In this study, we determined the pharmacokinetic parameters of three commonly used generic NRTIs in HIV/TB co-infected patients who were also receiving 4-drug anti-tuberculosis therapy in resource-limited settings. To our knowledge, this is the first study to describe the steady-state pharmacokinetic profiles of generic lamivudine, zidovudine and stavudine in patients with TB/HIV coinfection on concurrent therapy. Our study is also the first to identify a significant pharmacogenetic relationship between a common UGT2B7 allele and zidovudine clearance in human patients and also zidovudine glucuronidation in human liver.
To date, published studies of the pharmacokinetics of generic antiretroviral agents in resource-limited settings have not included patients taking known inducers or inhibitors of cytochrome P450 enzymes and drug transporters.11, 12, 29
Consequently, it is not known if these patients would achieve similar bioavailability of these drugs. We found the steady-state plasma AUC of lamivudine to be 0.98 times and 1.1 times that reported for generic and brand-name lamivudine (respectively) in HIV-infected Ugandan patients who were given lamivudine/stavudine/nevirapine (Triomune®, Cipla, Goa, India) and the patented versions of the drugs (Epivir®, GlaxoSmithKline, Research Triangle Park, NC, USA; Zerit®, Bristol Myers Squibb, Princeton, NJ, USA; Viramune®, Boehringer Ingelman, Columbus, OH, USA).11
Furthermore, the steady-state plasma AUC of stavudine was 1.5 and 1.3 times that reported for generic and brand-name stavudine (respectively) in the above-mentioned study.11
We were not able to identify published studies that have evaluated zidovudine pharmacokinetics in HIV-infected black Africans. However the zidovudine AUC in our patients was 0.78 and 0.84 of values reported for generic and brand name zidovudine (respectively) in HIV-negative Indian women.29
These comparisons to published data suggest that lamivudine and stavudine concentrations in the TB/HIV co-infected patients in present study were at least similar to (or perhaps slightly higher than, in the case of stavudine) other patients that were not taking TB medications. The 14 to 21% lower zidovudine AUC in our patients is consistent with induction of zidovudine clearance by rifampin, though it is lower than the 47 – 50% reduction previously reported.5, 6
The apparent minimal effect of rifampin on zidovudine AUC in our study could be due to population differences between our patients and the historical controls and/or the effect of concurrent medications such as trimethoprim/sulfamethoxazole. Trimethoprim with or without sulfamethoxazole has been shown to decrease zidovudine renal clearance,30, 31
and could have in part minimized the induction effect of rifampin in our study population.
We observed high variability in zidovudine clearance (over 100% coefficient of variation) relative to the clearance of stavudine and lamivudine. The mechanism underlying this high variability of zidovudine is not well understood but may involve polymorphisms in genes encoding proteins involved in zidovudine disposition, including UGT2B7
. In previous work using our human liver bank, we determined that the most common UGT2B7 nonsynonymous SNP (c.802C>T, UGT2B7
*2) was not associated with altered zidovudine glucuronidation or UGT2B7 protein expression.23
These findings were independently confirmed in a more recent study of human liver bank samples by Peterkin et al.32 UGT2B7
gene re-sequencing by several groups have identified other polymorphisms, many of them in linkage disequilibrium within discrete haplotype blocks.27, 28
Consequently in addition to UGT2B7
*2, we also assayed for the common SNP c.735A>G (defining the UGT2B7
*1c allele) and showed higher in vivo
clearance and also faster hepatic glucuronidation of zidovudine in individuals carrying this allele. Such similar results in two clearly distinct populations (i.e. HIV-infected Ghanaian patients and white American liver donors) suggest that these genotype-phenotype associations are unlikely to have occurred simply by chance. Rather, it implies that the c.735A>G SNP, or perhaps another variant in significant linkage disequilibrium with this SNP (in both white and black populations), may provide a mechanistic explanation for enhanced UGT2B7
An important and novel finding of this study is that c.735A>G (UGT2B7
*1c) was significantly associated with higher zidovudine glucuronidation activity as well as UGT2B7 protein content in a human liver bank, confirming the in vivo
association between UGT2B7
*1c and zidovudine clearance. This association is also confirmed in part by a recent study by Innocenti et al
that showed about 45% faster 3-hydroxy-glucuronidation of morphine (another UGT2B7 substrate) in human livers that carried the intron 1 SNP (IVS1 +985A>G).24
This latter SNP is in significant linkage disequilibrium with c.735A>G and also the exon 4 SNP (c.1062C>T). Although they did not measure hepatic UGT2B7 protein content, they did evaluate UGT2B7 mRNA content and splicing. Similar to our findings with zidovudine, they also found no effect of UGT2B7
*2 on morphine 3-glucuronidation or on UGT2B7 mRNA expression.24
However, in contrast to our study, where UGT2B7 mRNA levels were unchanged, they found higher UGT2B7 mRNA expression in carriers of the intron 1 SNP. This discrepancy may relate to the mechanism by which these noncoding SNPs may alter gene expression through alteration of mRNA splicing regulatory elements, as was proposed by Innocenti et al.24
Several splice variants of UGT2B7 have been observed, and it is possible that our mRNA assay differed from Innocenti’s assay in the splice forms quantified.24
For example, our mRNA assay may have quantified total UGT2B7 mRNA forms including those that were aberrantly spliced and predicted to result in truncated UGT2B7 protein.
