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Pharmacokinetic Properties of Nucleoside/Nucleotide Reverse Transcriptase Inhibitors
  Part II
JAIDS Journal of Acquired Immune Deficiency Syndromes: Volume 37 Supplement 1 1 September 2004
Piliero, Peter J MD*
From *Department of Medicine, Albany Medical College, Albany, NY.
Options for antiretroviral therapy in patients infected with HIV continue to expand as new drugs are integrated into treatment regimens. Nonetheless, nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs/NtRTIs) remain the backbone of highly active antiretroviral therapy (HAART). With the approval of emtricitabine in 2003, there are now 8 Food and Drug Administration (FDA)-approved NRTIs/NtRTIs. Several of these agents are effective as once-daily therapy, including didanosine, lamivudine, extended-release stavudine (FDA approved, but not currently available), tenofovir DF, and emtricitabine. Recent results from pharmacokinetic and clinical trials indicate that another NRTI, abacavir, may also be effective as a once-daily therapy, and FDA approval of once-daily dosing is anticipated. NRTIs are inactive as administered, requiring anabolic phosphorylation within target cells to achieve their antiretroviral effects. All NRTIs are converted to nucleoside triphosphates, which serve as the active metabolites (the NtRTI, tenofovir DF, only requires conversion to the diphosphate form). Frequency of drug administration is closely related to the pharmacokinetic properties of a drug. The key parameter is the half-life; however, the plasma elimination half-life of the NRTIs/NtRTIs as administered is of little use in developing a dosing schedule. Rather, the intracellular half-life of the nucleoside triphosphate is the relevant parameter. This article reviews the pharmacokinetic properties, particularly those of the various phosphorylation steps, of the NRTIs/NtRTIs.
There is considerable clinical interest on the part of both clinicians and HIV-infected patients in simplifying antiretroviral drug regimens. Numerous approaches have been suggested for reducing the therapeutic burden experienced by patients who need to take >=3 antiretroviral drugs each day, many of which require multiple pills per dose and multiple doses per day. A major consequence of such complex regimens is low adherence to therapy, which is believed to be improved with use of simpler drug regimens. Low adherence clearly decreases the ability of antiretroviral drugs to control HIV replication. Numerous studies have shown that very high levels of adherence are necessary to achieve adequate HIV suppression. Therefore, the ideal antiretroviral regimen should be simple, effective, and well tolerated.
In addition to the use of fixed-dose combinations that limit pill count, another approach to simplification of antiretroviral therapy is the development of drugs that have long half-lives and thus require only a single daily dose. The most important criterion for development of a once-daily formulation is the ability of the drug to maintain antiviral activity over the entire 24-hour period between doses. In its Guidelines for the Use of Antiretroviral Agents in HIV-Infected Adults and Adolescents, the Panel on Clinical Practices for Treatment of HIV Infection endorses once-daily regimens, but only with a backbone of nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs/NtRTIs) with pharmacokinetic profiles that justify once-daily use.1 A number of antiretroviral drugs are currently Food and Drug Administration (FDA) approved for once-daily administration, including the NRTIs didanosine (ddI), lamivudine (3TC), extended-release stavudine (d4T XR), emtricitabine (FTC), and the NtRTI tenofovir (TDF). Abacavir (ABC) is another NRTI that has the potential for once-daily administration based on pharmacokinetic data but that is not yet FDA approved for use in this fashion.1 Once-daily coformulations of ABC/3TC and TDF/FTC are in development; FDA approval of both coformulations is expected in 2004 or 2005.
This article focuses on the pharmacokinetic properties of the currently available NRTIs/NtRTIs and how these properties help to define their dosing schedules.
Adherence to therapy is of critical importance for the long-term success of the treatment of HIV infection, and suboptimal adherence to highly active antiretroviral therapy (HAART) is associated with virologic failure and drug resistance. Many factors play a role in adherence to therapy, including the number of pills to be taken, the frequency of dosing, meal requirements, regimen convenience, tolerability of the medications, psychosocial support, and patient education. Once- or twice-daily administration of antiretroviral agents is appealing, as it may increase adherence. The major pharmacokinetic requirement for once- or twice-daily administration is that the intracellular concentration (in the case of NRTIs and NtRTIs) or plasma concentration (in the case of NNRTIs and protease inhibitors [PIs]) of the antiretroviral agent or its active metabolite remains above the minimal concentration that can inhibit viral replication during the entire 12- or 24-hour dosing interval. The drugs currently approved for once-daily administration include the NRTI/NtRTIs ddI, FTC, 3TC, d4T XR, and TDF, with ABC under consideration for approval; the NNRTI efavirenz (EFV); and the PIs atazanavir (ATV), amprenavir/ritonavir (APV/r), and fosamprenavir/ritonavir (FPV/r). Most other antiretroviral agents are approved for twice-daily use.
