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	The Pharmacology of Antiretroviral Nucleoside and Nucleotide Reverse Transcriptase Inhibitors: Implications for Once-Daily Dosing         JAIDS Journal of Acquired Immune Deficiency Syndromes Supplement 1 1 August 2005   Back, David J PhD-; Burger, David M PharmD, PhD; Flexner, Charles W MD; Gerber, John G MD§   From the -Department of Pharmacology, University of Liverpool, Liverpool, United Kingdom; Department of Clinical Pharmacology, University Medical Centre Nijmegen, Nijmegen, The Netherlands; Departments of Medicine, Pharmacology, Molecular Sciences, and International Health, The Johns Hopkins University School of Medicine, Bloomberg School of Public Health, Baltimore, MD; and §Departments of Medicine and Pharmacology, University of Colorado Health Sciences Center, Denver, CO.   This independent continuing medical education activity was supported by an educational grant from Gilead Sciences, Inc.   Summary: The trend toward once-daily dosing in HIV antiretroviral therapy is based on the association between adherence, treatment outcome, and patient preferences. Patients prefer simpler treatments, fewer pills, less frequent dosing, and no food restrictions. When a regimen meets a patient's preferences, the patient is more likely to be adherent, and with good adherence, the regimen is more likely to be effective.   Nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) have been a prime focus for developing once-daily therapies primarily because they form the backbone of most current regimens. Within the NRTI class, however, drugs differ in their pharmacokinetic properties, such as plasma and intracellular half-lives, and thus in their suitability for once-daily dosing.   For example, newer NRTIs, such as tenofovir and emtricitabine, combine longer plasma half-lives with longer intracellular half-lives, prolonging exposure and the period of pharmacologic activity. Of equal importance, the clinical impact of systemic and intracellular interactions between concomitant drugs defines which once-daily drugs may be combined in once-daily regimens.   To construct simplified and effective therapies for individual patients, clinicians require an understanding of the plasma and intracellular pharmacokinetic properties of NRTIs and how these properties determine a drug's appropriateness for once-daily dosing and placement within a once-daily regimen.   ARTICLE TEXT Successful long-term control of HIV replication requires a high degree of adherence to the dosing schedule, such that approaching 100% of doses are taken on time. A recent survey revealed that HIV-positive patients receiving antiretroviral therapy (ART) reported that they would prefer an antiretroviral combination that could be taken once a day, does not involve a large number of pills, and is without dietary requirements.1 This is supported by a meta-analysis of virologic outcome data from clinical trials of various ART regimens that found a significant correlation between lower pill burden and treatment efficacy.2   The pharmaceutic industry is alert to patient preference and its implication on adherence, and therefore has recognized compact once-daily dosing as an important feature of new drug development. With the exception of enfuvirtide, only once-daily anti-HIV drugs have been launched since 2001. Notably, in August 2004, the US Food and Drug Administration (FDA) approved 2 coformulated nucleoside analogue combinations-abacavir with lamivudine and tenofovir with emtricitabine-intended for once-daily administration. In addition, a coformulation of tenofovir, emtricitabine, and efavirenz in a single once-daily pill is in development. This clear emphasis on once-daily drugs is indicative of a trend in ART to simplify treatment regimens by developing drugs and drug combinations that can be taken once daily in an effort to improve not only adherence but treatment outcome.   Despite the emphasis on once-daily drugs, it is not a given that every existing antiretroviral can be rereleased in a once-daily form or that every newly developed antiretroviral can routinely be labeled as a once-daily drug. The rationale for dosing some antiretroviral drugs every 24 hours is based on each drug's pharmacokinetic properties. For example, a drug must have a half-life sufficiently long to ensure against the development of resistance during trough periods or the occasional missed dose. A drug's half-life must also be similar to that of other once-daily drugs so as to be matched within a symmetric regimen. Not all drugs have these or other appropriate pharmacokinetic properties. Every drug is unique.   Three approaches have been used to develop once-daily drugs. The first approach is to develop a new drug whose molecule inherently has the right pharmacokinetic parameters. Examples of this approach are efavirenz and tenofovir. The second approach is to adapt the dose of a twice-daily formulation to achieve once-daily pharmacokinetics. An example of this type of once-daily drug is a protease inhibitor (PI) boosted with ritonavir to enhance its pharmacokinetics, such as lopinavir boosted with ritonavir. The final approach is to adapt the actual formulation of a twice-daily drug to achieve once-daily pharmacokinetics by altering the absorption kinetics, for example, stavudine-extended release (XR; FDA approved but not yet available).   Given the achievement of a once-daily drug by any of these means, the drug can be used only if it can be combined with other appropriate drugs without harmful interaction. Furthermore, the value of its once-daily pharmacokinetics can be fully appreciated only if the drug can be combined with other once-daily drugs in a purely once-daily regimen.   As a class, nucleoside reverse transcriptase inhibitors (NRTIs), which include nucleosides and nucleotides, have been a prime focus for developing once-daily therapies, primarily because they form the backbone of virtually all currently used regimens. Within the NRTI class, however, drugs differ in their pharmacokinetic properties, and thus in their suitability for once-daily dosing.   To offer patients the benefit of once-daily drugs and regimens, clinicians must understand the plasma and intracellular pharmacokinetic properties of antiretroviral drugs and how these properties determine a drug's appropriateness for once-daily dosing and place within a once-daily regimen. This understanding is the foundation on which clinicians can construct simplified and effective therapies for individual patients.   IMPORTANCE OF ADHERENCE   - Better adherence is associated with better therapeutic outcome.   - For optimal adherence and patient satisfaction, less frequent dosing is usually better.   - Low pill burden per dose and absence of food restrictions also improve adherence.   - The degree of adherence required for optimal response to once-daily drugs is not yet known and likely varies for different classes of drugs.   - When selecting a once-daily regimen for a particular patient, the clinician must take into account factors that would support optimal adherence within that patient's lifestyle.   Adherence is the primary reason behind the enthusiasm for once-daily dosing among patients and clinicians. Adherence to therapy is significant because of its strong association with successful therapy.3   To facilitate adherence, a treatment regimen should meet patient preferences as closely as possible, although still retaining efficacy. When patients are asked to name the components of an ideal treatment regimen, their responses speak to simplicity and ease: low pill count, low frequency (preferably once daily), no food restrictions, and no consequence for the occasional missed dose (ie, forgiveness). These responses suggest that patients want a treatment regimen that fits into their lifestyle. Findings from a multi-instrument survey support that conclusion: 73% of HIV-infected patients (n = 536) claimed that a once-daily regimen was more compatible with their lifestyle than a twice-daily regimen (P < 0.001).4   In another survey, Moyle1 asked 504 HIV-infected patients across 5 large European countries to identify their preferred regimen characteristics. Their responses showed a preference for once-daily regimens and low pill burdens (Fig. 1). With a total of 3 pills per day, 93% of patients preferred a once-daily regimen compared with 7% who preferred a twice-daily regimen. Likewise, with a total of 4 pills per day, the preference for once-daily compared with twice-daily dosing was 84% versus 16%. With a total of 8 or more pills per day, however, the preference shifted in favor of twice-daily dosing.   Although patients may prefer a simple low-frequency and/or low-pill-burden regimen, is there evidence that this sort of regimen increases adherence? Published studies in HIV therapy as well as in other therapeutic areas provide answers to that question.   In a meta-analysis of adherence in a variety of diseases, adherence was found to be negatively associated with dose frequency. Claxton et al5 identified 76 studies across a range of therapeutic areas that measured adherence via electronic monitoring. The mean dose-taking adherence (ie, the prescribed number of doses that were taken each day) increased as the number of daily doses decreased. In addition, the mean dose-timing adherence (ie, the doses taken within the prescribed time intervals) in the 14-study subset that measured dose timing showed increased adherence as the number of daily doses decreased.   Within HIV therapy, 2 studies evaluated regimen preference and associated adherence: CNA3014 and AI455135. Vibhagool et al6 (CNA3014) conducted a 48-week, randomized, open-label study of efficacy, safety, and adherence among 342 treatment-naive HIV-infected patients receiving abacavir twice-daily plus lamivudine/zidovudine twice daily versus indinavir thrice daily plus lamivudine/zidovudine twice daily. In a subgroup of this study (n = 143), Jordan et al7 measured the satisfaction associated with these 2 regimens through patient self-completion of a 9-item HIV Treatment Satisfaction Questionnaire. Self-reported adherence exceeded 95% in 72% of patients receiving the twice-daily therapy compared with 46% of those receiving thrice-daily therapy (P < 0.001). Furthermore, the only covariates associated with >95% adherence were satisfaction and less frequent therapy.8   In AI455135, Boyle et al9 compared adherence and satisfaction among treatment-experienced HIV-infected patients (n = 300) receiving a once-daily versus twice-daily regimen in a 48-week, open-label, randomized trial. Patients were randomized 2:1 to once-daily stavudine-XR/lamivudine/efavirenz or a continued standard of care (SOC) regimen that was at least as frequent as twice daily. At 12 weeks, dose-taking and dose-timing adherence (measured by electronic monitoring) was significantly greater in the once-daily group: 96% versus 83%, respectively, for dose-taking adherence (P < 0.001) and 86% versus 55%, respectively, for dose-timing adherence.   