Plasma Protein Binding Determination for Unstable Ester Prodrugs: Remdesivir and Tenofovir Alafenamide.

Remdesivir (RDV) and tenofovir alafenamide (TAF) are prodrugs designed to be converted to their respective active metabolites. Plasma protein binding (PPB) determination of these prodrugs is important for patients with possible alteration of free fraction of the drugs due to plasma protein changes in renal impairment, hepatic impairment, or pregnancy. However, the prodrugs' instability in human plasma presents a challenge for accurate PPB determination. In this research work, two approaches were used in the method development and qualification for PPB assessment of RDV and TAF. For RDV, dichlorvos was used to inhibit esterase activity to stabilize the prodrug in plasma during equilibrium dialysis (ED). The impact of dichlorvos on protein binding was evaluated and determined to be insignificant by comparing the unbound fraction (fu) determined by the ED method with dichlorvos present and the fu determined by an ultrafiltration method without dichlorvos. In contrast to RDV, TAF degradation in plasma is ∼3-fold slower, and TAF stability cannot be improved by dichlorvos. Fit-for-purpose acceptance criteria for the TAF PPB method were chosen, and an ED method was developed based on these criteria. These two methods were then qualified and applied for PPB determinations in clinical studies.

The determination of Sulfobutylether ß-Cyclodextrin Sodium (SBECD) by LC-MS/MS and its application in remdesivir pharmacokinetics study for pediatric patients.

SBECD (Captisol®) with an average degree of substitution of 6.5 sulfobutylether functional groups (SBE = 6.5), is a solubility enhancer for remdesivir (RDV) and a major component in Veklury, which was approved by FDA for the treatment of patients with COVID-19 over 12 years old and weighing over 40 kg who require hospitalization. SBECD is cleared mainly by renal filtration, thus, potential accumulation of SBECD in the human body is a concern for patients dosed with Veklury with compromised renal function. An LC-MS/MS method was developed and validated for specific, accurate, and precise determination of SBECD concentrations in human plasma. In this method, the hexa-substituted species, SBE6, was selected for SBECD quantification, and the mass transition from its dicharged molecular ion [(M-2H)/2]2-, Molecular (parent) Ion (Q1)/Molecular (parent) Ion (Q3) of m/z 974.7/974.7, was selected for quantitative analysis of SBECD. Captisol-G (SBE-γ-CD, SBE = 3) was chosen as the internal standard. With 25 µL of formic-acid-treated sample and with a calibration range of 10.0-1000 µg/mL, the method was validated with respect to pre-established criteria based on regulatory guidelines and was applied to determine SBECD levels in plasma samples collected from pediatric patients during RDV clinical studies.

Validation of LC-MS/MS methods for determination of remdesivir and its metabolites GS-441524 and GS-704277 in acidified human plasma and their application in COVID-19 related clinical studies.

Remdesivir (RDV) is a phosphoramidate prodrug designed to have activity against a broad spectrum of viruses. Following IV administration, RDV is rapidly distributed into cells and tissues and simultaneously metabolized into GS-441524 and GS-704277 in plasma. LC-MS/MS methods were validated for determination of the 3 analytes in human plasma that involved two key aspects to guarantee their precision, accuracy and robustness. First, instability issues of the analytes were overcome by diluted formic acid (FA) treatment of the plasma samples. Secondly, a separate injection for each analyte was performed with different ESI modes and organic gradients to achieve sensitivity and minimize carryover. Chromatographic separation was achieved on an Acquity UPLC HSS T3 column (2.1 × 50 mm, 1.8 µm) with a run time of 3.4 min. The calibration ranges were 4-4000, 2-2000, and 2-2000 ng/mL, respectively for RDV, GS-441524 and GS-704277. The intraday and interday precision (%CV) across validation runs at 3 QC levels for all 3 analytes was less than 6.6%, and the accuracy was within ±11.5%. The long-term storage stability in FA-treated plasma was established to be 392, 392 and 257 days at -70 °C, respectively for RDV, GS-441524 and GS-704277. The validated method was successfully applied in COVID-19 related clinical studies.

