Characterisation of the absorption, distribution, metabolism, excretion and mass balance of doravirine, a non-nucleoside reverse transcriptase inhibitor in humans.

Absorption, distribution, metabolism and elimination of doravirine (MK-1439), a novel non-nucleoside reverse transcriptase inhibitor, were investigated. Two clinical trials were conducted in healthy subjects: an oral single dose [14 C]doravirine (350 mg, ∼200 µCi) trial (n = 6) and an intravenous (IV) single-dose doravirine (100 µg) trial (n = 12). In vitro metabolism, protein binding, apparent permeability and P-glycoprotein (P-gp) transport studies were conducted to complement the clinical trials. Following oral [14 C]doravirine administration, all of the administered dose was recovered. The absorbed dose was eliminated primarily via metabolism. An oxidative metabolite (M9) was the predominant metabolite in excreta and was the primary circulating metabolite (12.9% of circulating radioactivity). Following IV administration, doravirine clearance and volume of distribution were 3.73 L/h (95% confidence intervals (CI) 3.09, 4.49) and 60.5 L (95% CI 53.7, 68.4), respectively. In vitro, doravirine is not highly bound to plasma proteins (unbound fraction 0.24) and has good passive permeability. The metabolite M9 was generated by cytochrome P450 3A (CYP3A)4/5-mediated oxidation. Doravirine was a P-gp substrate but P-gp efflux is not expected to play a significant role in limiting doravirine absorption or to be involved in the elimination of doravirine. In conclusion, doravirine is a low clearance drug, primarily eliminated by CYP3A-mediated metabolism.

Efavirenz reduces renal excretion of lamivudine in rats by inhibiting organic cation transporters (OCT, Oct) and multidrug and toxin extrusion proteins (MATE, Mate).

Efavirenz (EFV) is a non-nucleoside reverse transcriptase inhibitor used in first-line combination antiretroviral therapy (cART). It is usually administered with nucleoside reverse transcriptase inhibitors (NRTI), many of which are substrates of OCT uptake solute carriers (SLC22A) and MATE (SLC47A), P-gp (MDR1, ABCB1), BCRP (ABCG2), or MRP2 (ABCC2) efflux transporters. The aim of this study was to evaluate the inhibitory potential of efavirenz towards these transporters and investigate its effects on the pharmacokinetics and tissue distribution of a known Oct/Mate substrate, lamivudine, in rats. Accumulation and transport assays showed that efavirenz inhibits the uptake of metformin by OCT1-, OCT2- and MATE1-expressing MDCK cells and reduces transcellular transport of lamivudine across OCT1/OCT2- and MATE1-expressing MDCK monolayers. Only negligible inhibition of MATE2-K was observed in HEK-MATE2-K cells. Efavirenz also reduced the efflux of calcein from MDCK-MRP2 cells, but had a rather weak inhibitory effect on Hoechst 33342 accumulation in MDCK-MDR1 and MDCK-BCRP cells. An in vivo pharmacokinetic interaction study in male Wistar rats revealed that intravenous injection of efavirenz or the control Oct/Mate inhibitor cimetidine significantly reduced the recovery of lamivudine in urine and greatly increased lamivudine retention in the renal tissue. Co-administration with efavirenz or cimetidine also increased the AUC0-∞ value and reduced total body clearance of lamivudine. These data suggest that efavirenz is a potent inhibitor of OCT/Oct and MATE/Mate transporters. Consequently, it can engage in drug-drug interactions that reduce renal excretion of co-administered substrates and enhance their retention in the kidneys, potentially compromising therapeutic safety.

An accelerated background subtraction algorithm for processing high-resolution MS data and its application to metabolite identification.

