Pharmacokinetic Modeling of Lamivudine and Zidovudine Triphosphates Predicts Differential Pharmacokinetics in Seminal Mononuclear Cells and Peripheral Blood Mononuclear Cells.

The male genital tract is a potential site of viral persistence. Therefore, adequate concentrations of antiretrovirals are required to eliminate HIV replication in the genital tract. Despite higher zidovudine (ZDV) and lamivudine (3TC) concentrations in seminal plasma (SP) than in blood plasma (BP) (SP/BP drug concentration ratios of 2.3 and 6.7, respectively), we have previously reported lower relative intracellular concentrations of their active metabolites, zidovudine triphosphate (ZDV-TP) and lamivudine triphosphate (3TC-TP), in seminal mononuclear cells (SMCs) than in peripheral blood mononuclear cells (PBMCs) (SMC/PBMC drug concentration ratios of 0.36 and 1.0, respectively). Here, we use population pharmacokinetic (PK) modeling-based methods to simultaneously describe parent and intracellular metabolite PK in blood, semen, and PBMCs and SMCs. From this model, the time to steady state in each matrix was estimated, and the results indicate that the PK of 3TC-TP and ZDV-TP in PBMCs are different from the PK of the two in SMCs and different for the two triphosphates. We found that steady-state conditions in PBMCs were achieved within 2 days for ZDV-TP and 3 days for 3TC-TP. However, steady-state conditions in SMCs were achieved within 2 days for ZDV-TP and 2 weeks for 3TC-TP. Despite this, or perhaps because of it, ZDV-TP in SMCs does not achieve the surrogate 50% inhibitory concentration (IC50) (as established for PBMCs, assuming SMC IC50 = PBMC IC50) at the standard 300-mg twice-daily dosing. Mechanistic studies are needed to understand these differences and to explore intracellular metabolite behavior in SMCs for other nucleoside analogues used in HIV prevention, treatment, and cure.

Systemic delivery of gemcitabine triphosphate via LCP nanoparticles for NSCLC and pancreatic cancer therapy.

Nucleoside analogs are a significant class of anti-cancer agent. As prodrugs, they terminate the DNA synthesis upon transforming to their active triphosphate metabolites. We have encapsulated a biologically activate nucleotide analog (i.e. gemcitabine triphosphate (GTP)), instead of the nucleoside (i.e. gemcitabine) derivative, into a novel Lipid/Calcium/Phosphate nanoparticle (LCP) platform. The therapeutic efficacy of LCP-formulated GTP was evaluated in a panel of human non-small-cell lung cancer (NSCLC) and human pancreatic cancer models after systemic administrations. GTP-loaded LCPs induced cell death and arrested the cell cycle in the S phase. In vivo efficacy studies showed that intravenously injected GTP-loaded LCPs triggered effective apoptosis of tumor cells, significant reduction of tumor cell proliferation and cell cycle progression, leading to dramatic inhibition of tumor growth, with little in vivo toxicity. Broadly speaking, the current study offers preclinical proof-of-principle that many active nucleotide or phosphorylated nucleoside analogs could be encapsulated in the LCP nanoplatform and delivered systemically for a wide variety of therapeutic applications.

Organic cation transporters OCT1 and OCT2 determine the accumulation of lamivudine in CD4 cells of HIV-infected patients.

PURPOSE: Identifying factors that determine concentrations of antiretroviral drugs in CD4 cells are important for improving therapeutic efficacy. Experimental models indicate that the nucleoside reverse transcriptase inhibitor lamivudine is transported by the organic cation transporters 1 and 2 (OCT1 and OCT2, respectively). Here, we tested whether OCT1 and OCT2 contribute to the uptake of lamivudine into native CD4 cells of human immunodeficiency virus (HIV)-infected individuals. METHODS: CD4 cells obtained by non-activated cell sorting from 35 individuals with HIV-1 infection were incubated with lamivudine (10 µM, 30 min), and intracellular concentrations of lamivudine and its active metabolite lamivudine triphosphate were determined by liquid chromatography tandem mass spectrometry. The expression of OCT1 and OCT2 mRNA was measured by quantitative real-time polymerase chain reaction (PCR). A model of OCT2-transfected CD4 cells was established for mechanistic investigations. RESULTS: Intracellular concentrations of lamivudine and its active metabolite lamivudine triphosphate showed strong linear correlations with each other and with the CD4 mRNA expression of OCT1 and OCT2 (r > 0.80). Coincubation with protease inhibitors (ritonavir, nelfinavir) that inhibit OCT1 and OCT2 yielded decreased intracellular concentrations of lamivudine and lamivudine triphosphate. Incubation of CD4 cells from healthy donors transfected with an OCT2 expression vector yielded increased concentrations of lamivudine and lamivudine triphosphate. CONCLUSION: Our studies indicate a role of OCT1 and OCT2 for the cellular accumulation of lamivudine in HIV-infected individuals.