An LC-MS/MS method for determination of tenofovir (TFV) in human plasma following tenofovir alafenamide (TAF) administration: Development, validation, cross-validation, and use of formic acid as plasma TFV stabilizer.

Tenofovir disoproxil fumarate (TDF) and tenofovir alafenamide (TAF) are both tenofovir (TFV) prodrugs, with the same active intracellular metabolite, TFV-diphosphate (TFV-DP). TAF delivers TFV-DP to target cells more efficiently and at lower doses than TDF, thereby substantially reducing systemic exposure to TFV, which results in improved bone and renal safety relative to TDF. As such, the method developed for the determination of TFV following TAF administration involved two key differences from determination of TFV following TDF administration. First, human plasma samples (500 µL) immediately upon collection were treated with 20% formic acid (40 µL) (plasma: formic acid ratio of 100:8) to minimize hydrolysis of TAF to TFV, and thereby avoided overestimation of TFV concentrations. Second, various TFV validation tests were conducted in the presence of TAF to mimic the high TAF:TFV ratios in clinical samples collected within ~2 h after dosing. The method for determination of TFV was developed and validated at a US lab and followed FDA and EMA guidelines. To support global clinical studies of TAF, the method was cross-validated (one-way) between the US lab and a China lab and was successfully used for TFV determination in plasma samples from a clinical study that involved healthy Chinese subjects.

The determination of human peripheral blood mononuclear cell counts using a genomic DNA standard and application in tenofovir diphosphate quantitation.

A fluorescent quantitation method to determine PBMC-derived DNA amounts using purified human genomic DNA (gDNA) as the reference standard was developed and validated. gDNA was measured in a fluorescence-based assay using a DNA intercalant, SYBR green. The fluorescence signal was proportional to the amount (mass) of DNA in the sample. The results confirmed a linear fit from 0.0665 to 1.17 µg/µL for gDNA, corresponding to 2.0 × 106 to 35.0 × 106 cells/PBMC sample. Intra-batch and inter-batch accuracy (%RE) was within ±15%, and precision (%CV) was <15%. Benchtop stability, freeze/thaw stability and long term storage stability of gDNA in QC sample matrix, PBMC pellets samples, and pellet debris samples, respectively, as well as dilution linearity had been established. Consistency between hemocytometry cell counting method and gDNA-based counting method was established. 6 out of 6 evaluated PBMC lots had hemocytometry cell counts that were within ±20% of the cell counts determined by the gDNA method. This method was used in conjunction with a validated LC-MS/MS method to determine the level of tenofovir diphosphate (TFV-DP), the active intracellular metabolite of the prodrugs tenofovir alafenamide (TAF) and tenofovir disoproxil fumarate (TDF), measured in PBMCs in clinical trials of TAF or TDF-containing fixed dose combinations.

Confirmation of the drug-drug interaction potential between cobicistat-boosted antiretroviral regimens and hormonal contraceptives.

BACKGROUND: Cobicistat (COBI), a CYP3A inhibitor, is a pharmacokinetic enhancer that increases exposures of the HIV protease inhibitors (PIs) atazanavir (ATV) and darunavir (DRV). The potential drug interaction between COBI-boosted PIs and hormonal contraceptives, which are substrates of intestinal efflux transporters and extensively metabolized by CYP enzymes, glucuronidation and sulfation, was evaluated. METHODS: This was a Phase I, open-label, two cohort (n=18/cohort), fixed-sequence study in healthy females that evaluated the drug-drug interaction (DDI) between multiple-dose ATV+COBI or DRV+COBI and single-dose drospirenone/ethinyl estradiol (EE). DDIs were evaluated using 90% confidence intervals of the geometric least-squares mean ratios of the test (drospirenone/EE+boosted PI) versus reference (drospirenone/EE) using lack of DDI boundaries of 70-143%. Safety was assessed throughout the study. RESULTS: 29/36 participants completed the study. Relative to drospirenone/EE alone, drospirenone area under the plasma concentration versus time curve extrapolated to infinity (AUCinf) was 1.6-fold and 2.3-fold higher, and maximum observed plasma concentration (Cmax) was unaltered, upon coadministration with DRV+COBI and ATV+COBI, respectively. EE AUCinf decreased 30% with drospirenone/EE + DRV+COBI and was unchanged with ATV+COBI + drospirenone/EE, relative to drospirenone/EE alone. Study treatments were generally well tolerated. The majority of adverse events were mild and consistent with known safety profiles of the compounds. CONCLUSIONS: Consistent with COBI-mediated CYP3A inhibition, drospirenone exposure increased following coadministration with COBI-containing regimens, with a greater increase with ATV+COBI. Thus, clinical monitoring for drospirenone-associated hyperkalaemia is recommended with DRV+COBI and ATV+COBI should not be used with drospirenone. Lower EE exposure with DRV+COBI may be attributed to inductive effects of DRV on CYP enzymes and/or intestinal efflux transporters (that is, P-gp) involved in EE disposition.

