Human pharmacokinetics prediction with an in vitro–in vivo correction factor approach and in vitro drug-drug interaction profile of bictegravir, a potent integrase-strand transfer inhibitor component in approved biktarvy^® for the treatment of HIV-1 infection

Abstract

1. Bictegravir (BIC) is a potent small-molecule integrase strand-transfer inhibitor (INSTI) and a component of Biktarvy^®, a single-tablet combination regimen that is currently approved for the treatment of human immunodeficiency virus type 1 (HIV-1) infection. The in vitro properties, pharmacokinetics (PK), and drug-drug interaction (DDI) profile of BIC were characterised in vitro and in vivo.

2. BIC is a weakly acidic, ionisable, lipophilic, highly plasma protein-bound BCS class 2 molecule, which makes it difficult to predict human PK using standard methods. Its systemic plasma clearance is low, and the volume of distribution is approximately the volume of extracellular water in nonclinical species. BIC metabolism is predominantly mediated by cytochrome P450 enzyme (CYP) 3A and UDP-glucuronosyltransferase 1A1. BIC shows a low potential to perpetrate clinically meaningful DDIs via known drug metabolising enzymes or transporters.

3. The human PK of BIC was predicted using a combination of bioavailability and volume of distribution scaled from nonclinical species and a modified in vitro-in vivo correlation (IVIVC) correction for clearance. Phase 1 studies in healthy subjects largely bore out the prediction and supported the methods used. The approach presented herein could be useful for other drug molecules where standard projections are not sufficiently accurate.

 

Keywords:

• Bictegravir
• HIV-1
• single-tablet combination regimen
• human pharmacokinetics prediction
• in vitro–in vivo correction factor
• drug-drug interaction potential

Introduction

Bictegravir (BIC; Figure 1) is a potent small-molecule human immunodeficiency virus type 1 (HIV-1) integrase strand-transfer inhibitor (INSTI) with a high barrier to resistance in vitro (Tsiang et al. 2016; Santoro et al. 2020). Biktarvy^®, the once-daily, single-tablet, fixed-dose combination comprising BIC (50 mg), emtricitabine (FTC; 200 mg), and tenofovir alafenamide (TAF; 25 mg) has demonstrated good safety and tolerability, noninferior efficacy, and no treatment-emergent resistance in multiple phase 2 and 3 studies in HIV-1 infected, treatment-naive adults and in phase 3 switch studies in HIV-1 infected, virologically suppressed adults (Gallant et al. 2017; Sax, DeJesus et al. 2017; Sax, Pozniak et al. 2017; Daar et al. 2018; Molina et al. 2018). Biktarvy is indicated as a complete regimen for the treatment of HIV-1 infection in patients who have no antiretroviral treatment history or to replace the current antiretroviral regimen in those who are virologically suppressed (HIV-1 RNA <50 copies/ml) and on a stable antiretroviral regimen, with no history of treatment failure and no known resistance to any component of Biktarvy (EMA 2018). This therapy has no restrictions for food and few for drug-drug interactions (DDIs). Biktarvy is a preferred regimen for initial HIV-1 therapy and for patients switching regimens (European AIDS Clinical Society 2021; Panel on Antiretroviral Guidelines for Adults and Adolescents 2021).

Figure 1. Chemical structure of BIC.

An important process in any drug development program is the prediction of human pharmacokinetics (PK) from in vitro data and nonclinical in vivo data. Multiple methods have been proposed and used for predicting in vivo clearance (Obach et al. 1997). However, several factors can impact the accurate prediction of in vivo clearance, such as species differences in metabolic enzymes, interaction with transporters, and metabolic pathways (Di et al. 2013). Additionally, protein binding is known to impact the hepatic clearance of drugs (Jansen 1981; Berezhkovskiy 2012).

This report presents the in vitro properties and DDI profile of BIC, as well as the PK characterisation of this molecule in nonclinical species and humans. BIC was found to be highly bound to plasma proteins in humans and all nonclinical species tested and exhibited low in vivo clearance in nonclinical species. As such, this report describes an approach that was developed to successfully predict the human PK of BIC, by incorporating both in vitro-in vivo correlation (IVIVC) and protein binding.

Materials and methods

Extensive nonclinical characterisation of BIC was performed using in vitro and in vivo assays. A brief description of methodology is provided in this section; additional experimental details are provided in the Supplemental Material.

Reagents and compounds

BIC and GS-224337 (internal standard for mass spectrometry-based analysis) were synthesised by Gilead Sciences (Foster City, CA). The chemical structure of BIC is shown in Figure 1. [¹⁴C]BIC and [³H]BIC were provided by ViTrax (Placentia, CA), and the radioactive purity of both compounds was >99%. Radiolabelled triethyl amine ([¹⁴C]TEA) was purchased from PerkinElmer (Waltham, MA). All other chemicals or reagents were purchased from Sigma-Aldrich (St. Louis, MO; Gillingham, United Kingdom), VWR International (West Chester, PA), EMD Chemicals (Gibbstown, NJ), Toronto Research Chemicals (North York, ON, Canada), or Thermo Fisher Scientific (Carlsbad, CA), unless specified otherwise.

Studies to inform human PK prediction

In Vitro studies

Appropriate controls were included for each assessment and are detailed in the Supplemental Material. Concentrations of analytes were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for in vitro studies with unlabelled BIC, and high-performance liquid chromatography (HPLC) with radioflow detection for in vitro studies with radiolabelled BIC.

The physicochemical properties of BIC were measured using published methods; the distribution coefficient at pH 7.4 (logD_7.4) was determined using a reversed-phase HPLC method (Lombardo et al. 2001), and the pK_a was determined using a capillary electrophoresis method (Analiza, Cleveland, OH). The bidirectional membrane permeability of BIC (10 and 88 µM) was assessed in human colorectal adenocarcinoma (Caco-2) cell monolayers in a 24-well transwell plate assay.

The extent of BIC binding to pooled plasma (n = 3) from rats, dogs, monkeys, and humans was assessed by equilibrium dialysis for 3 h at 37 °C by QPS, LLC (Newark, DE). Pooled plasma (1 ml) containing BIC (2 μM) and compound-free phosphate buffer (1 ml) were placed into opposite sides of assembled dialysis cells separated by a semipermeable membrane (Nest Group, Southborough, MA). BIC concentrations in each cell were determined by LC-MS/MS, and quantification was by peak area ratios. The percent of BIC unbound in plasma dialysed against phosphate buffer was calculated.