Pharmacokinetics of lamivudine and lamivudine-triphosphate after administration of 300 milligrams and 150 milligrams once daily to healthy volunteers: results of the ENCORE 2 study.

There is interest in evaluating the efficacy of lower doses of certain antiretrovirals for clinical care. We determined here the bioequivalence of plasma lamivudine (3TC) and intracellular 3TC-triphosphate (3TC-TP) concentrations after the administration of two different doses. ENCORE 2 was a randomized crossover study. Subjects received 3TC at 300 and 150 mg once daily for 10 days (arm 1; n = 13) or vice versa (arm 2; n = 11), separated by a 10-day washout. Pharmacokinetic (PK) profiles (0 to 24 h) were assessed on days 10 and 30. Plasma 3TC and 3TC-TP levels in peripheral blood mononuclear cells were quantified by high-performance liquid chromatography-tandem mass spectrometry. Within-subject changes in PK parameters (the area under the concentration-time curve from 0 to 24 h [AUC(0-24)], the trough concentration of drug in plasma at 24 h [C(24)], and the maximum concentration of drug in plasma [C(max)]) were evaluated by determining the geometric mean ratios (GMRs) adjusted for study arm, period, and intra-individual variation. Regimens were considered bioequivalent if the 90% confidence interval (90% CI) fell within the range of 0.8 to 1.25. A total of 24 subjects completed the study. The GM (90% CI) 3TC AUC(0-24)), expressed as ng·h/ml, for the 300- and 150-mg doses were 8,354 (7,609 to 9,172) and 4,773 (4,408 to 5,169), respectively. Bioequivalence in 3TC PK following the administration of 300 and 150 mg was not demonstrated: the GMRs for AUC(0-24), C(24), and C(max) were 0.57 (0.55 to 0.60), 0.63 (0.59 to 0.67), and 0.56 (0.53 to 0.60), respectively. The GM (90% CI) 3TC-TP AUC(0-24) values (pmol·h/10(6) cells) for the 300- and 150-mg doses were 59.5 (51.8 to 68.3) and 44.0 (38.0 to 51.0), respectively. Bioequivalence in 3TC-TP PK following the administration of 300 and 150 mg was not demonstrated: the GMRs for AUC(0-24), C(24), and C(max) were 0.73 (0.64 to 0.83), 0.82 (0.68 to 0.99), and 0.70 (0.61 to 0.82), respectively. We found that 3TC at 150 mg is not bioequivalent to the standard regimen of 300 mg, indicating that saturation of cytosine phosphorylation pathways is not achieved at a dose of 150 mg.

Joint population pharmacokinetic analysis of zidovudine, lamivudine, and their active intracellular metabolites in HIV patients.

The population pharmacokinetic parameters of zidovudine (AZT), lamivudine (3TC), and their active intracellular metabolites in 75 naïve HIV-infected patients receiving an oral combination of AZT and 3TC twice daily as part of their multitherapy treatment in the COPHAR2-ANRS 111 trial are described. Four blood samples per patient were taken after 2 weeks of treatment to measure drug concentrations at steady state. Plasma AZT and 3TC concentrations were measured in 73 patients, and among those, 62 patients had measurable intracellular AZT-TP and 3TC-TP concentrations. For each drug, a joint population pharmacokinetic model was developed and we investigated the influence of different covariates. We then studied correlations between the mean plasma and intracellular concentrations of each drug. A one-compartment model with first-order absorption and elimination best described the plasma AZT concentration, with an additional compartment for intracellular AZT-TP. A similar model but with zero-order absorption was found to adequately described concentrations of 3TC and its metabolite 3TC-TP. The half-lives of AZT and 3TC were 0.81 h (94.8%) and 2.97 h (39.2%), respectively, whereas the intracellular half-lives of AZT-TP and 3TC-TP were 10.73 h (69%) and 21.16 h (44%), respectively. We found particularly a gender effect on the apparent bioavailability of AZT, as well as on the mean plasma and intracellular concentrations of AZT, which were significantly higher in females than in males. Relationships between mean plasma drug and intracellular metabolite concentrations were also highlighted both for AZT and for 3TC. Simulation with the model of plasma and intracellular concentrations for once- versus twice-daily regimens suggested that a daily dosing regimen with double doses could be appropriate.