The emergence of fixed-dose combinations and once-daily regimens is a response to patient preference for simpler, more convenient regimens. In a community-based survey on patient awareness and interest in once-daily antiretroviral regimens, 80% of 536 HIV-infected patients indicated they were most likely to remember to take all of their antiretroviral doses with a once-daily regimen, compared with 63% with a twice-daily regimen. Seventy-three percent of the patients felt that once-daily regimens would fit better in their daily lives than twice-daily regimens. Claxton et al carried out a literature review of 76 studies measuring associations between dosing frequency and medication adherence. The mean dose-taking adherence was 71%. Adherence declined as the number of daily doses increased from 1 dose (79%) to 4 doses (51%). Adherence was significantly greater for once-daily vs. 3-times-daily, once-daily vs. 4-times-daily, and twice-daily vs. 4-times-daily regimens. Simpler regimens requiring less frequent dosing resulted in better adherence across a variety of therapeutic classes.
If a dose of a drug is delayed or missed, low drug levels may allow an increase in viral replication and the selection of resistant virus. The outcome of missing doses depends on the pharmacology of the antiretroviral drug (ie, trough plasma concentration Cmin, elimination half-life, intracellular drug concentrations, and concentration that inhibits 50% of viral replication [IC50] of the HIV isolate of an individual patient). A greater Cmin/IC50 ratio and a longer drug half-life increase the likelihood that the Cmin will remain greater than the IC50 even when one dose is missed. However, if the ratio is low, missing a dose may result in inadequate drug exposure over a defined period, which may lead to a higher probability of developing drug resistance. The forgiveness factor, or the time during which the concentration of a drug remains above the viral IC50 following a delayed or missed dose, depends not on the number of times a day the drug is taken, but on its pharmacokinetic properties.
Nucleoside and nucleotide analogues are prodrugs that must be metabolized intracellularly to phosphorylated compounds to exert their activity. Currently, 8 NRTIs have been approved by the FDA for treatment of HIV infection. The phosphorylation of NRTIs to the active triphosphate form occurs in several steps and creates a pool of dideoxynucleoside analogue triphosphates (ddNTPs) that compete with endogenous deoxynucleotide triphosphates (dNTPs) for substrate binding to reverse transcriptase. The one currently available NtRTI, TDF, undergoes only 2 phosphorylation steps to the diphosphate, which is its clinically active metabolite. NRTI phosphorylation makes use of the nucleotide synthesis and nucleoside phosphorylation pathways within the cell. The intracellular concentration of nucleoside analogue triphosphates is regulated by positive or negative feedback mechanisms on >=1 enzymes in the phosphorylation pathway. One or more steps in the phosphorylation pathways can be rate limiting in formation of active drug. The error-prone nature of reverse transcriptase, the affinity of ddNTP, and the ratio of ddNTP to endogenous dNTP all contribute to the functional ability of the reverse transcriptases to incorporate the triphosphate of the nucleoside analogue into nascent proviral DNA, which results in chain termination.
Since NRTIs require intracellular activation, it has been hypothesized that the intracellular level of the active triphosphate metabolite of NRTIs might be a better predictor of virologic effectiveness than plasma drug levels. This hypothesis has recently been proved by the demonstration that intracellular concentrations of the active triphosphate metabolite of 3TC and d4T, rather than levels of unchanged drugs in plasma, correlated with virologic response in HIV-infected patients.
Because NRTIs must be phosphorylated by cellular enzymes to their active moieties, it is to be expected that a number of cellular factors affect intracellular metabolism. These cellular influences can be divided broadly into intrinsic (cell type, cell cycle, intracellular ddNTP:dNTP ratio) and external (activation state, infection status) factors. Studies with NRTIs indicate that differential phosphorylation kinetics and the amount of phosphorylated products in different cell types are related to expression of cellular kinases, efficiency of cellular kinases, intracellular dNTP concentration, incubation time, and intracellular ddNTP/dNTP ratio. The kinetics of thymidine nucleoside analogue phosphorylation are somewhat dependent on the cell cycle, at least in stimulated lymphocytes. A close association has been observed between the intracellular ddNTP/dNTP ratio and the anti-HIV activity of NRTIs. A high ratio produces a greater antiviral effect, which is consistent with competition between ddNTP and dNTP for incorporation into DNA being shifted in favor of ddNTP, resulting in greater inhibition of RT. Because dNTP pools vary depending on the activation state of the cell, the ratio also is affected by this variable.8 The NRTIs may be classified into 2 groups. Cell-activation-dependent drugs such as zidovudine (ZDV) and d4T are preferentially phosphorylated, yield higher ddNTP/dNTP, and exert more potent antiretroviral activity in activated than in resting cells. In contrast, cell-activation-independent NRTIs, including ddI, zalcitabine (ddC), and 3TC, produce higher ddNTP/dNTP ratios and exert more potent antiretroviral activity in resting cells.