The whole blood to plasma ratio (BPR) of BIC (0.5 μM; n = 3) was determined in rats, dogs, monkeys, and humans. Binding of BIC (3 μM; n = 2) to pooled mixed-sex human hepatic microsomal fraction (0.5 mg protein/ml) was also assessed.

The metabolism of BIC in cryopreserved hepatocytes from rats, dogs, monkeys, and humans was also performed (Frontage Laboratories, Exton, PA). [¹⁴C]BIC (20 μM) was incubated in a 24-well plate containing pooled cryopreserved hepatocyte suspensions (1 million cells/ml) from male rats, dogs, monkeys, and humans (Triangle Research Labs, Research Triangle Park, NC; XenoTech, Lenexa, KS) at 37 °C for 4 h under 5% CO₂:95% air. Metabolite profiling and identification were performed using an HPLC system coupled to UV, mass spectrometry, and radioactivity detectors.

The rate of hepatic metabolism of [³H]BIC (1 μM) was assessed in pooled hepatic microsomal fractions (1 mg/ml protein) from male rats, dogs, monkeys, and mixed-sex humans in the presence of NADPH regenerating system (BD Biosciences, Woburn, MA) and uridine diphosphate glucuronic acid. Formation of radioactive metabolites from [³H]BIC was quantified using peak area of radioactivity. The determined in vitro half-life (t_1/2) values were used to calculate predicted hepatic clearance (Obach et al. 1997). Predicted hepatic extraction was then calculated by comparison of predicted hepatic clearance to hepatic blood flow. A compound was considered stable if the reduction of substrate concentration was <10% over the course of the incubation; the extrapolated t_1/2 corresponded to >395 min in microsomal fractions.

Cytochrome P450 enzyme (CYP) reaction phenotyping was determined by incubating [³H]BIC (2 μM) with insect cell microsomal fractions containing baculovirus-expressed human CYP enzymes (1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4, and 3A5) coexpressed with human CYP reductase (BD Biosciences) in 0.1 M phosphate buffer (pH 7.4) at 37 °C for 45 min. Formation of radioactive metabolites from [³H]BIC was quantified using peak area of radioactivity. UDP-glucuronosyltransferase (UGT) reaction phenotyping was determined by incubating BIC (5 μM) with insect cell microsomal fractions containing baculovirus-expressed human UGT enzymes (1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17) in 0.1 M phosphate buffer (pH 7.4) at 37 °C for 60 min. Quantification of BIC-glucuronide formation was achieved using LC-MS/MS.

Nonclinical in vivo studies

An overview of the purpose and design of nonclinical studies is provided in Supplemental Table 1. All nonclinical studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the U.S. National Institutes of Health and were approved by the Institution’s Animal Care and Use Committee or local equivalent.

Table 1. In vitro ADME properties of BIC.

Molecular weight	449.38 Da
pKa	8.3
LogD7.4	2.1
Kinetic Solubility (pH 7)	88 µM
Caco-2 Papp (× 10^-6cm/s)	BIC (10 µM): 6.2, 27.2, 4.4
AB, BA, ER	BIC (88 µM): 14.8, 22.6, 1.5
Biopharmaceutics Classification System	Class 2
	Rat	Dog	Cynomolgus Monkey	Rhesus Monkey	Human
Blood to plasma ratio (mean)	0.58	0.60	0.65	0.62	0.64
Plasma protein binding (fu,plasma; mean unbound %)	0.01	1.24	0.31	0.32	0.25
Liver microsomal protein binding (mean unbound %)	NA	NA	NA	NA	86.3
Rate of metabolism (t1/2, min)^a	49	108	63	76	194
CLint (l/h/kg)^a	1.72	0.35	0.59	0.49	0.19
Predicted hepatic extraction (%)	29	16	27	18	13

AB: apical to basolateral permeability; BA: basolateral to apical permeability; BIC: bictegravir; CL_int: intrinsic clearance; ER: efflux ratio; f_u,plasma: fraction unbound in plasma; NA: not applicable; P_app: apparent permeability coefficient.

^aDetermined in hepatic microsomes fortified with NADPH and UDPGA (see materials and methods).

BIC PK was determined in rats, dogs, and monkeys (Agilux Laboratories, Worcester, MA; Covance Laboratories, Madison, WI; MPI Research, Mattawan, MI) following single-dose intravenous infusion for 30 min or oral administration. The dosing vehicle was 5% ethanol, 55% polyethylene glycol 300, and 40% water for both intravenous administration in rats, dogs, and monkeys, and for oral administration in rats. The oral dosing vehicle for dogs and monkeys was 30% Captisol (Ligand, San Diego, CA) in water. Serial blood samples were collected, and BIC plasma concentrations were quantified using an LC-MS/MS method.

Human PK prediction

The in vitro hepatic metabolic clearance of BIC was assessed from the rates of metabolism of [³H]BIC in pooled hepatic microsomal fractions from rats, dogs, monkeys, and humans, as described previously. The stability of BIC in hepatic microsomal fractions, expressed as in vitro t_1/2 values, was scaled using the well-stirred liver model (Obach et al. 1997) to predict hepatic metabolic clearance (without plasma protein binding restriction). To predict in vivo clearance, well-stirred model-based methods were applied to the in vitro predicted hepatic clearance in nonclinical species with and without consideration of binding to plasma and microsomal proteins. The human volume of distribution at steady state (V_ss) was predicted from the V_ss observed from all evaluated nonclinical species. The absorption rate constant (k_a) was assessed following administration of BIC tablets in pentagastrin-pretreated fasted dogs, which is a standard nonclinical model to evaluate the pharmacokinetics of human oral formulations (Lentz, 2008; Kesisoglou, 2014). In pentagastrin-pretreated fasted dogs the stomach pH is similar to that in humans and PK comparator studies showed similar trends in changes of plasma exposure profiles of orally dosed drugs (Zane et al. 2014). These studies support the hypothesis that the k_a in pentagastrin-pretreated dogs is an appropriate approach for predicting k_a in fasted humans. The oral bioavailability was estimated from the observed bioavailability following oral administration of BIC solution to rat, dog, and monkey (Supplemental Table 1).