Quantitation of intracellular triphosphate metabolites of antiretroviral agents in peripheral blood mononuclear cells (PBMCs) and corresponding cell count determinations: review of current methods and challenges.

INTRODUCTION: Peripheral blood mononuclear cells (PBMCs) are a critical component of the immune system and the target cells for human immunodeficiency virus, type 1 (HIV-1) infection. Nucleoside/nucleotide analogs for the treatment of HIV infection are prodrugs that require cellular activation to triphosphate (TP) metabolites for antiviral activity. A reliable method of PBMC isolation and subsequent cell counting, as well as an accurate bioanalytical determination of the TPs in PBMCs are important for understanding the intracellular pharmacokinetic (PK) of the TPs and its correlation with plasma PK, the drug effect, and dose determination. Areas covered: The authors review the challenges and solutions in PBMC sample collection, sample processing, cell lysis, cell counting methods, analyte extraction, and liquid chromatography/tandem mass spectrometry (LC-MS/MS) quantitative analysis of the nucleoside reverse transcriptase inhibitor-triphosphate (NRTI-TP) metabolites, and analogs. Expert opinion: Analyzing large numbers of clinical PBMC samples for determination of NRTI-TPs and analogs in PBMCs requires not only a validated LC-MS/MS bioanalytical method but also reliable methods for PBMC isolation, counting, cell lysis, and analyte recovery, and an approach for assessing analyte stability. Furthermore, a simple, consistent, and validated cell counting method often involves DNA quantitation of the PBMCs samples collected from clinical studies.

Pharmacokinetics and Safety of Velpatasvir and Sofosbuvir/Velpatasvir in Subjects with Hepatic Impairment.

BACKGROUND: The pharmacokinetics and safety of velpatasvir, a potent pangenotypic hepatitis C virus NS5A inhibitor, were evaluated in two hepatic impairment studies: a phase I study in hepatitis C virus-uninfected subjects and a phase III study (ASTRAL-4) in hepatitis C virus-infected patients. METHODS: In the phase I study, subjects with moderate or severe hepatic impairment (Child-Pugh-Turcotte Class B or C), and demographically matched subjects with normal hepatic function received a single dose of velpatasvir 100 mg. Pharmacokinetics and safety assessments were performed, and pharmacokinetic parameters were calculated using non-compartmental methods and summarized using descriptive statistics and compared statistically by geometric least-squares mean ratios and 90% confidence intervals. In ASTRAL-4, subjects with decompensated cirrhosis (Child-Pugh-Turcotte Class B) were randomized to receive treatment with either sofosbuvir/velpatasvir ± ribavirin for 12 weeks or sofosbuvir/velpatasvir for 24 weeks. Pharmacokinetic and safety assessments were performed and pharmacokinetic parameters were calculated using a non-compartmental analysis and summarized using descriptive statistics and were compared to pharmacokinetics from ASTRAL-1 [subjects without cirrhosis or with compensated (Child-Pugh-Turcotte Class A) cirrhosis]. RESULTS: In the phase I study, plasma exposures (area under the concentration-time curve) were similar in subjects with Child-Pugh-Turcotte Class B (n = 10) or Child-Pugh-Turcotte Class C hepatic impairment (n = 10) compared with normal hepatic function (n = 13). Percent free velpatasvir was similar in subjects without or with any degree of hepatic impairment. In the phase III study, velpatasvir overall exposure (area under the concentration-time curve over the 24-h dosing interval; AUCtau) was similar and sofosbuvir exposures were higher (~ 100%) for patients with Child-Pugh-Turcotte Class B hepatic impairment compared with the ASTRAL-1 population, which was not considered clinically relevant. CONCLUSIONS: No sofosbuvir/velpatasvir dose modification is warranted for patients with any degree of hepatic impairment.