Once-daily and twice-daily dosing of ART was compared in a study recently reported by Arribas et al.10 Using a validated questionnaire and a visual analog scale, adherence and satisfaction, respectively, were measured in 978 patients. Effectiveness was measured by viral load testing. Adherence and satisfaction were significantly better in patients receiving once-daily dosing compared with patients receiving twice-daily dosing. No significant differences in percentages of patients achieving a nondetectable viral load were seen in the once-daily group versus the twice-daily group.   A study by Bartlett et al2 correlated the antiretroviral pill burden with durability of virologic response using data from a meta-analysis of clinical trials in which treatment-naive HIV-infected patients received triple ART.2 At week 48, pill count was significantly negatively associated with plasma HIV RNA 50 copies/mL (P = 0.0085).   Adherence and Once-Daily Dosing: Issues to Consider   With evidence that simpler regimens are not only preferred by patients but are associated with improved adherence and, subsequently, outcome, the advantage of once-daily regimens seems to be established. The evidence, however, should be considered with the following caveats.   Established adherence thresholds may not apply to once-daily regimens. Adherence is a marker for ongoing presence of an antiviral agent in the blood. If a person is adherent (ie, taking drugs as prescribed), it is generally assumed that plasma and intracellular levels of the drug are at a sufficient level for viral suppression (although there is always the caveat of marked interpatient variability). A historical >95% target for adherence was established for use with older regimens, however, before the availability of newer or once-daily drugs or regimens.11 Applying the 95% threshold of adherence to once-daily dosing, patients could miss no more than 1 day each month at the maximum to remain adherent. This is a high expectation for any patient and one that may not have to be placed on patients receiving once-daily regimens. The pharmacokinetics of newer less frequently dosed drugs are more forgiving, and therefore may require adherence <95%. No data are yet available on this issue, however.   Not every drug or drug regimen can be adapted to once-daily dosing, despite the adherence advantage. Pharmacokinetic characteristics of an individual drug must ensure that there is a sufficient trough-level concentration (Ctrough) of the drug to protect against developing viral replication, and thus drug resistance. Furthermore, the forgiveness of the drug must be suited to once-daily dosing, allowing minimal compromise to efficacy by the occasional missed dose. Not every drug can meet these criteria.   The impact of total pill burden at a single dose is unknown. The survey conducted by Moyle1 showed that patients preferred to take no more than 6 pills at a single time. More data are needed to realize the full impact of this factor on once-daily dosing and adherence, however.   Food restrictions in relation to the administration of a particular drug affect adherence. Drugs that may be taken without regard to food are more easily integrated into daily life compared with drugs that must be taken with food or drugs that must be taken without food. In addition, dissimilar food restrictions of once-daily drugs within the same regimen make the regimen more complex and may compromise adherence.   Summary   For optimal adherence, once-daily dosing is the best dosing regimen possible with currently available agents. Selection of agents within a once-daily regimen, however, requires knowledge of the pharmacokinetics of the drugs and their suitability for once-daily combinations.   OVERVIEW OF NUCLEOSIDE REVERSE TRANSCRIPTASE INHIBITOR DISPOSITION Key Messages   - Equivalency between dosing frequencies (ie, once-daily vs. twice-daily dosing) is evaluated by comparing pharmacokinetic parameters, such as area under the curve (AUC), minimum concentration (Cmin), and maximum concentration (Cmax).   - It remains difficult to assess the relations between plasma pharmacokinetics, intracellular pharmacokinetics, and antiviral dynamics. Understanding these relationships, however, is important in designing regimens with optimal efficacy.   - Newer NRTIs combine longer plasma half-lives with longer intracellular half-lives and have the advantage of prolonged exposure, thereby reducing the consequence of an occasional missed dose. A long plasma half-life acts as a reservoir for uptake into the cell. In addition, a long intracellular half-life causes a postexposure effect after there is no longer drug in the plasma.   - Clinicians can use half-life data to inform their decisions on once-daily drugs.   - Symmetric drug combinations are preferable, with drugs of similar elimination half-lives (T1/2 ) combined within a single regimen. When a patient needs to discontinue the regimen, all drugs disappear from the plasma at the same rate, thus preventing periods of mono- or dual therapy.   Defining the plasma and intracellular pharmacokinetic properties of individual NRTIs is the basis for understanding the once-daily capability of each NRTI. These properties help to prove equivalence of exposure when a once-daily dosing regimen is compared with an SOC dosing regimen, identify drug-drug interactions, and establish thresholds of potency.   Overview of Plasma Pharmacokinetics of Nucleoside Reverse Transcriptase Inhibitors   NRTIs share a common class definition and intracellular mode of action against HIV-1 reverse transcriptase as well as some similar pharmacokinetic characteristics. Pharmacokinetic differences do exist within the class, however. Each NRTI thus must be considered a unique drug with a unique pharmacokinetic profile.   Influence of Food on Absorption   All NRTIs, with the exception of didanosine, may be taken without consideration of food (Table 1).12-19 This lack of food restrictions increases their simplicity of use for the patient. For the clinician, it more readily facilitates combination of NRTIs into once-daily regimens. Didanosine should be taken in the fasted state unless it is combined with tenofovir. When given as part of a tenofovir-containing regimen, however, the enteric-coated (EC) formulation of didanosine may be given with or without food and a dose reduction made (more detailed discussion to follow).   Pharmacokinetic Parameters   In general, NRTIs have a relatively high bioavailability (F), usually >80% (Table 2).12-19 The exceptions are zidovudine, didanosine, and tenofovir. Tenofovir's relatively low bioavailability is caused by the fact that it is administered as the prodrug tenofovir disoproxil fumarate, a fatty acid conjugate. Once the prodrug has reached the circulation, tenofovir is released. By definition, however, the bioavailability of tenofovir is calculated using the oral disoproxil prodrug as the reference. Drugs with low bioavailability often have a large variability in bioavailability, leading to variable plasma levels. As such, high bioavailability is a positive pharmacokinetic parameter. Absorption is fast for all NRTIs, with time to maximum concentration (Tmax) in the plasma occurring within 1 to 2 hours after administration. Rapid absorption leads to high peak levels, which may be associated with drug toxicity.   The T1/2, or plasma half-life, is a key area of difference among the NRTIs (see Table 2).12-19 For the older nucleosides, the T1/2 is relatively short. This rapid elimination was certainly an important factor in relation to the frequent dosing associated with early regimens. Newer NRTIs, such as emtricitabine and tenofovir, have significantly longer plasma half-lives (10 and 17 hours, respectively), thus offering the potential for less frequent dosing.   A drug's half-life not only helps to determine frequency of dosing but helps to determine drug symmetry, an important pharmacokinetic principle in drug combinations. Drugs with similar half-lives are considered symmetric. They may be administered at the same or equivalent intervals; likewise, they are eliminated within the same or equivalent intervals. A regimen containing drugs without symmetry may be problematic when the regimen is stopped. This is discussed in further detail in the last section of this article.   Nucleosides, in general, are not highly bound to proteins in the plasma (see Table 2).11-18 All but abacavir, lamivudine, and zidovudine are >90% unbound to protein (Fu). Low protein binding results in the absence of drug-drug interactions based on protein binding displacement. With the exception of abacavir, most NRTIs have a significant fraction that is excreted unchanged in the urine (Fe): 30% to 70% is typical, with 86% for emtricitabine. Drugs that are significantly excreted by the kidney require dose adjustment in patients with renal dysfunction (please refer to the prescribing information for the individual products).   Defining the Relation Between Plasma and Intracellular Pharmacokinetics   NRTIs are anabolized intracellularly by sequential phosphate additions. This process of phosphorylation yields the active drug moiety, which is the phosphorylated form of the parent drug. Because the phosphorylated form is the active form, the level of the intracellular drug triphosphate is more clinically relevant than the plasma level of the parent drug. Unfortunately, determination of intracellular active drug triphosphate concentrations is difficult to perform and not clinically practical. Therefore, the establishment of a relation between drug levels in both compartments (plasma and intracellular) could provide justification for using plasma drug levels, which are relatively simple to perform, as a surrogate marker for intracellular drug levels.   Clinicians generally agree that monitoring NRTI plasma levels is not currently indicated as a surrogate marker for intracellular levels, because there is an inconsistent relation between plasma levels of the NRTI and the intracellular level of the phosphorylated drug. Despite the lack of a consistent relation, however, it is reasonable to assume that some degree of relation exists between plasma and intracellular levels and that an as yet undefined relation must be explored and considered when evaluating once-daily dosing.   Technical Challenge of Measuring Intracellular Pharmacokinetics   A major challenge in establishing this relation is the lack of adequate technical capacity for measuring intracellular levels. The technical challenge of these measurements has limited their performance to only a few active research groups in the world, and there is no validation of results across laboratories. The technical challenges have also limited studies using these measurements to only a few data points per patient. In addition, questions remain regarding differences between sample sources: cell cultures, healthy volunteers, and HIV-infected patients. Given the accepted difficulty of performing intracellular measurements and the limited data available on the intracellular phosphorylated NRTI, it is difficult to judge the reliability of intracellular pharmacokinetic data. The technical challenge is compounded by the fact that intracellular data for a single NRTI cannot be extrapolated to all other NRTIs. All NRTIs are uniquely metabolized intracellularly, and only well-designed pharmacokinetic studies using intracellular measurement technology for a specific NRTI can define each NRTI's intracellular pharmacokinetic parameters.   