Human Single and multiple ascending dose PK study

Study Design

BIC PK following single and multiple ascending oral doses of BIC was evaluated in a phase 1, double-blind, randomised, placebo-controlled, single-centre study in healthy male and female subjects. The study (GS-US-141-1218) was approved by an institutional review board before initiation and was conducted in accordance with recognised international scientific and ethical standards, including, but not limited to, the International Conference on Harmonisation guideline for Good Clinical Practice and the principles embodied in the Declaration of Helsinki. Written informed consent was obtained from all subjects. An overview of the design of this study is presented in Supplemental Table 1.

Screening evaluations began no later than 28 days prior to the Day −1 visit. Eligible subjects were randomised 6:2 within each cohort to receive either BIC or placebo. In part A of the study, single doses of BIC or placebo were administered in cohorts 1 to 6 on Day 1 under fasted conditions. Dosing in a subsequent cohort began no earlier than 7 days after single-dose administration in the previous cohort. Each cohort was followed through Day 14. In part B, BIC or placebo was administered once daily on Day 1 through Day 14 under fasted conditions to cohorts 1 to 5. Each cohort in part B was followed through Day 23.

Subjects

Subjects were healthy, non-smoking men or nonpregnant, nonlactating women, with no significant medical history, normal renal function, and normal 12-lead ECG. Subjects were negative for hepatitis B/C and HIV and had refrained from blood donation or other activities that could have affected absorption, distribution, metabolism, and excretion (ADME) assessments.

Subjects were ineligible if they engaged in substance or alcohol abuse that could have potentially interfered with their safety or compliance or had taken any over the counter or prescription medication with the exception of vitamins, hormonal contraceptive medications, acetaminophen, or ibuprofen within 28 days of initiating dosing with study drug.

In part A of the study, 48 subjects (26 males and 22 females) were randomised into the study to receive either a single oral dose of BIC immediate release tablet at doses of 5, 25, 50, 100, 300, or 600 mg (6 subjects each) or placebo (2 subjects each). The median age was 35 years (range: 20–45), median body mass index was 27 kg/m² (range: 19–30), and median estimated glomerular filtration rate (Cockcroft-Gault method) was 122 ml/min (range: 91–211). In part B of the study, 40 subjects (22 males and 18 females) were randomised into the study to receive either multiple oral doses of BIC 5, 25, 50, 100, or 300 mg (6 subjects each) or placebo (2 subjects each). The median age was 36 years (range: 23–45), median body mass index was 27 kg/m² (range: 21–30), and median estimated glomerular filtration rate (Cockcroft-Gault method) was 129 ml/min (range: 93–186).

Treatment, sampling, and bioanalysis

BIC tablets were available as the BIC sodium salt, with each tablet containing 5, 25, or 100 mg of BIC. In addition, each tablet contained microcrystalline cellulose, lactose monohydrate, crospovidone, sodium stearyl fumarate, polyvinyl alcohol, polyethylene glycol, titanium dioxide, talc, and iron oxide yellow. Study drug was administered with 240 ml of water. In part A, study drug was administered orally on Day 1 following an overnight fast. Subjects continued to fast until 4 h postdose or until after collection of the 4-hour post dose blood sample. In part B, study drug was administered orally on Day 1 through Day 14 at approximately the same time each day following an overnight fast. Subjects continued to fast until 3 h post dose on all dosing days (or until after collection of the 3-hour postdose blood sample on Days 1 and 7). Blood samples were collected at prespecified time points (Supplemental Table 1). Additional blood samples were obtained at early termination, if applicable, and at the follow-up visit. For part B, the 24-hour postdose sample was collected prior to dosing on Days 2 and 8. Predose trough samples were collected on Days 4, 5, 6, 10, 11, 12, and 14. BIC concentrations in plasma were quantified using a validated LC-MS/MS method; details are provided in the Supplemental Material.

Pharmacokinetic Analysis

Plasma PK parameters for BIC in the nonclinical and healthy subject studies were estimated via noncompartmental analysis using the Phoenix WinNonlin software version 6.3 (Pharsight Corporation, Mountain View, CA). Details on the estimated PK parameters are provided in the Supplemental Material.

Studies to inform Drug-Drug interaction assessments

CYP and UGT inhibition and phenotyping

The potential for BIC to inhibit the major human drug metabolising CYP enzymes (1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4) was assessed by incubating BIC (0–100 μM) or control inhibitors (Supplemental Material) with pooled mixed-sex human liver microsomes and NADPH in the presence of probe substrates for individual enzymes (Cyprotex, Macclesfield, UK). The potential for BIC to inhibit UGT1A1 was assessed in duplicate using the method of Fisher with minor modifications (Fisher et al. 2000). BIC (0–300 µM) or control inhibitor (atazanavir or ritonavir) was incubated with pooled mixed-sex hepatic microsomal protein (0.3 mg/ml; BD Biosciences), alamethicin (15 µg/ml), uridine diphosphate glucuronic acid (5 mM), and the probe substrate, β-oestradiol (17 µM). The production of enzyme-specific metabolites was determined by LC-MS/MS. Half-maximal inhibitory concentration (IC₅₀) values were calculated by nonlinear regression of the enzyme activity versus inhibitor concentration data.

The potential for BIC-mediated, mechanism-based inhibition of CYPs (1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3 A) was assessed using a 2-stage incubation protocol at Cyprotex. BIC (final concentration 100 μM) was added to pooled mixed-sex human hepatic microsomal fractions diluted in 50 mM potassium phosphate buffer (pH 7.4), and incubation was carried out at 37 °C for 30 min in the presence or absence of 1 mM NADPH as the CYP cofactor. The second stage was used to assay the remaining enzyme activity by addition of the enzyme-specific substrate, and enzyme-specific metabolites were quantified by LC-MS/MS. Inhibition was calculated as percent change in the ratio of the metabolite peak area ratio values obtained following preincubations in the absence and presence of NADPH. Additionally, an IC₅₀ shift analysis was performed at Xenotech in order to determine the potency of BIC (0−200 μM) and controls as inhibitors of human hepatic midazolam 1′-hydroxylase activity in pooled mixed-sex microsomal fractions (0.05 mg protein/ml). Quantification of the 1′-hydroxymidazolam metabolite was by LC-MS/MS.

Induction

The induction potential of BIC (1, 3, 10, 30, and 60 μM) on CYP enzymes, UGT1A1, and P-glycoprotein (P-gp) was assessed by mRNA quantification using freshly isolated primary human hepatocytes from 3 separate adult donors (2 females and 1 male) using established methods at QPS, LLC. Appropriate positive and negative controls were included in each assessment (Supplemental Material).

