persistent hepatotoxicity and ATV was discontinued in both cases.

Of the 130 women with viral load data available, 104 (80%) had <50 copies/mL at delivery. As would be expected, of the subset that started atazanavir in pregnancy (n=25), the proportion with an undetectable viral load increased with time on treatment: 29% and 72% with <12 and >12 weeks of HAART respectively.

Of the 133 deliveries with data available, 64 (48%) were by elective caesarean, and half the remainder by vaginal delivery and the other half emergency caesarean (both 26%). Pre-term delivery <37 weeks occurred in 13/130 infants (10%).

The 94 infants with neonatal bilirubin measured had a median 71umol/L(range 3-258 umol/L). Three neonates had phototherapy; 1 polycythaemic (bilirubin 258 umol/L); 1 infant haemolytic anaemia (bilirubin 109 umol/L) and 1 no other cause (bilirubin 194 umol/L). There was 1 congenital cardiac abnormality.

There was only one transmission, reported to be in utero, to a mother with a history of poor adherence.

The investigators concluded from this case series (the largest to date) that atazanavir is well tolerated in pregnancy with low toxicity and discontinuation rates. This applied to women conceiving on atazanavir, starting in pregnancy or switching. The infant safety data are reassuring.

comment

Data on atazanavir PK during pregnancy at the PK Workshop, from the IMPAACT investigators in the US, reported in this issue of HTB, suggest that the dose of ATV/r should be increased to 400/100mg during the third trimester and the same considered during the second. The dose used in this study was 300/100 mg for the majority of the 149/155 women who received ATV boosted.

Currently, in the UK, most caregivers of pregnant women stick to the standard dose and some use TDM. However in this cohort only 17 women had routine third trimester TDM and 11 because of elevated viral load, despite it being available (though some don’t think it is evidence based or cost effective). Although the third trimester levels in patients on tenofovir might be of concern, these investigators do not report treatment failure.

In this study women who conceived on HAART did very well and did not loose virological control in the third trimester.

The 80% rate with <50 copies/mL at delivery among women initiating HAART in pregnancy improved with longer duration of treatment, as would be expected.

This case note review goes back to 2004, so a good proportion of women will have been treated prior to last years report emphasising the importance of earlier initiation of HAART in pregnancy at higher viral loads to ensure viral suppression and in turn greater choice over mode of delivery.

Reference:

Samuel M et al. Atazanavir in pregnancy: a report of 155 cases. 17th Annual BHIVA Conference, 6–8 April 2011, Bournemouth. Oral abstract O25.

The number of HIV positive patients accessing care in the UK who are aged over 50 years old has more than tripled between 2000-2009 from 2,432 to 12,063. Twenty percent of adults presenting for HIV care are now over 50. Previous cohort data showed an increased incidence of co-morbidities in the this patient group in relation to diabetes, hyperlipidaemia, cardiovascular and bone disease. [1, 2]

This study focused on the 504 patients aged over 50 out of a cohort of 2,700 patients attending Guys and St.Thomas’ Hospital as of 1 December 2010. [3]

Median age was 54 years (range: 50-83, IQR: 52-59), 76.4% (n=385) were male, 54.8% (n=276) were white, 38.3% (n=193) were black, 47.4% (n=239) were MSM. Median age at diagnosis was 46 years (range: 22-82, IQR: 40-52) and the median time since diagnosis was 9 years (range: <1-28, IQR: 5-14). Of the group, 35.3% (n=166) were aged 50 or over at diagnosis.

The CD4 count at diagnosis was available for 298 patients: 216 cells/mm3 (range: 3-1100, IQR: 79-401), of which 70.8% (n=211) had a CD4 count <350 cells/mm3 (50.2% of whom were <50 at diagnosis), 24.2% (n=72) had a CD4 count of 201-350 cells/mm3 at diagnosis (48.6% of whom were <50 at diagnosis) and 46.6% (n=139) had a CD4 count of <200 cells/mm3 (51.1% of whom were <50 at diagnosis).

Their current treatment included 46% on NNRTI-based regimens, 36% on PI-based regimens and 11% on other regimens, 5% were HAART naïve, 1% not currently on HAART and 1% not documented. The median time on ARVs was 7 years (IQR: 3-11 years) and 55% of the group are still on their first prescribed ARV combination.

The cardiovascular health of these patients showed a median 10 year Framingham cardiovascular risk score of 12.3%. Hyperlipidaemia was present in 44% of patients (with or without a statin or other lipid lowering agent). Currently 15.7% of the group are smokers and 11.3% have diabetes. Cardiovascular events were seen in 7.3% (n=37) of patients including 7 myocardial infarctions, 6 strokes or transient ischemic attacks, 8 positive coronary angiograms. Of these 37 people, 6 (16%) were current smokers, 29 (78.4%) were on statins and 5 (13.5%) were diabetics.

Bone health is always a concern in anyone over 50. In this study 134/504 patients (26.6%) have had a DEXA scan. Of those 134 people, 70 (52.2%) patients had results showing reduced BMD, 22 (16.4%) had a diagnosis of osteoperosis, 48 (35.8%) had a diagnosis of osteopenia, 8 (11.4%) were current smokers, 6 (8.6%) had concurrent renal disease.

Renal disease was documented in 39 patients (7.7%) and of these, 8 (20.5%) were currently on dialysis, 6 (15.4%) had documented HIVAN and 4 (10.3%) were diabetics.

Mental health problems were documented in 154/504 patients (30.6%), 104 (67.5%) of which had depression cited in their medical notes and 41 (8.1%) had documented memory impairment.

In conclusion, 35.3% of patients at Guy’s and St Thomas’ were aged 50 or over at diagnosis and 41.9% of patients aged over 50 presented with a CD4 count of <350 cells/mm3. This group have been found to have multiple co-morbidities affecting cardiovascular, renal, bone and mental health. Future work to follow on from this study will include comparisons between HIV positive patients over 50 and HIV negative patients over 50.

comment

This report provides important cross-sectional data on the changing demographics at many clinics and the high rates of comorbidities and polypharmacy associated with ageing with HIV.

It is notable that only 16% of serious cardiovascular event and only 11% of reduced bone density events occured in current smokers and that the smoking rate for this cohort was only 16%. This perhaps indicates that patients have already made proactive lifestyle changes but still remain at high risk of residual complications.

References:

1. Onen NF et al. Aging and HIV infection: a comparison between older HIV-infected persons and the general population. HIV Clin Trials. 2010;11(2):100–109. doi: 10.1310/hct1102-100.
2. Hasse et al. Aging and Non-HIV-associated Co-morbidity in HIV+ Persons: The SHCS. 18th Conference for Retroviruses and Opportunistic Infections. 2011. Poster abstract 792.
   http://www.retroconference.org/2011/Abstracts/40790.htm
3. Williams H. Co-morbidity and late presentation – findings from an over 50s cohort. 17th Annual BHIVA Conference, 6–8 April 2011, Bournemouth. Oral abstract O31.

Globally there are an estimated 2.1 million children under 15 infected with HIV. In the UK, the Collaborative HIV Paediatric Study (CHIPS) estimates the number of HIV-positive children to be 1,645 as of March 2010. Of these, 65% are over 10 years. A total of 262 have now been transferred to adult care.

As HAART has only been available since 1996, this is the first generation born with HIV to have survived to adulthood. As a result the long-term outcomes of HIV infection from birth and HIV treatment in childhood is still relatively uncertain.

This study aimed to examine the health outcomes of a cohort of 58 young people with perinatally acquired HIV infection that were originally part of the 900 clinic at St Mary’s Hospital and are now in adult care. The 900 clinic provides services for young people who were diagnosed with HIV when they were children and have been treated at St Mary’s Hospital since their diagnosis.

This was a case note review of all HIV-positive young people seen at the 900 clinic between January 2006-2011.

The median age of transfer from paediatric care in this cohort was 17.2 years (range 16.3 – 18.6) and the current median age was 20.6 (range 16.9 – 26.1) years. Overall outcomes of the 58 young people includes 5 (9%) who were transferred to local adult services, 51 who were still patients at the 900 clinic and 2 (4%) died (one 20 year old female due to MDR end-stage HIV disease and a 21 year old female due to nephropathy and sepsis who had declined ART). There was no loss to follow up and 7 of the patients included in the study had babies.

Of the 51 patients currently at the 900 clinic, their median current CD4 count was 425 cells/mm3 (IQR: 30-1140). More specifically, 22% had a CD4 count of <200 cells/mm3, 8% had a CD4 count of 200-350 cells/mm3 and 69% had a CD4 count of >350 cells/mm3. Of the 51 patients, 5 (10%) were ART naïve, 14 (27%) were on NNRTI-based regimens, 18 (35%) were on PI-based regimens, 2(4%) were on triple NRTI-based regimens and 12 (24%) had stopped ART.

Focusing on the 34 patients currently on ART, the median CD4 count was 480 with 6 patients had a CD4 count of <200 cells/mm3 (of which 3 had detectable viral loads), 2 had a CD4 count of 200-350 (of which 1 had a detectable viral load) and 26 had a CD4 count of >350 (of which 1 had a detectable viral load).