The plasma concentrations of NRTIs do not correlate with clinical efficacy or toxicity; these agents must be phosphorylated to become active against HIV infection. Thus, characterization of the pharmacokinetic parameters of the intracellular metabolites of NRTIs provides a better understanding of drug activity, which could lead to the development of more rational dose regimens in the HIV-infected population.
Thymidine Analogues

ZDV and d4T, the 2 thymidine analogues, are phosphorylated by the same 3 steps to the active triphosphate. In the first step, thymidine kinase catalyzes the formation of the monophosphate. In the second step the monophosphate is phosphorylated by thymidylate kinase to the diphosphate. Finally, the diphosphate is converted to the triphosphate by the enzyme nucleoside diphosphate kinase. Despite similar phosphorylation pathways, however, their phosphorylation kinetics differ significantly.
AZT is 3¢-azido-2¢,3¢-dideoxthymidine. It is usually given at a dosage of 300 mg twice daily. After oral administration, ZDV has a bioavailability of 60-70%. Distribution of ZDV exceeds total body water, with steady-state values of approximately 1.6 L/kg after IV administration. The half-life of the drug has ranged from 1-2 hours in various studies of HIV-infected patients. ZDV is metabolized to its glucuronide in the liver, kidney, and intestinal mucosa. Because of the extensive glucuronidation of ZDV, other drugs that are also glucuronidated or that inhibit this process cause an increase in ZDV plasma levels. Fourteen percent of the parent compound and 74% of the glucuronide have been recovered from the urine after oral administration in normal subjects. Renal excretion of ZDV is by both glomerular filtration and active tubular secretion.
ZDV, like other nucleoside analogues, is converted intracellularly to its triphosphate. The rate-limiting step is the conversion of ZDV to ZDV monophosphate, resulting in an accumulation of the monophosphate and the diphosphate within the cell. These intermediates competitively inhibit not only the normal substrate, thymidine, but also themselves. This inhibition causes a decrease in the thymidine triphosphate levels in the cells. Although the ability of ZDV triphosphate to inhibit HIV reverse transcriptase is 100 times greater than its ability to inhibit host DNA polymerase, the low concentration of thymidine triphosphate in the cell increases the chance that ZDV triphosphate will be used by DNA polymerase, thus causing inhibition of DNA synthesis and potential toxicity.
An AIDS Clinical Trials Group study published in 1994 evaluating the formation and elimination of total phosphorylated ZDV in peripheral blood mononuclear cells (PBMCs) included 21 asymptomatic HIV-infected patients during their first 24 weeks of therapy. This study suggested that metabolism of ZDV to its active intracellular forms may be saturable in some patients, is poorly correlated with plasma concentrations, and diminishes over time. The half-life of intracellular phosphorylated ZDV was twice that of plasma ZDV (4 vs. 2 hours), which suggested that an every-8-hour dosing regimen was feasible.
Recently, Rodriguez et al described methodology to measure the intracellular concentrations of ZDV triphosphate in PBMCs that combines an anion-exchange solid-phase extraction procedure with high-performance liquid chromatography and tandem mass spectrometry for analysis. With this method, intracellular ZDV triphosphate was measured in blood samples from 6 HIV-infected patients treated with ZDV administered at a dosage of 300 mg twice daily. Intracellular concentrations ranged between 42-193 fmol/106 cells. The same method was subsequently used for simultaneous in vivo determination of the ZDV and 3TC triphosphate pharmacokinetic profiles in HIV-infected patients receiving HAART. The median intracellular half-life of ZDV triphosphate was approximately 11 hours.
Finally, Ruane et al evaluated the pharmacodynamic effects of ZDV 600 mg once daily vs. 300 mg twice daily in HIV-infected patients over a 14-day dosing period. There was a trend toward a greater reduction in HIV RNA in the twice-daily dosing group. The slower decline in the once-daily group may have been due to the longer time required for intracellular phosphorylation to the triphosphate form. In a companion study, the intracellular area under the concentration-time curve (AUC) and time to peak plasma concentration (Cmax) of ZDV triphosphate were lower for the once-daily group. Although these findings raise concerns about once-daily dosing of ZDV, it is also possible that the antiviral effect of ZDV would not be compromised when it is used in combination with other potent antiretroviral agents.
D4T is 2¢3¢-didehydro-3¢deoxythymidine. This drug is currently available only for twice-daily administration. A new, extended-release formulation (Zerit XR, Bristol-Myers Squibb Co., Princeton, NJ) recently was approved for once-daily administration but is not commercially available at this time. Maximal plasma concentrations of d4T are achieved within 2 hours of oral administration and increase linearly as the dose increases, with an absolute bioavailability approaching 100%. There appears to be no accumulation of d4T in the plasma. The drug distributes into total body water and appears to enter cells by nonfacilitated diffusion. d4T is cleared quickly by both renal and nonrenal processes. In one study of 22 HIV-infected patients given 0.67, 1.33, 2.67, or 4 mg/kg of d4T, mean peak concentrations of 1.2-4.2 mg/L were attained. Thirty-four to 41% of an oral dose was excreted as unchanged drug in the urine. The mean values for plasma elimination half-life ranged from 1-1.6 hours.