BACKGROUND: Metabolite identification without radiolabeled compound is often challenging because of interference of matrix-related components. RESULTS: A novel and an effective background subtraction algorithm (A-BgS) has been developed to process high-resolution mass spectral data that can selectively remove matrix-related components. The use of a graphics processing unit with a multicore central processing unit enhanced processing speed several 1000-fold compared with a single central processing unit. A-BgS algorithm effectively removes background peaks from the mass spectra of biological matrices as demonstrated by the identification of metabolites of delavirdine and metoclopramide. CONCLUSION: The A-BgS algorithm is fast, user friendly and provides reliable removal of matrix-related ions from biological samples, and thus can be very helpful in detection and identification of in vivo and in vitro metabolites.

Profile of disposition, tissue distribution and excretion of the novel anti-human immunodeficiency virus (HIV) agent W-1 in rats.

The purpose of this study was to characterize the disposition, distribution, excretion and plasma protein binding of 6-benzyl-1-benzyloxymethyl-5-iodouracil (W-1) in rats. Concentrations of W-1 within biological samples were determined using a validated high performance liquid chromatography method. The plasma protein binding of W-1 was examined by equilibrium dialysis method. After oral administration of W-1 (50, 100 and 200 mg/kg, respectively) in self-microemulsifying drug delivery system formulation, the pharmacokinetic parameters of W-1 were as follows: the peak plasma concentrations (C max) were 0.42, 1.50 and 2.55 µg/mL, the area under the curve (AUC0-t) were 0.89, 2.27 and 3.96 µg/h mL and the plasma half-life (t 1/2) were 5.15, 3.77 and 3.77 h, respectively. Moreover, the prototype of W-1 was rapidly and extensively distributed into fifteen tissues, especially higher concentrations were detected in intestine, stomach and liver, respectively. The plasma protein binding of W-1 in rat, beagle dog and human were in the range of 97.96-99.13 %. This study suggested that W-1 has an appropriate pharmacokinetics in rats, such as rapid absorption, moderate clearance, and rapid distribution to multiple tissues. Those properties provide important information for further development W-1 as an anti-HIV-1 drug candidate.

Clinical pharmacology of tenofovir clearance: a pharmacokinetic/pharmacogenetic study on plasma and urines.

The HIV virus and hepatitis B virus nucleotide reverse transcriptase inhibitor tenofovir has been associated with proximal tubular toxicity; the latter was found to be predicted by plasma concentrations and with single-nucleotide polymorphisms in transporters-encoding genes. A cross-sectional analysis in adult HIV-positive patients with estimated creatinine clearance >60 ml min-1 was performed. Twelve-hour plasma and urinary tenofovir concentrations and single-nucleotide polymorphisms in several transporter-encoding genes were analysed. In 289 patients 12-h tenofovir plasma, urinary and urinary to plasma ratios were 69 ng ml-1 (interquartile range 51.5-95), 24.3 mg ml-1 (14.3-37.7) and 384 (209-560). At multivariate analysis estimated creatinine clearance, protease inhibitors co-administration and SLC28A2 CT/TT genotypes were independently associated with plasma tenofovir exposure; ABCC10 GA/AA genotypes and protease inhibitor co-administration were independently associated with the urinary to plasma tenofovir ratio. Tenofovir clearance was associated with genetic polymorphisms in host genes and with co-administered drugs: if confirmed by ongoing studies these data may inform treatment tailoring and/or dose reductions.

In Vivo Profiling and Distribution of Known and Novel Phase I and Phase II Metabolites of Efavirenz in Plasma, Urine, and Cerebrospinal Fluid.