As far as complications of disease and treatment were concerned, 2 patients required gastrostromy tubes to help adherence, 6 (12%) had severe lipodystrophy (5 requiring surgery and 1 injectable fillers), 11 patients had a history of mental health problems (this included 4 patients who had intentional overdoses requiring admission to hospital) and 7 (14%) who had been prescribed antidepressants at some point.

Of the 51 patients currently at the 900 clinic, 13 (25%) had been admitted to adult inpatient services for a median duration of 9 days (range 3-133), 2 for OIs (PCP and MAI), 4 following overdoses and 1 for CVA and osteonecrosis.

The investigators concluded that after 2 decades of living with HIV, 20% of the patients at the 900 clinic have severe immunosuppression (CD4 <200), 25% have required hospital admission an 3% died. There were high rates of co-morbidity, lipodystrophy and depression. There are also a small group of young people who remain off ART with low CD4 counts. Overall 85% of those on ART currently have undetectable viral loads.

comment

The high rate of suppression on HAART (>85%) is a significant achievement given complicated treatment histories. Complicated balance for people still of treatment given the focus of long-term uncontrolled viraemia in adult patients, but also balanced with concern for long-term complications including cardiovascular and bone health.

Rate of psychiatric-related morbidity is especially concerning.

Reference:

Wan T et al. Health outcomes for young adults with perinatally acquired HIV-1 infection following transfer to adult services. 17th Annual BHIVA Conference, 6–8 April 2011, Bournemouth. Oral abstract O31.

Chelsea and Westminster Hospital reported results on the feasibility of using calcaneal stiffness as an indicator for osteoporosis in HIV-positive patients. [1]

Currently, a DEXA scan remains the gold standard measure of Bone Mineral Density (BMD) with a T-score of -1 and above being normal -1 to -2.5 indicating osteopaenia and <-2.5 indicating osteoperosis. However, DEXA scans are not available in all clinics, require extra patient attendance and radiation safety approval and cost around £60.

Several studies have previously shown a significant difference in BMD and fracture prevalence in both HIV-positive men and women. A population-based study by Triant et al. reported a high level of significance (p=0.0001 in men and p=0.002 in women) in bone fractures between over 2,200,000 HIV-negative patients and 8,525 HIV-positive patients. [2]

Scourfield et al at Chelsea and Westminster Hospital have found that a calcaneal ultrasound test using the GE-Achilles Insight machine is able to calculate the calcaneal stiffness index (CSI) and estimate the t-score. In HIV-negative people, CSI is currently used to predict hip fracture risk and vertebral fracture in post-menopausal women. However, this is not yet used in HIV-positive people.

The test is portable, takes 15 seconds to perform with minimal inter-operator variability. Osteopenia is most readily apparent in parts of the skeleton with high bone turnover as found in highly trabeculated, weight-bearing bones. The calcaneus (heel bone) is ideal as it is easily accessible, comfortable for the patient and weight-bearing.

The study aimed to correlate CSI with DEXA scan results in an HIV-positive population to assess the value and cost-effectiveness of using the GE-Achilles Insight machine as a screen tool for osteoperosis.

CSI measurements using the GE-Achilles Insight were performed on 100 people at random who had undergone a DEXA scan within the last 6 months. Their median age was 51 years (IRQ: 46-58), 85% were male, positive for a median of 15 years (IQR: 11-20) and with a BMI of 24 (IQR 21-26). Ethnicity of participants was 83% white Caucasian, 10% black African, 4% SE Asian, 1% black Caribbean, 1% Middle Eastern and 1% Indian. CSI scores were analysed to determine the optimum sensitivity. A cost-effectiveness analysis was then conducted.

The DEXA scan showed a prevalence of 15% osteoporosis and 55% osteopaenia. The CSI t-score showed 67% positive (estimated t-score =<-1.0) and 43% negative (estimated t-score >-1.0). This meant in the CSI t-score had 100% sensitivity but only 51% specificity compared to the DEXA scan. The positive predictive value of a CSI score of =<-2.5 for osteoporosis was 30%.

The cost-effectiveness analysis concluded that CSI is reliable and cost-effective method. If the 100 study participants involved in this study who had all previously undergone the DEXA scan had been screened first with CSI using a cut-off value of =<-1.0 this would have resulted in all cases of osteoporosis identified and 43 fewer DEXA scans which amounts to a saving of £2,795 (at an average cost of £65 per scan). However, due to the lower level of specificity, this would also have meant that 19 cases of oesteopenia were missed.

comment

The cost of the GE-Achilles Insight is only £12,000 and for a minimal investment the considerable ongoing concerns of high levels of currently undiagnosed bone disease could be easily addressed in every large clinic.

EACS guidelines recommend repeat DEXA screening every 3-5 years in patients at higher risk of BMD and include HIV as a risk factor when using the FRAX calculator and age over 50 years as a risk factor if not using FRAX. The financial and health saving appear significant given aging positive population.

CSI t-scores are also quicker, easier and less inconvenient for the patient. Patient volunteers or staff without medical training can also perform these to reduce cost further.

References:

1. A Scourfield et al. The use of calcaneal stiffness index to screen for osteoporosis in HIV-infected individuals. 17th Annual BHIVA Conference, 6–8 April 2011, Bournemouth. Oral abstract O33.
2. Triant VA et al. Fracture prevalence among human immunodeficiency virus (HIV)-infected versus non-HIV-infected patients in a large U.S. healthcare system. J Clin Endocrinol Metab 2008;93:3499-3504.
3. European AIDS Clinical Society (EACS). Guidelines: Prevention and Management of Non-infectious Co-morbidities in HIV.
   http://www.europeanaidsclinicalsociety.org/guidelinespdf/2_Non_Infectious_Co_Morbidities_in_HIV.pdf

There is no internationally agreed definition of a long-term non-progressor (LTNP), in recent years more usefully referred to as a slow progressor, or an HIV controller (HIC). As a result several different interpretations of what parameters can make someone a LTNP or a HIC exist and these change both between and within countries. Either way these atypical patients are an important group to research as they have the potential to increase our understanding of HIV pathogenesis.

Mandalia and colleagues performed a retrospective review of the Chelsea and Westminster patient database to determine the prevalence of LTNPs or HICs and their associated rates of progression.

In this case LTNPs and HIC were defined as being HIV-1 positive for >7 years, with a CD4 T cell count >=450 cells/mm3, CD4 T-cell slope >=0 since entry to cohort, no OIs and naïve to ART. LTNPs were defined as having varying HIV-1 RNA plasma load which is mostly low but detectable whereas HICs maintain undetectable levels of viral load.

The study cohort looked at all patients registered from January 1988 to February 2010. CD4 count slope was derived from a random intercept model. A stable CD4 T-cell count was defined as patients whose CD4 T-cell count slope was >=0. Survival analyses were used to estimate time to HIV progression and data were censored at the most recent visit to the clinic.

Out of a total cohort of 14,227 patients, only 12 people were LTNPs and 1 person was a HIC. These 13 patients account for 0.38% of the total cohort that is in line with the results from a similar cohort in France where 0.4% of patients were LTNPs (although their inclusion criteria was slightly different).

Of the 1,204 patients who had been HIV positive for over 7 years with no history of ART, 965 had a CD4 count <450 cells/mm3 whilst 239 had a CD4 count of >=450 cells/mm3. The median time of disease progression in the cohort with CD4 count <450 was 4.0 years (IQR: 1.0-7.3), while those in the cohort with CD4 count >=450 had a median time to progression of 6.2 years (IQR: 2.0–9.6). The difference in time taken for disease progression was statistically significant (p=<0.001) suggesting that patients whose CD4 counts remain within “normal” range progress less rapidly.

Reference:

Mandalia S et al. Frequency and characteristics of long-term non-progressors and HIV controllers in the Chelsea and Westminster HIV cohort. 17th Annual BHIVA Conference, 6–8 April 2011, Bournemouth. Oral abstract O28.

A study by Ashby and colleagues aimed to characterise the neurocognitive function of young adults with perinatally acquired HIV (paHIV) infection and compare them to their HIV negative siblings or family members as aged matched controls.

The study had two arms, group 1 was made up of 33 perinally infected HIV-positive young people aged 16-25, group 2 was a control group of 14 HIV-negative young people matched by age who were aware of their family member’s HIV status. Both groups completed a series of computerised neurocognitive tests, prospective and retrospective memory questionnaires (PRMQ) and the International HIV Dementia Scale (IHDS) testing. There was an additional sub-study, which involved Magnetic Resonance Spectroscopy (MRS) scanning of which all candidates were eligible. The MRS sub-study used 8 participants from group 1 and 4 from group 2.

The two groups were evenly matched in terms of demographics as shown in Table 1.