ZDV and d4T differ significantly with respect to phosphorylation kinetics. The first step in phosphorylation of the 2 drugs, in which a monophosphate derivative is produced, is catalyzation by cytosolic thymidine kinase. This step is rate limiting for d4T but not for ZDV. Thymidine kinase has a similar Km (concentration of substrate that leads to half-maximal velocity) value for its natural substrate thymidine and for ZDV, but d4T is a poor substrate, with up to a 700-fold increase in Km. Thus, since the rate-limiting step is formation of d4T monophosphate, most of the d4T in cells is not phosphorylated.
The results of pharmacokinetic studies reported by Dudley et al, Cheer and Goa,and Kaul et al support once-daily administration of d4T XR. The pharmacokinetics and efficacy of d4T XR were evaluated in healthy subjects and HIV-infected patients in a multiple-dose study in which the drug was given as a single daily oral dose of 100 mg for 9 consecutive days. In 16 subjects, d4T XR sustained plasma concentrations in the therapeutic range over a 24-hour dosing interval, with low intersubject and intrasubject variability. The pharmacokinetics of d4T XR given once daily were also compared with those of the standard twice-daily formulation of d4T in 16 antiretroviral-naive HIV-infected individuals; each drug was given in combination with twice-daily 3TC and once-daily EFV. The pharmacokinetic profile of the 2 formulations indicates that drug exposure with once-daily d4T XR approximates that with standard twice-daily d4T after a single dose and at steady state. The apparent terminal elimination half-life of d4T XR is 10.3 hours.
Cytosine Analogues
3TC, FTC, and ddC, the 3 cytidine analogues, are phosphorylated by the same 3 steps to the active triphosphate. In the first step, deoxycytidine kinase catalyzes the formation of the monophosphate. In the second step, the monophosphate is phosphorylated by cytidine monophosphate/deoxycytidine monophosphate kinase to the diphosphate. Finally, the diphosphate is converted to the triphosphate by the enzyme nucleoside diphosphate kinase.
3TC, the (-)-enantiomer of 2¢,3¢-dideoxy,3¢-thiacytidine, is a dideoxy analogue of cytidine. The recommended oral dose is 150 mg twice daily or 300 mg once daily in combination with other antiretroviral agents. The drug is rapidly absorbed after oral administration, with a Cmax of 0.5-1.5 hours. The absolute bioavailability is approximately 82% in adults, and the mean apparent volume of distribution is approximately 1.3 L/kg after IV administration. Systemic clearance of single IV doses averages approximately 0.3 L/h/kg.
The mean pharmacokinetic parameters of 3TC after oral administration of 150 mg twice daily and 300 mg once daily, respectively, were Cmax, 2077 and 3461 ug/L; Cmin, 332 and 146 ug/L; elimination half-life, 6.1 and 7.9 hours; time to Cmax, 1.6 and 2.2 hours; average concentration over the dosage interval (Cav), 711 and 705 ug/L; and AUC over 2 dosage intervals (24 hours), 17,085, and 16,644 ug.h/L. There were statistically significant differences (P < 0.05) between the 2 schedules for Cmax and Cmin values, whereas no significant differences were observed for the other parameters. The investigators concluded that once-daily and twice-daily administration of the same total daily dose of 3TC led to a similar exposure in plasma.
Moore et al evaluated the pharmacokinetics of 3TC phosphorylation in PBMCs from 10 HIV-infected patients who were receiving treatment with a regimen of 3TC/ZDV. In this crossover study, patients were randomly assigned to receive 3TC 150 mg twice a day or 300 mg twice a day for 14 days. The median half-life of intracellular 3TC triphosphate was 15.3 hours for the 150-mg dose and 16.1 hours for the 300-mg dose, a nonsignificant difference. The rate-limiting step was conversion of 3TC diphosphate to 3TC triphosphate.
With combined solid-phase extraction and high-performance liquid chromatography with UV detection, levels of 3TC nucleotides were determined in the PBMCs of an HIV-infected patient on a regimen that included 150 mg of 3TC twice daily. Levels were 0.61, 0.68, and 0.20 ng/106 cells for 3TC triphosphate, diphosphate, and monophosphate, respectively. 3TC triphosphate had a long intracellular half-life of 15.5 hours. The average intracellular level of 3TC triphosphate from the above-described kinetics was 1.33 ng/106 cells. Intracellular AUC0-10, a measure of exposure, was estimated to be 13.7 ng/106 cells · h.
