Time-dependent changes in HIV nucleoside analogue phosphorylation and the effect of hydroxyurea

Hoggard, Patrick Ga; Kewn, Stephena; Maherbe, Anlieb; Wood, Robinb; Almond, Lisa Ma; Sales, Sean Da; Gould, Jaynec; Lou, Yuc; De Vries, Corryd; Back, David Ja; Khoo, Saye Ha; for the CHARM Study Group

Clinical Science

Background: Nucleoside analogues are activated to their triphosphates, which compete with endogenous deoxynucleoside triphosphate (dNTP) pools to inhibit HIV reverse transcriptase. Hydroxyurea has been administered with nucleoside analogues to modulate intracellular dNTP pools and thus the ratio of drug triphosphate : endogenous triphosphate.

Objectives: To examine changes in drug activation over time and investigate the effects of hydroxyurea on intracellular phosphorylation of antiretroviral nucleoside analogues.

Patients: A total of 229 HIV-infected individuals receiving abacavir, lamivudine and zidovudine were randomly assigned to receive or not nevirapine and hydroxyurea. Twenty-four patients were recruited to an observational substudy measuring intracellular drug triphosphate and dNTP concentrations at 0, 2, 6, 12, 24 and 48 weeks.

Methods: Drugs were extracted from isolated peripheral blood mononuclear cells before analysis of endogenous dNTP and drug triphosphates by primer extension assays.

Results: Twenty-two out of 24 patients were followed to completion of the substudy. Hydroxyurea had no demonstrable effect on endogenous dNTP or drug triphosphate levels at any timepoint. However, the ratio of zidovudine triphosphate to endogenous deoxythymidine triphosphate was significantly increased with hydroxyurea. A significant decrease in lamivudine triphosphate (3TCTP) and the 3TCTP : endogenous deoxycytidine triphosphate ratio was seen over 48 weeks. In five patients who failed therapy in the first 24 weeks, significantly reduced 3TCTP was seen.

Conclusion: Hydroxyurea does not affect measurable pools of endogenous nucleosides in vivo. Decreased lamivudine phosphorylation over time may provide a novel pharmacological explanation for the mechanism of resistance to this drug.

From the aDepartment of Pharmacology and Therapeutics, University of Liverpool, Liverpool L69 3GE, UK; bDepartment of Medicine, Somerset Hospital, Green Point, Cape Town 8051, South Africa; cGlaxo-SmithKline Research and Development, Greenford, Middlesex UB8 0HE, UK; and dInternational AIDS Therapy Evaluation Centre, University of Amsterdam, 1105 AZ Amsterdam, the Netherlands.

Correspondence to Dr Patrick Hoggard, Department of Pharmacology and Therapeutics, University of Liverpool, Ashton Street, Liverpool L69 3GE, UK. Tel: +44 0151 794 5919; fax: +44 0151 794 5656; e-mail: patrick@liverpool.ac.uk.

Received: 14 February 2002; revised: 17 July 2002; accepted: 7 August 2002.

Article Outline
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Nucleoside analogues are essential components of a standard antiretroviral regimen for the treatment of HIV [1,2], and in addition drugs such as lamivudine may be used to treat chronic hepatitis B infection. The antiviral activity of nucleoside analogues is dependent upon the intracellular formation of triphosphate metabolites (dideoxynucleoside triphosphate) [3,4], which compete against corresponding endogenous triphosphates (deoxynucleoside triphosphate; dNTP) to inhibit DNA synthesis. Although the absolute level of drug triphosphate will influence its antiviral activity, it is ultimately the balance of competition between drug triphosphate and each corresponding dNTP (the ratio of drug triphosphate : dNTP) that most accurately reflects the competition for the enzyme.