Development of a population simulation model for HIV monotherapy virological outcomes using lamivudine.

Current highly active antiretroviral therapy (HAART) requires the use of combinations of three drugs to minimize the early emergence of drug-resistant HIV strains. Therefore, long-term monotherapy data with new agents are unavailable. However, the development of computer models for Monte-Carlo-type simulations of antiviral monotherapy, which incorporate HIV infection dynamic distributions from previously studied populations, together with pharmacokinetics and pharmacodynamic parameters of the new agent, could serve as an important tool. The nucleoside lamivudine (3TC) was used as a representative drug to standardize an improved pharmacodynamic and infection dynamic monotherapy model. 3TC plasma concentration versus time profiles was used to drive the cellular accumulation of 3TC-triphosphate (TP) in primary human lymphocytes in the model, over a 16 week period. The fraction of HIV reverse transcription inhibited was calculated using the median inhibitory concentration and intracellular 3TC-TP levels. Virus loads and activated CD4+ T-cell counts were generated for 2,200 theoretical individuals and compared with the outcomes of an actual 3TC monotherapy trial at the same dose. Pharmacokinetic variance alone did not account for the interindividual HIV-load variability. However, selection of appropriate distributions of the various pharmacokinetic and infection dynamics parameters produced a similar range of virus load reductions to actual observations. Therefore, once parameter and variance distributions are standardized, this modelling approach could be helpful in planning clinical trials and predicting the antiviral contribution of each agent in a HAART modality.

Pharmacokinetic evaluation of gemcitabine and 2',2'-difluorodeoxycytidine-5'-triphosphate after prolonged infusion in patients affected by different solid tumors.

BACKGROUND: The study determined pharmacokinetic parameters, toxicity profile and preliminary clinical activity of gemcitabine administered i.v. at different infusion rates in patients with a range of solid tumors. PATIENTS AND METHODS: Twenty patients were enrolled for both pharmacokinetic and clinical studies. Gemcitabine 300 mg/m(2) was administered during 1 h, 2 h or 3 h, and as a conventional dose of 1000 mg/m(2) during 30 min infusion. Administration was on days 1, 8 and 15 every 4 weeks. RESULTS: Patients were randomly assigned to one of the four arms. After 30 min infusion of 1000 mg/m(2) gemcitabine the plasma concentration remained above the saturation level of 10-20 microM, whereas after 1, 2 or 3 h infusion 300 mg/m(2) gemcitabine it remained below the saturation level for most of the time (being in the range 2.5-10 microM). Gemcitabine triphosphate was determined in the four arms in white blood cells; for infusion times from 0.5 to 3 h there was a progressive enhancement of gemcitabine triphosphate levels. In all evaluable patients the toxicity was mild, myelosuppression being the main toxicity. No grade 3 or 4 toxicities occurred. Clinical response was similar in patients receiving 300 mg/m(2) gemcitabine in 2 and 3 h and in the 1000 mg/m(2) arm. CONCLUSIONS: 300 mg/m(2) gemcitabine during 3 h infusion produced the highest accumulation of gemcitabine triphosphate. Thus, to achieve the highest possible gemcitabine triphosphate level, prolonged infusion time would appear to be more important than a high dose administered as a short infusion. However, there was no substantial difference in toxicity or antitumoral activity in the all different patient groups.

Intracellular pharmacokinetics of tenofovir diphosphate, carbovir triphosphate, and lamivudine triphosphate in patients receiving triple-nucleoside regimens.