Yuen et al conducted a single-center, randomized, 2-way, crossover study to compare the steady-state pharmacokinetics of 3TC in plasma and those of 3TC triphosphate in PBMCs. Sixty healthy subjects received 3TC 300 mg once daily for 7 days and 150 mg twice daily for 7 days. Steady-state plasma 3TC pharmacokinetics following the once- and twice-daily regimens were bioequivalent with respect to the AUC from 0-24 hours at steady state (AUC24,ss) and average plasma 3TC concentration at steady state (Cave,ss) over the dosing interval. Steady-state intracellular 3TC triphosphate pharmacokinetics after the once- and twice-daily regimens were bioequivalent with respect to AUC24,ss, Cave,ss, and maximal 3TC concentration. Overall, the results of this study suggest that for key AUC-related parameters, 3TC 300 mg once daily is pharmacokinetically equivalent to 3TC 150 mg twice daily.
Zalcitabine- ddC
Zalcitabine, or 2¢,3¢-dideoxycytidine, does not appear to undergo significant metabolism. The pharmacokinetics of single doses of ddC were studied in 8 symptomatic HIV-infected patients. The results of this pilot study indicate that ddC is rapidly and extensively absorbed when administered as an oral tablet or solution to fasting patients with AIDS or AIDS-related complex. It is also rapidly eliminated, with a half-life of 1-2 hours. Because of its 3-times-daily dosing requirement, limited potency, and potential for adverse effects, this agent is not among the recommended NRTIs in current treatment guidelines and is rarely used in clinical practice today. There are no apparent differences in the absorption or elimination of ddC between the 0.5- and the 5-mg oral doses.
Emtricitabine FTC
FTC, the NRTI most recently approved by the FDA, is a fluorinated nucleoside analogue of cytosine; its chemical name is 5-fluoro-1-(2R,5S)[2-(hydroxymethol)-1,3-oxathiolan-5-yl]cytosine. The drug, which is similar in many ways to 3TC, has in vitro activity against HIV-1 that, depending on the in vitro methodology, is similar to or approximately 4- to 10-fold more potent than that of 3TC, although this difference does not appear to correlate with the antiviral effect of these 2 agents. Mean IC50 values for FTC range from 0.0014-0.14 nmol/L, compared with 0.002-2.5 mM for 3TC. The mean plasma elimination half-life of FTC after a single dose is about 8-10 hours in HIV-infected patients. However, after multiple doses of the drug at a dose of 200 mg daily, the intracellular half-life was approximately 39 hours.
FTC, like other nucleoside analogues, undergoes intracellular phosphorylation by various cellular kinases to its 5¢-monophosphate and diphosphate as well as the active triphosphate, which competitively inhibits reverse transcriptase by being incorporated into the viral genome, causing termination in DNA chain elongation. Darque et al28 determined the intracellular level of FTC 5¢-triphosphate in human PBMCs from HIV-infected patients using a method combining solid-phase extraction and high-performance liquid chromatography. Levels of FTC triphosphate were determined in patients receiving oral doses of 25, 100, or 200 mg twice daily, or 100 or 200 mg once daily. Results from this study demonstrated that high intracellular levels of FTC triphosphate were associated with better suppression of plasma HIV RNA. When normalized to the number of PBMCs of the samples, levels of triphosphate in the 1-, 3-, 6-, 9-, and 12-hour samples were 0.20, 1.48, 2.02, 2.26, and 1.52 pmol/106 cells, respectively.
In a number of trials, combination therapy that includes FTC 200 mg once daily with 2 other antiretroviral agents has shown similar efficacy to triple therapy that includes 3TC 150 mg twice daily. The combination of FTC + ddI has been shown to be more effective than d4T + ddI with regard to achieving or maintaining durable suppression of HIV levels after 24-48 weeks of therapy.
Guanosine Analogue

Abacavir sulfate, a structural analogue of the purine guanine, is (1S,cis)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol. In HIV-infected patients, AUC and Cmax under fasting conditions are dose dependent. With single doses of ABC from 100-1200 mg, mean AUCs increased from 1.1 to 33.1 mg/L · h and Cmax increased from 0.6 to 9.6 mg/L. With doses of 200 or 400 mg 3 times daily for 4 weeks, mean AUCs were 4.2 mg/L · h and 7.1 mg/L · h, respectively, and Cmax values were 2.2 mg/L and 3.3 mg/L. After single or multiple doses, Cmax was attained after a mean of 0.7-1.7 hours, and the mean half-life was 0.8-1.5 hours. The main route of excretion is renal.