Efflux Transport

BIC (10 μM) was assessed as a potential substrate or inhibitor of P-gp or breast cancer resistance protein (BCRP) using P-gp- and BCRP-transfected Madin-Darby canine kidney strain II (MDCKII) cells. The apparent permeability coefficient, non-specific binding, and compound instability were assessed by estimating the recovery in the experiment.

P-gp and BCRP inhibition by BIC (0.33–80 μM) or positive control (Supplemental Material) were assessed in P-gp- and BCRP-overexpressing MDCKII cells in the presence of fluorescent substrates calcein AM (10 μM) and pheophorbide A (1 μM), respectively. Following incubation, fluorescence changes were quantified, and IC₅₀ values were calculated.

Uptake Transport

BIC was evaluated as a potential substrate or inhibitor of uptake transporters organic anion transporting polypeptide (OATP) 1B1 and 1B3 using wild-type and human OATP1B1- and OATP1B3-transfected Chinese hamster ovary (CHO) cells. Uptake rates of BIC (1 μM) and positive and negative control compounds (0.1 μM atorvastatin and 10 μM antipyrine, respectively) were determined in the presence and absence of a known OATP inhibitor (rifampicin, 40 μM) using an oil spin assay. For the inhibition assay, BIC (0.11–80 μM) or positive control inhibitor rifampicin (10 μM) was spiked into assay buffer containing a fluorescent substrate (Fluo 3, 2 μM). Following incubation, fluorescence changes were quantified, and IC₅₀ values were calculated using GraphPad Prism 5 (GraphPad Software, San Diego, CA).

Renal Transport

To assess organic cation transporter (OCT) 2 inhibition, BIC (0.014−10 µM) or positive control dolutegravir (DTG) (10 μM) was incubated with nontransfected or OCT2-transfected MDCKII cells in the presence of [¹⁴C]TEA. Multidrug and toxin extrusion protein 1 (MATE1) inhibition was assessed at Solvo Biotechnology (Budaors, Hungary). BIC (0.11−80 µM) or positive control (100 μM quinidine) was incubated with nontransfected or MATE1-transfected CHO cells in the presence of 5 µM [¹⁴C]TEA. Samples were analysed by liquid scintillation, and fractional transport activities were calculated. Transport inhibition assays for organic anion transporter (OAT) 1, OAT3, OCT1, and bile salt export pump (BSEP) were performed at Solvo Biotechnology. To measure OAT1 and OCT1 transporter inhibition, BIC (0.14−100 µM) or positive control (200 μM benzbromarone for OAT1; 100 μM verapamil for OCT1) was incubated with transporter-overexpressing CHO cells in the presence of appropriate probe substrates (5 µM [³H]para-aminohippurate for OAT1; 5 µM [¹⁴C]TEA for OCT1). To assess OAT3 inhibition, BIC (0.14–100 µM) or positive control (200 μM probenecid) was incubated with OAT3-overexpressing Flp-In 293 cells in the presence of probe substrate (1 µM [³H]estrone-3-sulfate). Transporter-specific accumulation of the probe substrates in cells was measured by liquid scintillation/fluorescence reader and compared to its accumulation in the absence of BIC under the same assay conditions. To assess BSEP inhibition, BIC (0.1–100 μM) or positive control (20 μM cyclosporine A) was incubated with Spodoptera frugiperda ovarian cell (Sf9) membrane vesicle preparations (total protein: 50 μg/well) and probe substrate (2 µM [³H]taurocholate) in the absence or presence of ATP. Fractional transport activities were calculated, and IC₅₀ values were calculated using GraphPad Prism.

Results

In Vitro properties

A summary of the in vitro properties of BIC are presented in Table 1. BIC is a lipophilic molecule with one ionisable weak acid functional group. In Caco-2 cells, the permeability of BIC was high (apparent permeability coefficient ≥6.2 × 10⁻⁶cm/s), but BIC exhibited a concentration-dependent increase in apical to basolateral permeability and a decrease in efflux ratio indicating saturable efflux transport. Consequently, the influence of efflux transport was considered unlikely to be significant for BIC absorption at a therapeutic oral dose.

In equilibrium dialysis studies, BIC was found to be highly bound in rat, dog, monkey, and human plasma (>98% bound). There was little binding of BIC to human liver microsomes. The BPR was <1 and similar across all species tested; BIC was not preferentially distributed into erythrocytes.

The rates of metabolism (t_1/2, min) of BIC in hepatic microsomal fractions obtained from humans and nonclinical species are presented in Table 1. BIC exhibited high metabolic stability in rat, dog, monkey, and human liver microsomal fractions, with predicted hepatic extractions <30% of the hepatic blood flow. The predicted human hepatic metabolic clearance, without consideration of plasma binding, was low (0.17 l/h/kg; Table 1).

The percentages of parent drug and identified metabolites following incubation with [¹⁴C]BIC (20 μM) in cryopreserved hepatocytes are provided in Table 2. BIC was primarily metabolised via hydroxylation (3 variants), N-dealkylation, glucuronidation, and glucose conjugation. BIC was found to be minimally metabolised in rat and human hepatocytes, with metabolites accounting for <10% of total radioactivity following incubation for 4 h. BIC appeared to be more extensively metabolised in dog hepatocytes, with metabolites accounting for >20% of total radioactivity in the 4-hour incubation extract. The most extensive metabolism of BIC among the species was observed in the monkey hepatocytes, with total metabolites accounting for >47% of radioactivity in the 4-hour incubation extracts. All human metabolites were also observed in nonclinical species. CYP3A4 and 3A5 were the only two human recombinant CYP enzymes that metabolised BIC; no turnover was observed with other CYP enzymes. Among the 12 individual recombinant expressed human UGT enzymes that were screened, the recombinant human UGT1A1 formed the largest quantity of BIC-glucuronide under the conditions tested, with lesser quantities also generated by UGT1A3, 1A8, and 1A9.

Table 2. Summary of metabolites of BIC detected in cryopreserved hepatocytes.