These factors offer a basis for understanding the great variability between intracellular pharmacokinetic data measured in different laboratories as well as between in vitro and in vivo measurements. It is important for clinicians to be aware of these technical challenges so as to properly evaluate appropriate study data.   Limited Evidence for Relation Between Plasma and Intracellular Pharmacokinetics   The plasma concentration of a drug serves as a reservoir for intracellular uptake and phosphorylation. Although the plasma and intracellular compartments are separate, it is reasonable to expect that one affects the other. Evidence to prove this, however, is limited and seems to vary according to the specific drug.   Anderson et al21 demonstrated a relation between plasma and intracellular lamivudine levels. In a study of 33 treatment-naive HIV-infected patients receiving zidovudine/lamivudine/indinavir, a direct linear correlation was found between the concentration of lamivudine in the plasma and lamivudine-triphosphate (TP) inside the cell. The relation was independent of time after dose. In the same article, however, there was no such correspondence between plasma and intracellular zidovudine levels. In contrast, early reports from the NRTI monotherapy era indicated weak plasma/intracellular relations for zidovudine and didanosine.22   Weller et al23 demonstrated an association between plasma abacavir exposure and its antiviral effect (an intracellular activity) using pharmacodynamic modeling. The AUC and Cmax significantly correlated with a change in HIV RNA from baseline to the end of a 12-week monotherapy period (P < 0.05).   A relation between plasma and intracellular pharmacokinetics also may be inferred from the fact that increased plasma exposure, attributable to a drug-drug interaction, may produce clinically relevant toxicity within the cells and tissues. An example is the intracellular bone marrow toxicity that results from increased plasma exposure to zidovudine.   Establishing Equivalence Between Once-Daily and More Frequent Dosing   Although pharmacokinetic findings from a study of a single drug cannot be extrapolated to other NRTIs, pharmacokinetic studies of once-daily lamivudine, zidovudine, and abacavir illustrate the pharmacokinetic principles involved in assessing plasma and intracellular equivalence of once-daily dosing compared with twice-daily dosing and in determining the appropriateness of a drug for once-daily dosing.   Lamivudine: Once-Daily Versus Twice-Daily Dosing   The steady-state plasma pharmacokinetics of 150 mg of lamivudine administered twice daily and 300 mg of lamivudine administered once daily were studied by Bruno et al24 in a nonblind sequential pharmacokinetic study enrolling 13 HIV-infected patients. The plasma pharmacokinetic profile was assessed first over a 12-hour period after 7 days of twice-daily dosing. The following day, once-daily dosing was begun, and the pharmacokinetic profile was again assessed after 7 days but over a 24-hour period. The differences between the 2 regimens in terms of mean T1/2, average steady-state concentration (Cavg), and AUC were not statistically significant. In contrast, the differences between plasma concentrations at peak and trough levels (Cmax and Cmin, respectively) were statistically significant. The Cmax was 75% higher and the Cmin was 50% lower with once-daily dosing compared with twice-daily dosing. Despite the differences in Cmax and Cmin, the equivalence in AUC between the 2 dosing frequencies demonstrates that once-daily and twice-daily dosing of lamivudine produces equivalent exposure in the plasma.   In a study of 60 healthy volunteers, Yuen et al25 compared the steady-state pharmacokinetics of lamivudine in plasma with its intracellular active anabolite, lamivudine-TP. Lamivudine-TP was measured in peripheral blood mononuclear cells (PBMCs) after 7 days of 300-mg once-daily dosing and again after 7 days of 150-mg twice-daily dosing. The plasma pharmacokinetic findings were similar to those of Bruno et al.24 The differences between plasma concentrations at peak and trough levels were statistically significant, with a 66% higher Cmax and a 53% lower Cmin with once-daily dosing compared with twice-daily dosing. The Cavg and AUC were equivalent, however, and the T1/2 was not determined. Intracellular pharmacokinetics of lamivudine-TP showed equivalence in terms of AUC, Cmax, and Cavg; however, there was a large interpatient variation in the AUC.25 Trough levels were 18% to 24% lower with once-daily dosing compared with twice-daily dosing but remained within the range of antiviral efficacy. The intracellular half-life was 18.4 hours, consistent with once-daily dosing. Although the correlation between plasma and intracellular NRTI levels remains unclear, these findings are consistent with those of Anderson et al21 regarding lamivudine.   Interpretation of Lamivudine Study Results   The studies by Bruno et al24 and Yuen et al25 show higher plasma Cmax and lower Cmin with the once-daily regimen compared with the twice-daily regimen. It is difficult to know the clinical implication of this lower plasma Cmin, because the antiviral activity of an NRTI occurs inside the cell. The consideration that the drug in plasma acts as a reservoir for intracellular uptake, however, would suggest that a lower Cmin would offer less drug uptake into the cells, less phosphorylation, and thus less active drug. Yet, AUC data as well as data showing the drug concentration needed to inhibit viral replication by 90% (IC90) suggest that this is not the case. Bruno et al24 and Yuen et al25 showed equivalence between once-daily and twice-daily dosing in terms of plasma exposure (AUC). Therefore, it could be expected that the total intracellular exposure would also be the same, resulting in equivalent phosphorylation and an equivalent amount of active drug. In fact, the study by Yuen et al,25 the only study to measure intracellular concentrations, did demonstrate an intracellular equivalence of exposure between the 2 dosing regimens. In addition, although the once-daily plasma Cmin levels in both studies were below those of the twice-daily regimen, the levels are within the previously reported IC90 range of 0.01 to 0.46 mg/L.26 The significance of the lower plasma Cmin and whether the higher plasma Cmax can compensate for this remain unknown.   Although the findings of Bruno et al24 and Yuen et al25 were qualitatively similar in terms of the relation of once-daily versus twice-daily dosing, there were significant quantitative differences in the data. The total exposure of lamivudine was 2-fold higher in the 12 HIV-infected patients studied by Bruno et al24 compared with the 60 healthy volunteers studied by Yuen et al25 for once-daily and twice-daily dosing. The reason for this difference is unknown, but it may be attributable to differences in drug pharmacokinetics between HIV-infected and healthy individuals. On average, HIV-infected patients have a greater degree of peripheral lymphocyte activation than do healthy volunteers. Turriziani et al27 reported significantly higher and more variable levels of the nucleoside activator thymidine kinase in people with HIV infection than in healthy volunteers, regardless of zidovudine or lamivudine substrate.   This difference in activation provides a rationale for the difference in the pharmacology of a drug in an HIV-infected patient compared with a healthy volunteer. If increased activation results in a greater level of phosphorylation, a greater level of phosphorylated active NRTI might be expected in an HIV-infected patient than in a healthy volunteer. The result may be advantageous in that it might mean a greater anti-HIV effect. It may, however, also be disadvantageous in that it might mean greater toxicity if the increased activation extends to all body cells. There are no data addressing the potential benefit or risk of increased activation.   Zidovudine: Once-Daily versus Twice-Daily Dosing   Ruane et al28 compared the pharmacokinetics and viral efficacy of once-daily zidovudine (600 mg) and twice-daily zidovudine (300 mg) in an open-label study enrolling 32 treatment-naive HIV-infected patients. At day 14, daily measurements of HIV RNA showed that the group receiving the once-daily regimen had a lower mean reduction in HIV RNA from baseline compared with the group receiving the twice-daily regimen: -0.59 log10 copies/mL versus -0.85 log10 copies/mL. The viral load reduction was also slower in the once-daily group. The intracellular AUC and Cmax of zidovudine-TP at steady state were 41% and 16% lower, respectively, in the once-daily regimen compared with the twice-daily regimen (GlaxoSmithKline, data on file).   Contrasting these findings with those of the lamivudine study conducted by Yuen et al25 again highlights the uniqueness of each NRTI. Unlike lamivudine, zidovudine taken once daily did not produce an intracellular AUC or Cmax equivalent to twice daily. This finding, in addition to the inferior antiviral effect, precludes zidovudine from being considered a once-daily drug. A pharmacokinetic rationale for the finding may point to the uniqueness of zidovudine's phosphorylation pathway, in which the phosphorylation of the monophosphate to the diphosphate is the rate-limiting step. Consequently, if the cell is exposed to a higher dose of zidovudine at a single time, it could prevent formation of the active triphosphate form. The experience with zidovudine once-daily dosing underscores the importance of detailed knowledge of pharmacokinetic principles and the need for clinical studies to determine the possibility of once-daily dosing.   Abacavir: Once-Daily versus Twice-Daily Dosing   To date, there has been no comparative pharmacokinetic study of once-daily and twice-daily abacavir alone. The results of 2 studies evaluating levels of carbovir-TP (the intracellular active form of abacavir) support the use of once-daily abacavir in HIV-infected patients, however. Harris et al29 examined the pharmacokinetics of carbovir-TP over 24 hours in PBMCs from 5 patients receiving 600 mg of abacavir once daily. Study findings suggested an estimated intracellular half-life of greater than 12 hours, with carbovir-TP levels greater than 100 fmol/106 cells over the entire 24-hour period. Piliero et al30 also studied the pharmacokinetics of abacavir and intracellular carbovir-TP in PBMCs from 20 patients on a stable regimen containing twice-daily abacavir. On the day of the study, the evening dose was not given, thereby allowing 24-hour sampling after a dose. The mean carbovir-TP half-life in this study was determined to be 20.6 hours, supporting once-daily dosing as a viable option.   