OBJECTIVE: To evaluate the potential for a pharmacologic mechanism to explain suboptimal virologic responses observed in a triple-nucleoside only regimen containing tenofovir disoproxil fumarate (TDF), abacavir (ABC), and lamivudine (3TC). METHODS: This was a prospective evaluation of intracellular concentrations and pharmacokinetics of tenofovir diphosphate (TFV-DP), carbovir triphosphate (CBV-TP), and lamivudine triphosphate (3TC-TP) in patients on triple-nucleoside regimens. Fifteen patients on a stable TDF plus ABC plus a third nucleoside reverse transcriptase (RT) inhibitor (3TC [n = 13], stavudine [n = 2]) regimen discontinued TDF or ABC, replacing it with a nonnucleoside RT inhibitor or protease inhibitor. Peripheral blood mononuclear cells were collected after the last dose of TDF or ABC at baseline and over 12 to 96 hours as well as at days 14 and 28 after discontinuation. Nucleotide concentrations were measured directly using liquid chromatography with tandem mass spectrometry; changes after ABC or TDF discontinuation would provide evidence of an intracellular drug interaction. RESULTS: Intracellular nucleotide concentrations of the continued drugs were unaffected when TDF or ABC was discontinued. Intracellular levels of TFV-DP exhibited less inter- and intrapatient variability than CBV-TP or 3TC-TP. TFV-DP also had persistent intracellular levels on TDF discontinuation (median half-life of 150 hours, range: 60 to >175 hours). CBV-TP concentrations fell to below the limit of detection in all patients by 72 hours after the last ABC dose in accordance with a median half-life of 18 hours (range: 12-19 hours). CONCLUSIONS: An intracellular drug interaction does not explain the suboptimal viral response in patients treated with the nucleoside-only regimen of TDF, ABC, and 3TC.

Potent antiviral effect of reverset in HIV-1-infected adults following a single oral dose.

Reverset (2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine, RVT) is a potent inhibitor of HIV-1 replication in cell culture, with a 90% effective concentration at or below 1 microM. In vitro, RVT retains its activity against isolates harbouring mutations in the reverse transcriptase (RT) gene that otherwise confer resistance to lamivudine and/or zidovudine. The pharmacokinetics and safety of single oral doses of RVT (10-200 mg) were evaluated in an initial Phase I clinical trial. The viral load changes were determined on 18 HIV-1-infected antiretroviral therapy-naive subjects that were randomized into three cohorts, each cohort consisting of three study periods. The subjects received up to two oral doses of active drug and one placebo dose with a 1-week washout period separating the three study periods. Quantification of viral RNA was performed on the pre-dose, 12, 24 and 48 h post-dose plasma samples. A single oral dose of RVT to antiretroviral-naive subjects significantly reduced plasma viral load by 0.45 +/- 0.10 log10 copies/ml (P=0.0003). A mean drop of 0.37 +/- 0.12 log10 copies/ml (P=0.001) was obtained at the lowest dose of 10 mg. Sequence analysis of the HIV-1 RT gene performed before and after RVT dosing detected no genotypic changes in this short-term study. The viral RT gene of one subject had at predose the following genotype: L41 + N103 + C181 + W210 + D215, indicating prior exposure to zidovudine and non-nucleoside analogues, and anticipating high-level resistance against these agents. A single 10 mg RVT dose resulted in a viral load drop of 0.61 +/- 0.05 log10 providing evidence that a viral strain with the indicated genotype is susceptible to RVT.

Effect of mycophenolate mofetil on the pharmacokinetics of antiretroviral drugs and on intracellular nucleoside triphosphate pools.