Efavirenz (EFV) is principally metabolized by CYP2B6 to 8-hydroxy-efavirenz (8OH-EFV) and to a lesser extent by CYP2A6 to 7-hydroxy-efavirenz (7OH-EFV). So far, most metabolite profile analyses have been restricted to 8OH-EFV, 7OH-EFV, and EFV-N-glucuronide, even though these metabolites represent a minor percentage of EFV metabolites present in vivo. We have performed a quantitative phase I and II metabolite profile analysis by tandem mass spectrometry of plasma, cerebrospinal fluid (CSF), and urine samples in 71 human immunodeficiency virus patients taking efavirenz, prior to and after enzymatic (glucuronidase and sulfatase) hydrolysis. We have shown that phase II metabolites constitute the major part of the known circulating efavirenz species in humans. The 8OH-EFV-glucuronide (gln) and 8OH-EFV-sulfate (identified for the first time) in humans were found to be 64- and 7-fold higher than the parent 8OH-EFV, respectively. In individuals (n = 67) genotyped for CYP2B6, 2A6, and CYP3A metabolic pathways, 8OH-EFV/EFV ratios in plasma were an index of CYP2B6 phenotypic activity (P < 0.0001), which was also reflected by phase II metabolites 8OH-EFV-glucuronide/EFV and 8OH-EFV-sulfate/EFV ratios. Neither EFV nor 8OH-EFV, nor any other considered metabolites in plasma were associated with an increased risk of central nervous system (CNS) toxicity. In CSF, 8OH-EFV levels were not influenced by CYP2B6 genotypes and did not predict CNS toxicity. The phase II metabolites 8OH-EFV-gln, 8OH-EFV-sulfate, and 7OH-EFV-gln were present in CSF at 2- to 9-fold higher concentrations than 8OH-EFV. The potential contribution of known and previously unreported EFV metabolites in CSF to the neuropsychological effects of efavirenz needs to be further examined in larger cohort studies.

Development and Validation of a Sensitive LC-MS-MS Method for the Determination of Adefovir in Human Serum and Urine: Application to a Clinical Pharmacokinetic Study.

A rapid and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of adefovir (PMEA,9-(2-phosphonylmethoxyethyl) adenine) concentration in human serum and urine. The analysis was performed on a negative ionization electrospray mass spectrometer via multiple reaction monitoring. The monitored transitions were set at m/z 272.0 → 134.0 and m/z 276.0 → 149.8 for PMEA and internal standard, respectively. After protein precipitation, samples were separated by high-performance liquid chromatography on a reversed-phase Dikma Diamonsil C18 (250 × 4.6 mm; 5 µm) column with a mobile phase of 0.1 mM ammonium formate buffer-methanol. The calibration curves were linear over the serum concentration range 0.5-1,000 ng/mL and urine concentration range 2.0-1,000 ng/mL. The intra- and interday precision values of PMEA in both serum and urine were lower than 18.16% for low quality control and 13.70% for medium and high quality control. The accuracy, recovery, matrix factor and stability were also within the acceptable limits. The developed method was successfully applied to the pharmacokinetic study of following oral administration of single dose of pradefovir mesylate (10, 30, 60, 90 and 120 mg) and adefovir dipivoxil (10 mg) to healthy Chinese volunteers.

Water-compatible molecularly imprinted polymer as a sorbent for the selective extraction and purification of adefovir from human serum and urine.

A molecularly imprinted polymer has been synthesized to specifically extract adefovir, an antiviral drug, from serum and urine by dispersive solid-phase extraction before high-performance liquid chromatography with UV analysis. The imprinted polymers were prepared by bulk polymerization by a noncovalent imprinting method that involved the use of adefovir (template molecule) and functional monomer (methacrylic acid) complex prior to polymerization, ethylene glycol dimethacrylate as cross-linker, and chloroform as porogen. Molecular recognition properties, binding capacity, and selectivity of the molecularly imprinted polymers were evaluated and the results show that the obtained polymers have high specific retention and enrichment for adefovir in aqueous medium. The new imprinted polymer was utilized as a molecular sorbent for the separation of adefovir from human serum and urine. The serum and urine extraction of adefovir by the molecularly imprinted polymer followed by high-performance liquid chromatography showed a linear calibration curve in the range of 20-100 µg/L with excellent precisions (2.5 and 2.8% for 50 µg/L), respectively. The limit of detection and limit of quantization were determined in serum (7.62 and 15.1 µg/L), and urine (5.45 and 16 µg/L). The recoveries for serum and urine samples were found to be 88.2-93.5 and 84.3-90.2%, respectively.