The phosphorylation pathway of ABC differs from that of all other NRTIs. The first step in the conversion of ABC to its active metabolite, carbovir triphosphate, is phosphorylation to ABC monophosphate by adenosine phosphotransferase. This step is followed by deamination by a cytosolic enzyme to form carbovir monophosphate, which undergoes 2 subsequent phosphorylations, to the diphosphate by guanylate kinase and to the triphosphate by nucleoside diphosphate kinase and other enzymes. Unlike most other nucleoside analogues, no rate-limiting steps are present in the phosphorylation pathway. Carbovir triphosphate competes with endogenous 2¢-deoxyguanosine triphosphate (dGTP) for incorporation into the nucleic acid chain, and after incorporation, terminates DNA chain growth.
Although ABC is metabolized intracellularly to its 5¢-monophosphate in CD41 CEM human T-lymphoblastoid cell lines and in human peripheral blood lymphocytes, the diphosphate and triphosphate metabolites are not formed. The only triphosphate found in cells incubated with ABC is that of the guanine analogue (2)-carbovir. CD41 CEM cells incubated with ABC or carbovir for 48 hours accumulated carbovir triphosphate to levels of 0.28 mM and 0.38 mM, respectively. The elimination kinetics of carbovir triphosphate produced from either ABC or carbovir were essentially identical. Disappearance of intracellular carbovir triphosphate was monophasic, with a half-life of 3.3 hours. Carbovir triphosphate is a selective inhibitor of HIV reverse transcriptase. The inhibition constant (Ki) for inhibition by carbovir triphosphate of incorporation of dGTP into DNA by HIV-1 reverse transcriptase is 21 nmol/L.
Results of 2 recent studies evaluating levels of carbovir triphosphate intracellularly support the use of once-daily ABC in HIV-infected patients. Kewn et al examined the pharmacokinetics of carbovir triphosphate over 24 hours in PBMCs from 6 HIV-infected patients who were receiving ABC at a dose of 300 mg twice daily as part of their combination regimen. Levels of intracellular carbovir triphosphate ranged from <20-374 fmol/106 cells (50 fmol/106 cells = 100 nM) and were measurable throughout the 24-hour study period, with the highest levels found between 6-8 hours. This finding suggests a long half-life for carbovir triphosphate within cells. Since the Ki for carbovir triphosphate inhibition by incorporation of dGTP into DNA by reverse transcriptase has been reported to be 21 nM, these new data point to an above-inhibitory level of carbovir triphosphate within the cell throughout the 24-hour interval. Using the same assay method, Harris et al36 determined the intracellular levels of carbovir triphosphate in 5 HIV-positive patients receiving ABC 600 mg once daily for 5-17 months as part of a HAART regimen. The half-life of carbovir triphosphate was estimated to be >12 hours. With the exception of 1 patient with lower carbovir triphosphate levels at 3 time points, patients had intracellular carbovir triphosphate levels greater than 100 fmol/106 cells (>200 nM) over the entire 24-hour period.
Using a hollow-fiber pharmacodynamic model system, Drusano et al examined ABC dosing and ascertained the impact of the administration schedule on the activity of the drug. The antiviral effect and relationship to pharmacokinetics of a continuous infusion of ABC over 24 hours were compared with those of once-daily and twice-daily dosing of ABC. All 3 dosing regimens produced similar ABC exposure and antiviral effect. The findings of this study indicate that it is the AUC that drives the antiviral effect of ABC and that once-daily administration of ABC will produce similar results.
Piliero et al studied the pharmacokinetics of ABC and the intracellular pharmacokinetics of carbovir triphosphate in blood and mononuclear cells from 20 patients who had been on a stable antiretroviral regimen containing ABC dosed twice daily (either as ABC or a triple combination of ABC + 3TC/ZDV twice daily) for at least 6 weeks. On the day of pharmacokinetic sampling, patients were given only 1 dose of ABC in the morning (the evening dose was not given); those on the triple-combination regimen were given combination 3TC/ZDV in the evening. Pharmacokinetic samples were obtained within 30 minutes prior to the morning dose and 2, 4, 8, 12, 16, and 24 hours after dosing. The observed ABC terminal half-life in plasma was 2.59 hours, which was modestly higher than the value at 1.5 hours previously reported. The carbovir triphosphate concentration time profile was a flat terminal curve from 8-12 hours through 24 hours. The mean carbovir triphosphate half-life was 20.6 hours. This prolonged carbovir triphosphate half-life is much longer than that previously observed in in vitro studies (3.3 hours) and may be due to a newer assay that directly measures carbovir triphosphate or due to a saturation step and pooling of precursors (carbovir monophosphate or carbovir diphosphate). The mean steady state Cmax,ss, from time 0 to last measurable concentration, and 24-hour concentration were 29.7, 21.1, and 14.9 fmol/106 cells, respectively. The intracellular carbovir triphosphate concentrations observed in this study were approximately 2-fold higher than the reported Ki value for dGTP into DNA by HIV-1 reverse transcriptase of 21 nM throughout the 24-hour interval, with concentrations of approximately 40 nM. The investigators concluded from these results that once-daily dosing of ABC is a viable option.