Of the 33 young people in group 1 who were HIV-positive, the median CD4 count was 444 cells/mm3 (IQR: 174-725), median CD4% was 21%, 18 (55%) young people had a HIV viral load of <50 copies/mL. The median age at diagnosis was 5 years (IQR: 0-9) with the median number of years since diagnosis of 15 (IQR: 13-20). Currently 26 (79%) of young people were on ARVs with the median age of starting ARVs being 13 years (IQR: 8-13) and the number of years since starting ARVs being 8.5 years (IQR: 4-13).

The results are shown in Tables 2 and 3. Results are mean scores unless otherwise stated. The only significantly different result from neurocognitive panel of tests between the two groups was the Prospective and Retrospective Memory Questionnaire (PRMQ) test where group 2 scored lower than group 1.

However, the MRS sub-study results showed significant differences between the two groups in terms of concentrations of Chol/Cr and MI/Cr. This indicates significant increases in cerebral metabolite inflammatory factors despite the fact that 5/8 people in group 1 had a plasma viral load of <50 cells/mm3.

This year the BHIVA audit focused on the Immigration Removal Centres (IRCs) linked to Hillingdon Hospital in Uxbridge. It included eleven IRCs in the UK that dealt with people at all stages of an asylum application.

The audit was a retrospective review of medical notes from all patients seen between January 2008 and January 2010 and included all routine clinical data. The audit included patient characteristics, comparisons between practice and BHIVA/NAT advice on care for HIV positive detainees and comparisons between practice and recommended clinical guidelines.

The study included 116 patients referred to Hillingdon Hospital and a further 18 (16%) who were not seen because of deportation, release, transfer to another IRC or delays due to IRC regulations. Of the 18, 50% (9) were HIV positive. A total of 60/116 patients referred were HIV positive. Of these, 85% were on ARVs with prior care in 36 different UK centres. Their median age was 33 years (IQR: 28, 41). The median number of visits was 5 (IQR: 2, 6).

Following detention, 24 patients (39%) were deported, 20 (33%) released, 11 (18%) still detained at the time of analysis and 6 (10%) had unknown outcome. Of the 44 people removed from the IRC (24 deported and 20 released), only 8 (18%) were given prior notice (5 were given 1 week notice, 2 were given 2 weeks notice and 1 was dying). Of the 8 who had preparation for removal, none had a 3-month contingency supply of ARVs, 5 (63%) received information about HIV support facilities in onward destination and 4 (50%) had a medical summary letter.

Aspects of health care in detention centres where information was not available due to lack of records included missed ARV doses at time of arrest and detention, treatment interruption after detention, standards of medical care in detention.

The study concluded that the IRCs audited were not able to meet BHIVA/NAT standards for detainees. These included unbroken access for ARVs, availability of past medical information, preparation for removal of HIV positive detainees. It found that 82% of patients were not given prior notice of removal and of the 18% who were given notice the time was inadequate to meet the standard recommendations.

The authors recommended that data collection become standardised in centres for asylum seekers, that there is a national audit of IRC medical services, and greater advocacy to empower patients and medical staff to make BHIVA/NAT advice mandatory.

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The Detention Centres Services Operating Standards Manual states that asylum seekers “must have available to them the same range and quality of services as the general public receives from the National Health Service”. This is clearly not happening.

A similar report by Medical Justice looking at the clinical care of detainees with HIV released in 2011 concluded: “This report provides evidence to suggest that immigration detainees living with HIV have been subjected to practices which, in other circumstances, would be considered unacceptable. Our records indicate that breaches of the NAT/BHIVA advice are routine; they occur intentionally in some cases, and as a result of inadequacies in others. Taken together, these breaches amount to a system of care, which is frequently detrimental to the health and well-being of those detained for immigration purposes. The UKBA claim that they are neither willing nor able to enforce the provisions of the NAT/BHIVA advice within immigration detention.”

How we treat failed asylum seekers awaiting deportation is to our national shame.

References:

1. Sabapathy K et al. An audit of HIV care provision for Immigration Removal Centre patients. 17th Annual BHIVA Conference, 6–8 April 2011, Bournemouth. Oral abstract O14.
   http://www.bhiva.org/documents/Conferences/Bournemouth2011/Presentations/110407/O14KalpanaSabapathy.pdf
2. Jon Burnett, Eden Fessahaye, and Anna Stopes, Detained and Denied: The clinical care of immigration detainees living with HIV. Medical Justice.
   http://www.medicaljustice.org.uk/images/stories/reports/d%26d.pdf

Please visit these index pages for full coverage from the workshop.

http://www.hiv-druginteractions.org/LatestArticlesContent.aspx?ID=543

http://www.natap.org/2011/Pharm/Pharm.htm

Abstracts from the workshop, published in Reviews in Antiviral Therapy & Infectious Diseases 2011_3 are available to download from the meeting website.

http://www.virology-education.com/index.cfm/t/Workshop_Materials/vid/34792068-01CF-4196-986A8271C014BBD7

Selected presentations are also available online.

http://regist2.virology-education.com/2011/12HIV_PK/13_April.html

Articles included in this issue of HTB are:

• Quad reduces levels of oral contraceptives
• Elvitegravir/cobicistat and acid reducing agents
• Lopinavir/r reduces levels of rifabutin
• Cobicistat has little impact on CYP2D6 and CYP2B6
• Atazanavir levels indicate need for higher dose during pregnancy
• Once-daily 150-mg maraviroc explored in people taking atazanavir/ritonavir
• Lopinavir troughs lower in children on once-daily dose with efavirenz
• Dose optimisation: 50 mg ritonavir-boosting, 3TC dosing and raltegravir once-daily

This open‐label fixed‐sequence study investigated the pharmacokinetics of a combined oral contraceptive containing 25 ug ethinylestradiol and norgestimate with a fixed dose combination tablet containing elvitegravir, cobicistat, FTC and TDF (“Quad”).

Steady‐concentrations of ethinylestradiol, norelgestromin (the active metabolite of norgestimate), EVG and cobicistat were determined in 15 HIV‐negative women receiving the oral contraceptive alone or with the “quad” tablet. Coadministration decreased the AUC of ethinylestradiol by ~25% and increased the AUC of norelgestromin by ~2‐fold. Concentrations of elvitegravir and cobicistat were within the range of values observed in previous studies. Changes in progesterone and FSH were similar in both treatment phases, but changes in LH were greater in the combination phase.

In light of the decrease in ethinylestradiol, it is recommended that when coadministered with the “Quad” tablet, oral contraceptives should contain at least 30 μg ethinylestradiol.

Reference:

German P et al. EVG/cobicistat/FTC/TDF and oral contraceptives Pharmacokinetic interaction between norgestimate/ethinyl estradiol and EVG/COBI/FTC/TDF single tablet regimen. Oral abstract: O_17.

The effects of omeprazole (20 mg once daily) or famotidine (40 mg once daily) on the pharmacokinetics of EVG and cobicistat were studied in HIV-negative subjects (n=11 per group).

When omeprazole was administered 2 hours prior to elvitegravir and cobicistat the AUC and Cmax of elvitegravir increased by 10% and 16%, but those of cobicistat decreased by 8% and 10%. Separating omeprazole and EVG and cobicistat by 12 h had no effect (<10% change) on the AUC or Cmax of elvitegravir or cobicistat. Administration of famotidine 12 h apart from elvitegravir and cobicistat had no effect (<10% change) on the AUC or Cmax of elvitegravir or cobicistat.

Similar results were observed in a separate study (n=16) of the simultaneous coadministration of famotidine with elvitegravir and cobicistat. No dosing restrictions are necessary on the administration of EVG and cobicistat with proton pump inhibitors.

Based on the available data, elvitegravir and cobicistat should be administered simultaneously with, and/or 12 hours after, dosing of H2 receptor antagonists.

Reference:

Mathias A et al. Effect of acid reducing agents on the relative bioavailability and pharmacokinetics of cobicistat boosted elvitegravir. 12th International Workshop on Clinical Pharmacology of HIV Therapy, 13–15 April 2011, Miami. Poster abstract: P_13

This preliminary study investigated rifabutin and LM565 (25 O desacetyl rifabutin) exposures in 14 HIV/TB coinfected patients starting LPV/r (400/100 mg twice-daily) and rifabutin (150 mg 3 times weekly). Control values for rifabutin alone (300 mg once-daily) were obtained prior to starting LPV/r. Control values for LPV/r alone were obtained 10 weeks after stopping rifabutin. Pharmacokinetic parameters (median, range) are shown in the Table 1.

Rifabutin AUC was below the target of 4.5 μg.h/ml in 42% of patients at week 2 of therapy and in 28% of patients at week 6 of therapy. The change in LM565 AUC was significantly greater than the change in rifabutin AUC. When given with LPV/r, rifabutin 150 mg three times weekly may result in low rifabutin concentrations.

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This study highlights the importance of the active metabolite when rifabutin is given with LPV/r.

Reference:

Cusato M et al.  Pharmacokinetic evaluation of rifabutin and its active metabolite LM565 coadministered with lopinavir/r in HIV infected patients. 12th International Workshop on Clinical Pharmacology of HIV Therapy, 13–15 April 2011, Miami. Oral abstract: O_14.