Drug-Drug Interaction Studies Between Hepatitis C Virus Antivirals Sofosbuvir/Velpatasvir and Boosted and Unboosted Human Immunodeficiency Virus Antiretroviral Regimens in Healthy Volunteers.

Background: Combining antiviral regimens in the hepatitis C virus (HCV)/human immunodeficiency virus (HIV)-coinfected population can be complex as they share overlapping mechanisms for elimination that may result in drug interactions. The pharmacokinetics, safety, and tolerability of sofosbuvir/velpatasvir (SOF/VEL) with multiple antiretroviral (ARV) regimens were evaluated. Methods: Healthy volunteers were enrolled into 2 phase 1, open-label, randomized, multiple-dose, cross-over studies. SOF/VEL and ARV regimens were administered alone and in combination; ARVs (and pharmacokinetic enhancers) included atazanavir (ATV), cobicistat (COBI), darunavir (DRV), dolutegravir (DTG), efavirenz (EFV), elvitegravir (EVG), emtricitabine (FTC), lopinavir (LPV), raltegravir (RAL), rilpivirine (RPV), ritonavir (RTV), tenofovir alafenamide (TAF), and tenofovir disoproxil fumarate (TDF). Geometric least squares means ratios (coadministration:alone) and 90% confidence intervals were constructed for area under the plasma concentration-time curve over the dosing interval, maximum concentration, and trough, for all analytes. Safety and tolerability were also evaluated. Results: In total, 237 participants were enrolled. No clinically relevant differences in the pharmacokinetics (PK) of SOF, SOF metabolite GS-331007, or VEL were observed other than an approximate 50% decrease in VEL exposure when administered with EFV/FTC/TDF. No clinically relevant differences in the PK of ARVs were observed when administered with SOF/VEL. Study treatments were well tolerated, including no observed creatinine clearance changes during evaluation of TDF-containing regimens. Conclusions: SOF/VEL and ARV regimens including ATV, COBI, DRV, DTG, EVG, FTC, LPV, RAL, RPV, RTV, TAF, or TDF may be coadministered without dose adjustment. Use of SOF/VEL with EFV-containing regimens is not recommended due to an approximate 50% reduction in VEL exposure.

Pharmacokinetics and Safety of Tenofovir Alafenamide in HIV-Uninfected Subjects with Severe Renal Impairment.

Tenofovir alafenamide (TAF) is an oral prodrug of tenofovir (TFV) that has greater stability in plasma than TFV disoproxil fumarate (TDF) and circulates as intact TAF, resulting in the direct and higher lymphatic loading of and exposure to TFV diphosphate, the active moiety. Unlike TFV, TAF is minimally eliminated in urine. The pharmacokinetics (PK) of TAF and TFV in HIV-uninfected subjects with severe renal impairment and matched healthy controls were evaluated. Subjects with severe renal impairment (RI; estimated glomerular filtration rate [eGFR], 15 to 29 ml/min) and controls (eGFR, ≥90 ml/min) matched for age, gender, and body mass index received a single dose of TAF at 25 mg. Blood and urine samples for TAF and TFV PK determinations were collected over 7 days postdosing, and subjects were followed up at 14 days. A total of 14 renally impaired subjects and 13 control subjects enrolled and completed the study. The TAF maximum observed concentration in plasma (Cmax) and the area under the concentration-versus-time curve (AUC) extrapolated to infinite time (AUCinf) were 79% and 92% higher, respectively, in subjects with severe RI than the controls, primarily due to higher absorption. The TFV Cmax and AUCinf were 2.8-fold and 5.7-fold higher, respectively, in subjects with severe RI than the controls. In subjects with severe RI, TAF at 25 mg provided a TFV AUC 10 to 40% lower than that from historical TDF-based TFV exposures in subjects with normal renal function. There were no discontinuations due to adverse events. In subjects with severe RI receiving TAF at 25 mg, TAF exposures were higher than those for the controls; these differences are unlikely to be clinically meaningful. TFV exposures were higher than those for the controls but lower than the exposures in nonrenally impaired subjects on TDF-based regimens.