Analyte^a	Identity	Fraction of Radiochromatogram (%)
		Rat	Dog	Monkey	Human
BIC	Parent	91.5	78.7	52.4	93.9
M305	N-dealkylation	1.7	8.7	2.4	1.2
M465a	Hydroxylation-1	1.2	1.4	2.7	ND
M465b	Hydroxylation-2	ND	0.2	11.6	0.6
M465c	Hydroxylation-3	ND	3.6	ND	ND
M611	Glucose conjugation	ND	0.8	4.4	ND
M625	Glucuronide conjugation	5.2	6.6	21.7	4.3
M641	Hydroxylation/glucuronidation	ND	ND	4.1	ND
Total	−	99.6	100	99.3	100

BIC: bictegravir; ND: not detected.

^aAnalyte metabolite identification numbers correspond to their molecular weight (e.g. M305 = metabolite with 305 Da molecular weight).

Nonclinical in vivo studies

Mean plasma concentration-time profiles of BIC (0.5 mg/kg) following a single intravenous infusion in rats, dogs, and monkeys are displayed in Figure 2(A), and the mean plasma PK parameters are presented in Table 3. Following intravenous administration in rats, dogs, and monkeys, BIC PK profile revealed a short distribution phase followed by a monophasic elimination, low clearance (0.1−1.3% of hepatic blood flow), and the V_ss ranged from 0.09 to 0.22 l/kg, which is approximately the volume of extracellular water in nonclinical species.

Figure 2. Plasma PK profile of BIC following a single-dose administration in rat, dog, and cynomolgus monkey (n = 3, mean ± S.D.). (A) Intravenous infusion (30 min) at a dose of 0.5 mg/kg; (B) oral solution administration at a dose of 0.5 mg/kg (rat) and 1 mg/kg (dog, monkey). Cyno: cynomolgus monkey.

Table 3. Nonclinical plasma PK parameters for BIC following a single-dose administration (n = 3, mean ± S.D.).

Species	Intravenous^a					Oral^b
	AUCinf (nM•h)	CL (L/hr/kg)	Vss (L/kg)	t1/2 (h)	MRT (h)	F (%)
Sprague-Dawley rat	549000 ± 45700	0.0020 ± 0.0002	0.13 ± 0.008	54.3 ± 14.4	62.0 ± 6.7	22.8 ± 7.8
Beagle dog	58,700 ± 17,700	0.022 ± 0.006	0.15 ± 0.02	5.34 ± 0.18	7.10 ± 1.32	41.8 ± 13.9
Cynomolgus monkey	49,400 ± 12,400	0.024 ± 0.007	0.095 ± 0.010	3.58 ± 0.23	4.16 ± 0.93	73.8 ± 40.3
Rhesus monkey	43,000 ± 5,050	0.026 ± 0.003	0.11 ± 0.02	3.76 ± 0.76	4.36 ± 1.30	NA

AUC_inf: area under the plasma concentration-time curve extrapolated to time infinity; CL: plasma clearance; F: oral bioavailability; MRT: mean residence time; NA: not applicable; t_1/2: estimated plasma elimination half-life; V_ss: volume of distribution at steady state.

^aIntravenous dose: 0.5 mg/kg infused over 30 min in all species.

^bOral dose: 0.5 mg/kg in rat; 1.0 mg/kg in dog and monkey.

Mean plasma concentration-time profiles of BIC (0.5 or 1.0 mg/kg) following a single oral dose in rats, dogs, and monkeys are displayed in Figure 2(B), and the mean plasma PK parameters are presented in Supplemental Table 2. BIC was quickly absorbed following oral administration, reaching observed peak plasma concentration (C_max) within 4 h postdose. The oral bioavailability of the BIC solution formulation was moderate to high (42−74%; Table 3). The mean k_a following administration of BIC tablets in pentagastrin-pretreated fasted dogs ranged from 1.5 to 2 h⁻¹.

Human Pharmacokinetics

Pharmacokinetic Prediction

Human hepatic clearance was predicted using well-stirred model-based method. In vitro hepatic clearance was scaled; (a) with no correction, (b) with correction for plasma binding, and (c) with correction for plasma and microsomal binding. The application of these methods on the in vitro clearance determined in rats, dogs, and monkeys with and without consideration of binding resulted in predicted hepatic clearance values that were markedly different than the in vivo measured clearance in these nonclinical species. Specifically, hepatic clearance was overpredicted when binding was not considered (Table 4) across all species and underpredicted when binding was considered in three out of the four nonclinical species (Table 5). An inverse correlation was observed between the plasma unbound fraction and the ratio of predicted to measured clearance (henceforth referred to as in vitro-in vivo correction factor [IVIV-CF]; Table 4) across nonclinical species. Rat had the lowest plasma unbound fraction (0.01%) and showed the largest discrepancy between predicted and observed values (IVIV-CF = 605); dog had the highest plasma unbound fraction (1.24%) and showed the lowest fold difference (IVIV-CF = 15). Cynomolgus monkey was the species with a fraction of unbound BIC in plasma mostly similar to humans (0.31% and 0.25% in monkey and human plasma, respectively). For this reason, the IVIV-CF in humans was assumed to be similar to the ratio of predicted to measured clearance in monkeys and this single-species approach did not need additional correction for binding. Therefore, an IVIV-CF of 25 was applied to the predicted human clearance, which was close to the IVIV-CF observed in cynomolgus monkey (22-fold). Applying this 25-fold factor to the uncorrected in vitro human clearance of 0.17 l/h/kg resulted in a projected in vivo human clearance of 0.007 l/h/kg.

Table 4. Comparisons between in vitro predicted clearance and in vivo measured clearance.

Species	fu,plasma (%)	In Vivo CL (L/hr/kg)		IVIV-CF^b
		Measured	Predicted^a
Rat	0.01	0.0020	1.21	605×
Dog	1.24	0.022	0.29	15×
Cynomolgus monkey	0.31	0.024	0.43	22×
Rhesus monkey	0.32	0.03	0.41	14×
Human	0.25	0.007 (projected)	0.17	25× (projected)

CL: clearance; f_u,plasma: fraction unbound in plasma; IVIV-CF: in vitro-in vivo correction factor.

^aPredicted without plasma or liver microsomal protein binding correction.

^bIVIV-CF = predicted CL/measured CL.

Table 5. Correlations between in vitro predicted clearance with plasma and liver microsomal protein binding applied and in vivo measured clearance.

Species	fu,plasma (%)	In Vivo CL (l/h/kg)
		Measured	Predicted with Plasma Binding^a	Predicted with Plasma and Liver Microsomal Binding^b
Rat	0.01	0.0020	0.00042	0.00049
Dog	1.24	0.022	0.02097	0.02407
Cynomolgus monkey	0.31	0.024	0.00492	0.00569
Rhesus monkey	0.32	0.03	0.00725	0.00838
Human	0.25	0.007 (projected)	0.00320	0.00369

CL: clearance; f_u,plasma: fraction unbound in plasma.