Hydroxyurea has been evaluated for a possible role in combination therapy for HIV infection [5,6]. Hydroxyurea has little or no direct antiretroviral activity per se but in-vitro data showed that a combination of nucleoside reverse transcriptase inhibitors and hydroxyurea is synergistic without increasing cellular toxicity [7]. Hydroxyurea inhibits cellular ribonucleotide reductase [8], resulting in a decrease in endogenous dNTP synthesis in the presence of hydroxyurea. This will shift the competition between the drug and endogenous triphosphates to favour incorporation of the drug into the proviral chain.

Hydroxyurea is cheap and affordable. Its use has been proposed in resource poor countries [9], where as part of combination therapy it may enhance the efficacy of treatment or allow the sparing of antiretroviral drugs, which will in turn drive costs down.

Here we report on a substudy of a large randomized multi-centre trial evaluating the effect of antiretroviral combination therapy [10]. The primary objective of the study was to investigate the effect of hydroxyurea on the ratio of intracellular drug and endogenous triphosphates in HIV-infected previously antiretroviral-naive patients receiving a triple nucleoside analogue regimen with or without nevirapine at intervals over 48 weeks.

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Materials and methods

Study design

The CHARM study is a large multi-centre trial evaluating the effect of antiretroviral nucleoside analogue combination therapy in 229 patients [10]. Male and female HIV-1-infected patients (with HIV-1-RNA values greater than 5000 copies/ml between week −12 and week −4) who were antiretroviral naive or had received less than 2 weeks previous nucleoside reverse transcriptase inhibitor-based therapy were eligible for inclusion. Patients also had to understand and provide written informed consent to participate in the study. All subjects were started on a triple nucleoside analogue regimen of zidovudine, lamivudine and abacavir, [combivir (zidovudine 300 mg twice a day plus lamivudine 150 mg twice a day) plus abacavir 300 mg twice a day]. Subjects were then randomly assigned to receive or not nevirapine (200 mg twice a day) and secondly to receive or not hydroxyurea (500 mg twice a day).

Patients were excluded if they were considered unlikely to complete the 72-week study period, or unlikely to comply with dosing schedules/protocol evaluations. Patients with pancreatitis or neuropathy in the past 6 months, hepatic dysfunction, renal failure or treatment with radiation therapy/cytotoxic chemotherapeutic agents/immunomodulating agents within 30 days of study drug administration were also excluded.

The present report describes an observational pharmacokinetic substudy in 24 subjects with HIV infection (plasma HIV-1-RNA level greater than 5000 copies/ml; CD4 cell count greater than 150) recruited at a single site (Somerset Hospital, Cape Town, South Africa).

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Sampling for intracellular pharmacokinetics

Subjects attended the clinic on the day of sampling. Adherence to the randomized antiretroviral medication was quantified as the percentage of doses that should have been taken. Details of the previous dose and time of the last meal were recorded. Blood samples were collected for triphosphate determination in peripheral blood mononuclear cells (PBMC) at baseline (week 0, 0 h). After pre-dose (0 h) measurements, medications were taken simultaneously. Dosing was observed and the time recorded. Samples were then taken at 2, 6, 12, 24, and 48 weeks at both 0 h (pre-dose) and 2 h post-dose (thought to correspond approximately to the time of maximal nucleoside analogue plasma concentration) [11]. PBMC were isolated from venous blood (64 ml), collected into eight heparinized Vacutainer CPT tubes (Beckton Dickinson, Oxford, UK). After washing in phosphate-buffered saline (10 ml), an aliquot (100 μl) was removed to determine cell densities (Coulter counter, Coulter, High Wycombe, UK). Cell pellets were extracted (6 h) in 60% methanol (2 ml; v/v in 50 mM Tris-HCl; pH 7.4), and then dried by rotary evaporation and stored at −20°C before shipment to the University of Liverpool.