Drug-drug interaction studies with TDF and ddI, described below, have shown that TDF increases ddI exposure; however, there appears to be no such interaction between TDF and ABC. In a pilot study conducted by Kearney et al, 8 healthy subjects received a single 300-mg dose of ABC alone and following multiple dosing of TDF at steady state to maximize the interaction potential with ABC. The results of the study indicate that compared with historical pharmacokinetic data for the 2 drugs, the plasma pharmacokinetics of ABC were not significantly affected by TDF, nor were the pharmacokinetics of TDF affected by a single dose of ABC. Hawkins et al40 studied interactions between ABC and TDF in vivo. Cells were collected from 15 HIV-infected patients receiving TDF, ABC, and a third NRTI. Median intracellular concentrations of the phosphorylated forms of ABC and TDF were similar at baseline (141 and 87.2 fmol/106 cells, respectively) compared with day 28 (120 and 89.6 fmol/106 cells, respectively). The authors concluded that these data in patients indicate no intracellular interaction between TDF and ABC.
Clinical trial data on ABC as a once-daily drug are accumulating. Data from one such study were reported in a presentation at the 1st IAS Conference on HIV Pathogenesis and Treatment in 2001. Researchers from Thailand reported the results of a randomized 3-arm pilot study of once-daily ABC plus 3TC in 151 treatment-naive patients. The 3 arms were ZDV, 3TC, and ABC, each in standard twice-daily doses; 3TC 300 mg once daily plus ZDV and ABC, both 300 mg twice daily; and ABC 600 mg once daily plus ZDV 300 mg and 3TC 150 mg, both twice daily. The 48-week results from this study indicate equivalent antiviral activity for the 3 treatment arms, which suggests that the use of once-daily dosing of 3TC and ABC is equal in efficacy to twice-daily dosing of these drugs.41 More recent studies have provided further evidence that ABC once daily, when combined with 3TC and EFV, is efficacious and equivalent to twice-daily dosing in achieving virologic suppression (see article by Ruane and DeJesus in this issue).
Adenine Analogues

ddI, a structural analogue of the purine nucleoside inosine, is 2¢,3¢-dideoxinosine. The drug is available as buffered tablets, as delayed-release enteric-coated (EC) capsules, as a powder, and as a pediatric suspension. Originally, one of the major limitations of ddI was its poor solubility at low pH values. This problem was overcome by the formulation of a buffered tablet for clinical use. The oral bioavailability of the drug was found to be dose dependent: bioavailability is 27% for once-daily administration, compared with 36% for twice-daily administration. The volume of distribution of ddI is approximately 1 L/kg, and the serum elimination half-life ranges from 0.6-2.8 hours.
Intracellularly, ddI is converted to dideoxyinosine monophosphate by a cytosolic 5¢ nucleotidase. Dideoxyinosine monophosphate is then converted to dideoxyadenosine monophosphate by adenylate synthetase and adenylate lyase, followed by further phosphorylation to the diphosphate by adenylate kinase and, possibly, other enzymes. Adenylate kinase also catalyzes conversion of dideoxyadenosine diphosphate to 2¢3¢-dideoxyadenosine 5¢-triphosphate. 2¢,3¢-dideoxyadenosine-5¢triphosphate competes with deoxyadenosine triphosphate for the active binding site on HIV reverse transcriptase. Its incorporation into viral DNA halts viral chain elongation and terminates viral replication owing to the absence of a 3¢-hydroxyl group, which is present in the natural substrate and is required for nucleic acid replication and DNA elongation. 2¢3¢-dideoxyadenosine-5¢-triphosphate has an intracellular half-life o 8-40 hours, which is the rationale for once-daily dosing of ddI.
Clinical support for once-daily dosing was provided by a pharmacokinetic study from the Netherlands. In a randomized, crossover study, Hoetelmans et al evaluated the plasma pharmacokinetics of ddI during once-daily or twice-daily dosing for 7 days, followed by 7 days of the other dosing regimen, in 19 HIV-infected patients on ddI-containing HAART regimens. The total daily doses of ddI in both regimens were identical. The investigators found no significant differences between the 2 regimens in any of the pharmacokinetic parameters measured. The authors concluded that once-daily dosing of ddI leads to exposure in plasma similar to twice-daily dosing and that the results of this study supported once-daily administration of this drug.
Additional support for antiretroviral combinations that include once-daily therapy with ddI has been provided by the results of a study by Maggiolo et al, who compared a once-a-day HAART regimen with 2 conventional twice-a-day regimens. A total of 102 antiretroviral-naive patients were randomly assigned to receive either once-a-day treatment with EFV, ddI, and 3TC; a low pill burden regimen of EFV once daily plus 3TC/ZDV twice daily, or a high pill burden regimen of NFV twice daily plus 3TC/ZDV twice daily. The results of the study indicated that once-a-day HAART with ddI + 3TC + EFV is an effective alternative to twice-a-day regimens.