^aPredicted with plasma protein binding correction.

^bPredicted with plasma and liver microsomal protein binding correction. Human liver microsomal protein binding (unbound, 86.3%; Table 1) applied across all species.

The mean V_ss of BIC measured in nonclinical species ranged from 0.095 to 0.13 l/kg, which approximated the volume of extracellular water in each species. For a human weighing 70 kg, the volume of extracellular water is approximately 14 litres. Therefore, the V_ss of BIC in humans was projected to be 0.2 l/kg.

The oral bioavailability of BIC ranged from 23% to 74% in nonclinical species, with an average value of 46%. The oral bioavailability in humans was projected to be 50%, which also suggested a fraction absorbed F_a×F_g (fraction absorbed × fraction escaping gut-wall elimination) of 0.5 since hepatic extraction was predicted to be insignificant. The mean k_a following administration of BIC tablets in pentagastrin-pretreated fasted dogs ranged from 1.5 to 2 h⁻¹; therefore, the mean k_a of BIC in humans was projected to be 2 h⁻¹. The plasma concentration-time profile was simulated following multiple-dose administration of BIC 100 mg (once daily for 7 days). The projected steady-state mean PK profile of BIC is displayed in Figure 3. The projected human PK parameters of BIC, including area under the plasma concentration-time curve over the dosing interval (AUC_tau), observed concentration at the end of the dosing interval (C_tau), and t_1/2 are summarised in Table 6.

Figure 3. Clinical steady state mean PK profile following repeat dosing of BIC 100 mg once daily for 7 days in healthy subjects. Projected PK (dashed line) and observed PK (solid line) are shown (n = 6, mean ± S.D.).

Table 6. Plasma PK summary of BIC following single- and multiple-dose administration of BIC 100 mg in healthy subjects.

Parameter	Observed PK (n = 6, mean ± S.D.)			Projected PK
	Single Dose	Multiple Dose		Multiple Dose
		Day 1	Day 7	Day 7
Cmax (ng/ml)	6998 ± 2529	6248 ± 1676	9397 ± 1954	5890
Ctau (ng/ml)	NA	NA	3145 ± 820	2754
AUCinf (h•ng/ml)	163,028 ± 39,549	NA	NA	NA
AUCtau (h•ng/ml)	NA	NA	126,786 ± 30,010	102,403
AUC0-24h (h•ng/ml)	NA	79,774 ± 15,062	NA	NA
Tmax (h)^a	2.3 (1.5, 3.0)	2.5 (2.0, 3.0)	1.8 (1.5, 3.0)	1.8
t1/2 (h)^a	18.9 (18.0, 20.1)	NA	NA	19.8
CL/F (l/h)^b	0.656 ± 0.216	NA	NA	NR
Vz/F (l)^b	19.8 ± 11.409	NA	NA	NR
Accumulation ratio of AUC (%)^c	NA	NA	159 ± 19	NR

AUC: area under the plasma concentration-time curve; AUC_0-24h: area under the plasma concentration-time curve from time 0 to 24 h following the 1^st dose; AUC_inf: AUC from time 0 to infinity; AUC_tau: area under the plasma concentration-time curve over the dosing interval (24 h) following the Day 7 dose; CL/F: apparent oral clearance; C_max: observed peak plasma concentration; C_tau: observed concentration at the end of the dosing interval; NA: not applicable; NR: not reported; T_max: time to C_max; V_z/F: apparent volume of distribution.

^aData presented as median (quartile 1, 3).

^bAssuming F = 50% and a body weight of 70 kg, the predicted human CL and V_ss is 0.980 L/h and 28 L respectively.

^cAccumulation ratio of AUC = AUC_tau on Day 7/AUC_0-24h on Day 1.

Observed Human PK

The observed steady-state mean PK profiles of BIC following repeat once-daily administration of BIC 100 mg is displayed in Figure 3. Plasma PK parameters of BIC are summarised in Table 6. The median time to reach C_max (T_max) of BIC in plasma was 2.3 h following single-dose administration of BIC 100 mg, and the median t_1/2 was 18.9 h. The mean apparent oral clearance and volume of distribution were very low, consistent with in vitro and nonclinical PK data. Following administration of BIC 100 mg once daily for 7 days, the shape of the PK profile observed on Days 1 and 7 was similar to that observed following single-dose administration. On Day 7, the median T_max was 1.8 h and the mean accumulation ratio was 159%. The PK results following single ascending doses of BIC 5, 25, 50, 300, or 600 mg and multiple ascending doses of BIC 5, 25, 50, or 300 mg in healthy subjects were previously reported (Zhang, Custodio et al. 2017).

Drug-Drug interactions

A summary of in vitro DDI assessments is presented in Table 7; detailed results are presented in Supplemental Tables 3–6. BIC had little or no inhibitory effect on the activities of any of the CYP isoforms (IC₅₀ values of >100 μM for all CYPs) and was a very weak inhibitor of human UGT1A1, with a calculated IC₅₀ of 176 μM (Table 7). The potential for BIC to be a mechanism-based inhibitor of the human CYP enzymes was also assessed. BIC showed no meaningful change in inhibitory potency against CYP1A2, 2B6, 2C8, 2C9, 2C19, or 2D6, and exhibited substrate-dependent inhibition of CYP3A, determined with midazolam 1′-hydroxylase (% change relative to control of 39.8%). The effect on CYP3A activity was confirmed using the IC₅₀ shift protocol (Supplemental Figure 1). There was little difference in the effects of BIC without preincubation (direct format assay) and with preincubation in the absence of the NADPH (time-dependent format assay). Preincubation in the presence of NADPH increased the apparent inhibitory potency of BIC ≥3.1-fold, from an IC₅₀ of >200 to 64.3 μM (95% confidence interval: 39.7−104.1 μM), suggesting that BIC is a weak mechanism-based inhibitor of human CYP3A (kinetic inhibition constant for inactivation [K_I] >100 μM). Further determination of kinetic parameters was not possible due to solubility limitations and weak activity. No time-dependent inhibition was observed for any of the other enzymes evaluated.

Table 7. In vitro PK drug interaction assessment of BIC.