Pharmacokinetics of Co-Formulated Elvitegravir/Cobicistat/Emtricitabine/Tenofovir Disoproxil Fumarate After Switch From Efavirenz/Emtricitabine/Tenofovir Disoproxil Fumarate in Healthy Subjects.

BACKGROUND: Elvitegravir (EVG), a HIV integrase inhibitor, is metabolized primarily by CYP3A, and secondarily by UGT1A1/3; Efavirenz (EFV), a HIV non-nucleoside reverse transcriptase inhibitor, is metabolized by Cytochrome P450 (CYP) 2B6 and induces CYP3A and uridine diphosphate glucuronosyltransferase (UGT) with residual effects post discontinuation because of long T1/2 (40-55 hours). This study evaluated the pharmacokinetics after switching from efavirenz/emtricitabine/tenofovir disoproxil fumarate (EFV/FTC/TDF) to elvitegravir/cobicistat/emtricitabine/tenofovir disoproxil fumarate (EVG/COBI/FTC/TDF). METHODS: Healthy subjects (n = 32 including n = 8 CYP2B6 poor metabolizers) received EVG/COBI/FTC/TDF (150/150/200/300 mg) on days 1-7, and after a washout, received EFV/FTC/TDF (600/200/300 mg) on days 15-28 and switched to EVG/COBI/FTC/TDF (150/150/200/300 mg) for 5 weeks (days 29-62). Pharmacokinetic assessments occurred on days 7, 28, 35, and 42; trough samples (Ctrough) were collected periodically until day 63. Safety was assessed throughout the study. RESULTS: Twenty-nine subjects completed with 3 adverse events leading to discontinuation; no grade ≥3 adverse events were reported. Post-EFV/FTC/TDF, mean EVG area under concentration (AUCtau) was 37% and 29% lower and mean Ctrough ∼3- and ∼5-fold above IC95, respectively, on days 35 and 42, and 7-8-fold above IC95 by 5 weeks. COBI AUCtau returned to normal by day 42. EVG glucuronide, GS-9200, AUCtau was higher (46% and 32% on days 35 and 42, respectively) postswitch. CYP2B6 poor metabolizers displayed higher EFV AUCtau and Cmax (125% and 91%, respectively) versus non-poor metabolizers, and lower EVG and COBI exposures. EFV Ctrough was >IC90 (10 ng/mL) in all subjects postswitch. FTC and tenofovir (TFV) exposures were unaffected. CONCLUSIONS: After EFV/FTC/TDF to EVG/COBI/FTC/TDF switch, EVG and/or EFV exposures were in an active range. These findings support further evaluation of switching regimens in HIV-1 patients.

Population Pharmacokinetics of Boosted-Elvitegravir in HIV-Infected Patients.

Elvitegravir (EVG) is an HIV strand transfer integrase inhibitor approved for the treatment of HIV infection as a part of antiretroviral regimens containing cobicistat (COBI) or ritonavir (RTV) as a booster. The population pharmacokinetics of EVG in treatment-naive and -experienced HIV patients was determined, and the effects of demographic, biometric, and formulation covariates on EVG pharmacokinetics (PK) were evaluated. Data from 31 clinical studies (25 in healthy subjects, 6 phase 1b to phase 3 in HIV-1-infected patients) with COBI-boosted EVG studies (as EVG/co or EVG/COBI/FTC/TDF single-tablet regimen) or RTV-boosted EVG studies (EVG/r) were analyzed using NONMEM. The effect of the covariates age, sex, race, health status (healthy volunteers vs HIV patients), weight, body mass index (BMI), body surface area (BSA), creatinine clearance (estimated GFR), and formulation were evaluated. EVG PK, with COBI or RTV, was described by a 2-compartment model, with first-order absorption and elimination and an absorption lag time. A statistically significant, but not clinically relevant, effect of BSA on EVG clearance (CL) was observed. Coadministration of atazanavir or lopinavir with EVG/r had an effect on EVG CL consistent with the known interaction with these agents. No other covariate had a meaningful effect on EVG PK. EVG PK was well described in a population PK model with HIV-infected patients, with low PK variability and no relevant effect of demographic or biometric covariates.