Polymorphisms in other genes associated with zidovudine disposition such as those encoding drug transporters have the potential to explain the variability in zidovudine clearance observed in this study and warrant future evaluation. A trend (P
=0.06) for elevated peripheral blood mononuclear cell zidovudine triphosphate levels was observed in carriers of the ABCC4
(MRP4) c.3724G>A variant.4
The clinical relevance of the association between UGT2B7
genetic variation and zidovudine metabolism and disposition is as yet unclear. Zidovudine like all NRTIs require intracellular phosphorylation to form the active triphosphate moieties that exert clinical effect.33
While zidovudine pharmacologic variability affects treatment response,34
no relationship between plasma zidovudine concentrations and clinic effect has been demonstrated. However, percent change in CD4 cell count and rate of decline in HIV RNA in plasma during zidovudine-containing therapy were related to intracellular concentrations of zidovudine triphosphate.35, 36
Furthermore, a higher total intracellular concentration of the phosphorylated moieties of zidovudine was associated with increased probability of reduced hemoglobin levels.37
As the relationship between zidovudine plasma exposure and intracellular triphosphate concentrations is currently not predictable, further studies are needed to define the influence of UGT2B7*1c
carriers status on intracellular concentrations of zidovudine-triphosphate, as well as clinical effect (efficacy and toxicity).
We also investigated potential patient factors that may influence the pharmacokinetic profile of each of the drugs evaluated. Except for a possible small effect of sex on stavudine CL/F normalized for body weight and age on lamivudine CL/F normalized for body weight, patient demographics did not appear to influence drug exposure in our population. However, these results should be interpreted with caution, as the study was not powered sufficiently to detect relatively small differences. The effect of sex on stavudine clearance is not known as few studies have explored the influence of sex on the disposition of antiretrovirals,38
and the largest and most comprehensive previous pharmacokinetic study of 81 patients included only 7 women.8
The observed influence of age on lamivudine CL/F needs to be validated in a larger population. Though, it has been shown that children younger than 12 years old tend to have higher values for lamivudine CL/F than children aged 12 – 18 years old, age has not been shown to influence lamivudine CL/F in adults.39
There are no known interactions between lamivudine and zidovudine or stavudine.40, 41
The lower AUC of lamivudine observed in the patients who received the co-formulated tablets lamivudine/zidovudine compared to the separate lamivudine tablets given with stavudine could be due to the significant differences in body weight or BMI between the two groups. Clearance and half-life of lamivudine did not differ between the two groups to suggest differences in elimination of the drug.
We recognize that our study had some limitations. The size of the study population was small and so failure to find significant relationships between pharmacokinetic parameters of some of these drugs and patient factors such as age, sex, BMI does not exclude the existence of significant relationships. Comparison of drug exposure in our study patients to historical controls rather than an appropriate control that did not receive rifampin-containing TB therapy does not allow us to_exclude the effect of population differences. The differences in lamivudine exposure between patients who received lamivudine/zidovudine and lamivudine/stavudine should also be interpreted with caution as it may be due to differences in disease state, as patients who had hemoglobin < 8g/dL were preferentially given stavudine. Rifampin is known to induce the activity of UGT2B7,5, 6
and trimethoprim has been shown to decrease renal clearance of zidovudine by 58% during coadministration but had no influence on the ratio of zidovudine glucuronide to zidovudine.30, 31
Although, these patients were on several concurrent medications including rifampin and trimethoprim-sulfamethoxazole, we do not think the concurrent medications confounded the relationship between UGT2B7 polymorphism and zidovudine metabolism as all patients received these medications at the time of initiation of HAART and at the time of PK testing.
Despite the above limitations, this pilot study provides important pharmacokinetic data on commonly used generic NRTIs in HIV/TB co-infected patients receiving concurrent TB therapy, and identifies a novel association between UGT2B7 genetic variation and zidovudine metabolism and disposition. The influence of UGT2B7*1c carriers status on intracellular concentrations of zidovudine and it anabolites, as well as clinical effect (efficacy and toxicity) needs to be investigated as it may have implications for individualized therapy or dosing of zidovudine.