Cobicistat, the boosting agent being developed for combination with the integrase inhibitor elvitegravir and protease inhibitors, had little impact on two key drug-metabolizing enzymes (CYP2D6 and CYP2B6) or on the drug transporter P-glycoprotein, according to results of a three-part study by Gilead Sciences [1].

In a phase 2 trial, a once-daily single pill coformulation of elvitegravir/cobicistat plus tenofovir/emtricitabine controlled viral replication as well as Atripla, the once-a-day one-pill combination of efavirenz, tenofovir, and emtricitabine, through 48 weeks in previously untreated people [2]. Average elvitegravir trough concentration in that study exceeded the protein binding-adjusted 95% inhibitory concentration of elvitegravir by a factor of 10. In a 44-person pharmacokinetic study, cobicistat boosted elvitegravir to levels equivalent to those reached with ritonavir boosting [3]. Unlike ritonavir, cobicistat has no anti-HIV activity.

Cobicistat is a strong inhibitor of CYP3A, but earlier studies showed that it does not inhibit the enzymes CYP1A2, CYP2C8, CYP2C9, or CYP2C19 and that it is a weak inhibitor of CYP2D6 and P-glycoprotein. The new study evaluated the impact of cobicistat on drugs whose metabolism is strongly affected by CYP2D6 (desipramine), CYP2B6 (efavirenz), or P-glycoprotein (digoxin).

Researchers enrolled 10 people in the desipramine group, 25 in the digoxin group, and 18 in the efavirenz group. Study participants took 150 mg of cobicistat or the probe drug (desipramine, efavirenz, or digoxin) in one of two sequences: (1) cobicistat once daily for 9 day, then one dose of cobicistat and one dose of the probe drug, then a washout period with no drugs, than one dose of the probe drug, or (2) one dose of the probe drug, a washout, cobicistat once daily for 9 days, then one dose of cobicistat and one dose of the probe drug.

Nine people originally assigned to desipramine, 22 assigned to digoxin, and 17 assigned to efavirenz completed the study. Ages averaged 35 in the desipramine group, 33 in the digoxin group, and 33 in the efavirenz group. Respective average weights were 76 kg, 71 kg, and 74 kg. About two thirds of volunteers were white.

Most treatment-emergent toxicities were mild (grade 1), except for a single case of treatment-emergent gastroesophageal reflux after 9 days in the digoxin group that required withdrawal from the study.

Coadministration of cobicistat with desipramine led to a 58% to 65% increase in desipramine area under the concentration-time curve (AUC) and to a 24% increase in desipramine maximum concentration (Cmax). Efavirenz Cmax fell about 13% with cobicistat, while digoxin Cmax rose about 41%. Efavirenz and digoxin AUC changed little with cobicistat. When taken with the probe drugs, cobicistat reached levels similar to those recorded in earlier studies of this boosting agent.

The Gilead team concluded that cobicistat may be classified as a weak CYP2D6 inhibitor since desipramine exposure rose less than 2-fold with cobicistat. The lower efavirenz Cmax and higher digoxin Cmax with cobicistat appear not to require dose modifications.

The investigators believe that additional drug-drug interaction studies are not needed to explore the impact of cobicistat on drugs affected by CYP2D6, CYP2B6, or P-glycoprotein.

References:

1. German P et al. The effect of cobicistat on cytochrome P450 2D6, 2B6 and P-glycoprotein using phenotypic probes. 12th International Workshop on Clinical Pharmacology of HIV Therapy, 13–15 April 2011, Miami. Oral abstract O_01.
2. Cohen C et al. Randomized, phase 2 evaluation of two single-tablet regimens elvitegravir/cobicistat/emtricitabine/tenofovir disoproxil fumarate versus efavirenz/emtricitabine/tenofovir disoproxil fumarate for the initial treatment of HIV infection. AIDS. 2011;25:F7-F12.
3. German P et al. Pharmacokinetics and bioavailability of an integrase [inhibitor] and novel pharmacoenhancer-containing single-tablet fixed-dose combination regimen for the treatment of HIV. J Acquir Immune Defic Syndr. 2010;55:323-329.

An atazanavir/ritonavir dose of 400/100 mg daily during the third trimester of pregnancy yielded concentrations equivalent to concentrations with 300/100 mg daily after delivery in women taking the protease inhibitors (PIs) with or without tenofovir [1]. IMPAACT P1026s investigators recommended 400/100 mg for the third trimester, regardless of tenofovir use, and suggested it should also be considered for the second trimester.

Previous work by the IMPAACT team found that plasma exposure of ritonavir-boosted atazanavir decreased by about 30% during pregnancy and by an additional 30% when tenofovir was part of the regimen [2]. Those findings led the researchers to propose raising the atazanavir/ritonavir dose from 300/100 mg to 400/100 mg during pregnancy. The new study assessed the impact of the higher dose.

This study is part of IMPAACT P1026s, an ongoing nonblinded trial of antiretroviral pharmacokinetics in HIV-positive pregnant women that includes women taking atazanavir/ritonavir. With or without tenofovir as part of their regimen, these women took 300/100 mg of atazanavir/ritonavir once daily during the second trimester, 400/100 mg once daily during the third trimester, and 300/100 mg from delivery through 2 weeks postpartum. The IMPAACT investigators conducted intensive 24-hour sampling at steady state during the second and third trimester and postpartum.

Atazanavir pharmacokinetic targets were the estimated 10th percentile area under the concentration-time curve (AUC) of 29.4 mcg*h/mL in nonpregnant adults and a 24-hour concentration of 0.15 mcg/mL. The researchers also collected maternal and umbilical cord blood samples at delivery and measured infant bilirubin concentrations 24 to 48 hours and 4 to 6 days after birth.

Of the 59 study participants, 31 took atazanavir/ritonavir with tenofovir and 28 without tenofovir. Twenty-three women were Hispanic, 16 black, 16 Asian, 3 white, and 1 with unrecorded race. Median age and weight at delivery were 29.7 years and 71.4 kg. Median gestational age was 38 weeks and median birth weight 3088 grams.

Among women taking atazanavir/ritonavir with or without tenofovir, median AUC and trough concentrations, and numbers of women who met AUC and trough targets in each trimester showed better exposure with the 400/100-mg dose than with 300/100 mg during pregnancy, see Table 1.

Median atazanavir cord blood concentration measured 0.15 mcg/mL (range below detection to 1.33), and median ratio of cord blood to maternal delivery concentration was 0.18 (range 0.03 to 4.08). Among 48 women with a detectable atazanavir concentration at delivery, median atazanavir concentration stood at 1.38 mcg/mL (range 0.18 to 5.63). Median ratio of cord blood/maternal atazanavir was 0.14 (range 0.02 to 4.08).

The researchers recorded 37 grade 3 or 4 maternal adverse events, including 23 elevated bilirubin values. All bilirubin levels in infants were normal.

The IMPAACT investigators concluded that atazanavir clearance is increased during the second and third trimesters. Atazanavir exposure improved from the second to the third trimester, when the dose rose from 300/100 mg to 400/100 mg once daily. After delivery, atazanavir concentrations with the 300/100-mg dose equaled or exceeded concentrations with the higher dose in the third trimester.

The researchers proposed that 400/100 mg of atazanavir/ritonavir provides adequate atazanavir exposure during the third trimester “and should be considered during the second trimester as well.”

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The authors commented that their data support use of 400/100 atazanavir/ritonavir dosing in both the second and third trimester.

The FDA is expected ask for a change in the SPC but we haven’t yet heard what the EMA will do.

References:

1. Mirochnick M, Stek A, Capparelli EV, et al. Pharmacokinetics of increased dose atazanavir with and without tenofovir during pregnancy. 12th International Workshop on Clinical Pharmacology of HIV Therapy, 13–15 April 2011, Miami. Oral abstract O_10.
2. Mirochnick M, Best BM, Stek AM, et al. Atazanavir pharmacokinetics with and without tenofovir during pregnancy. JAIDS. 2011;56:412-419.
   http://journals.lww.com/jaids/Fulltext/2011/04150/Atazanavir_Pharmacokinetics_With_and_Without.5.aspx

Etravirine may greatly lower concentrations of maraviroc when the drugs are taken without a ritonavir-boosted protease inhibitor (PI), according to results of a retrospective, multicenter French analysis of antiretroviral-experienced people [1]. Maraviroc concentrations were low regardless of whether this CCR5 antagonist was dosed at 300 or 600 mg twice daily.

Etravirine, the most recently licensed nonnucleoside, induces the CYP3A4 enzyme, while maraviroc is a CYP3A4 substrate. As a result, giving the drugs together may result in lower-than-normal maraviroc concentrations. Thus, a maraviroc dose of 600 mg twice daily is recommended with etravirine. Combinations of antiretrovirals with different mechanisms and with activity against virus resistant to the first three antiretroviral classes are favoured constituents of salvage regimens, which often exclude PIs because of resistance or toxicities.

