Rapid drop in mitochondria in fat biopsies within four weeks of d4T use

1 December 2012. Related: Side effects.

Muirgen Stack, HIV i-Base

A study by Menezes and colleagues from the University of the Witwatersrand, Johannesburg, published in HIV Medicine, was stopped by the DSMB due to the negative impact of d4T (stavudine) on mitochondria after only four weeks of use. [1]

This was a prospective, open-label, randomised, controlled trial comparing standard and low-dose d4T with tenofovir, designed to assess early differences in adipocyte mitochondrial DNA (mtDNA) copy number, gene expression and metabolic parameters in HIV positive, black South African patients.

Recruitment to the trial was stopped early after a data safety and monitoring board (DSMB) analysis of the first 60 patients demonstrated the group given d4T at both standard and low doses showed a greater fall in the mean mtDNA copies/cell than those in the arm receiving tenofovir.

A total of 60 HIV positive, treatment naive adults were enrolled between September 2008 and December 2009. They were randomly assigned 1:1:1 to receive 3TC (lamivudine) and efavirenz, plus: standard weight-based dose of d4T (30 mg if < 60 kg or 40 mg if > 60 kg) according to the then South African guidelines [2], or a low dose of d4T (20 mg if < 60 kg or 30 mg if > 60 kg), or tenofovir (300 mg).

Inclusion criteria included an absolute neutrophil count (ANC) >750 cells/mm3, haemoglobin >7.0 g/dL (70 mg/mL) and a platelet count >50,000 platelets/mm3. Patients also needed an alanine and aspartate aminotransferase measurement and an alkaline phosphatase measurement < 2.5 x the upper limit of normal (ULN) and a total bilirubin measurement < 2.5 x ULN. Patients were excluded if they were pregnant or breastfeeding and if they had received any antiretroviral drugs (including post exposure prophylaxis) other than single-dose nevirapine within the past six months.

Baseline characteristics including age and BMI were well matched between the three arms, although this was a cohort that was largely female (85%) and black (98%). All patients had a CD4 count below 200 cells/mm3, with 78% being at WHO stage 1 and 3% at stage 4.There was no statistically significant difference in the markers of inflammation (leptin, adiponectin) and lipid (HDL, LDL and triglycerides) and glucose metabolism between the arms at baseline.

At weeks 0 and 4, subcutaneous fat biopsies from the supra-ilac region were performed under local anaesthetic on fasted subjects. Total DNA was extracted at the same time points from adipose tissue. This allowed for determination of mitochondrial and nuclear gene copy numbers. The nuclear genes assayed included the peroxisome proliferator-activated receptor (PPAR)-gamma coactivator 1-alpha (PGC1), nuclear respiratory factor-1 (NRF1) and mitochondrial transcription factor-A (TFAM). Other genes measured included those involved in mitochondrial energy metabolism: cytochrome c oxidase subunit III (COX3), cytochrome c oxidase subunit IV (COX4) and mitochondrial cytochrome B (MTCYB). Two nuclear genes involved in lipid metabolism, leptin (LEP) and lipoprotein lipase (LPL), were also assayed.

The authors found a 29% decrease in the mean mtDNA copies/cell from baseline to four weeks in the standard-dose (30-40 mg) d4T arm (P < 0.05), and a 32% decrease in the low-dose (20-30 mg) d4T arm (P < 0.005), when compared with the tenofovir arm, which only had a 4% decrease in the mean mtDNA copies/cell.

For each individual dose of d4T (20, 30 and 40 mg), there was also a drop in mean mtDNA copy number (22, 35 and 31% respectively) vs. tenofovir (300 mg) (4%) at 4 weeks of HAART. The decrease in mtDNA copy number for both d4T 30 and 40 mg doses was significantly higher than for tenofovir 300 mg (P < 0.005 and P < 0.05, respectively). The drop in mtDNA copy number with the d4T 20 mg dose was not significant when compared with tenofovir (P = 0.40).

Only two of the genes assayed had their expression levels significantly altered by stavudine. NRF1 and MTCYB had a greater fall in expression (mean±SD) with the standard (-0.21±0.32 and -0.2±0.43) but not the low dose (0.0±0.51 and -0.11±0.5) of d4T dose when compared with the tenofovir 300 mg arm (0.11±0.26 and 0.16±0.42), (P < 0.05). There were no statistically significant changes in the markers of inflammation and lipid and glucose metabolism with any of the drug doses.

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These findings supporting the policy to switch from d4T to tenofovir in first line public sector HAART made by the South African government in 2010. [3]

Despite the advance of newer ARVs with better side effect profiles, the issue of cost is used by some countries to continue d4T use. Although d4T is being phased out, it is estimated to still be used by approximately 50% of people on treatment globally. Dose reduction (from the standard 40 mg twice daily to 30 mg or 20 mg twice daily) has been proposed as an interim measure that may have similar efficacy, but reduced toxicity. [4] However, other studies have shown that serious side effects continue at significant levels even with a lower dose, and that routine management often fails to detect and switch patients early, leading to peripheral neuropathy and lipoatrophy that is irreversible. [5]

Despite the study stopping enrolment early, limiting its statistical power, it still produced clear findings. There is an early association between mitochondrial depletion and stavudine therapy in this black South African population compared to minimal effect on copy number and related gene expression in patients using tenofovir. The rapid onset of these changes (four weeks) after starting stavudine supports the recent changes in South African guidelines to preferentially use tenofovir.

Combined with the reduced expression of NRF1 and MTCTYB, the authors speculate a role for these changes in the pathology of NRTI toxicity and an associated pre-requisite for lipodystrophy. However, whether these changes were transient or remained throughout the course therapy is less clear due to the lack of measurements past four weeks.

Finally, whilst the lowest dose of d4T (20 mg) was associated with non-significant alterations in mtDNA and gene expression levels compared to tenofovir 300 mg, this may be related to the study size. This should not be seen as supporting the safety of the reduced dose.

References:

1. Menezes CN et al. The early effects of stavudine compared with tenofovir on adipocyte gene expression, mitochondrial DNA copy number and metabolic parameters in South African HIV-infected patients: a randomized trial. HIV Medicine (4 October 2012, early view).
   http://onlinelibrary.wiley.com/doi/10.1111/j.1468-1293.2012.01054.x/abstract
2. National Antiretroviral Treatment Guidelines, First Edition. National Department of Health (2004).
   http://southafrica.usembassy.gov/media/2004-doh-art-guidelines.pdf
3. Clinical guidelines for the Management of HIV & AIDS in adults and adolescents. National Department of Health South Africa (2010).
   http://www.who.int/hiv/pub/guidelines/south_africa_art.pdf
4. Hill A et al. Systematic review of clinical trials evaluating low doses of stavudine as part of antiretroviral treatment. Expert Opin Pharmacother (2007) 8:679-688.
   http://www.ncbi.nlm.nih.gov/pubmed/17376022
5. Koegl C, Wolf E, Postel N et al. Low dose stavudine: as effective as standard dose but less side effects. 9th EACS, Warsaw. 25-29 October 2003. Abstract 9.8/5.
   http://www.aegis.org/conferences/eacs/2003/284.html