Methanolic extracts were re-extracted with perchloric acid (0.4 N; 200 μl; 4°C) before the quantification of endogenous triphosphates was carried out by template primer extension assays as described previously [12,13]. After endogenous triphosphate measurement, drug triphosphates were quantified using template primer extension assays as already described [12,14] using standard curves containing either carbovir triphosphate (CBVTP; 0, 0.1, 0.2, 0.4 and 0.6 pmol); lamivudine triphosphate (3TCTP; 0, 0.05, 0.1, 0.2, 0.4 and 0.8 pmol) or zidovudine triphosphate (ZDVTP; 0, 0.0125, 0.25, 0.5, 0.1 and 0.2 pmol).

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Statistical methods

Despite numerous in-vitro studies, no in-vivo data have been published on the effect of hydroxyurea on nucleoside analogue phosphorylation. At the time of the design of this observational study, clinical triphosphate data were limited to zidovudine. Using these data with a standard deviation of 50% for intracellular concentrations of ZDVTP [15], we calculated that the recruitment of 21 patients would give a power of 80% to detect a difference of 30% in the intracellular concentrations of drug and endogenous triphosphates between baseline and other timepoints (two-sided α = 0.05). Drug and endogenous triphosphates were quantified by interpolation from standard curves (r2 > 0.95). Values were then standardized to pmoles per million PBMC. Triphosphate ratios (drug : endogenous) were also calculated. As a result of evidence of non-normality, statistical analysis was performed on log-transformed intracellular triphosphate pharmacokinetic data. Drug and endogenous triphosphate data are expressed as means with 95% confidence intervals for descriptive summary. Ratio data are expressed as means with 90% confidence intervals. A comparison of intracellular triphosphate concentrations or ratios between the with and without hydroxyurea treatment groups were examined using the geometric least squares mean ratios with 90% confidence intervals obtained from SAS PROC MIXED models with fixed factors of weeks, hour, hydroxyurea and nevirapine and subject as random effect for each triphosphate pharmacokinetic parameter. Hour was included in the earlier model to test whether there was a significant difference between the pre-dose (0 h) and post-dose (2 h) over the 48 weeks. As there was no statistical significant difference in the MIXED effect model, in order to reduce variability these samplings were combined (mean) to investigate longitudinal changes. Statistical analysis for changes in intracellular phosphate levels with time was also performed by Cuzick's test for trend. Differences in the intracellular triphosphates between patients responding and failing treatment was assessed using the Mann–Whitney U test.

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Intracellular phosphorylation was analysed in 24 patients (11 receiving hydroxyurea). Baseline characteristics are shown in Table 1. There were two withdrawals: one subject withdrew consent after baseline measurements and another withdrew after 6 weeks, leaving 22 patients (10 receiving hydroxyurea) with serial phosphorylation measurements to 48 weeks. Viral loads and CD4 cell counts were monitored to 72 weeks. Three patients stopped taking abacavir at week 2 as a result of hypersensitivity reactions but continued (on zidovudine, lamivudine and nevirapine) to 48 weeks; these patients showed typical results and were included in all analyses.

We observed marked interindividual variability in endogenous dNTP and drug triphosphates (Fig. 1 and Fig. 2). These are consistent with previous studies using either template primer or high-performance liquid chromatography-based assays [12,13,16–19]. We also observed marked variability in the formation of drug triphosphate over time (Fig. 3).

The results from PROC MIXED models, after adjusting for week and nevirapine, show that hydroxyurea had no detectable effect upon the formation of endogenous dNTP at any timepoint (Table 2). Similarly, conversion to the active drug triphosphates were not significantly different in the presence or absence of hydroxyurea (Table 3). The competition of lamivudine and abacavir for the enzyme reverse transcriptase (measured by the ratio of drug triphosphate : endogenous triphosphate) was unaltered by the addition of hydroxyurea to the triple drug nucleoside analogue regimen (Table 2; Fig. 2). However, the addition of hydroxyurea resulted in a small increase in ZDVTP : deoxythymidine (deoxythymidine triphosphate; dTTP) (Table 3). These models also show that there were significant incremental trend increases for deoxyguanosne triphosphate (dGTP), deoxycytidine triphosphate (dCTP), deoxyadenosine triphosphate (dATP) and a significant decline for 3TCTP and 3TCTP/dCTP over the 48 weeks (all P values < 0.001). Using nevirapine, only the CBVTP : dGTP ratio had significantly increased (P values < 0.001). However, this significant effect increased (P value < 0.001), but this could be by chance, as there is no interpretation for that effect.