To assess the impact of etravirine on maraviroc in treatment-experienced people, French investigators conducted this retrospective multicenter study of 67 people taking etravirine and maraviroc with or without one or two nucleosides, with or without raltegravir, and without a PI. The researchers excluded people taking other drugs likely to interact with maraviroc, and they excluded people with undetectable maraviroc levels. They figured trough concentrations (Ctrough, collected 12 hours after dosing) and Cave (any post dose concentration), and they set the target Ctrough at 75 ng/mL.

The 49 men (73%) and 18 women studied had a median age of 50 (IQR 46 to 55) and a median weight of 62 kg (IQR 61 to 70). Thirty people (45%) were taking 300 mg of maraviroc twice daily, 37 (55%) were taking 600 mg twice daily, and 50 (50%) were also taking raltegravir. Everyone took 200 mg of etravirine twice daily. The investigators analysed 106 samples to calculate Cave and 82 to calculate Ctrough.

In people taking maraviroc at 300 mg twice daily, median Cave and Ctrough were 63 ng/mL (IQR 29 to 127) and 53 ng/mL (IQR 27 to 84); for people taking 600 mg twice daily, median Cave and Ctrough were 62 ng/mL (IQR 35 to 90) and 59 ng/mL (IQR 36 to 84).

For the whole study group, 62% of Cave values and 67% of Ctroughs stood below the target of 75 ng/mL. Among people taking 300 mg of maraviroc twice daily, 60% of Cave values and 71% of Ctroughs were under 75 ng/mL. Among people taking 600 mg twice daily, 64% of Cave values and 65% of Ctroughs were under 75 ng/mL. Even when the Ctrough cutoff was 50 ng/mL, 39% of Ctroughs were suboptimal, regardless of maraviroc dose. So in this population, doubling the maraviroc dose did not lower the risk of subtherapeutic troughs.

Median etravirine Ctrough was 696 ng/mL (IQR 474 to 1068), which is about 180-fold higher than the protein binding-adjusted 50% effective concentration for etravirine against wild-type (nonmutant) virus. Median etravirine Ctrough did not differ significantly between people taking 300 mg of maraviroc twice daily and people taking 600 mg twice daily (813 and 679 ng/mL).

“Surprisingly,” the French team concluded, “maraviroc Ctroughs were very similar” with both maraviroc doses and “quite lower” than concentrations seen when the regimen contained a ritonavir-boosted PI. Those findings suggest that increasing the maraviroc dose has “a modest effect” on CYP3A4 induction by etravirine.

Two small cases series of treatment-experienced people starting maraviroc, etravirine, and raltegravir suggest that the combination can promote a good virologic response through 48 weeks [2,3].

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The method of determining Cavg here is different from that used previously by Pfizer. There was considerable discussion about maraviroc target concentrations.

References:

1. Solas C et al. Pharmacokinetic interaction between maraviroc and etravirine: a multicentre study in HIV-patients receiving an antiretroviral regimen without PI. 12th International Workshop on Clinical Pharmacology of HIV Therapy, 13–15 April 2011, Miami. Oral abstract O_13.
2. Youngblood C et al. Use of maraviroc-, raltegravir-, and etravirine-containing regimens in treatment-experienced patients: a case-series study. J Int Assoc Physicians AIDS Care (Chic). 2011 Mar 23. Epub ahead of print.
3. Nozza S et al. Raltegravir, maraviroc, etravirine: an effective protease inhibitor and nucleoside reverse transcriptase inhibitor-sparing regimen for salvage therapy in HIV-infected patients with triple-class experience. Letter to editor. AIDS. 2010;24:924-928.

HIV-positive people taking standard-dose atazanavir/ritonavir plus 150 mg of maraviroc once-daily had lower maraviroc maximum concentrations (Cmax) and average concentrations (Cavg) than HIV-negative people taking 300 mg of maraviroc twice daily without atazanavir/ritonavir. But in this pharmacokinetic modeling study, maraviroc minimum concentration (Cmin) and effective constant concentration (ECC) were similar in the two groups.

Clinical researchers are interested in a regimen combining the CCR5 antagonist maraviroc with atazanavir/ritonavir and excluding reverse transcriptase inhibitors. But finding the appropriate maraviroc dose is complicated because atazanavir/ritonavir raises maraviroc concentrations through inhibition of the CYP3A4 enzyme and drug transporters. And modeling maraviroc pharmacokinetics has proved difficult because of interpatient and intrapatient variability in rate and extent of maraviroc absorption.

Pharmacokinetic data from antiretroviral-experienced people suggested lower maraviroc exposure than predicted by a drug interaction study in healthy volunteers taking 300 mg of maraviroc twice daily with atazanavir/ritonavir (study A4001025) [2].

To further assess maraviroc’s interaction with atazanavir/ritonavir, Pfizer researchers used a semiphysiologic maraviroc PK model to analyse maraviroc concentrations in two groups: (1) 12 healthy volunteers who took 300 mg of maraviroc twice daily without atazanavir/ritonavir in study A4001025 [2], and (2) 58 HIV-positive antiretroviral-naive people who took 150 mg of maraviroc plus 300/100 mg of atazanavir/ritonavir, both once daily, in study A4001078.

The Pfizer team used data from the volunteers to estimate the impact of atazanavir/ritonavir on intrinsic maraviroc clearance and extent of absorption. Then they applied these values to a “rich” set of data involving 145 samples from 15 people with HIV and a “sparse” data set involving 138 samples from 57 people with HIV. The Pfizer investigators used a nonlinear mixed model with two absorption compartments and four disposition compartments. The model introduced absorbed maraviroc into a liver compartment in which atazanavir/ritonavir inhibited metabolic clearance and absorption transporters. The model scaled renal clearance at a baseline creatinine clearance of 120 mL/min.

In the 12 HIV-negative volunteers taking 300 mg of maraviroc twice daily, atazanavir/ritonavir reduced population intrinsic clearance of maraviroc from 90 to 14 L/h, while maraviroc absorption rose from 83.3% to 98.5%. These changes led to the following predicted changes: (1) a 2.5-fold increase in maraviroc Cmax, (2) a 4.6-fold increase in 12-hours maraviroc area under the concentration-time curve, and (3) a 10-fold increase in Cmin.

At the same intrinsic clearance, the 150-mg once-daily maraviroc dose in HIV-positive people resulted in 93.5% absorption. Compared with the healthy volunteers taking 300 mg of maraviroc twice daily without atazanavir/ritonavir, the sparse-data 57-person HIV group taking 150 mg once daily with the protease inhibitors had a lower maraviroc Cmax (591 versus 933 ng/mL) and a lower Cavg (170 versus 213 ng/mL). But Cmin was higher in the HIV group than in HIV-negative volunteers (43 versus 38 ng/mL), as was ECC (95 versus 89 ng/mL).

Analysis of maraviroc concentrations in the 15-person rich-data HIV sample yielded similar results: Cmax 614 ng/mL, Cavg 170 ng/mL, Cmin 44 ng/mL, and ECC 96 ng/mL. However, intrinsic maraviroc clearance in the rich-data analysis was 9 L/h and absorption 69%.

Compared with a maraviroc dose of 300 mg twice daily without atazanavir/ritonavir, the Pfizer researchers concluded that in HIV-positive people a dose of 150 mg once daily with atazanavir/ritonavir “produced less peak-trough fluctuation with very similar Cmin and ECC values.” The investigators believe this result “implies similar efficacy for the maraviroc component of any such combination.”

References:

1. Weatherley B et al. Modeling of maraviroc pharmacokinetics in the presence of atazanavir/ritonavir in healthy volunteers and HIV-1-infected patients. 12th International Workshop on Clinical Pharmacology of HIV Therapy, 13–15 April 2011, Miami. Abstract P_05.
2. Abel S et al. Effects of CYP3A4 inducers with and without CYP3A4 inhibitors on the pharmacokinetics of maraviroc in healthy volunteers. Br J Clin Pharmacol. 2008;65 Suppl 1:38-46.
   http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2311406/?tool=pubmed

Children who switched from twice- to once-daily lopinavir/ritonavir plus efavirenz had a low lopinavir trough concentration in a small Thai study [1]. Troughs were not as low during twice-daily lopinavir/ritonavir dosing with or without efavirenz or with once-daily lopinavir/ritonavir without efavirenz. All children maintained an undetectable viral load, and those with low troughs had a dose increase.

In the United States once-daily lopinavir/ritonavir is licensed for antiretroviral-naive adults and for adults with fewer than three lopinavir-related resistance mutations. But data are sparse on the once-daily lopinavir/ritonavir tablet for children. To fill that pharmacokinetic gap, researchers in Thailand mounted a pilot study involving children already taking twice-daily lopinavir/ritonavir with or without the nonnucleoside efavirenz.

The 12 study participants had maintained a viral load below 40 copies for at least 3 months with twice-daily lopinavir/ritonavir. When children enrolled in the pilot trial, researchers collected blood samples over 12 hours to measure lopinavir levels. Then all children switched to an equivalent dose of once-daily lopinavir/ritonavir. Two weeks after the switch, the investigators collected blood samples over 24 hours.