Evaluating Pharmacologic Activity of a Dosing Regimen   Usually, the plasma T1/2 is the pharmacokinetic parameter used to indicate the potential for once-daily dosing. This is a simplification, however, because plasma is not the compartment of action, and there are more factors at play. It thus may be more helpful to focus on the length of exposure (ie, whether a drug remains pharmacologically active for a prolonged period after dosing). The longer the drug is present in a pharmacologically active concentration, the more forgiving the drug is to the occasional missed dose.   The plasma and intracellular half-lives are factors in prolonging exposure.12,13,17-19,25,29-33 A long plasma half-life acts as a reservoir for uptake into the cell. A long intracellular half-life causes a postexposure effect after there is no longer drug in the plasma. Even a drug with a short plasma half-life, however, may be appropriate for once-daily dosing if it does not have dose-limiting toxicity. Plasma half-life is thus not categorically the measure of pharmacokinetic activity. Among the current once-daily drugs, tenofovir has the longest plasma and intracellular half-lives; therefore, it has the most prolonged exposure after a single dose.   Threshold of Potency   Anderson et al21 determined the intracellular concentration threshold for optimal activity of zidovudine-TP to be 30 fmol/106 cells and that of lamivudine-TP to be 7.0 pmol/106 cells, showing that patients who have intracellular levels of these 2 agents above these levels have a higher likelihood of achieving antiviral response. It should be noted, however, that these thresholds were not associated with a specific time point but were ascertained from averaged random samples. It is not known whether this lamivudine threshold applies to once-daily dosing.   Thresholds of potency may be quite difficult to ascribe to NRTIs in triple therapy that includes other drug classes. In these regimens, the NNRTI or PI may be the most active component with potential for antiviral synergy, obscuring the pharmacokinetic-pharmacodynamic relations of the NRTI.   Summary   Equivalency between once-daily dosing and more frequent dosing is evaluated by comparing pharmacokinetic parameters. Newer NRTIs, such as tenofovir and emtricitabine, combine longer plasma half-lives with longer intracellular half-lives; thus, they have the advantage of prolonged exposure. Clinicians can use half-life data to decide on dosing frequencies of drugs.   NUCLEOSIDE REVERSE TRANSCRIPTASE INHIBITOR-DRUG INTERACTIONS   There are only a limited number of pharmacokinetic drug interactions involving NRTIs, because most NRTIs are renally cleared, do not involve the CYP450 hepatic enzyme system, and are metabolized only to a limited extent. As a class, therefore, their potential of being involved in clinically significant pharmacokinetic interactions is lower than that of PIs and nonnucleoside reverse transcriptase inhibitors (NNRTIs).   Systemic Nucleoside Reverse Transcriptase Inhibitor Interactions Key Messages   - Lopinavir/ritonavir increases tenofovir exposure by approximately 30%. This is considered clinically unimportant, except in patients with impaired renal function.   - Tenofovir increases exposure to didanosine, resulting in potential didanosine-related clinical consequences. A dose reduction of didanosine, when coadministered with tenofovir, compensates for the increased exposure.   - Atazanavir should be boosted with ritonavir when used in a tenofovir-containing regimen, because tenofovir reduces atazanavir exposure.   - There are no known systemic drug interactions involving emtricitabine.   Most clinically significant drug interactions of NRTIs have been known for some time and do not require review (Bristol-Myers Squibb Company, data on file) (Table 5).19,34-53 For example, reports of systemic interactions between zidovudine and probenecid, rifampin, and valproic acid date back in the literature to 1989, 1993, and 1994, respectively. Recent drug interaction data focus primarily on tenofovir because it is the only new NRTI with known interactions. Tenofovir has been studied with 18 different agents, 16 of which had no clinically significant drug-drug interactions.54-56 Tenofovir-related interactions include the impact that lopinavir/ritonavir has on tenofovir, which is clinically irrelevant, as well as the impact that tenofovir has on didanosine and atazanavir. Clinicians should monitor renal function more frequently if the underlying glomerular filtration rate is reduced when using tenofovir with lopinavir/ritonavir. There are no described drug interactions with emtricitabine. Like lamivudine, this drug is mainly eliminated by the kidneys, and drug interaction studies with tenofovir, stavudine, famciclovir, and indinavir showed no significant change in emtricitabine exposure or exposure to the other drugs.12   Impact of Tenofovir on Atazanavir   Tenofovir coadministered with unboosted atazanavir (400 mg once daily) lowers atazanavir exposure by approximately 25%, requiring boosting of atazanavir. This was shown in a study of healthy volunteers receiving atazanavir, EC-didanosine, and tenofovir. A secondary objective of the study was to evaluate changes in the pharmacokinetics of tenofovir and atazanavir when the 2 drugs were coadministered (n = 36) (Bristol-Myers Squibb Company, data on file).39 Kaul et al39 found atazanavir exposure to be decreased when 400 mg of atazanavir is coadministered with 300 mg of tenofovir and food compared with atazanavir given alone. In some patients, the decrease in atazanavir Cmin secondary to tenofovir may result in a suboptimal atazanavir concentration, and thus virologic failure. There are no published data, however, that correlate the drug-drug interaction with virologic outcome.   When coadministering atazanavir and tenofovir, it is necessary to increase the exposure of atazanavir. Boosting a 300-mg dose of atazanavir with 100 mg of ritonavir seems to compensate for the negative impact of tenofovir (Bristol-Myers Squibb Company, data on file).53 Trough levels of atazanavir boosted with ritonavir (300 mg/100 mg) are 4 times higher than those of unboosted atazanavir (400 mg).57 Although the exposure of boosted atazanavir is also decreased by tenofovir, the greater exposure offered by the boosting renders the decrease clinically irrelevant when dealing with PI-susceptible virus infection.   A recent study by Kruse et al,57 however, does not confirm the decrease in atazanavir exposure by the presence of tenofovir. A total of 178 plasma samples were collected from people taking atazanavir with or without tenofovir, but the timing of the dose of atazanavir in relation to blood collection was not rigorous. Data showed similar trough levels of boosted atazanavir regardless of tenofovir coadministration. The Ctrough, however, varied substantially among these subjects. Despite these observations, most clinicians believe it prudent to give atazanavir only in the boosted form when using it in a tenofovir-containing regimen.   Impact of Tenofovir on Didanosine   Multiple studies have demonstrated the interaction between tenofovir and didanosine. Kearney et al42 found that the exposure to didanosine increases approximately 44% when the buffered tablet formulation of didanosine is given in the fasting state 1 hour before tenofovir (n = 30). A similar increase (48%) was observed in a subsequent study by Kearney et al41 when EC-didanosine was given in the fasting state 2 hours before tenofovir (n = 28). In the same study, EC-didanosine given simultaneously with food and tenofovir further increased the exposure (60%). Because increased exposure to didanosine can result in the development of pancreatitis and/or peripheral neuropathy, this degree of drug-drug interaction can be of concern.   Assessments of didanosine dose reduction instruct that when tenofovir is also included in the regimen, a lower dose of didanosine (250 mg) compensates for the increased exposure.17,40,43,58 There is debate among clinicians, however, whether patients weighing <60 kg should have an even greater dose reduction (ie, 200 mg). There are no data available yet on this issue. Recent early treatment failures associated with regimens containing tenofovir and didanosine (more detailed discussion to follow) suggest caution in using these 2 agents in combination.   The mechanism for this interaction is believed to involve purine phosphorylase, a key enzyme required for the metabolic breakdown of didanosine. Findings from enzymatic inhibition assays point to inhibition of this enzyme by the phosphorylated metabolites of tenofovir, resulting in increased circulating didanosine.51 This mechanism is discussed in greater detail in the section on intracellular NRTI interactions.   Cellular toxicity may also result from didanosine-tenofovir coadministration. A retrospective analysis showed a significant decrease in CD4, CD8, and total lymphocyte cell counts, despite an undetectable viral load, in patients receiving didanosine and tenofovir (n = 302).59 More than 50% of patients had a decrease of 100 CD4 cells at 48 weeks. Patients not receiving coadministered didanosine and tenofovir did not experience the decrease but instead had a steady increase in cell counts. Data from the study do not point to one drug or the other as the cause of the decrease. Patients who received only one or the other drug (but not both) did not experience the decline in cell counts. The mechanism for this toxicity is unclear, although a role for guanosine triphosphate has been put forward.60 The decrease in CD4 cell count suggests worsening immunodeficiency, but there are no clinical data to correlate with this laboratory observation.   Summary   Drug-drug interactions can result in an increase or decrease in exposure to the drugs. Understanding the therapeutic indices of the drugs in question and knowledge of the plasma concentration of the drug necessary for maximal efficacy are important determinants of the significance of these drug-drug interactions. For a drug with a high therapeutic index, like lamivudine, a 2-fold increase in exposure is not clinically significant, but for a drug with a low therapeutic index, like didanosine, even small increases in exposure can result in excess toxicity.   Intracellular Nucleoside Reverse Transcriptase Inhibitor Interactions Key Messages   - Intracellular interactions of clinical impact do occur within the NRTI class of antiviral agents.   - An interaction that decreases a drug's intracellular phosphorylation also decreases its antiviral activity. Such a decrease must be considered when making dosing decisions.   - Ribavirin and didanosine should not be coadministered because of didanosine-related mitochondrial toxicity.   - Although in vitro data suggest a decrease of antiviral activity when ribavirin and zidovudine are coadministered, data from clinical trials show no loss of efficacy.   NRTIs are in their active form only after phosphorylation-the process of adding sequential phosphate groups-inside the cell, as previously discussed. The level of phosphorylation is a key component in the therapeutic index of an NRTI. As seen in Figure 5, the probability of effect (y-axis), in terms of viral inhibition (dotted-curved line) and toxicity (solid-curved line), is a function of intracellular NRTI phosphorylation (x-axis).61 Therefore, factors that increase or decrease phosphorylation have a significant effect on the efficacy and toxicity of a particular NRTI in a particular patient. One of the factors that affect phosphorylation, and therefore a regimen's efficacy and toxicity, is intracellular drug-drug interaction.   Factors Influencing Nucleoside Reverse Transcriptase Inhibitor Intracellular Metabolism   Because NRTIs must be phosphorylated to their active anabolites by cellular enzymes, it is not surprising that multiple cellular factors affect the intracellular metabolism of these drugs. These cellular influences can be broadly divided into intrinsic and external factors. Examples of intrinsic factors include cell type, cell cycle, and intracellular ratio of drug triphosphate to endogenous nucleoside triphosphate (ddN-TP/dN-TP; the building blocks for the cell's DNA replicative process), whereas examples of extracellular factors include activation status of the cell and infection status. In the following discussion, triphosphates are referred to as the active form of the drug. In the case of tenofovir, a phosphate group is already in place in situ; consequently, the active form is usually referred to as a diphosphate (ie, having the addition of 2 more phosphates). Ultimately, however, tenofovir exerts its pharmacologic effect because there are 3 phosphate groups on the molecule, as do all the NRTIs.   The ddN-TP/dN-TP ratio within the cell actually determines the activity of the drug. The drug triphosphate inside the cell competes with the corresponding endogenous triphosphate for binding to HIV reverse transcriptase and subsequent integration into the complementary viral DNA chain. Consequently, with respect to a number of intracellular interactions, it is the alteration of the ddN-TP/dN-TP ratio that drives the efficacy or toxicity of the drug.   Other cellular factors have been shown, at least in vitro, to alter drug metabolism. The activation state of the cell may change the relation between drug triphosphate and endogenous triphosphate.61 For example, in activated cells, the ratio of zidovudine-TP/dN-TP and stavudine-TP/dN-TP is greater than in resting cells.62 This is thought to be attributable to changes in the cell cycle, where increased expression of thymidine kinase is seen during the S-phase. Conversely, didanosine, lamivudine, abacavir, and tenofovir have a more favorable ddN-TP/dN-TP ratio in resting cells. Resting cells, such as monocytes and macrophages, have low levels of endogenous triphosphates, resulting in low catalytic activity of HIV reverse transcriptase. Consequently, less drug phosphorylation may be required for the same antiviral effect.   Although cell culture studies in vitro have shown that intracellular phosphorylation of the NRTIs is similar in control and HIV-infected cells, in vivo studies have shown that phosphorylation of zidovudine in PBMCs isolated from HIV-infected patients is greater than in PBMCs isolated from healthy volunteers. Furthermore, in patients with decreased CD4 cell counts, zidovudine phosphorylation was greater in resting PBMCs, suggesting that HIV infection results in an increase in drug phosphorylation as well as in the activation state.61 There are also some recent data suggesting that women have higher phosphorylation rates than men.61   Interactions between drugs inside the cell may increase or decrease the level of phosphorylation of a single or both drugs. To understand this, it is helpful to consider the specific phosphorylation pathway through which an NRTI becomes activated.   Activation of Nucleoside Analogues   The activation of an NRTI follows a phosphorylation pathway unique to its specific backbone nucleoside or nucleotide analogue. For example, nucleosides that are thymidine analogues follow a phosphorylation pathway different than nucleosides that are guanosine analogues. With reference to Figure 6, it can be seen which drugs share similar enzyme pathways. Laboratory and clinical data suggest that it may be better to coadminister 2 nucleoside analogues with different activation pathways rather than those with shared enzymes so as to ensure maximal anti-HIV activity. For example, coadministration of zidovudine and stavudine, both thymidine analogues, results in a 90% decrease in the ratio of stavudine-TP to dN-TP, a decrease that is consistent with a reduction of antiviral effect.63 Other shared pathway interactions have been documented in the literature. It is important for clinicians to be aware of these limitations of combining certain NRTIs.   When combining NRTIs that share the same activation pathway, it is important to know their effects on intracellular phosphorylation (not just on plasma levels), because this affects the regimen's antiviral activity and toxicity. An intracellular interaction that alters the concentration of the drug triphosphate may not necessarily be detected by a corresponding alteration in the plasma drug level. This is supported by a recent trial with the investigational drug SPD754 and lamivudine. SPD754 is a deoxycytidine analogue that shares a common activation pathway with lamivudine. The correlation of SPD754's plasma and intracellular triphosphate levels has been established by Adams et al.64 Bethell et al65 conducted a 2-part pharmacokinetic study to evaluate the effects of SPD754 and lamivudine on each other's phosphorylation and antiviral activity. The intracellular concentration of SPD754-TP was markedly reduced (4-6-fold) in the presence of lamivudine, with a resulting decline in antiviral activity. In contrast, there was no effect on the plasma pharmacokinetics and there was no intracellular effect on lamivudine-TP. The clear clinical implication is that these drugs should not be used in the same regimen.   The general principle that NRTIs within the same pathway are candidates for interaction must be acknowledged with some caveats. As Table 8 demonstrates, not all interactions result in decreased anti-HIV activity.26,51,59,63,65-75 In the case of tenofovir and didanosine, adenosine analogues, there is no evidence of an intracellular interaction. This may be secondary to the fact that the addition of the first phosphate is the rate-limiting step in the formation of nucleoside triphosphate and tenofovir already contains the first phosphate.   Didanosine-Tenofovir   Tenofovir has been shown to increase plasma levels of didanosine. To determine whether the impact extends to intracellular levels of drug triphosphate as well, Robbins et al26 compared in vitro levels of drug in human PBMCs. The phosphorylation of didanosine to dideoxyadenosine- triphosphate (ddA-TP), the active phosphorylated form of didanosine, was measured in resting and simulated PMBCs to which 5 M of tenofovir or placebo had been added. The converse was also measured: the phosphorylation of tenofovir in resting and stimulated cells to which didanosine (2 M and 20 M) or placebo had been added. The phosphorylated forms of each drug were measured using radiolabeled tenofovir and didanosine with high-performance liquid chromatography after incubation. The presence of either drug did not affect the amount of the other drug in the phosphorylated form; in other words, the amount of tenofovir did not affect the concentration of phosphorylated didanosine. Neither did didanosine affect the concentration of phosphorylated tenofovir.   Robbins et al26 concluded that there seemed to be no intracellular drug interaction between didanosine and tenofovir at the level of phosphorylation, despite the systemic increase in didanosine concentrations. This finding suggests that adjustment of the didanosine dosage to account for the increased systemic exposure when coadministered with tenofovir may be sufficient.   A recently published study by Ray et al51 probed further into the intracellular mechanism potentially responsible for the increased didanosine exposure when it is coadministered with tenofovir as well as when it is coadministered with allopurinol or ganciclovir. The study focused on the role of PNP in these interactions. PNP is a ubiquitous enzyme found throughout the body, particularly in red blood cells and liver cells. It is responsible for the breakdown of didanosine-the only antiviral agent cleared by PNP-before elimination by the kidneys. With no significant change in the renal clearance of didanosine despite the increased plasma exposure, it was postulated that the inhibition of PNP might be the molecular site of interaction.   To explore this hypothesis, Ray et al51 performed in vitro absorption, enzymatic, and metabolic assays to study the cells' permeability to didanosine, the substrate specificity of PNP, and the validity of its inhibition as a mechanism for the increased exposure of didanosine as well as to confirm intracellular PNP-dependent didanosine degradation. Study results showed that tenofovir, allopurinol, and ganciclovir all inhibited PNP-dependent didanosine breakdown in the cellular metabolic assay (Fig. 7). The decreased breakdown allows the plasma concentration of didanosine to increase; hence, a dose adjustment is warranted. It should be noted that this interaction is specific to didanosine and does not involve other NRTIs.   Nucleoside Reverse Transcriptase Inhibitors: Ribavirin   Potential interactions between NRTIs and ribavirin are of timely interest because of ribavirin's extensive use among the large number of patients coinfected with hepatitis C virus (HCV) and HIV. Worldwide, an estimated 25% of the 40 million individuals infected with HIV are also coinfected with HCV.76 Typically, anti-HCV therapy includes up to 48 weeks of peginterferon alfa coadministered with ribavirin. Ribavirin is used with caution in coinfected patients, however. Independent of ART, there is concern for the development of dose-dependent hemolytic anemia, the primary adverse effect of ribavirin. Associated with ART, there is the potential for a ribavirin-NRTI drug interaction, which, in turn, raises concerns about antiviral efficacy and toxicity.   Ribavirin and Anti-HIV Efficacy   Data from in vitro studies show that ribavirin modulates triphosphate formation for some nucleosides. Specifically, ribavirin has an antagonistic effect on pyrimidine nucleoside analogues (stavudine, lamivudine, zidovudine, emtricitabine, and zalcitabine) by inhibiting their intracellular phosphorylation. Ribavirin inhibits total zidovudine phosphorylation in vitro by causing an increase in the intracellular formation of endogenous triphosphate.75 This decreases cellular thymidine kinase activity, resulting in reduced zidovudine phosphorylation.   