Pharmacokinetics and safety of boosted elvitegravir in subjects with hepatic impairment.

Elvitegravir (EVG), an HIV strand transfer integrase inhibitor, is metabolized primarily via cytochrome P450 3A4 (CYP3A) and secondarily via glucuronidation. The pharmacokinetics (PK) and safety of cobicistat (COBI)-boosted EVG (EVG/co) were evaluated in subjects with impaired liver function. The enrolled subjects had stable moderate liver impairment (n = 10; Child-Pugh-Turcotte [CPT] class B) or were healthy controls (n = 10) matched for age (±5 years), gender, and body mass index (±15%). EVG/co (150/150 mg) was administered once daily for 10 days, followed by pharmacokinetic (PK) sampling. Safety was assessed throughout the study. EVG and COBI exposures were compared between the impairment and control groups, with a ≥100% increase considered clinically relevant. EVG and COBI protein binding was also measured. All enrolled subjects completed the study. The treatment-emergent adverse event (AE) incidences were comparable between the groups; all study drug-related AEs were mild. The geometric mean ratio (90% confidence interval [CI]) for EVG area under the concentration-time curve over the dosing interval (AUCtau) and maximum observed plasma concentration (Cmax) were 135% (103%, 177%) and 141% (109%, 183%), respectively. The corresponding values for COBI were 99.8% (76.0%, 131%) and 86.1% (65.4%, 113%), respectively, indicating no clinically relevant change in exposure. No correlations were observed between the EVG and COBI exposures versus CPT score. The EVG- and COBI-free fractions were similar between groups. EVG and COBI do not require dose adjustment in moderate or mild liver impairment, as no clinically relevant PK changes were observed for EVG or COBI in this special population. No PK or safety data are available for EVG or COBI in subjects with severe hepatic impairment.

Effect of food on rilpivirine/emtricitabine/tenofovir disoproxil fumarate, an antiretroviral single-tablet regimen for the treatment of HIV infection.

The effect of food on rilpivirine/emtricitabine/tenofovir disoproxil fumarate single-tablet regimen (STR) was evaluated in healthy subjects. Subjects (N = 24) received rilpivirine/emtricitabine/tenofovir disoproxil fumarate (25/200/300 mg) under fasted or fed conditions (light [390 kcal, 12 g fat]; standard [540 kcal, 21 g fat]) followed by pharmacokinetic (PK) sampling. The 90% confidence interval (CI) of the geometric mean ratio for rilpivirine, emtricitabine, tenofovir exposure was estimated for fed versus fasted dosing and light versus standard meal, with equivalence boundaries of 80 - 125%. Safety was assessed throughout study. Twenty-three subjects completed the study; one discontinued due to protocol violation. Adverse events were mild to moderate. Emtricitabine PK was unaffected. Tenofovir AUCinf was 38% and 28% higher, respectively, with standard and light meal versus fasted. Rilpivirine AUCinf and Cmax were 16% and 26% higher with a standard, and 9% and 34% with a light meal, respectively, versus fasted. Compared to standard meal, the lower limit of rilpivirine AUClast and AUCinf when taken with the light meal were narrowly below the equivalence bounds (79.9 and 79.2, respectively), rilpivirine Cmax was narrowly above (129). Rilpivirine/emtricitabine/tenofovir disoproxil fumarate should be administered with food, which can be a standard or light meal.

Pharmacokinetics of cobicistat boosted-elvitegravir administered in combination with rosuvastatin.