Study group ages ranged from 9.3 to 17.7 years (median 13.1), weight from 26.8 to 50.3 kg (median 40.8), CD4 count from 456 to 1239 (median 699), and CD4 percent from 16% to 31% (median 23%). Five of six children taking lopinavir/ritonavir with efavirenz and six taking the protease inhibitors without efavirenz completed both pharmacokinetic evaluations. Among children not taking efavirenz with the PIs, five were taking tenofovir/lamivudine and one zidovudine/didanosine.

Children combining lopinavir/ritonavir with two nucleosides and without efavirenz took lopinavir doses ranging from 255 to 283 mg/m(2) (median 271) with twice-daily dosing and from 514 to 570 mg/m(2) (median 544) with once-daily dosing. These children had the following areas under the concentration-time curve (AUC), maximum concentrations (Cmax), and trough concentrations (C12h or C24h) before and after switching to once-daily lopinavir/ritonavir. See Table 1.

Children combining lopinavir/ritonavir with efavirenz took lopinavir doses ranging from 273 to 338 mg/m(2) (median 303) with twice-daily dosing and from 538 to 645 mg/m(2) (median 612) with once-daily dosing. They had the following lopinavir concentrations before and after switching to once-daily lopinavir/ritonavir. See Table 1.

* P <0.05 for once vs twice daily concetrations within group (not between group)

For all 11 children who completed the study, the geometric mean ratio of lopinavir AUC once daily/twice daily was 1.01 (90% confidence interval 0.85 to 1.21). For Cmin, the geometric mean ratio of lopinavir once daily/twice daily was 0.21 (90% confidence interval 0.09 to 0.48). When taking lopinavir/ritonavir twice daily, all children had a lopinavir 12-hour (trough) concentration above 1 mcg/mL. After the switch to once-daily dosing, 5 of 6 children taking lopinavir without efavirenz and 0 of 6 taking lopinavir with efavirenz had a trough above 1.0 mcg/mL. The 7 children with a 24-hour lopinavir concentration below that cutoff after the switch to once-daily dosing had their dose increased by 20% to 30% after 12 weeks. Troughs remained low in 4 of these 7 children after the dose increase.

With once-daily lopinavir/ritonavir, median efavirenz concentrations were 62.6 (range 36.2 to 197.2) mcg x hr/mL for AUC, 4.1 (range 3.1 to 10.8) mcg/mL for Cmax, and 1.7 (range 0.9 to 6.0) mcg/mL) for Cmin.

All children maintained a viral load below 40 copies/mL through 24 weeks of once-daily lopinavir/ritonavir. No lopinavir/ritonavir side effects emerged, as might be expected in a group already tolerating twice-daily lopinavir/ritonavir well.

Several published studies have addressed lopinavir/ritonavir pharmacokinetics with once-daily dosing [2-4] or with twice-daily dosing with efavirenz [5-7]. In Canada a study of 7 children with a median age of 9.8 years found similar pharmacokinetics with once- and twice-daily dosing and no observable difference in tolerability [2]. A Netherlands study of 19 children with a median age of 4.5 found evidence that a lopinavir/ritonavir dose of 460/115 mg/m(2) “leads to mean pharmacokinetic parameters comparable to data of 800/200 mg lopinavir/ritonavir once daily in adults, although the variability observed in the trough levels is much higher in children” [3]. Another Dutch study of 15 children with a median age 11.8 years found that a lopinavir/ritonavir dose of 300/75 mg/m(2) twice daily compensates for the enzyme-inducing effect of efavirenz given at 14 mg/kg once daily.

References:

1. Chokephaibulkit K et al. Pharmacokinetics of lopinavir/r tablets administered once versus twice daily with/without efavirenz in antiretroviral treatment experienced children. 12th International Workshop on Clinical Pharmacology of HIV Therapy, 13–15 April 2011, Miami. Abstract P_19.
2. la Porte C et al. Pharmacokinetics and tolerability of once- versus twice-daily lopinavir/ritonavir treatment in HIV-1-infected children. Antivir Ther. 2009;14:603-606.
3. van der Lee M et al. Pharmacokinetics of a once-daily regimen of lopinavir/ritonavir in HIV-1-infected children. Antivir Ther. 2006;11:439-445
4. Rosso R et al. Lopinavir/ritonavir exposure in treatment-naive HIV-infected children following twice or once daily administration. J Antimicrob Chemother. 2006;57:1168-1171.
5. King JR et al. Steady-state pharmacokinetics of lopinavir/ritonavir in combination with efavirenz in human immunodeficiency virus-infected pediatric patients. Pediatr Infect Dis J. 2009;28:159-161.
6. Bergshoeff AS et al. Increased dose of lopinavir/ritonavir compensates for efavirenz-induced drug-drug interaction in HIV-1-infected children. J Acquir Immune Defic Syndr. 2005;39:63-68.
7. Fraaij PL et al. Safety and efficacy of a NRTI-sparing HAART regimen of efavirenz and lopinavir/ritonavir in HIV-1-infected children. Antivir Ther. 2004;9:297-299.

The issue of dose optimisation is important for several reasons, ranging from finding the right dose for an individual patient that achieves the highest probability of therapeutic success with the lowest risk of adverse events, to as a strategy to decrease the cost of antiretroviral therapy in order to increase the delivery of therapy to more individuals. A plenary/round table discussion on ‘Dose optimisation and simplification’ focused on the decrease cost to increase access issue.

Here are some compelling reasons to pursue this research: “over 90% of HIV-infected individual live in very poor countries where the cost of antiretroviral treatment can still be a major barrier to access” (Andrew Hill, Liverpool University); “antiretrovirals consume 50% of the drug budget for South Africa…the need to reduce antiretroviral prices is becoming more pressing as ART scale up continues in low- and middle-income countries” (Gary Maartens, University of Cape Town); and “while 5 million individual are currently accessing ARV for treatment, 10 million more require therapy” (Steve Becker, Bill & Melinda Gates Foundation).

Atazanavir drug levels similar with 50 mg or 100 mg ritonavir boosting

Abstract P-31 is a good example of one dose optimisation strategy. This study investigated ATV concentrations in 12 healthy volunteers when given with either 100 mg or 50 mg of RTV for 10 days. [1]

The AUC of ATV when given with 50 mg of RTV was 47.09 mg*h/L and was 50.62 mg*h/L with 100 mg of RTV; the 90% CI of the ratio was 82.5 to 116.38. The ATV trough concentrations were 0.59 mg/L with 50 mg of RTV and 0.79 mg/L with 100 mg; all trough concentrations were above 0.15 mg/L, which is the suggested threshold concentration.

There was also no difference in ATV Cmax with the 2 RTV doses. In the 10-day treatment periods, total and LDL cholesterol significantly increased with the 100 mg RTV dose, whereas there were no significant changes with the 50 mg RTV dose.

These data in HIV-negative volunteers indicate atazanavir exposure was equivalent when given with either 100 mg or 50 mg of RTV, but there were fewer adverse effects on lipid metabolism with the 50 mg RTV dose. These data provide a basis for further studies in HIV-infected persons as a strategy to minimise RTV-associated adverse effects, and as a strategy to decrease the costs of therapy.

3TC dose reduction to 150 mg once-daily fails to achieve sufficient intracellular levels

While the atazanavir/ritonavir example above provides an example of a dose optimisation lead worth pursuing, two other abstracts are examples of strategies that do not.

The first, abstract O-05 evaluated whether a 3TC dose of 150 mg once daily, compared with the usual 300 mg once daily dose, achieved bioequivalent intracellular concentrations of the active metabolite, 3TC-triphosphate. [2]

24 HIV-negative volunteers participated in this study.

The geometric mean 24-hour AUC of 3TC-triphosphate from the 300 mg dose was 59.5 pmol/106 cells; this value for 150 mg was 44.0 pmol/106 cells. The geometric mean 24-hour concentrations were 1.49 pmol/106 cells and 1.23 pmol/106 cells for the 300 mg and 150 mg 3TC doses, respectively.

The geometric mean ratios for AUC and trough concentration were 0.73 and 0.82, respectively, indicating that the 150 mg dose was not bioequivalent to the 300 mg dose.

This was a reasonable study to perform. It was possible the intracellular formation of 3TC-triphosphate might be saturable, and thus lower doses would achieve intracellular concentrations the same as higher doses. This study showed, however, this was not the case. Because bioequivalence was not demonstrated, there is no pharmacologic basis to pursue a lower 3TC dose as a dose optimisation strategy.

Once-daily raltegravir PK

The QDMRK study compared once vs twice daily raltegravir in 770 treatment-naive persons demonstrated once daily therapy was inferior to twice daily, with overall proportions of subjects with HIV-RNA <50cpm at week 48 of 83.2% vs. 88.9%, respectively. PK data were provided at CROI and demonstrated raltegravir trough concentrations with 800 mg once daily were substantially lower than 400 mg twice daily dose.

At the PK Workshop, additional PK data were provided in abstract O-09. [3]

Ctrough values, from the 24-hour intensive PK studies, were lower for once daily RAL: the geometric mean was 40nM for 800mg once daily versus 257nM for 400 mg twice daily. The trough concentration were similarly lower from the spare/population PK data: the geometric mean was 83nM for once daily and was 380nM for twice daily.