Assessment	Protein
Inhibition – reversible	CYPs 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3 A: IC50 > 100 µM
	UGT1A1: IC50 = 176 µM
Inhibition – time dependent	CYPs 1A2, 2B6, 2C8, 2C9, 2C19, or 2D6: none
	CYP3A: KI > 100 μM
Induction – mRNA expression	CYPs 1A2, 2C8, 2C9: none; P-gp: 5.76-fold at 60 µM BIC
	CYP2B6: EC50 = 103 μM; CYP3A4: EC50 = 19 μM
Transporter – substrate	P-gp and BCRP: yes
Transporter – inhibition	P-gp, BCRP, BSEP: IC50 > 80 µM
	OATP1B1, OATP1B3, OCT1, OAT1: IC50 > 80 µM
	OCT2: IC50 = 0.42 µM; OAT3: IC50 = 55 µM; MATE1: IC50 = 8.0 µM

BCRP: breast cancer resistance protein; BSEP, bile salt export pump; CYP: cytochrome P450 enzyme; EC₅₀: half-maximal effective concentration; IC₅₀: half-maximal inhibitory concentration; K_I: kinetic inhibition constant; MATE: multidrug and toxin extrusion; OAT: organic anion transporter; OATP: organic anion-transporting polypeptide; OCT: organic cation transporter; P-gp: P-glycoprotein; UGT: UDP-glucuronosyltransferase.

Treatment with BIC (1–60 µM) led to no significant increases (<2-fold) in CYP2C8 and 2C9 mRNA or CYP1A2 mRNA and activity (Table 7). BIC was a very weak inducer of CYP2B6, with concentration-dependent mRNA increases of up to 4.74-fold at 60-µM BIC; however, no increase in CYP2B6 activity was observed in any of the donors. BIC was a weak inducer of CYP3A4, with concentration-dependent mRNA increases of up to 16.7-fold at 60-µM BIC; however, only small increases (up to 2.6-fold) in CYP3A activity were observed. P-gp mRNA increased 5.76-fold at 60-µM BIC, with no P-gp mRNA increases observed at lower BIC concentrations.

In vitro, BIC was a substrate of human P-gp and BCRP transporters, as the efflux ratios were increased in P-gp- and BCRP-overexpressing MDCKII cells compared with nontransfected cells (Supplemental Table 5). BIC was not a substrate of OATP1B1 or OATP1B3 uptake transporters, as the rates of uptake in OATP1B1- and OATP1B3-overexpressing CHO cells (43 and 41 pmol/min/10⁶ cells, respectively) were comparable to the rate of uptake in nontransfected cells (48 pmol/min/10⁶ cells).

In vitro, BIC did not inhibit OATP1B1-, OATP1B3-, or OAT1-mediated transport when tested at concentrations up to 80 μM for OATP1B1 and OATP1B3 and 100 μM for OAT1 (Table 7). BIC showed weak inhibition of P-gp (20%), BCRP (6%), BSEP (46%), OCT1 (13%), and OAT3 (64%; IC₅₀ = 55 µM) and concentration-dependent inhibition of OCT2 (94%; IC₅₀ = 0.42 μM) and MATE1 (79%; IC₅₀ = 8 μM) at the highest concentrations tested (10 μM for OCT2; 80 μM for P-gp, BCRP, OATP1B1, OATP1B3, and MATE1; and 100 μM for BSEP, OCT1, OAT1, and OAT3).

Discussion

This report provides a comprehensive assessment of the nonclinical in vivo PK and in vitro ADME properties of BIC. BIC is a weakly acidic (pK_a = 8.3), lipophilic (LogD_7.4 = 2.1) BCS class 2 (high permeability, low solubility) molecule with an ionisable functional group and exhibits high plasma protein binding. The report also describes an example of a human prediction approach that may be applied to other molecules with significant differences in the in vitro predicted clearance and the in vivo observed clearance.

A variety of methods are available for prediction of human PK, ranging from those that are based largely upon extrapolation from PK in nonclinical species (e.g. allometric scaling) to bottom-up methods, such as physiologically based PK (PBPK) that predicts PK from basic enzymology and known human anatomy and physiology (Lavé T et al. 1999; Obach RS 1999; Mahmood I et al. 2003; Jones RD et al. 2011; Poulin P et al. 2011; Ring BJ et al. 2011; Chen Y et al. 2012; Grime KH et al. 2013). Allometric scaling methods assume homogeneity in PK processes across species and fail when there are clear species differences in drug disposition (Huang Q and Riviere JE 2014). Accurate prediction of the plasma time profile and PK parameters using a bottom-up PBPK modelling requires accurate prediction of CL which is subject to the same pitfalls we encountered when scaling in vitro to in vivo CL. Since the intravenous PK in nonclinical species showed a minimal distribution phase, monophasic elimination, V_ss similar to extracellular water and the acidic nature of BIC it is unlikely that a PBPK approach to prediction of V_ss would be markedly different. Intermediate between these two approaches are IVIVC methods that combine in vitro data, such as metabolic stability and protein binding, with PK data from nonclinical species. For human PK prediction for BIC, we employed a hybrid approach with cross-species scaling of volume of distribution and bioavailability, but a more mechanistic approach for prediction of elimination. Studies in nonclinical species indicated that excretion would be minimal, and metabolism (particularly hepatic metabolism) would be the dominant elimination process.

There are a variety of methods for using in vitro hepatic metabolic stability data to predict in vivo clearance (e.g. well-stirred liver, parallel tube, dispersion, distributed and tanks in series models), and the predicted intrinsic clearance for BIC was low by all these models. Various well-stirred model-based methods without and with protein binding were considered. Protein binding of BIC was high in plasma, with a percent unbound of <0.5% in all species tested except for in dogs (1.24%), including 0.25% unbound in humans. It seems that dog represents an outlier with regard to f_u,plasma. However, the ratio between dog and human f_u,plasma is 4.96; instead the ratio of human to rat f_u,plasma is much higher (25), suggesting that rat is indeed behaving differently. In human plasma, the low free fraction of BIC is primarily accounted for by binding to serum albumin with relatively little interaction of BIC with human α1-acid glycoprotein protein (Gilead internal data), as would be expected for an acidic molecule. The mechanism for the higher protein binding in rat compared to other species is not known. The binding to human microsomes was low (86.3% unbound); thus, consideration of microsomal binding had little impact on predicted clearance. BIC showed minimal partitioning into erythrocytes with a BPR close to 0.6 in all species tested and therefore a BPR correction was not applied for clearance prediction. Comparing in vivo clearance in nonclinical species with predicted in vitro clearance, we found that direct application of plasma free fraction to intrinsic clearance led to underprediction while ignoring plasma free fraction led to overprediction. We thus applied a model where a scaling factor was derived for clearance from nonclinical in vivo data and the corresponding in vitro data. This method was ultimately proven to be sufficiently accurate and led to the selection of BIC.