Molecularly imprinted nanoparticles prepared by miniemulsion polymerization as a sorbent for selective extraction and purification of efavirenz from human serum and urine.

A molecularly imprinted polymer (MIP) has been synthesized in order to specifically extract efavirenz from serum and urine by dispersive solid-phase extraction following by HPLC-UV analysis. The imprinted nanoparticles were prepared by miniemulsion polymerization method using efavirenz as template molecule and methacrylic acid as functional monomer. Molecular recognition properties, binding capacity and selectivity of the MIPs were evaluated and the results revealed that the obtained MIPs had high specific retention for efavirenz in aqueous medium. The MIP was used as a molecular sorbent for the separation of efavirenz from human serum and urine. The extraction of efavirenz by MIP coupled with HPLC analysis showed a linear calibration curve in the range of 50-300 µg/L with exellent precisions (3.66% and 4.6% for 100 and 300 µg/L respectively). The limit of detection (LOD) and limit of quantification (LOQ) were determind in serum (17.3 and 57.5 µg/L) and urine (10.6 and 36.2 µg/L). The maximum recoveries for serum and urine samples were found to be 95.2% and 92.7% respectively. Due to the high precision and accuracy, this method may be the UV-HPLC choice with MIP extraction for bioequivalence analysis of efavirenz in serum and urine.

Randomized, placebo-controlled single-ascending-dose study to evaluate the safety, tolerability and pharmacokinetics of the HIV nucleoside reverse transcriptase inhibitor, BMS-986001, in healthy subjects.

The objectives of this study were to evaluate the safety, tolerability and pharmacokinetics (PK) of BMS-986001 as a single oral dose in healthy male subjects. Sixty-four healthy male subjects were randomized to receive a single dose of BMS-986001 or placebo in this single-blind, placebo-controlled, sequential ascending-dose study. There were eight treatment groups (10, 30, 100, 300, 600, and 900 mg fed; and 100 and 300 mg fasted) of eight subjects each (BMS-986001 n = 6/placebo n = 2). BMS-986001 was well tolerated, with no serious adverse events (AEs), deaths, or discontinuations due to AEs reported. AEs were experienced by 14.6% of subjects receiving BMS-986001; however, these did not appear to be dose related and were not considered to be related to study drug. BMS-986001 was rapidly absorbed and exhibited a linear dose-exposure relationship across the dose range studied. PK appeared similar whether administered with or without food. Administration of BMS-986001 as a single dose was generally safe and well tolerated. A linear dose-exposure relationship was seen across all doses studied, with no apparent food effect. Further clinical development is warranted.

Excretion and metabolism of lersivirine (5-{[3,5-diethyl-1-(2-hydroxyethyl)(3,5-14C2)-1H-pyrazol-4-yl]oxy}benzene-1,3-dicarbonitrile), a next-generation non-nucleoside reverse transcriptase inhibitor, after administration of [14C]Lersivirine to healthy volunteers.

Lersivirine [UK-453,061, 5-((3,5-diethyl-1-(2-hydroxyethyl)(3,5-14C2)-1H-pyrazol-4-yl)oxy)benzene-1,3-dicarbonitrile] is a next-generation non-nucleoside reverse transcriptase inhibitor, with a unique binding interaction within the reverse transcriptase binding pocket. Lersivirine has shown antiviral activity and is well tolerated in HIV-infected and healthy subjects. This open-label, Phase I study investigated the absorption, metabolism, and excretion of a single oral 500-mg dose of [14C]lersivirine (parent drug) and characterized the plasma, fecal, and urinary radioactivity of lersivirine and its metabolites in four healthy male volunteers. Plasma C(max) for total radioactivity and unchanged lersivirine typically occurred between 0.5 and 3 h postdose. The majority of radioactivity was excreted in urine (approximately 80%) with the remainder excreted in the feces (approximately 20%). The blood/plasma ratio of total drug-derived radioactivity [area under the plasma concentration-time profile from time zero extrapolated to infinite time (AUC(inf))] was 0.48, indicating that radioactive material was distributed predominantly into plasma. Lersivirine was extensively metabolized, primarily by UDP glucuronosyltransferase- and cytochrome P450-dependent pathways, with 22 metabolites being identified in this study. Analysis of precipitated plasma revealed that the lersivirine-glucuronide conjugate was the major circulating component (45% of total radioactivity), whereas unchanged lersivirine represented 13% of total plasma radioactivity. In vitro studies showed that UGT2B7 and CYP3A4 are responsible for the majority of lersivirine metabolism in humans.