Significant relationships were also found between raltegravir concentrations and virologic response. For example, the odds ratio for achieving HIV-RNA below 400 or 50 copies/mL increased with increasing concentrations, and the odds of virologic failure decreased with increasing raltegravir concentrations. From the presentation at the meeting, no clear breakpoint or threshold raltegravir concentrations could clearly be identified.

From the data, it looks that a trough concentration less than approximately 60 nM increases the risk of virologic failure.

These data indicate raltegravir and the investigational integrase inhibitors elvitegravir and dolutegravir all exhibit pharmacodynamic relationships between trough concentrations and virologic response.

This raltegravir study is a clear example of a potential hazard for dose optimisation efforts: for all antiretrovirals, there is some threshold concentration you can’t go below without increasing the risk of virologic failure for a patient.

References:

All references are to the Programme and Abstracts for the 12th International Workshop on Clinical Pharmacology of HIV Therapy, 13–15 April 2011, Miami.

1. Estévez J et al. No changes in atazanavir exposure when boosted with 100 mg or 50 mg of ritonavir in health volunteers. Poster abstract P_31.
2. Else L et al. Pharmacokinetics of plasma lamivudine (3TC), and its active intracellular anabolite 3TC-triphosphate (3TC-TP) over a 24 hour dosing interval following administration of 3TC 300 mg and 150 mg once daily (od) to HIV-negative healthy volunteers. The ENCORE2 Study. Oral abstract O_05.
3. Wenning L et al. PK/PD analyses for QDMRK, a phase III study of the safety & efficacy of once versus twice-daily raltegravir in treatment-naïve HIV-infected patients. Oral abstract O_09.

The following report is an edited summary of a rapid summary from Jules Levin whose natap.org website posted numerous studies from this meeting.

• Rapid report: state of HCV policy, treatment, access

Abstracts from the oral and poster sessions are available online at the following links:

http://www1.easl.eu/easl2011/program/Orals/

http://www1.easl.eu/easl2011/program/Posters/

A limited number of web casts are due to be posted online but were not yet available when HTB went to press.

http://www.natap.org

This has been a landmark meeting with the leading developments being the oral late breaker in the last clinical session late yesterday afternoon where BMS reported 4/11 genotype 1 non-responders achieved SVR24 cure using only the two oral BMS HCV drugs (the protease inhibitor BMS-650032 and the NS5A inhibitor BMS-790052). [1]

This was proof of concept that we can cure at least some patients without PEG-IFN and ribavirin.

We know that genotype-1a will not respond as well to therapies coming out in the near future, genotype-1b responds better. In the BMS study and others genotype-1a comprised more of the failures.

Patients with viral failure quickly added PEG-IFN plus ribavirin and all responded with undetectable HCV viral load. This was reported in November 2010 at American Association for the Study of Liver Diseases conference (AASLD) and is equally important. BMS also reported that using the QUAD therapy (BMS-650032, BMS-790052 plus PEG-IFN and ribavirin) 9/10 patients achieved SVR24 cure and the outlying patient had <LLOQ at week 24 post treatment undetectable on retesting 35 days later.

BMS also reported impressive data on their peg-lambda interferon in a study comparing it to Pegasys with it showing safety benefit and also surprisingly activity benefit as well, the programme is moving ahead. [2]

The second major story at EASL was the development of potent nucleotides by the small biotech company Pharmasset that appear to have a high barrier to resistance. So far no resistance has been seen and this is a key to the success of these drugs. Pharmasset reported three studies on PSI-938 and PSI-7977 in combination without PEG-IFN and ribavirin in patients with genotype-1. More than 90% patients essentially achieved undetectable over relatively short-term follow up. [3]

They also reported a nucleotide in combination with PEG-IFN and ribavirin that also displayed essentially 100% undetectability in patients in early follow up, and they reported similar results for people with genotype 2 and 3. [4, 5]

Taking these two developments together, the cure data and the potency of these nucleotides, we can perhaps cure HCV with nucleotide therapy using short-term oral combination regimens.

Recently, BMS & Pharmasset announced a joint study that will combine the BMSNS5A with a Pharmasset nucleotide, 2 potent drugs expected to perform well together. Events are unfolding in an accelerated way now in HCV drug development coming out of this meeting.

BMS also reported the first SVR12 results in a dose-ranging study of its NS5A inhibitor BMS-790052 plus PEG-INF plus ribavirin vs PEG-INF plus ribavirin. SVR12 were 92% (11/12) receiving BMS-790052 vs 42% (5/12) receiving standard of care. [6]

Roche reported SVR data on their ritonavir-boosted HCV protease inhibitor danoprevir (RG7227) and on their nucleoside analogue polymerase inhibitor mericitabine (RG7128), both used in combination with PEG-IFN plus ribavirin. [7, 8]

Of particular note, this is the first-time data reported on low-dose ritonavir boosting danoprevir. In with HIV protease inhibitors, ritonavir-boosting provides better efficacy compared to unboosted PIs, and although HCV is different boosted HCV PIs may provide additional benefits.

The danoprevir/r results were under-reported due to many other headline studies but preliminary data after 12 weeks in null-responders reported undetectable viral load in 88% (14/16) of genotype-1b patients and 50% (4/8) of genotype 1a. Roche was the first company to report combination therapy with two oral drugs with the INFORM study but their development programme was delayed because of an ALT elevation observation seen with their PI. Ritonavir-boosting lowers the Cmax and appears to have resolved that issue development is continuing.

Numerous studies were reported by Vertex on telaprevir and by Merck on boceprevir regarding IL28B and other data on how the drug will be used in support of the phase 3 studies reported at AASLD in November. The FDA hearing is scheduled for April 2011 and both drugs are expected to be approved.

Pharmasset announced the start of a cutting edge study called Atomic which will look at various treatment schedules. [9]

Novartis presented SVR rates in a phase 2b study of a non-immunosuppressive cyclosporine-A derivative called alisporivir (DEBO25) used with PEG-IFN plus ribavirin in genotype-1 treatment-naïve patients of 76% rate compared to 55% for the PEG-IFN plus ribavirin. [10]

Alisporivir is the first in a new class of drugs called cyclophilin inhibitors. Unlike other compounds in development that target the hepatitis C virus directly, alispirovir, targets host proteins that the hepatitis C virus uses for replication. This therapy has particular appeal because not only does resistance appear hard to develop but cross-resistance to other classes of HCV drugs is unlikely. This increases options for non-responders who could use other experimental compounds. A phase 3 study in gentotype 1 naives is underway and study in non-responders is also planned. A Phase 2b trial looking at the potential of the agent in HCV patients with genotypes 2 and 3 is also underway. The host proteins are needed for replication in all types of HCV infection so there is potential for the agent to have broad activity in all six variations of HCV.

Tibotec had a few presentations on their once daily TMC435 protease inhibitor but as they have already entered Phase 3 having shown an impressive 84% SVRs in treatment-naïve patients in phase 2 and are on a fast track to get to the market. These included IL28B data and a couple of other posters including the ASPIRE Study with preliminary data in nonresponders and a poster of five patients treated with TMC435 monotherapy & retreated with TMC435 plus PEG-IFN and ribavirin showing they were at least in short-term followup able to reach undetectable. [11, 12, 13, 14]

Boerhinger Ingelheim reported phase 2 data on their protease inhibitor BI201335 plus PEG-IRN plus ribavirin in naives & treatment-experienced and announced publicly they are starting phase 3. [15]

Presidio had a poster on their NS5A development programme and issued an announcement about the clinical efficacy of their lead candidate PPI-461 in patients in an ongoing study. [16]

Conclusion

Drug development for hepatitis C is moving up to the next level.

At the meeting many companies reported important new study results. Second generation protease inhibitors are progressing but are still in earlier development stages. They will hopefully provide a higher barrier to resistance and have perhaps activity against some of the mutations the early generation PIs are associated with.

New NS5A inhibitors are in development by several companies in the wake of the potent first in class by BMS. Abbott reported clinical data in patients on their potent looking second-generation protease inhibitor (ABT-450) that will be boosted by low-dose ritonavir. Abbott also has NNRTIs and has reported on them previously but reported here on preclinical data on their own NS5A. So Abbott now has three classes of oral HCV drugs.

Intermune reported on a second-generation protease in development with Roche that appears to be active against the key protease mutation 155, and Intermune also reported for the first time on their own NS5A inhibitor.

Gilead reported 24 presentations at the meeting. This was their coming out party, as they reported follow-up clinical data on their four-drug combination of protease, NNRTI, PEG-IFN plus ribavirin, with all patients becoming undetectable in the follow up. Gilead also revealed the many new drugs they are developing in multiple classes including additional protease inhibitor, their own nucleotide and NS5A inhibitor. So Gilead now has several drugs in several different drug classes. And Gilead also reported on a TLR7 inhibitor that appears to be active against both hepatitis B and C, so this immune-based type of therapy may bring a new dimension to HBV therapy.