Statins are commonly used medications by HIV-1 patients. Elvitegravir/cobicistat/emtricitabine/tenofovir DF is a single tablet regimen for the treatment of HIV. The pharmacokinetic interaction between cobicistat-boosted elvitegravir (EVG/co) and rosuvastatin was evaluated. Breast cancer resistance protein (BCRP) and organic anion transporting polypeptide (OATP) 1B1 and 1B3 inhibition were assessed in vitro. Healthy subjects (N = 12) received a single dose of rosuvastatin 10 mg alone and in combination with EVG/co. Intensive pharmacokinetic sampling was conducted and safety was assessed throughout the study. Rosuvastatin pharmacokinetic exposure parameters were evaluated using 90% confidence intervals (CI) of the geometric mean ratio (GMR) of the test (combination) versus reference (rosuvastatin alone) using equivalence boundaries of 70-143% for AUCinf and 70-175% for Cmax . Elvitegravir and cobicistat inhibited BCRP and OATP in vitro, emtricitabine and TDF did not. Clinically, study treatments were well tolerated, with adverse events generally mild. Upon coadministration, rosuvastatin plasma concentrations increased (Cmax 89% higher), while AUCinf changes were modest (38% higher) and clinically nonrelevant, potentially driven by moderate inhibition of intestinal efflux by BCRP, and/or hepatic uptake by OATPs by EVG/co. Elvitegravir and cobicistat pharmacokinetics were comparable to historical data. Rosuvastatin may be coadministered with EVG/COBI/FTC/TDF without dose adjustment.

Idiosyncratic reactions and metabolism of sulfur-containing drugs.

INTRODUCTION: Idiosyncratic drug reactions (IDRs) that involve the formation of toxic metabolites followed by covalent binding to cellular proteins often go undiscovered until after post-marketing. The goal of this article is to review the current status of IDRs, potential mechanisms and the challenges associated with predicting drug toxicity. AREAS COVERED: The authors review the metabolic pathways of five select classes of sulfur-containing drugs (captopril, troglitazone, tienilic acid, zileuton, methimazole and sudoxicam) suggesting that bioactivation plays a crucial role in the occurrence of IDRs. The reader will gain further awareness that the sulfur atom can propagate as the bioactivation site for the formation of reactive and conceivably toxic metabolites. As such, it is the body's capacity to detoxify these drug products that may determine whether IDRs occur. EXPERT OPINION: Incomplete understanding of mechanisms culminating in IDR occurrence represents a monumental impediment toward their abrogation. Moreover, current technology utilized to predict their manifestation (including structure-toxicity relationships) is not infallible and thus, development of novel tools and strategies is indispensible. In an attempt to streamline clinical development and drug approval processes, consortiums have been instated under the US FDA Critical Path Initiative. Collectively, these parameters along with the availability of validated biomarkers and new/updated regulatory guidance could positively influence the outcome of drug toxicity profiles and direct future drug development.

The conduct of in vitro studies to address time-dependent inhibition of drug-metabolizing enzymes: a perspective of the pharmaceutical research and manufacturers of America.

Time-dependent inhibition (TDI) of cytochrome P450 (P450) enzymes caused by new molecular entities (NMEs) is of concern because such compounds can be responsible for clinically relevant drug-drug interactions (DDI). Although the biochemistry underlying mechanism-based inactivation (MBI) of P450 enzymes has been generally understood for several years, significant advances have been made only in the past few years regarding how in vitro time-dependent inhibition data can be used to understand and predict clinical DDI. In this article, a team of scientists from 16 pharmaceutical research organizations that are member companies of the Pharmaceutical Research and Manufacturers of America offer a discussion of the phenomenon of TDI with emphasis on the laboratory methods used in its measurement. Results of an anonymous survey regarding pharmaceutical industry practices and strategies around TDI are reported. Specific topics that still possess a high degree of uncertainty are raised, such as parameter estimates needed to make predictions of DDI magnitude from in vitro inactivation parameters. A description of follow-up mechanistic experiments that can be done to characterize TDI are described. A consensus recommendation regarding common practices to address TDI is included, the salient points of which include the use of a tiered approach wherein abbreviated assays are first used to determine whether NMEs demonstrate TDI or not, followed by more thorough inactivation studies for those that do to define the parameters needed for prediction of DDI.

Ophthalmic drug delivery considerations at the cellular level: drug-metabolising enzymes and transporters.