The effect of lopinavir/ritonavir on the renal clearance of tenofovir in HIV-infected patients.

We determined the effects of lopinavir/ritonavir on tenofovir renal clearance. Human immunodeficiency virus-infected subjects taking tenofovir disoproxil fumarate (TDF) were matched on age, race, and gender and were enrolled into one of the following two groups: group 1: subjects taking TDF plus lopinavir/ritonavir plus other nucleoside reverse transcriptase inhibitors (NRTIs); group 2: subjects taking TDF plus NRTIs and/or non-NRTIs but no protease inhibitors. Twenty-four-hour blood and urine collections were carried out in subjects for tenofovir quantification. Drug transporter genotype associations with tenofovir pharmacokinetics were examined. In 30 subjects, median (range) tenofovir apparent oral clearance, renal clearance, and fraction excreted in urine were 34.6 l/h (20.6-89.5), 11.3 l/h (6.2-22.6), and 0.33 (0.23-0.5), respectively. After adjusting for renal function, tenofovir renal clearance was 17.5% slower (P=0.04) in subjects taking lopinavir/ritonavir versus those not taking a protease inhibitor, consistent with a renal interaction between these drugs. Future studies should clarify the exact mechanism and whether there is an increased risk of nephrotoxicity.

Use of accelerator mass spectrometry to measure the pharmacokinetics and peripheral blood mononuclear cell concentrations of zidovudine.

The remarkable sensitivity of accelerator mass spectrometry (AMS) is finding many new applications in pharmacology. In this study AMS was used to measure [(14)C]-Zidovudine (ZDV) concentrations at the drug's site of action (peripheral blood mononuclear cells, PBMCs) following a dose of 520 ng (less than one-millionth of the standard daily dose) to a healthy volunteer. In addition, the pharmacokinetics of this microdose were determined and compared to previously published parameters for therapeutic doses. Microdose ZDV pharmacokinetic parameters fell within reported 95% confidence intervals or standard deviations of most previously published values for therapeutic doses. Blood, urine, stool, saliva, and isolated PBMCs were collected periodically through 96 h postdose and analyzed for ZDV and metabolite concentrations. The results showed that ZDV is rapidly absorbed and eliminated, has one major metabolite, and is sequestered in PBMCs. (14)C mass balance assessments indicated a significant portion of ZDV remained after 96 h with a much prolonged elimination half-life. Results of this study demonstrate the usefulness of microdosing and AMS as a tool for studying the pharmacokinetic characteristics, including PBMC concentrations, of ZDV and underscore the value of AMS as a tool with which to perform pharmacokinetic and mass balance studies using trace amounts of radiolabeled compound.

Pharmacokinetics and dosing recommendations of tenofovir disoproxil fumarate in hepatic or renal impairment.