GSK has also entered the field. They reported here on two new drugs, a NS5B polymerase inhibitor GSK248585 and GSK2336805, a NS5A inhibitor.

Patients with genotype 1a and IL28 CT and TT do not do as well. So a patient can test to see if they are CC, CT or TT and also if they are genotype-1a or 1b. Genotype 1a is an indication that response to therapy may not be as good, with lower SVR rates observed in several studies. Also CT & TT patients don’t do as well as CC patients in terms of SVR rates, so these appear to be important considerations in treatment decision-making.

The expectation is that we will be able in theory to cure HCV in all patients except for those who may be too sick. We are now moving to the next level of drug development where one oral HCV drug plus PEG-IFN and ribavirin is not enough. We need multiple oral HCV drugs in a regimen with or without PEG-IFN and ribavirin. The range of studies is currently looking at a broad range of approaches.

Drug resistance has also evolved to another level. The majority opinion emerging at EASL amongst leading academic and industry researchers, publicly and privately, is that resistance will not be a problem. Resistance is hoped to disappear after 1.5 to 2 years, as evidenced in the study reported here by Vertex on telaprevir. This would enable people to re-treat with the same protease inhibitor in combination with other classes of oral drugs, after having failed it with resistance mutations. To support this hope Tibotec reported retreating five patients who had received TMC435 monotherapy with TMC435 plus PEG-IFN and ribavirin and in the short-term they achieved undetectable viral load.

There is however a significant minority opinion who feel strongly that we do not yet know yet if this is true. Here it is important to stress that the majority view is only an opinion we do not know what will happen in the future. The worst case scenario is virologically failing patients remain on failing therapy and accumulate mutations and compensatory mutations that do not disappear or they disappear by a insensitive test and reappear after re-using a protease, as in HIV.

The telaprevir-Vertex resistance study above used resistance testing that had a population sensitivity cut-off of 20%, so resistance of less than 20% could not be seen. The Tibotec example also can be undercut because the PEG-IFN plus ribavirin could have been doing all the work. This can only be demonstrated in clinical studies of recycling compounds in people with treatment failure. This type of study is not easy to design and implement.

Another issue being discussed in the wake on much data reported here on how IL28B affects treatment outcomes. Surprisingly, there was a lot of talk about using a lead-in of PEG-IFN and ribavirin to block use of boceprevir or telaprevir in Europe, in the sense that if a patient had a good PEG-IFN and ribavirin response at week 4, they could continue on this and not add boceprevir or telaprevir. This scenario is not in the best interest of patient outcomes as treatment would still be 48 rather than 24 weeks.

At panel discussion on global HCV included Jake Liang from the US NIH and John Ward from the US CDC who did not provide any information suggesting US federal government money will be provided. The HHS HCV Strategy plan is expected to be publicly unveiled in May. There are no federal, state or city large-scale HCV testing & screening programmes. There are no discussions that address large-scale care in HCV, similar to the Ryan White Care Act for HIV.

comment

This year EASL as significant in terms of a change in direction for the management of HCV as the World AIDS Conference in Vancouver 1996 was for the management of HIV. 2011 is the year that heralds triple combination therapy for ‘difficult to manage’ genotype 1 HCV infection. At a preliminary FDA hearing at the end of April 2011, both first-generation NS3/4 protease inhibitors that have completed phase 3 studies (boceprevir from Merck and telaprevir from Vertex/Jannsen-Cilag) received unanimous support from the FDA expert panel and will shortly receive formal FDA approval for treatment in combination with PEG-IFN and ribavirin for genotype 1 HCV infection.

There are, however, a number of unanswered questions for these two drugs. These included whether it will be possible to use a shorter duration of treatment for all patients or will some groups need to use response-guided lengths of therapy? How will clinicians manage the complexities of response-guided therapy? Will virology laboratories manage to turn around HCV viral load results in time? How will we mange potential side effects in clinical practice (anaemia for boceprevir, rash for telaprevir)?

In addition to these, the questions surrounding the emergence of resistance mutations and their significance for future treatment options remain unanswered. Although data from both boceprevir and telaprevir studies suggest that the majority of mutations disappear by 2-3 years post therapy, the potential re-emergence and cross-resistance with the use of second- and third-generation PIs thereby reducing the efficacy of future PI-containing treatment options remains a concern for some.

Whilst we grapple with these issues and learn how to use the first-generation NS3/4 inhibitors, a plethora of other DAAs–PIs, NS5b polymerase inhibitors (both nucleosides and non-nucleosides) and a few NS5a inhibitors–are getting ready to go into phase 3 studies. The next question that whether PEG-IFN and ribavirin can still be considered as standard-of-care therapy for genotype 1 HCV infection?

For HIV doctors treating co-infected patients, although there is some preliminary data with telaprevir in triple combination (CROI 2011), data with boceprevir is yet to be presented. Over the next months, whilst we wait further data and formal approval of the first two anti-HCV protease inhibitors, the key issue to consider is who should we treat now with PEG-IFN and ribavirin and who should wait for triple therapy?

References:

Unless stated otherwise reports and links are to natap.org. Many of these reports include a selection of presentation slides.

1. Lok A et al. HCV cured without peginterferon/ribavirin with two oral HCV drugs.
   http://natap.org/2011/EASL/EASL_28.htm
2. Investigational compound PEG-interferon lambda achieved higher response rates with fewer flu-like and musculoskeletal symptoms and cytopenias than PEG-interferon alfa in phase IIb study of 526 treatment-naive hepatitis C patients. BMS press release. (03 April 2011).
   http://natap.org/2011/EASL/EASL_31.htm
3. Lawitz E et al. Once daily dual nucleotide combination of PSI-938 and PSI-7977 provides 94% HCV RNA < LOD at day 14: first purine/pyrimidine clinical combination data (the Nuclear Study).
   http://natap.org/2011/EASL/EASL_07.htm
4. Nelson DR et al. PSI-7977 QD plus PEG/RBV in HCV GT1: 98% rapid virologic response, complete early virologic response: the PROTON study.
   http://natap.org/2011/EASL/EASL_06.htm
5. Larezari J et al. PROTON study: PSI-7977 QD with PEG/RBV: 12-week safety, RVR, cEVR, & SVR12 in treatment-naïve patients with HCV GT2 or GT3.
   http://natap.org/2011/EASL/EASL_22.htm
6. Pol S et al. First report of SVR12 for a NS5A replication complex inhibitor, BMS-790052, in combination with pegIFNα-2a and RBV: phase IIA trial in treatment-naive HCV genotype 1 subjects.
   http://natap.org/2011/EASL/EASL_13.htm
7. Rouzier R et al. Activity of danoprevir plus low-dose ritonavir in combination with peginterferon alfa-2a (40KD) plus ribavirin in previous null responders.
   http://www.natap.org/2011/EASL/EASL_21.htm
8. Pokros P et al. First SVR data with the nucleoside analogue polymerase inhibitor mericitabine (RG7128) combined with peginterferon/ribavirin in treatment-naive HCV G1/4 patients: interim analysis from the JUMP-C trial.
   http://natap.org/2011/EASL/EASL_30.htm
9. Pharmasset initiates phase 2b ATOMIC trial of PSI-7977 for multiple HCV genotypes. Pharmasset press release. (30 March 2011).
   http://www.natap.org/2011/EASL/EASL_04.htm
10. Flisiak R et al. Once daily alisporivir (DEB025) plus Peg-IFN-alfa-2A/ ribavirin results in superior sustained virologic response (SVR24) in chronic hepatitis C genotype 1 treatment-naïve patients – The ESSENTIAL study.
    http://natap.org/2011/EASL/EASL_08.htm
11. Zeuzem S et al. The ASPIRE Trial: TMC435 in treatment-experienced patients with genotype 1 HCV infection who have failed previous PegIFN/RBV treatment: Week 24 interim analysis.
    http://natap.org/2011/EASL/EASL_18.htm
12. Medivir announces positive phase 2b 48-week (SVR24) interim results of TMC435 in treatment-naive patients chronically infected with genotype-1 hepatitis C virus. Medvir press release. (22 February 2011).
    http://www.natap.org/2011/HCV/022211_02.htm
13. Tibotec starts global phase 3 clinical trials studying TMC435 in adults with chronic genotype 1 HCV. Tibotec press release. (18 February 2011).
    http://www.natap.org/2011/HCV/022211_01.htm
14. Medivir kiicks off phase 1a trial of the hepatitis C polymerase inhibitor TMC649128. Medvir press release. (16 February 2011).
    http://www.natap.org/2011/HCV/021711_01.htm
15. Sulkowski M et al. SILEN-C1: sustained virological response (SVR) and safety of BI 201335 combined with peginterferon alfa 2a and ribavirin (PegIFN/RBV) in treatment-naïve patients with chronic genotype-1 HCV infection.
    http://natap.org/2011/EASL/EASL_20.htm
16. Presidio Pharmaceuticals, Inc. announces positive results in a phase 1b clinical trial of PPI-461, a novel, potent broadly-active hepatitis C virus (HCV) inhibitor. Presido press release.
    http://natap.org/2011/EASL/EASL_03.htm