No significant change in the intracellular concentration of endogenous dCTP, dGTP and dTTP was seen over the 48 weeks (Cuzick's trend test P = 0.136, 0.087, and 0.860, respectively). However, dATP increased with time (P = 0.003). During the course of this study there was no significant decrease in the triphosphate metabolites of abacavir and zidovudine (Cuzick's trend test P = 0.333 and 0.880, respectively). The results for dATP, 3TCTP and the 3TCTP : dCTP ratio were in agreement with parametric MIXED effect models. Both 3TCTP and the 3TCTP : dCTP ratio significantly decreased over the 48 weeks of the study (Cuzick's trend test P < 0.0001 and < 0.0001, respectively). Intracellular 3TCTP at 48 weeks was significantly lower than initial measurements [2.19; 95% confidence interval (CI) 1.66–2.88 at 2 weeks versus 1.08; 95% CI 0.81–1.44 at 48 weeks]. Similarly, the 3TCTP : dCTP ratio at 48 weeks was significantly lower than initial measurements (3.87 pmol/106 cells; 95% CI 2.91–5.13 pmol/106 cells at 2 weeks versus 1.64 pmol/106 cells; 95% CI 1.23–2.20 pmol/106 cells at 48 weeks).

Although the dose taken in the pharmacokinetic study was observed, it is possible that lower adherence may have an impact on intracellular triphosphate levels. However, the removal of patients with less than 80% adherence from the 3TCTP analysis resulted in similar decreases in 3TCTP and the 3TCTP : /dCTP ratio over the 48 weeks (Cuzick's trend test P < 0.0001 and < 0.0001, respectively). Intracellular 3TCTP in more adherent patients (> 80%) at 48 weeks remained significantly lower than initial measurements (2.32; 95% CI 1.69–3.13 at 2 weeks versus 1.22; 95% CI 0.87–1.63 at 48 weeks).

This pharmacokinetic substudy was not powered to compare drug activation with clinical response. In the first 24 weeks of study, five patients developed virological failure (defined as < 1 log10 reduction in plasma HIV-1-RNA levels by week 12 or a confirmed plasma HIV-1-RNA level greater than 400 copies/ml after week 24). The median concentration of 3TCTP was significantly less (1.08 pmol/106 cells) in these patients compared with 1.83 pmol/106 cells in patients who remained virologically suppressed (P = 0.025; 95% CI for difference between medians −1.29 to −0.09). Similarly, the median ratio of 3TCTP : dCTP was 1.91 in virological failures and 3.03 in patients who remained suppressed (P = 0.038; 95% CI for difference between medians −2.36 to −0.08). No differences were noted for CBVTP or ZDVTP.

There was also marked within-individual variability; for example, there was a between two- and threefold variability in endogenous dNTP between the highest and lowest values within each patient over the study. There were also median sixfold, 13-fold and 11-fold differences in CBVTP, ZDVTP and 3TCTP concentrations, respectively, between the highest and lowest values within each patient over the time course of the study. The apparent decrease in 3TCTP with time will influence the latter, but within-individual variability could be partly caused by a lack of adherence over the study period.

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We failed to observe any changes in intracellular concentrations of any of the endogenous triphosphates with hydroxyurea throughout the study (Table 3). Neither was any change observed in dATP in PBMC from HIV patients receiving hydroxyurea in the absence of antiretroviral therapy [20]. In-vitro studies have suggested that the enhancement of didanosine activity [21,22] may be caused by the reduction of dATP by hydroxyurea [4,23,24].

