Two-dimensional LC-MS/MS determination of antiretroviral drugs in rat serum and urine.

A simple, rapid, reliable and highly sensitive on-line two-dimensional reversed-phase liquid chromatography-tandem mass spectrometric (2D-LC/MS/MS) method to determine antiretroviral drugs viz., abacavir (ABC), nevirapine (NVP) and indinavir (IDV) in rat serum and urine was developed and validated. The analytes were extracted on-line from rat serum and urine by a restricted access material (RAM) column and back-flushed into the reversed-phase C18 column for separation by LC. Detection was carried out by ESI-MS/MS. The developed method showed good selectivity, accuracy and precision for quantification of the antiretroviral drugs in rat serum and urine. Quantification limits for abacavir and nevirapine were 4.0 ng ml(-1), whereas for indinavir 4.7 ng ml(-1). The calibration graphs were linear in the range of 4-50 ng ml(-1)for abacavir, nevirapine and indinavir. The method was successfully applied to study the pharmacokinetics of antiretroviral in rats.

High-performance liquid chromatographic assay for abacavir and its two major metabolites in human urine and cerebrospinal fluid.

A simple, reversed-phase HPLC assay has been developed and validated to measure the HIV-1 reverse transcriptase inhibitor abacavir and its two major metabolites, a 5'-glucuronide and a 5'-carboxylate, in human urine and cerebrospinal fluid. Sample preparation involved centrifuging to minimize particulates, then diluting the supernatant before HPLC separation and ultraviolet detection at 295 nm. The method described was used successfully to measure concentrations of abacavir and its two major metabolites in urine and cerebrospinal fluid from HIV-1 infected subjects.

1592U89, a novel carbocyclic nucleoside analog with potent, selective anti-human immunodeficiency virus activity.

1592U89, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclo pentene-1-methanol, is a carbocyclic nucleoside with a unique biological profile giving potent, selective anti-human immunodeficiency virus (HIV) activity. 1592U89 was selected after evaluation of a wide variety of analogs containing a cyclopentene substitution for the 2'-deoxyriboside of natural deoxynucleosides, optimizing in vitro anti-HIV potency, oral bioavailability, and central nervous system (CNS) penetration. 1592U89 was equivalent in potency to 3'-azido-3'-deoxythymidine (AZT) in human peripheral blood lymphocyte (PBL) cultures against clinical isolates of HIV type 1 (HIV-1) from antiretroviral drug-naive patients (average 50% inhibitory concentration [IC50], 0.26 microM for 1592U89 and 0.23 microM for AZT). 1592U89 showed minimal cross-resistance (approximately twofold) with AZT and other approved HIV reverse transcriptase (RT) inhibitors. 1592U89 was synergistic in combination with AZT, the nonnucleoside RT inhibitor nevirapine, and the protease inhibitor 141W94 in MT4 cells against HIV-1 (IIIB). 1592U89 was anabolized intracellularly to its 5'-monophosphate in CD4+ CEM cells and in PBLs, but the di- and triphosphates of 1592U89 were not detected. The only triphosphate found in cells incubated with 1592U89 was that of the guanine analog (-)-carbovir (CBV). However, the in vivo pharmacokinetic, distribution, and toxicological profiles of 1592U89 were distinct from and improved over those of CBV, probably because CBV itself was not appreciably formed from 1592U89 in cells or animals (<2%). The 5'-triphosphate of CBV was a potent, selective inhibitor of HIV-1 RT, with Ki values for DNA polymerases (alpha, beta, gamma, and epsilon which were 90-, 2,900-, 1,200-, and 1,900-fold greater, respectively, than for RT (Ki, 21 nM). 1592U89 was relatively nontoxic to human bone marrow progenitors erythroid burst-forming unit and granulocyte-macrophage CFU (IC50s, 110 microM) and human leukemic and liver tumor cell lines. 1592U89 had excellent oral bioavailability (105% in the rat) and penetrated the CNS (rat brain and monkey cerebrospinal fluid) as well as AZT. Having demonstrated an excellent preclinical profile, 1592U89 has progressed to clinical evaluation in HIV-infected patients.

High-performance liquid chromatographic determination of 2',3'-didehydro-3'-deoxythymidine, a new anti-human immunodeficiency virus agent, in human plasma and urine.

Sensitive and selective high-performance liquid chromatographic techniques have been developed for the determination of 2'-3'-didehydro-3'-deoxythymidine, d4T (BMY-27857), in human plasma and urine. The methods had linear standard curves over the concentration ranges 0.025-25.0 and 0.5-100 micrograms/ml for the plasma and urine matrices, respectively. Both methods used solid-phase extraction for isolating d4T and the internal standard, thymidine oxetane, from the biological matrix. In addition, the analytical column, mobile phase, instrumentation and chromatographic conditions used for both methods were identical. The ultraviolet absorbance of the column effluent was monitored at 266 nm. Results of analysis of quality control samples indicated that the intra-assay precision values, as measured by percent relative standard deviation, were within 12 and 3%, and accuracy samples deviated less than 10 and 5% from nominal values for the plasma and urine assays, respectively.