A key large study by Gries et al67 investigated the effect of ribavirin on intracellular and plasma kinetics of nucleosides in patients who are coinfected with HIV and HCV. A subset of 48 coinfected patients from the AIDS PEGASYS Ribavirin International Coinfection Trial (APRICOT) participated in a prospective nested pharmacokinetic study. Eligible patients were receiving an anti-HIV regimen, including zidovudine/lamivudine or stavudine/lamivudine in steady state. Patients were randomized to receive peginterferon alfa-2a plus ribavirin (800 mg/d) or placebo. Blood samples were collected at 0, 2, 4, 6, 8, and 12 hours after the morning dose of anti-HIV drug and at baseline and after 8 to 12 weeks of treatment with ribavirin/peginterferon or placebo/peginterferon. Plasma levels of parent drug and intracellular levels of the drug triphosphate were quantified.   Study data supported the conclusion that ribavirin seemed not to perturb the intracellular metabolism of lamivudine, stavudine, or zidovudine or their corresponding endogenous nucleoside triphosphates. In addition, ribavirin seemed not to modify the plasma concentration-time profile of lamivudine, stavudine, or zidovudine. The clinical relevance of these findings is that ribavirin, as part of an interferon-based regimen for the treatment of HCV infection, in combination with stavudine, lamivudine, and zidovudine does not have a negative impact on anti-HIV efficacy. This finding is consistent with findings from large trials of coinfected patients receiving treatment with peginterferon and ribavirin. No loss of disease control has been observed in these trials.77-79 It is therefore important to note that in vitro studies (eg, the study by Sim et al75) are not always predictive of what happens in patients.   A number of questions arise from the study of Gries et al,67 however. What would be the effect of a higher dose (>800 mg/d) of ribavirin? Would 1200 mg/d produce a toxic effect? With an initial time point of 8 weeks, what happened before the first time point? What happened before the cell's homeostatic mechanism had already returned the state to normal? Indeed, some preliminary data suggest that higher doses of ribavirin may have an impact on intracellular phosphorylation.80   Ribavirin and Toxicity   Ribavirin decreases DNA synthesis when coadministered with an NRTI. In addition, NRTIs may decrease DNA synthesis, particularly that of mitochondrial DNA. Therefore, ribavirin plus an NRTI can have an additive antimitochondrial effect. The degree of mitochondrial DNA inhibition varies with the NRTI.   The mechanism by which didanosine develops a unique toxicity when coadministered with ribavirin has to do with ribavirin's own phosphorylation. Ribavirin is a guanosine analogue, which undergoes phosphorylation according to the guanine activation pathway.81 One of the major effects of its phosphorylation is the inhibition of inosine monophosphate dehydrogenase (IMPDH), which leads to elevated levels of ddA-TP, the metabolically active product of didanosine.82 Although this has a potentially synergistic anti-HIV effect, more importantly, it could result in toxic levels of ddA-TP for mitochondrial toxicity.   Mitochondrial toxicity in coinfected patients receiving didanosine and ribavirin was first reported by Lafeuillade et al71 in 2001. A recent report of a 2-year (January 2000-July 2002) longitudinal analysis of didanosine-related adverse events (hyperamylasemia, pancreatitis, hyperlactatemia/lactic acidosis, or neuropathy) was published by Moreno et al.73 Among 35 patients who received concomitant didanosine and ribavirin, 57% developed 1 or more adverse events after a mean of 87 days. Based on these findings, didanosine should not be used in an antiretroviral regimen for any patient also receiving ribavirin.   Hydroxyurea and Mycophenolic Acid   Hydroxyurea (HU) inhibits the enzyme ribonucleotide reductase, which is responsible for the reduction of cellular ribonucleotides to deoxyribonucleotides. Therefore, inhibition of this enzyme potentially results in decreased levels of didanosine-TP and endogenous triphosphate, despite in vitro data pointing to an enhanced anti-HIV effect when HU is combined with didanosine.83 Clinical data to date generally do not support a more favorable outcome. In fact, 1 randomized trial comparing didanosine/stavudine/efavirenz with and without HU was discontinued because of excessive drug toxicity in the HU-containing arm.84 More recently, results from the pharmacology substudy of the Compact Highly Active Antiretroviral Medication (CHARM) trial, a study in which patients received zidovudine/abacavir/lamivudine with and without HU, demonstrated that intracellular endogenous triphosphate levels were unaltered by HU.85   Mycophenolate mofetil (MMF) is hydrolyzed to its active metabolite, mycophenolic acid (MPA) in vivo. Like ribavirin, MPA also inhibits IMPDH, and in vitro studies have demonstrated that MPA can augment the anti-HIV activity of purine-based NRTIs.86 For example, abacavir and MPA have been observed to have a synergistic anti-HIV activity in vitro when used in combination. A number of clinical studies are currently in progress to investigate the effects of MPA when used in combination with NRTIs, specifically abacavir. Margolis et al86 concluded that in vivo modulation of the ratio of carbovir-TP and dN-TP may be achievable with doses of MMF 50% lower than those used in organ transplantation.   Summary   Clinicians must be aware of the interactions between antiretrovirals and between antiretrovirals and other concomitant drugs. Most of the interaction data, however, refer to PIs or NNRTIs-a reflection of the fact that these drugs are extensively metabolized by CYP450 enzymes. Our understanding of interactions involving NRTIs, especially at the phosphorylation level, is more limited and reflects, in part, the difficulty of being able to measure the drug phosphates in patient studies. It is thus vital for the clinician to be aware that interactions may not only theoretically occur but do occur in practice. More clinical studies are warranted to aid our understanding of NRTI disposition.   CLINICAL PHARMACOLOGIC PERSPECTIVE ON ONCE-DAILY REGIMENS AND NUCLEOSIDE REVERSE TRANSCRIPTASE INHIBITOR FAILURES   Key Messages   - The utility of once-daily regimens is different in treatment-naive patients compared with treatment-experienced patients because of resistance.   - The precise mechanism of early failure for some regimens is unclear. It is too early to discard all triple- or quadruple-NRTI regimens; more studies need to be done.   - The pharmacologic principle of symmetry (ie, matching drugs for pharmacokinetic properties) is important when combining antiretroviral drugs, primarily because of resistance concerns.   The potential benefit of once-daily therapy, in terms of simplicity and adherence, makes these regimens appealing to clinicians and patients. It can be a challenge, however, to determine if a once-daily regimen is appropriate for a particular patient, and if so, which regimen. The patient's virus and treatment history as well as the properties of the drugs and regimen must all be considered when deciding if once-daily therapy is appropriate for a particular patient.   Patient Considerations   Based on the pharmacokinetic principles discussed, there is a considerable difference in constructing a treatment regimen for a treatment-naive patient compared with a treatment-experienced patient. Although it may be a relatively simple task to develop a once-daily regimen for a treatment-naive patient, it can be difficult, if not impossible, to develop an effective once-daily regimen for a treatment-experienced patient. In the latter case, the choice of drugs and regimens is significantly narrowed. Because of these narrowed choices, the importance of strict adherence may be greater for a treatment-experienced patient than for a treatment-naive patient, because the cost of losing a regimen or drug class as a result of resistance is great.   Although once-daily regimens have been shown to improve adherence, some patients may have a misperception about the tolerance, or forgiveness, of a once-daily regimen for doses taken late or skipped. Missing a dose on a once-daily regimen has a potentially greater impact than missing a dose on a twice-daily regimen because of the prolonged time without exposure to drug. Some clinicians may choose to stay with a twice-daily regimen for treatment-experienced patients with the understanding that most patients miss an occasional dose. Alternatively, some clinicians may choose to reserve twice-daily regimens for only those treatment-experienced patients who have demonstrated poor adherence. Regimen forgiveness must be balanced against the perceived convenience of using once-daily drugs.   Drugs with pharmacokinetic properties (eg, more rapid systemic clearance) that result in lower concentrations at the end of a dosing interval (Cmin or Ctrough) may be more susceptible to resistance if a dose is missed or is taken late. This is dependent also on the inherent antiviral potency of the drug, the relative genetic barrier to resistance, and the other drugs in the regimen.   Clinical Considerations   Table 9 lists drugs that have pharmacokinetic properties that would support once-daily dosing. The listed drugs are FDA approved for once-daily administration (indicated with an asterisk) or are in sufficiently advanced clinical development, with published or reported studies, that a clinician might consider prescribing them for once-daily use.   Tenofovir   Tenofovir is the first antiretroviral NRTI developed from the outset and approved by the FDA as a once-daily drug, with plasma and intracellular half-lives of 17 and >60 hours, respectively.87 Tenofovir is currently approved for treating HIV infection in adults in combination with other antiretroviral agents. In antiretroviral-experienced patients, the addition of once-daily tenofovir to existing therapy resulted in a further sustained decrease in HIV plasma RNA concentrations of 0.65 to 0.87 log copies/mL compared with placebo after 48 weeks of treatment.87 Several large trials have confirmed the antiretroviral activity of tenofovir in 3-drug regimens with other agents, including other NRTIs, PIs, and NNRTIs.88-91 In a 3-year randomized, double-blind, comparison trial in which treatment-naive patients also received lamivudine and efavirenz, 300 mg of tenofovir administered once daily was as effective as 40 mg of stavudine administered twice daily and was better tolerated. Patients receiving tenofovir showed significantly fewer adverse effects associated with mitochondrial toxicity (lactic acidosis, peripheral neuropathy, and lipodystrophy) compared with patients receiving stavudine and had better total fasting lipid profiles.89 (Ed Note: tenofovir is associated with bone loss in patients, and previously found in pre-clinical animal studies).   