BACKGROUND: Tenofovir disoproxil fumarate is the prodrug of the acyclic nucleotide reverse transcriptase inhibitor tenofovir that is indicated for use in the treatment of HIV. Tenofovir is eliminated as unchanged drug in the urine, with a significant component of active tubular secretion. The aim of this study was to evaluate the pharmacokinetics of tenofovir in subjects with renal or hepatic impairment, both of which are common in HIV-infected individuals. PATIENTS AND METHODS: HIV seronegative and otherwise healthy subjects with varying degrees of renal or hepatic function were recruited, and tenofovir pharmacokinetics were evaluated over 48 hours (hepatic impairment study) and 96 hours (renal impairment study) following a single tenofovir disoproxil fumarate 300 mg dose. Subjects with hepatic dysfunction were categorised based upon Child-Pugh-Turcotte score, and subjects with renal impairment were categorised based upon their calculated creatinine clearance (CL(CR)) using the Cockcroft-Gault method. RESULTS: As expected for a renally eliminated drug, subjects with and without hepatic impairment displayed similar tenofovir systemic exposures without evidence of substantial alterations in drug disposition, and therefore no dosage adjustments were warranted in these patients. In contrast, in subjects with renal impairment, two distinct groups were observed: (i) subjects with CL(CR)>/=50 mL/min in whom tenofovir pharmacokinetics were similar to subjects with normal renal function; and (ii) subjects with CL(CR) <50 mL/min (moderate or severe impairment) in which tenofovir renal clearance was substantially reduced and thus drug exposures increased. Subjects with end-stage renal disease (ESRD) demonstrated no extrarenal route of tenofovir elimination. Simulations of once-daily or modified dosing regimens demonstrated the need for tenofovir disoproxil fumarate dose-interval adjustment to prevent unnecessary drug accumulation. In patients with ESRD, high-flux haemodialysis efficiently removed tenofovir, with an elimination rate of 134 mL/min and an extraction coefficient of 54%. CONCLUSION: No tenofovir disoproxil fumarate dose adjustment is warranted in the setting of hepatic impairment. Tenofovir disoproxil fumarate 300 mg every 48 hours in individuals with moderate renal impairment and twice weekly corresponding to every 72-96 hours in those with severe renal impairment is recommended in order to target steady-state tenofovir exposures consistent with those observed in subjects with normal renal function receiving tenofovir disoproxil fumarate 300 mg once daily. For subjects receiving thrice-weekly 4-hour maintenance haemodialysis sessions, tenofovir disoproxil fumarate 300 mg administered every 7 days after a haemodialysis session is recommended. HIV-infected patients with significant end-organ dysfunction should be monitored in accordance with clinical practice, including close management of their viral suppression and clinical chemistries.

Systemic and renal pharmacokinetics of adefovir and tenofovir upon coadministration.

Adefovir and tenofovir are nucleotide analogs that undergo renal secretion by the human renal organic anion transporter. The pharmacokinetics of tenofovir and adefovir following the administration of tenofovir disoproxil fumarate and adefovir dipivoxil alone and together were determined in 24 healthy subjects in an 8-day, open-label, fixed-sequence study. Subjects received oral doses of adefovir dipivoxil on days 1 and 8 and oral doses of tenofovir disoproxil fumarate on days 2 to 8. Pharmacokinetic sampling was performed on days 1, 7, and 8. The plasma pharmacokinetics of tenofovir and adefovir were unaltered upon coadministration. Furthermore, the renal clearances (CLrenal) of tenofovir and adefovir were unaffected by their coadministration. The plasma Cmax values of tenofovir and adefovir were 33-fold and 340-fold lower than their Km values for the human renal organic anion transporter. These results demonstrate that coadministration of tenofovir disoproxil fumarate and adefovir dipivoxil does not result in substantial changes to their individual pharmacokinetic profiles.

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.

Determination of stavudine in human plasma and urine by high-performance liquid chromatography using a reduced sample volume.