No significant difference in intracellular activation to the triphosphates of zidovudine, lamivudine and abacavir was seen with hydroxyurea, but a significantly higher ZDVTP : dTTP ratio was observed in patients receiving hydroxyurea. Because endogenous dTTP concentrations did not alter over time, the change in the ZDVTP : dTTP ratio is more likely to be the result of a cytostatic effect. Hydroxyurea is thought to arrest the cell cycle at the S-phase, which results in increased thymidine kinase activity [25].

In recently published studies, excess drug toxicity observed in the hydroxyurea arm outweighed any long-term improvement in virological suppression [26], and was also responsible for the discontinuation of a randomized trial comparing efavirenz/didanosine/stavudine with and without hydroxyurea [27]. The minimal alteration in the competition between drug and endogenous triphosphate with hydroxyurea may explain why intensification with this drug was not associated with a more favourable outcome. However, some authors argue that the risk : benefit ratio may still favour the use of hydroxyurea in certain situations, e.g. salvage regimens and resource-poor settings [2]. It is possible that hydroxyurea may also inhibit other intracellular or extracellular pathways involved in HIV replication, such as the inhibition of antigen-specific immune activation or arresting the cell-cycle [5,28].

Manipulation of the physiological maintenance of the cell may not be associated with long-term benefit. dNTP synthesis in vivo is regulated by many negative and positive feedback loops [29], and it is therefore possible that upregulation in the salvage pathway may conserve dNTP pools. Furthermore, as dNTP synthesis is essential for cell replication, the physiological regulation of dNTP pools would be expected in the long term.

A significant increase in dATP with time was seen in all patients over 48 weeks. As host kinases responsible for the activation of dNTP are subject to competition from the nucleoside analogues, it is possible that homeostatic mechanisms upregulate to maintain DNA synthesis. As the formation of dATP has no competition for activation (i.e. no antiretroviral dATP analogue), an increase in dATP may be seen. Increased dATP may have implications for future therapy with didanosine (whose activity is dependent on the ratio of its active dideoxyadenosine triphosphate to dATP).

The activation of zidovudine and abacavir did not change significantly throughout the 48 weeks sub-substudy, consistent with our previous observations that illustrated that zidovudine phosphorylation was not altered over time [19]. However, there was a significant decrease in both 3TCTP and the ratio of 3TCTP : dCTP over time (Fig. 2). Perhaps of greater importance is the fact that a similar decrease in 3TCTP and 3TCTP : dCTP with time was also observed in the more adherent patients (> 80% adherence). The clinical significance of these observations is uncertain. The optimal ratio of drug : endogenous triphosphate of the nucleoside analogues required for anti-HIV activity in vivo has not been defined, although a reduced ratio results in a decreased antiviral effect in vitro [30]. HIV resistance during lamivudine monotherapy emerges at a relatively rapid rate compared with other nucleoside analogues [31] and the downregulation of drug phosphorylation over the first few weeks of therapy may provide a pharmacological explanation for this phenomenon. It is interesting to note that, in the five patients failing therapy within the first 24 weeks, 3TCTP and the ratio of 3TCTP : dCTP were significantly reduced compared with patients without virological rebound. Four of these patients had less than 80% adherence to antiretroviral medication, but no temporal changes in ZDVTP concentrations were observed during this time despite their receiving combined zidovudine/lamivudine.

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A decrease in the intracellular concentration of lamivudine triphosphate was seen over the 48-week trial. The reduced intracellular activation of lamivudine may provide some explanation for the development of drug resistance. Second, hydroxyurea shows a minimal effect on the intracellular pharmacokinetics of the nucleoside analogues and their drug : endogenous triphosphate ratios. The results demonstrated that long-term in-vivo studies are required to assess the therapeutic long-term benefits of the modulation of intracellular pools.

Sponsorship: Financial support for the study was provided by Glaxo Smithkline Research and Development.

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Antiretroviral therapy; clinical trials; intracellular activation; nucleoside analogues; triphosphate

© 2002 Lippincott Williams & Wilkins, Inc.