In a study designed to evaluate a potential interaction between ddI and TDF, healthy subjects received ddI (buffered or EC) 400 mg once daily and TDF 300 mg once daily. Although simultaneous administration of TDF and ddI (buffered or EC) did not affect TDF pharmacokinetics, the AUC increased by 40% for buffered ddI and by 48% for ddI EC.47,48 Robbins et al49 conducted a study to determine whether TDF and ddI had a similar intracellular interaction in human PBMCs. Phosphorylation of 2 uM and 20 uM of ddI to dideoxyadenosine triphosphate was determined in resting and stimulated PBMCs in the presence or absence of 5 uM of TDF. Similarly, phosphorylation of 5 uM of TDF to TDF diphosphate was examined in the presence or absence of 2 uM and 20 uM of ddI. Tenofovir had no effect on intracellular levels of dideoxyadenosine triphosphate in quiescent or stimulated PBMCs with 2uM or 20 uM of ddI, nor did ddI alter levels of TDF diphosphate. Thus, no significant interactions were detected between TDF and ddI in human PBMCs as determined by the extent of formation of the phosphorylated anabolites. The results of the pharmacokinetic studies demonstrating increased plasma concentrations of ddI when the drug was given with TDF suggest that the combination increases ddI absorption. The lack of an apparent interaction between TDF and ddI in PBMCs of healthy humans in vitro at clinically relevant concentrations suggests that the altered processes for transport or metabolism that explain the systemic interaction are distinct from the processes that govern transport and metabolism in PBMCs. Thus, if the systemic interaction is the result of an alteration in the transport of ddI in the gastrointestinal tract or the inhibition of metabolism, then the responsible proteins must not be coexpressed in human PBMCs and gastrointestinal epithelial cells.
Tenofovir DF

TDF, a prodrug of tenofovir, was synthesized to enhance oral absorption and to improve cellular uptake of the drug. The chemical name of TDF is 9-[(R))-2-[[bis[[(isopropoxycarbonyl)oxy]methoxy]phosphinyl]methoxy]propyl]adenine fumarate (1:1). It is the first NtRTI approved for treatment of HIV infection. In vivo, TDF is hydrolyzed to tenofovir, an acyclic nucleoside phosphonate (nucleotide) analogue of adenosine 5¢-monophosphate, which is then phosphorylated to tenofovir diphosphate, the pharmacologically active metabolite. Tenofovir diphosphate inhibits HIV reverse transcriptase by competing with endogenous deoxyadenosine 5¢-diphosphate for incorporation in viral DNA.50,51 Unlike the NRTIs, which must undergo 3 phosphorylation steps for activation, tenofovir only needs to be converted to the diphosphate, which is the active form of the drug. As a result of the decreased phosphorylation requirement, tenofovir diphosphate is active in both dividing (activated) and nondividing (resting) cells.
In a phase I/II study, Barditch-Crovo et al53 assessed the pharmacokinetics of tenofovir in 49 HIV-infected patients who received oral TDF, in doses ranging from 75-600 mg/d, or placebo on day 1 and days 8-35. After a single oral dose, the median Cmax of TDF was proportional to the dose administered. The median steady-state Cmax and AUC were 326 ng/mL and 3020 ng · h/mL, respectively, in patients infected with HIV who received TDF 300 mg/d with food for 28 days. For the 300-mg and 600-mg dose cohorts, after Cmax was achieved, tenofovir concentrations in serum declined in a biphasic manner, with terminal half-lives between 12 and 15 hours.
The use of TDF once daily as part of a triple-combination regimen has been studied in several clinical trials (see article by Ruane and DeJesus in this issue). Long-term clinical data support the use of TDF in combination with 3TC and EFV in treatment-naive patients.54 This combination showed comparable efficacy and a superior safety profile compared with the combination of d4T + 3TC + EFV. Tenofovir DF has also shown efficacy when combined with FTC and LPV/r in treatment-naive patients.55 Studies of triple-nucleoside regimens containing TDF and 3TC with either ABC or ddI have shown poor efficacy and these combinations should be avoided.
The simplification of HAART regimens has been a high priority for many years. As the number of effective drugs increases, so does the number of possible effective regimens. The trend toward fixed-dose combinations and once-daily dosage forms of many antiretroviral drugs has provided welcome relief to patients. Not only is their medication burden simplified, but, as a consequence of improved adherence to therapy, they should experience better control of HIV and thus reduced morbidity. The search for new, more effective drugs with pharmacokinetic properties that permit once-daily dosing continues and should contribute to the improved outlook for HIV-infected patients.
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