Pharmacokinetic evaluation of (-)-6-aminocarbovir as a prodrug for (-)-carbovir in rats.

The recently synthesized carbocyclic 2',3'-didehydro-2',3'-dideoxy-6-deoxy-6-amino-guanosine [(-)6AC] was evaluated as a prodrug for carbovir, carbocyclic 2',3'-didehydro-2',3'-dideoxyguanosine [(-)CBV] in seven male Sprague-Dawley rats. A randomized three-way cross-over design was used. Rats were assigned to receive the following treatments: a 20 mg/kg (-)6AC infusion, 40 mg/kg (-)6AC orally, and a 20 mg/kg (-)CBV infusion. Blood samples were collected over 480 min, and urine was collected for up to 48 hr. A 2- to 3-day washout period was observed between treatments. Following i.v. infusion, (-)6AC concentrations in the blood declined rapidly in a monoexponential pattern with an elimination half-life of 11.3 +/- 3.3 min (mean +/- SD, n = 7). The time-averaged total body clearance was 115.7 +/- 32.6 ml/min/kg. The fraction of the dose excreted unchanged in urine was 0.28 +/- 0.06. The fraction of the (-)6AC dose metabolized to (-)CBV was 0.48 +/- 0.14. Following oral administration of (-)6AC, the bioavailability of (-)CBV was 46.2 +/- 9.9% (n = 6) in comparison with the bioavailability of approximately 20% previously obtained after an oral dose of (-)CBV. The Cmax of (-)CBV after a 40 mg/kg oral dose of (-)6AC was 1.65 +/- 0.7 micrograms/ml as compared with the previously reported Cmax of 1.00 microgram/ml obtained after a 60 mg/kg oral dose of (-)CBV. (-)6AC has considerable potential for the improvement of the extent of absorption of (-)CBV from oral dosing.

High-performance liquid chromatographic determination of 2',3'-didehydro-3'-deoxythymidine (d4T) in human and rabbit plasma and urine and its application to pharmacokinetic studies in the rabbit.

A rapid and sensitive liquid chromatographic assay for 2',3'-didehydro-3'-deoxythymidine (d4T) in plasma and urine is described. This assay uses thymidine oxetane (TO), a synthetic precursor of d4T, as internal standard. Sample preparation involves a simple extraction of plasma or urine with 5% isopropyl alcohol in methylene chloride. The method is specific and sensitive, allowing a linear response over a 2000-fold range of concentrations in human plasma (5 ng/ml to 10 micrograms/ml) and urine (50 ng/ml to 100 micrograms/ml). This assay, developed for human plasma and urine, is also applicable to rabbit samples with minor modification. Intravenous bolus doses of 10 mg/kg d4T to rabbits showed that the plasma concentration-time profile followed a biexponential decay. Estimates of the distribution and elimination half-lives were 6.7 +/- 0.9 and 51 +/- 6 min, respectively. The total-body and renal clearances were 23.4 +/- 3.6 and 8.82 +/- 3.9 ml/min.kg, respectively. That the renal clearance exceeds the glomerular filtration rate in the rabbit suggests that d4T is actively secreted in the renal tubule. The fraction excreted unchanged in the urine was 36 +/- 8%. Similar results were obtained in the same rabbits at steady state during constant-rate intravenous infusion. Noncompartmental analysis estimates of the MRT and Vdss were 46 +/- 5 min and 1.08 +/- 0.13 L/kg, respectively.

Pharmacokinetics of 3'-fluoro-3'-deoxythymidine and 3'-deoxy-2',3'-didehydrothymidine in rats.

Concentrations of 3'-fluoro-3'-deoxythymidine (FDT) and 3'-deoxy-2',3'-didehydrothymidine (D4T) in plasma declined in a biexponential fashion. Total clearance of D4T (1.75 +/- 0.22 liters/h/kg; mean +/- standard deviation) was significantly greater than that of FDT (1.19 +/- 0.19 liters/h/kg) owing to greater renal and nonrenal clearances of the former. Steady-state volumes of distribution of FDT (1.20 +/- 0.12 liters/kg) and D4T (1.07 +/- 0.15 liters/kg) were similar.

(-)-6-Aminocarbovir pharmacokinetics and relative carbovir exposure in rats.