Sensitive high-performance liquid chromatographic assays have been developed for the quantification of stavudine (2',3'-didehydro-3'-deoxythymidine, d4T) in human plasma and urine. The methods are linear over the concentration ranges 0.025-25 and 2-150 microg/ml in plasma and urine, respectively. An aliquot of 200 microl of plasma was extracted with solid-phase extraction using Oasis cartridges, while urine samples were simply diluted 1/100 with HPLC water. The analytical column, mobile phase, instrumentation and chromatographic conditions are the same for both methods. The methods have been validated separately, and stability tests under various conditions have been performed. The detection limit is 12 ng/ml in plasma for a sample size of 200 microl. The bioanalytical assay has been used in a pharmacokinetic study of pregnant women and their newborns.

Application of directly coupled LC-NMR-MS to the structural elucidation of metabolites of the HIV-1 reverse-transcriptase inhibitor BW935U83.

The human in vivo metabolism of the HIV-1 reverse transcriptase inhibitor 5-chloro-1-(2',3'-dideoxy-3'-fluoro-erythro-pentofuranosyl)uracil (BW935U83) was studied using 19F NMR spectroscopy, directly coupled LC-NMR and LC-NMR-MS. The number and relative proportions of the drug metabolites were obtained from 19F NMR spectra of whole human urine. The novel use of the continuous-flow 19F detected LC-NMR experiment yielded chromatographic retention times and 19F chemical shifts for the parent drug, the glucuronide conjugate of the parent and an early eluting polar metabolite. The parent drug and its glucuronide conjugate were easily characterised by directly coupled 1H LC-NMR spectroscopy and two-dimensional TOCSY experiments. The identification of the second metabolite was achieved using 19F NMR and directly coupled 1H LC-NMR-MS which furnished the molecular weight, and through the use of MS-MS techniques, information on the fragment ions. This species was identified as 3-fluoro-ribolactone.

Urinary metabolites of a novel quinoxaline non-nucleoside reverse transcriptase inhibitor in rabbit, mouse and human: identification of fluorine NIH shift metabolites using NMR and tandem MS.

1. The urinary metabolites of (S)-2-ethyl-7-fluoro-3-oxo-3,4-dihydro-2H-quinoxaline-carboxylic acid isopropylester (GW420867X) have been investigated in samples obtained following oral administration to rabbit, mouse and human. GW420867X underwent extensive biotransformation to form hydroxylated metabolites and glucuronide conjugates on the aromatic ring, and on the ethyl and isopropyl side-chains in all species. In rabbit urine, a minor metabolite was detected and characterized as a cysteine adduct that was not observed in mouse or man. 2. The hydroxylated metabolites and corresponding glucuronide conjugates were isolated by semi-preparative HPLC and characterized using NMR, LC-NMR and LC-MS/MS. The relative proportions of fluorine-containing metabolites were determined in animal species by 19F-NMR signal integration. 3. The fluorine atom of the aromatic ring underwent NIH shift rearrangement in the metabolites isolated and characterized in rabbit, mouse and human urine. 4. The characterization of the NIH shift metabolites in urine enabled the detection and confirmation of the presence of these metabolites in human plasma.

Urinary metabolites of a novel quinoxaline non-nucleoside reverse transcriptase inhibitor in dog, cynomolgus monkey and mini-pig.

1. The metabolism of (S)-2-ethyl-7-fluoro-3-oxo-3,4-dihydro-2H-quinoxalinecarboxylic acid isopropylester (GW420867X) has been investigated following oral administration to dog, cynomolgus monkey and mini-pig. 2. The urinary metabolites were isolated and characterized using semi-preparative HPLC, NMR and LC-MS/MS. The relative proportions of fluorine-containing metabolites were determined for each species by 19F-NMR signal integration. 3. The metabolite profiles for each species were similar, although the proportion of individual components varied, suggesting that similar metabolic pathways are involved in the biotransformation of GW420867X in the species studied. 4. The urinary metabolites indicated that the major routes of biotransformation included hydroxylation and subsequent glucuronic acid conjugation on the aromatic ring, and on the ethyl and isopropyl side chains. A component was observed in mini-pig urine that corresponded to hydroxylation and glucuronidation accompanied by loss of the fluorine atom.