OBJECTIVE: To study the effect of mycophenolate mofetil therapy on the pharmacokinetic parameters of a number of antiretroviral drugs, on intracellular pools of deoxycytidine triphosphate (dCTP) and deoxyguanosine triphosphate (dGTP), and on intracellular concentrations of the triphosphate of lamivudine (3TCTP). DESIGN: Randomised pharmacokinetic study. PARTICIPANTS: Nineteen HIV-1-infected patients. METHODS: Antiretroviral-naive men starting treatment with didanosine 400 mg once daily, lamivudine 150 mg twice daily, abacavir 300 mg twice daily, indinavir 800 mg twice daily, ritonavir 100 mg twice daily and nevirapine 200 mg twice daily were randomised to a group with or without mycophenolate mofetil 500 mg twice daily. After 8 weeks of therapy, the plasma pharmacokinetic profiles of mycophenolic acid (the active metabolite of mycophenolate mofetil), abacavir, indinavir and nevirapine, and triphosphate concentrations (dCTP, dGTP and 3TCTP) in peripheral blood mononuclear cells, were determined. RESULTS: Nine of the 19 patients received mycophenolate mofetil. There was no difference in plasma clearance of indinavir or abacavir between the two groups. The clearance of nevirapine was higher in patients using mycophenolate mofetil (p = 0.04). In 12 patients, of whom five also received mycophenolate mofetil, intracellular triphosphates were measured. There was no significant difference in intracellular dCTP, dGTP or 3TCTP concentrations between the two groups. CONCLUSION: In this small cohort of patients, mycophenolate mofetil therapy reduced the plasma concentration of nevirapine but had no effect on plasma concentrations of indinavir and abacavir. There were no consistent effects of mycophenolic acid on the intracellular concentrations of dCTP, dGTP or 3TCTP.

Antiviral dynamics and sex differences of zidovudine and lamivudine triphosphate concentrations in HIV-infected individuals.

OBJECTIVES: Nucleoside analog reverse transcriptase inhibitors (NRTI) are used in virtually all anti-HIV regimens. Clinical response depends on the intracellular formation of the pharmacologically active triphosphate moiety. Our objective was to quantify the pharmacological characteristics of zidovudine and lamivudine triphosphate in HIV-infected individuals. METHODS: Peripheral blood mononuclear cells were obtained at multiple planned intervals from antiretroviral-naive adults participating in a study of zidovudine, lamivudine and indinavir, and triphosphate levels were determined by immunoassay and high-performance liquid chromatography/mass spectrometry. Plasma HIV-RNA, CD4 cell counts, and plasma drug concentrations were collected over 18 months. Data were analysed using non-parametric, regression and time-to-event methods. RESULTS: Thirty-three subjects were evaluated. The estimated half-lives of zidovudine and lamivudine triphosphate were 7 and 22 h, respectively. Triphosphate concentrations were elevated in individuals with low baseline CD4 cell counts. Triphosphate concentrations in women were higher than in men by 2.3 and 1.6-fold for zidovudine and lamivudine, respectively. Women reached an HIV-RNA level under 50 copies/ml twice as fast as men. Zidovudine triphosphate above 30 fmol/10(6) cells was independently predictive of the time to under 50 copies/ml. Lamivudine triphosphate above 7017 fmol/10(6) cells was independently predictive of a longer virological response. Indinavir concentrations were related to antiviral responses in univariate analyses. CONCLUSION: Zidovudine and lamivudine triphosphate concentration thresholds were independently associated with the antiviral activity of zidovudine, lamivudine, and indinavir. The significantly elevated triphosphate concentrations in women and individuals with low baseline CD4 cell counts, groups that historically experience high rates of serious NRTI toxicities, provide a hypothesis for the pathogenesis of these events.

Development of a pharmacodynamic model for HIV treatment with nucleoside reverse transcriptase and protease inhibitors.

There is a need for models useful for predicting the efficacy of agents developed for treating human immunodeficiency virus (HIV) based on information obtained during the drug development process. A pharmacodynamic model that superimposes the pharmacokinetics of anti-HIV nucleoside reverse transcription (RT) and protease inhibitors over a previously published predator-prey model of HIV and CD4 dynamics was developed to address this need. This model was applied to in vitro measurements and patient-derived pharmacokinetics of the unbound antiviral drugs to simulate HIV-1 and CD4 counts versus time and dose. The primary mechanism for nucleoside RT inhibitors was assumed to be competitive inhibition of HIV-1-RT by the active nucleoside triphosphates (NTP). Cellular accumulation and breakdown rates of the NTP were estimated from previous in vivo pharmacokinetic studies. Median inhibition concentrations for the HIV-1 RT enzyme were estimated from previously published cell-free binding studies. The concentration of active protease inhibitor available for binding with HIV-1 protease was assumed equal to the unbound fraction in the plasma. The resulting simulations for mono- and dual nucleoside therapy with zidovudine and lamivudine single dose regimen with the protease inhibitor indinavir, produced similar HIV and CD4 response profiles to those reported in large Phase II and III clinical trials. Based on these findings this pharmacodynamic model can be applied to predict starting doses for a new agent based on simulated biological responses as a function of time for dosage regimens comprising one or two agents. However, the model overestimated the efficacy of highly effective drug combinations where all three agents are combined as in highly active anti-retroviral therapy.