The high plasma protein binding of BIC was also reflected in its volume of distribution which approximated the volume of extracellular water (0.095 to 0.13 L/kg) in nonclinical species. The transepithelial permeability of BIC was high. Although efflux transport was observed, passive transcellular diffusion was expected to be the primary mechanism for BIC intestinal absorption. Overall, these data support a high intestinal absorption potential for BIC in humans. The oral bioavailability of BIC ranged from 23% to 74% in nonclinical species and, considering the low systemic plasma clearance, is consistent with moderate to high absorption (F_a×F_g).

The human PK parameters of BIC predicted by the described method were consistent with the BIC PK parameters observed following once-daily dosing of BIC 100 mg for 7 days in healthy human subjects; the predicted values for AUC_tau, C_tau, and t_1/2 were all within 2-fold of the observed values. Thus, the human PK of BIC was successfully predicted based on this hybrid approach that incorporated both IVIVC and protein binding, indicating that the described method could be useful to consider for the human PK prediction of other molecules with similar physicochemical and PK properties.

The in vitro metabolism profiles of BIC in nonclinical species were consistent with those observed in human. All human metabolites were also found in nonclinical species. Oxidation, direct glucuronidation, and oxidation followed by phase II conjugation were the major metabolic pathways for BIC in rats, monkeys, and humans. The in vivo metabolism profiles were also consistent with in vitro profiles (Subramanian et al. 2023). The in vitro metabolism data showed that BIC clearance was mediated by hepatic metabolism to oxidative metabolites and a direct glucuronide conjugate. These reactions were mainly catalysed by CYP3A and UGT1A1. Since CYP3A and UGT1A1 play a major role in the elimination of BIC, the systemic exposure of BIC may be altered by inducers or inhibitors of these enzymes (EMA 2018; FDA 2018).

It is important to establish whether there is a potential for interactions between BIC and the metabolism of endogenous compounds or other drugs in vivo. At concentrations of up to 100 μM, BIC did not reversibly inhibit the metabolic functions of CYP enzymes, including CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, or 3A4, or UGT1A1 in human hepatic microsomal fractions. BIC was found to be a very weak (K_I >100 μM) mechanism-based inhibitor of human CYP3A, but this is unlikely to be clinically relevant as the computed K_I greatly exceeded the unbound steady state C_max in human plasma (approximately 34 nM). BIC was found to be a weak inducer of CYP3A4 in cultured human hepatocytes but presents a low potential as an inducer of CYP3A4 at clinically relevant concentrations because it is highly bound to plasma proteins (>99% bound in humans). The low potential for clinically meaningful DDIs was confirmed in dedicated clinical studies; the plasma PK of the CYP3A4-sensitive substrate midazolam and partial substrates velpatasvir and voxilaprevir were unaffected by coadministration with the BIC/FTC/TAF fixed-dose combination (Zhang, Custodio et al. 2017; EMA 2018; FDA 2018). Additionally, multiple-dose administration of BIC resulted in no change in the plasma t_1/2 of BIC (Zhang, Custodio et al. 2017), suggesting a lack of autoinduction, and did not affect the PK of norgestimate and ethinyl oestradiol. Collectively, these data indicate that BIC is not a clinically relevant inducer of CYP3A following oral dosing (Zhang, Custodio et al. 2017; EMA 2018; FDA 2018).

BIC was a substrate for intestinal efflux transporters P-gp and BCRP in vitro, and its intestinal absorption may be decreased by inducers or increased by coadministered inhibitors of P-gp and BCRP. BIC was not an inhibitor of the hepatic transporters OATP1B1, OATP1B3, OCT1, and BSEP, or the renal transporters OAT1 and OAT3 at clinically relevant concentrations in vitro. Although BIC was an inhibitor of renal transporters OCT2 and MATE1, clinical studies showed a lack of clinically relevant changes in the plasma exposure of the OCT2 and MATE1 substrate metformin following coadministration with the BIC/FTC/TAF fixed-dose combination with no effect on pharmacodynamic characteristics of metformin, such as glucose metabolism and active lactate and glucagon-like peptide 1 levels after an oral glucose tolerance test (Zhang, West et al. 2017; EMA 2018; FDA 2018).

Overall, the high potency and favourable PK profile of BIC make it readily amenable for inclusion in the small 15 × 8-mm fixed-dose combination tablet for Biktarvy (EMA 2018; FDA 2018). BIC has a favourable in vitro resistance profile and improved activity compared with all other INSTIs, including DTG (Tsiang et al. 2016; Smith et al. 2018). Structural differences between BIC and DTG result in BIC’s increased protein binding, lower clearance, increased solubility, and higher in vitro potency against select INSTI resistance mutations compared with DTG (Gallant et al. 2017). The comprehensive nonclinical characterisation and human PK presented in this report will also provide experimentally derived parameters for future modelling, such as PBPK descriptions of BIC in various settings.

Acknowledgements

The authors thank Gene Eisenberg, Jolyn Twelves, and Irene Lepist for their role in select in vitro assays; Mike Matles for select bioanalysis; and Philip Morganelli, Peter Pyun, and Haolun Jin for BIC and reference compound synthesis. The authors thank Agilux Laboratories, Covance Laboratories, and MPI Research for conducting the nonclinical in vivo PK studies. Authors thank Sibylle Wilbert for editorial assistance of this manuscript.

Disclosure statement

All authors are current employees except that BS, GR, HZ, JC, and JW are former employees. Parts of this work were previously presented (Wang J, Lazerwith S, Morganelli P, Pyun H, Jin H, Tang J, Matles M, Mwangi J, Wang K, Eisenberg G, Murray B, Rhodes G, Zhang H, Custodio J. 2017. Prediction of bictegravir human pharmacokinetics from protein binding and in vitro-in vivo correlation. Poster session presented at: 21st North American ISSX Meeting; Sep 24−28; Providence, RI).

Additional information

Funding

This work was supported by Gilead Sciences, Inc.