The potential for the metabolic conversion of (-)-6-aminocarbovir to (-)-carbovir, a potent reverse transcriptase inhibitor effective against human immunodeficiency virus, has been examined in male Sprague-Dawley rats. Plasma (-)-6-aminocarbovir concentrations declined rapidly in a biphasic manner following an iv bolus dose of 20 mg/kg. The total systemic clearance was 5.4 liter/hr/kg and the terminal t1/2 was 0.35 hr. Following iv dosing, approximately half of the dose was excreted into the urine and comprised equivalent quantities of (-)-carbovir and (-)-6-aminocarbovir. Orally administered (-)-6-aminocarbovir was rapidly absorbed (tmax of 0.39 hr and Cmax of 4.96 micrograms/ml) following a 60 mg/kg dose. Following oral administration, 32% of the dose was eliminated in the urine, and comprised (-)-carbovir (75%) and (-)-6-aminocarbovir (25%). The oral bioavailability of (-)-6-aminocarbovir was 46% by plasma AUC comparison and 33% based on urinary excretion data. Exposure to (-)-carbovir was lower following (-)-6-aminocarbovir dosing than observed following (-)-Carbovir dosing, by both the oral and iv routes.

Improved fluorometric high-performance liquid chromatographic assay for (-)-carbovir in rat blood and urine.

Carbovir is a carbocyclic guanosine analogue with potent in vitro activity against the human immunodeficiency virus. All of the activity resides in the (-)-enantiomer. An ion-paired liquid chromatographic assay for (-)-carbovir was developed on a Spherisorb C8 column with fluorescence detection (275 nm excitation, 345 nm emission). Guanosine nucleosides are fluorescent at a pH less than 2.5, and fluorescence detection resulted in a four-fold improvement in the limit of quantitation (0.039 microgram/ml) compared to the previously developed assay with ultraviolet detection. Standard curves were processed with an internal standard at (-)-carbovir concentrations of 0.039-40 micrograms/ml in whole rat blood with a solid-phase extraction technique. Total variability was less than 16% at all concentrations and less than 10% at concentrations greater than 0.3 microgram/ml. Within-day variability was less than 7.5% at concentrations greater than 0.3 microgram/ml. Urine was analyzed directly after dilution and an diethyl ether wash to remove impurities. The total coefficients of variation were less than 10% from 0.5-20 micrograms/ml in urine. The concentrations of (-)-carbovir in rat blood were detectable for as long as 8 h after intravenous and oral doses of 20 and 60 mg/kg, respectively.

Tissue distribution and metabolic disposition of zidovudine in rats.

The tissue distribution and metabolic fate of [5'-3H]zidovudine was studied in rats after a single dose of 10 mg/kg by gavage. The drug was absorbed rapidly and distributed into all tissues. Peak blood and tissue levels were observed 0.25 hr post-dose. The level of peak radioactivity in the stomach, intestine, liver, spleen, adrenals, and kidney was higher than in plasma, while in the heart, lung, thymus, lymph nodes, muscle, bone, and skin it was similar to that in plasma. Only in the testes and the brain the radioactivity was lower than in plasma. Blood and plasma radioactivity levels were nearly equivalent. A biphasic disappearance of radioactive material was observed in blood and plasma, as well as in most tissues, with a rapid decline in the early phase (0.25-4 hr) and a slower decline thereafter. The 0-24-hr urinary and fecal recoveries (mean +/- SD) of radioactive material were 78 +/- 14% and 20 +/- 9% of dose, respectively, indicating virtually complete recovery of the radioactive dose. Reversed-phase HPLC analysis indicated that approximately 88% of urinary radioactivity corresponded to unchanged zidovudine, with the remaining radioactivity accounted for by five metabolites. One of these urinary metabolites was identified as 3'-azido-3'-deoxy-5'-O-beta-D-glucopyranuronosylthymidine and another as 3'-amino-3'-deoxythymidine (AMT). The majority of fecal radioactivity (greater than 70%) corresponded to AMT. There is a component of biliary excretion in the disposition of zidovudine. At least 7% of a parenteral dose of zidovudine was secreted in the bile, primarily as 3'-azido-3'-deoxy-5'-beta-D-glucuronylazidothymidine, which may be a source of fecal AMT.

Disposition in animals of a new anti-HIV agent: 2',3'-didehydro-3'-deoxythymidine.

The pharmacokinetics and CNS penetration of the anti-human immunodeficiency virus agent 2',3'-didehydro-3'-deoxythymidine have been examined in CD-1 mice. The drug was rapidly cleared from plasma with a terminal half-life of 17 min after an iv bolus dose at 25 mg/kg. Oral absorption of 2',3'-didehydro-3'-deoxythymidine was rapid and complete (98% bioavailable) with plasma levels approximately the same as those measured after iv administration. Estimates of the total body clearance and apparent volume of distribution were 43 ml/hr and 19 ml, respectively. In the mouse, entry into the central nervous system was rapid but the concentrations were somewhat low. However, drug concentrations which were reported to be effective in inhibiting replication of the virus in cell culture, greater than 0.01 microM, could be measured in the brain after a single oral dose at 25 mg/kg. A study to examine the urinary excretion of the drug in CD rats, beagle dogs and cynomolgus monkeys showed that 2',3'-didehydro-3'-deoxythymidine was primarily renally excreted unchanged.