Emtricitabine   Emtricitabine was also developed from the outset as a once-daily agent based on its pharmacokinetic properties, including its 10-hour plasma half-life and its >39-hour intracellular half-life. Emtricitabine is FDA approved for treating HIV infection in adults in combination with other antiretroviral agents. In 2 small monotherapy trials, emtricitabine at a dose of 200 mg administered once daily produced a maximal mean drop in HIV plasma RNA concentration of 1.9 logs.31 Several large trials have confirmed the antiretroviral activity of emtricitabine in 3-drug regimens with other agents, including NRTIs, PIs, and/or NNRTIs.90-94 In 2 randomized comparison studies, emtricitabine- and lamivudine-based triple-combination regimens had similar long-term efficacy.31   Lamivudine   Lamivudine was originally marketed as a twice-daily drug with a recommended dose of 150 mg taken twice daily. This dosage was based in part on the short half-life (5-7 hours) of its parent compound in plasma. The intracellular half-life of lamivudine 5-TP, however, is 15 to 16 hours, and the drug is now approved by the FDA for 300-mg once-daily dosing.33 In a large, prospective, randomized trial, 554 treatment-naive patients received 150 mg of lamivudine twice daily or 300 mg once daily in combination with zidovudine and efavirenz.95 At the end of 48 weeks, these 2 regimens were indistinguishable in terms of virologic efficacy, CD4 cell response, and adverse effects. In 81 treatment-experienced patients who were fully suppressed on a twice-daily lamivudine regimen, a 300-mg once-daily regimen produced a similar magnitude of viral suppression at 24 weeks compared with the twice-daily regimen.96   Abacavir   Like lamivudine and didanosine, abacavir was originally developed as a twice-daily drug based on plasma pharmacokinetics of the parent compound (plasma half-life: 1.54 ± 0.63 hours). Given the much longer half-life of intracellular carbovir 5-TP (12-21 hours), however, once-daily dosing is pharmacologically rational. A comparative prospective trial has demonstrated that once-daily dosing of abacavir is as effective as twice-daily dosing,97 and on the basis of this trial, abacavir is now FDA approved for once-daily dosing. In addition, the FDA recently approved abacavir for once-daily dosing in a coformulation with lamivudine. (Ed Note: study of lypodystrophy, body changes, both abacavir and tenofovir have been found not to be associated with lipoatrophy (fat wasting). A longer term study of several years found that after switching to tenofovir fat loss had improved in patients who experienced fat loss, as evaluated by objective testing).   One consideration with abacavir in a once-daily regimen is the possibility of abacavir hypersensitivity syndrome, characterized by fever, abdominal pain, and rash. This syndrome occurs in 3% to 5% of recipients but can be fatal, especially if the patient is rechallenged with the drug.98 Recent data suggest that this syndrome is the consequence of a specific genetic polymorphism linked to HLA-B57 and the heat-shock locus Hsp70-Hom in the white population.99 There is no evidence that once-daily administration of abacavir alters the incidence of the hypersensitivity syndrome.   Didanosine   The intracellular triphosphate of didanosine, ddATP, has a half-life of 25 to 40 hours (although this is actually based on limited data), making once-daily dosing of this drug pharmacokinetically rational. In fact, didanosine was the first antiretroviral nucleoside approved for once-daily administration. Unless coadministered with tenofovir, however, didanosine must be administered in the fasted state, thereby creating at least a twice-daily regimen when combined with any antiretroviral that must be given with food. This is especially true when didanosine is given with most HIV PIs. Toxicity is a major concern with didanosine, particularly the possibility of peripheral neuropathy and pancreatitis, as has been discussed.   Stavudine-Extended Release TOP   Stavudine-XR has been approved by the FDA as a once-daily NRTI. At the time of this writing, however, it is not yet commercially available because of manufacturing difficulties. In a randomized prospective trial involving 783 patients, stavudine-XR given once daily produced the same virologic benefit as the standard twice-daily immediate-release formulation.100 Initial concerns that administering stavudine once daily might increase the frequency of side effects, especially peripheral neuropathy, have not been borne out by controlled clinical trials. In the same study, the incidence of adverse events was the same in once-daily and twice-daily dosing regimens.   Stavudine-Extended Release   Stavudine-XR has been approved by the FDA as a once-daily NRTI. At the time of this writing, however, it is not yet commercially available because of manufacturing difficulties. In a randomized prospective trial involving 783 patients, stavudine-XR given once daily produced the same virologic benefit as the standard twice-daily immediate-release formulation.100 Initial concerns that administering stavudine once daily might increase the frequency of side effects, especially peripheral neuropathy, have not been borne out by controlled clinical trials. In the same study, the incidence of adverse events was the same in once-daily and twice-daily dosing regimens.   Regimen Considerations   Recent studies have proven the danger of prescribing untested combinations of once-daily antiretroviral drugs. Several once-daily regimens have produced high rates of early virologic nonresponse or rapid failure with the development of drug resistance. To date, most of these failing regimens are triple-NRTI combinations.   Recent Experience With Once-Daily Nucleoside Reverse Transcriptase Inhibitor-Only Combination Regimens   A small pilot study by Farthing et al101 assessed the efficacy of a once-daily regimen of 600 mg of abacavir, 300 mg of lamivudine, and 300 mg of tenofovir in treatment-naive patients. At week 8, 9 (52%) of 17 patients had viral rebound after initial viral suppression.   The tenofovir/lamivudine/abacavir (ESS30009) trial reported by Gallant et al102 was a randomized, prospective, open-label, multicenter study evaluating once-daily regimens in 345 treatment-naive patients. The regimens consisted of 600 mg of efavirenz or 300 mg of tenofovir in combination with the fixed dose coformulation of 600 mg of abacavir and 300 mg of lamivudine. Because of early reports of poor antiviral efficacy from some investigators, an unplanned interim analysis at 8 weeks was conducted after most patients had completed 8 weeks of treatment.103 At week 8, 50 (49%) of 102 patients who were receiving the triple-NRTI regimen met the definition of virologic nonresponse (194 patients were at or beyond week 8 at this point). In contrast, only 5 (5.4%) of 92 of those receiving the regimen containing efavirenz met the failure definition. As a result, the triple-NRTI arm of this study was immediately terminated.   A single-site pilot study of 22 treatment-naive patients receiving the triple-NRTI once-daily regimen of 250 mg of EC-didanosine, 300 mg of lamivudine, and 300 mg of tenofovir was also terminated early because of poor efficacy.104,105 By week 12, 20 (91%) of 22 patients met the definition of virologic failure.   Landman et al106 conducted an abacavir/lamivudine/tenofovir (TONUS) pilot study in which treatment-naive patients received once-daily abacavir/lamivudine/tenofovir for 12 months. Like the other triple-NRTI trials discussed, this trial was also stopped after an unplanned interim analysis. Twelve of 36 patients met the definition of virologic failure.   Elion et al88 reported on an abacavir/lamivudine/zidovudine/tenofovir (COL40263) open-label multicenter trial in which a once-daily regimen of abacavir, lamivudine, zidovudine, and tenofovir was studied. Unlike the previously discussed all-NRTI regimens, this regimen produced virologic failure in only 10 (11%) of 88 treatment-naive patients at week 8, suggesting the possible benefit of a 4-drug regimen or the benefit of using zidovudine with tenofovir.   Why Did the Once-Daily Nucleoside Reverse Transcriptase Inhibitor-Only Combination Regimens Fail?   Much attention has been focused on the possible causes of the unacceptably high early virologic failure rates in the once-daily NRTI-only combination regimens described previously. Major possibilities have included unexpected pharmacokinetic drug interactions, an unexpected adverse pharmacodynamic interaction (ie, antagonism), inadequate concentrations of the drugs involved throughout the 24-hour dosing interval, and a low genetic barrier to resistance combined with inadequate drug concentrations.   Pharmacokinetic drug interactions are unlikely to explain these early treatment failures. Studies of interactions between tenofovir and abacavir107 and tenofovir and lamivudine42 show no significant pharmacokinetic interactions involving altered plasma concentrations of the parent drug. Although a systemic interaction exists between tenofovir and didanosine, resulting in an approximately 44% mean increase in the didanosine AUC when the 2 drugs are coadministered, this interaction does not explain the early virologic failure.42 Didanosine toxicity did not result in early drug discontinuation in the 2 studies cited. The phosphorylated forms of these drugs are not antagonistic in vitro, and their concentrations are not altered when combined.26 In addition, intracellular concentrations of the phosphorylated forms of tenofovir and abacavir are not altered when these agents are combined.32,108   Antagonism, or lack of pharmacologic synergy, is another possible cause of early treatment failure with once-daily NRTIs. Synergy is the property of combined drugs to inhibit a microorganism more than would be expected based on the effect of each drug if given individually. Usually, antimicrobial synergy requires that the drugs attack different molecular targets in the same organism. Until recently, however, antiretroviral combinations have been developed neglecting this principle, with the assumption that different agents in an antiretroviral combination work together by preventing resistance. Current US Department of Health and Human Services guidelines recommend that an initial combination antiretroviral regimen include drugs from more than 1 class.3 (Note that for the purposes of a synergy analysis, NNRTIs and NRTIs are different classes because they do, in fact, attack a different target in the virus, even though the targets are on the same enzyme.) These recommendations are based on the relatively weaker performance of triple-NRTI regimens as compared with regimens containing a PI or NNRTI plus NRTIs. It should be noted, however, that twice-daily regimens containing all NRTIs have performed much better than the once-daily regimens and have not been associated with substantial early treatment failure.109,110 This suggests that the reduced frequency of administration of the NRTIs in the once-daily regimens as well as the agents selected may have contributed to early failure.   