Zidovudine triphosphate and lamivudine triphosphate concentration-response relationships in HIV-infected persons.

OBJECTIVE: To quantitate intracellular concentrations of zidovudine and lamivudine triphosphate and explore relationships with virologic and immunologic responses to antiretroviral therapy. DESIGN: Eight antiretroviral-naive, HIV-infected persons with CD4 T cell counts > 100 x 10(6) cells/l, and HIV RNA in plasma > 5000 copies/ml participating in a prospective, randomized, open-label study of standard dose versus concentration-controlled therapy with zidovudine, lamivudine, and indinavir. METHODS: Peripheral blood mononuclear cells and plasma were collected frequently throughout the study for quantitation of intracellular zidovudine triphosphate and lamivudine triphosphate concentrations, and zidovudine and lamivudine concentrations in plasma. CD4 T cells and HIV RNA in plasma (Roche Amplicor Ultrasensitive Assay) were measured at baseline and every 4 weeks throughout the study. Relationships among intracellular and plasma concentrations, and CD4 T cells and HIV RNA in plasma were investigated with regression analyses. RESULTS: Significant relationships were observed between the intracellular concentrations of zidovudine triphosphate and lamivudine triphosphate and the baseline level of CD4 cells. Lamivudine triphosphate concentrations were related in a linear manner to the apparent oral clearance of lamivudine from plasma. A direct linear relationship was found between the intracellular concentrations of zidovudine triphosphate and lamivudine triphosphate. The percent change in CD4 cells during therapy and the rate of decline in HIV RNA in plasma were related to the intracellular concentrations of zidovudine triphosphate and lamivudine triphosphate. CONCLUSION: These studies into the intracellular clinical pharmacology of nucleoside reverse transcriptase inhibitors illustrate potential clinical implications as determinants of therapeutic success. Moreover, these findings provide several leads and a strong impetus for future investigations with nucleoside reverse transcriptase inhibitors particularly when given in combination and sequentially.

Affinity labelling of the tick-borne encephalitis virus RNA replicase proteins by 4-N-exo-base-substituted photoreactive CTP analogs.

4-N-exo-base-substituted photoreactive analogs of CTP were designed and synthesized. Two flavivirus proteins NS5 and NS3 are shown to be labelled after RNA synthesis in the presence of the analogs, irradiation by UV-light (313 nm) and subsequent [alpha-32P]NTP incorporation.

Pharmacologically directed design of the dose rate and schedule of 2',2'-difluorodeoxycytidine (Gemcitabine) administration in leukemia.

The objective of this study was to determine the dose rate of 2',2'-difluorodeoxycytidine (dFdC) that maximizes the accumulation of the active 5'-triphosphate (dFdCTP) in circulating leukemia cells during therapy. The investigational approach was to evaluate the relationship between plasma dFdC and the accumulation of dFdCTP by circulating leukemia cells during infusion of different dFdC dose rates in the same individuals. Four patients with relapsed leukemia were treated weekly with two or three consecutive infusions of 800 mg/m2, the first administered over 1 h, the second over 2 h, and the third over 3 h. Two patients, one with acute myelogenous leukemia and one with acute lymphocytic leukemia, received all three infusions, but thrombocytopenia prohibited infusion of the third dose to two patients with chronic lymphocytic leukemia. The average steady-state plasma dFdC levels, achieved within 15 min after the infusion began, were 43.8 microM during infusion of 800 mg/m2/h, 9.4 microM during infusion of 400 mg/m2/h, and 5.6 microM at 267 mg/m2/h. The median area under the concentration times time curve of dFdCTP in leukemia cells during infusion was increased 2.3- and 5.1-fold for the 2- and 3-h infusions, respectively. In vitro incubations of leukemia cells from the four patients with 2.5-100 microM dFdC for 1 h showed that the maximum cellular accumulation of dFdCTP was produced by 15-20 microM dFdC. We conclude that a dose rate of greater than 400 mg/m2/h was required to achieve plasma dFdC levels that supported the maximum rate of dFdCTP accumulation in leukemia cells.