Ophthalmic drugs typically achieve < 10% ocular bioavailability. A drug applied to the surface of the eye may cross ocular-blood barriers where it may encounter metabolising enzymes and cellular transporters before it distributes to the site of action. Characterisation of ocular enzyme systems and cellular transporters and their respective substrate selectivity have provided new insight into the roles these proteins may play in ocular drug delivery and distribution. Altered metabolism and transport have been proposed to contribute to a number of ocular disease processes including inflammation, glaucoma, cataract, dry eye and neurodegeneration. As ocular enzyme and transport systems are better characterised, their properties become an integral consideration in drug design and development.

Cytochrome P450 3A expression and activity in the rabbit lacrimal gland: glucocorticoid modulation and the impact on androgen metabolism.

PURPOSE: Cytochrome P450 3A (CYP3A) is an enzyme of paramount importance to drug metabolism. The expression and activity of CYP3A, an enzyme responsible for active androgen clearance, was investigated in the rabbit lacrimal gland. METHODS: Analysis of CYP3A expression and activity was performed on lacrimal gland tissues obtained from naïve untreated and treated New Zealand White rabbits. For 5 days, treated rabbits received daily administration of vehicle or 0.1% or 1.0% dexamethasone, in the lower cul-de-sac of each eye. Changes in mRNA expression were monitored by real-time RT-PCR. Protein expression was confirmed by Western blot. Functional activity was measured by monitoring the metabolism of CYP3A probe substrates-namely, 7-benzyloxyquinoline (BQ) and [3H]testosterone. RESULTS: Cytochrome P450 heme protein was detected at a concentration of 44.6 picomoles/mg protein, along with its redox partner NADPH reductase and specifically CYP3A6 in the naïve rabbit lacrimal gland. Genes encoding CYP3A6, in addition to the pregnane-X-receptor (PXR) and P-glycoprotein (P-gp) were expressed in the untreated tissue. BQ dealkylation was measured in the naïve rabbit lacrimal gland at a rate of 14 +/- 7 picomoles/mg protein per minute. Changes in CYP3A6, P-gp, and androgen receptor mRNA expression levels were detected after dexamethasone treatment. In addition, dexamethasone treatment resulted in significant increases in BQ dealkylation and CYP3A6-mediated [3H]testosterone metabolism. Concomitant increases in CYP3A6-mediated hydroxylated testosterone metabolites were observed in the treated rabbits. Furthermore, ketoconazole, all-trans retinoic acid, and cyclosporine inhibited CYP3A6 mediated [3H]testosterone 6beta hydroxylation in a concentration-dependent manner, with IC50 ranging from 3.73 to 435 microM. CONCLUSIONS: The results demonstrate, for the first time, the expression and activity of CYP3A6 in the rabbit lacrimal gland. In addition, this pathway was shown to be subject to modulation by a commonly prescribed glucocorticoid and can be inhibited by known CYP3A inhibitors.

Disposition and biotransformation of the acetylenic retinoid tazarotene in humans.

Oral tazarotene, an acetylenic retinoid, is in clinical development for the treatment of psoriasis. The disposition and biotransformation of tazarotene were investigated in six healthy male volunteers, following a single oral administration of a 6 mg (100 microCi) dose of [14C]tazarotene, in a gelatin capsule. Blood levels of radioactivity peaked 2 h postdose and then rapidly declined. Total recovery of radioactivity was 89.2+/-8.0% of the administered dose, with 26.1+/-4.2% in urine and 63.0+/-7.0% in feces, within 7 days of dosing. Only tazarotenic acid, the principle active metabolite formed via esterase hydrolysis of tazarotene, was detected in blood. One major urinary oxidative metabolite, tazarotenic acid sulfoxide, accounted for 19.2+/-3.0% of the dose. The majority of radioactivity recovered in the feces was attributed to tazarotenic acid representing 46.9+/-9.9% of the dose and only 5.82+/-3.84% of dose was excreted as unchanged tazarotene. Thus following oral administration, tazarotene was rapidly absorbed and underwent extensive hydrolysis to tazarotenic acid, the major circulating species in the blood that was then excreted unchanged in feces. A smaller fraction of tazarotenic acid was further metabolized to an inactive sulfoxide that was excreted in the urine.