A third possible explanation for early treatment failure is that the concentrations of drugs achieved throughout a 24-hour dosing interval are inadequate to suppress HIV replication completely. To achieve full antiviral effect, plasma drug concentrations and, in turn, the concentrations of active intracellular diphosphate or triphosphate must be kept at a level that fully suppresses HIV and prevents resistance.64 Factors that reduce plasma drug concentrations are thus a consideration when examining treatment failure. These factors include high inter- and intraindividual pharmacokinetic variability, inadequate drug concentrations at the end of a 24-hour dosing interval, drug-drug interactions, and adverse food effects. Past pharmacokinetic studies of the drugs included in the failed regimens, however, demonstrated that the half-lives of these drugs were likely adequate for once-daily dosing, with trough levels in the expected therapeutic range. The strongest argument against inadequate plasma and intracellular drug concentrations for these regimens is the fact that all these drugs have performed well in once-daily combinations with NNRTIs or PIs. Borderline drug concentrations for these NRTIs that were acceptable with more potent PIs and NNRTIs may have been inadequate in these single-class regimens, however. The possibility that concomitant NRTIs might reduce each other's plasma concentrations after prolonged administration was addressed in the TONUS study by Landman et al.106 Yet, pharmacokinetic testing at 1 month showed that 32 (86%) of 37 of patients had a trough Cmin, considered adequate for all 3 drugs.   Finally, it has been suggested that a low genetic barrier to resistance may exist for the failed triple-NRTI combinations. Early virologic failure after initial response, as seen in these studies, indicates the selection of preexisting mutant virus resistant to 1 or more of the administered drugs. The TONUS study found that 11 of the 12 patients with virologic failure had M184V and K65R mutations and 1 had the M184V mutation alone.106 The authors concluded that these findings support the hypothesis that the treatment failure in this study was the consequence of a low genetic barrier to resistance, allowing the selection of resistance mutations, such as K65R, that would not emerge in the presence of more potent PIs or NNRTIs.   Resistance testing conducted in the other failed triple-NRTI trials supports this hypothesis as well. In the ESS3009 study, all patients with early virologic failure for whom genotype information was available had the M184V mutation. In addition, 64% had both the M184V and K65R mutations.102 Jemsek et al105 reported that 100% of patients who failed treatment had M184I/V and 10 (50%) of 20 of these patients also had K65R. Resistance testing was available in 82 of the patients with treatment failure in ACTG 5095. The incidence of mutations was lower in this trial: 34% of patients had only M184V, 11% had M184V plus another resistance-associated mutation, and 2% had a resistance-associated mutation other than M184V.   These specific mutations are significant in assessment of the failure of these triple-NRTI therapies. The prevalence of K65R has risen since the approval of tenofovir, although it is still low (2%-4%) in treatment-experienced patients.111 The presence of K65R confers some degree of resistance to abacavir, didanosine, and tenofovir. In contrast, it makes the virus hypersusceptible to zidovudine. M184V confers resistance to lamivudine but also confers zidovudine hypersusceptibility. The hypothesis of a low genetic barrier to resistance suggests that the triple-NRTI therapies first selected out virus with the M184V mutation, followed by (or simultaneously with) selection of virus with the K65R mutation. These 2 mutations would render the virus resistant to lamivudine, abacavir, didanosine, and tenofovir. Triple-NRTI regimens containing these drugs would thus be more likely to produce treatment failure. If zidovudine (or stavudine) is included, however, the hypersusceptibility conferred by the K65R mutation preserves the inhibitory effects of that NRTI and prevents early treatment failure. Inclusion of a thymidine analogue (zidovudine or stavudine) in tenofovir-containing triple-NRTI regimens may thus prevent the development of K65R, but this possibility is still under investigation. There have been no prospective data to support this as the best clinical strategy for using tenofovir in a once-daily regimen.   In contrast, preliminary 96-week findings from Study 903, a 3-year, randomized, double-blind, active-controlled study of once-daily triple-NRTI regimens containing tenofovir in 600 treatment-naive patients, showed a much lower incidence of K65R and M184V mutations and a much lower rate of treatment failure.112 Enrolled patients received tenofovir or stavudine in combination with lamivudine and efavirenz. At week 96, 12.3% of patients met the definition of virologic failure and 2.7% and 0.6% of patients receiving tenofovir and stavudine, respectively, had the K65R mutation. This suggests that the use of a potent drug like efavirenz prevents HIV replication and negates the possible selection of the resistance mutations that occurs with the triple-NRTI once-daily regimen.   Conclusions From Recent Experience With Once-Daily Nucleoside Reverse Transcriptase Inhibitor-Only Combination Regimens   Until more is known about the mechanism underlying early virologic treatment failure in these trials, clinicians should probably not administer tenofovir in combination with didanosine and lamivudine or in combination with abacavir and lamivudine for treatment-naive or treatment-experienced patients unless other drugs are added to the regimen. Inclusion of a thymidine analogue is likely to potentiate a tenofovir-containing triple-NRTI regimen.   Recent Experience With Once-Daily Nonnucleoside Reverse Transcriptase Inhibitor Plus Nucleoside Reverse Transcriptase Inhibitor Regimens   Recently (November 2004), a Dear Health Care Provider letter was issued by Bristol-Myers Squibb Company113 warning of the risk of virologic failure in patients with a high baseline viral load when receiving didanosine, tenofovir, and efavirenz or nevirapine. This warning was based on recently conducted trials by Podzamzcer et al,114 the first reports implicating NNRTIs in combination with NRTIs in early failure. Study is ongoing to determine the mechanism and clinical implications of these failures.   Regimen Symmetry   Regimens containing drugs with similar half-lives are considered pharmacokinetically symmetric. They may be administered at the same or equivalent intervals; likewise, they are eliminated within the same or equivalent intervals. The issue of symmetry may be critical in terms of resistance and is of particular importance when a patient must stop a regimen. Although treatment reviews and guidelines as well as expert advice caution clinicians and patients never to stop treatment, the reality is that a number of patients do stop treatment.   Discontinuing a regimen that consists of drugs with different half-lives may result in eventual monotherapy of the drug with the longest half-life. This is of particular concern when using a drug with a long half-life and a low genetic barrier to resistance, such as nevirapine or efavirenz. Figure 8 illustrates this situation, in which the drugs in the discontinued regimen have dissimilar half-lives. Such prolonged monotherapy may have contributed to the unexpected high frequency of NNRTI resistance after single-dose nevirapine to prevent mother-to-child transmission of HIV.115 Several studies are under way to determine the length of time needed to cover the tail of nevirapine with different regimens after that single dose or after stopping combination therapy that includes nevirapine or efavirenz.115,116 Understanding the principle of regimen symmetry, the range of half-lives, and the concentrations achieved in relation to viral IC90 enables clinicians to determine prospectively how to stagger the drugs when stopping.   Coformulation   Coformulation continues to be used to simplify ART and is likely to play a role in future regimen selection. Coformulated drugs should have matched pharmacokinetic properties (ie, symmetry) to minimize the risk of resistance when drug doses are missed or taken late. With regard to nucleosides, the focus should include their phosphorylated intracellular anabolites as well as the parent drug. Coformulated abacavir/lamivudine and tenofovir/emtricitabine are examples of recently approved coformulations that are matched for pharmacokinetic properties of their intracellular nucleoside triphosphates.   Evidence-Based Regimens   Table 10 provides data on once-daily regimens that are classified as virologic successes or failures in clinical trials.88-94,97,101,102,105,106,113,114 Successful regimens are those in which 80% or more of subjects have undetectable viral loads after at least 24 weeks of treatment.   Summary   Several NRTIs have pharmacokinetic properties that support once-daily dosing. Unexpected early virologic failure has been seen with the once-daily triple-NRTI combinations of didanosine/lamivudine/tenofovir and abacavir/lamivudine/tenofovir. Combining these agents with a once-daily NNRTI or PI has not been associated with early virologic treatment failure.   Unanswered Questions   There are many issues that need further study:   - What is the optimal adherence threshold for once-daily regimens?   - What is the total pill burden patients are willing to take at a single time?   - What is the extent of increased cell activation associated with HIV infection?   - What is the relation between plasma and intracellular pharmacokinetics for all NRTIs?   - Is there differential phosphorylation in different cell types in vivo?   - Why have some once-daily regimens failed, despite lack of evidence of adverse systemic or intracellular interactions?   - Should tenofovir be routinely combined with a thymidine analogue to prevent emergence of the K65R resistance mutation?   CONCLUSION   Once-daily dosing and regimens improve the convenience and simplicity of treatment. The complexity of the pharmacokinetic parameters and interactions affecting this simplified treatment, however, must first be defined and managed. Each drug is unique, and not all drugs have the appropriate pharmacokinetic properties to qualify for once-daily dosing. Among the current once-daily drugs, tenofovir has the longest plasma and intracellular half-lives, and thus the most prolonged exposure. Until further research is completed, care providers should be cautious in using untested combinations of antiretroviral drugs.           View Older Articles   Back to Top   www.natap.org