Visual Overview
Abstract
Lenacapavir (LEN), a long-acting injectable, is the first approved human immunodeficiency virus type 1 capsid inhibitor and one of a few Food and Drug Administration-approved drugs that exhibit atropisomerism. LEN exists as a mixture of two class 2 atropisomers that interconvert at a fast rate (half-life < 2 hours) with a ratio that is stable over time and unaffected by enzymes or binding to proteins in plasma. LEN exhibits low systemic clearance (CL) in nonclinical species and humans; however, in all species, the observed CL was higher than the in vitro predicted CL. The volume of distribution was moderate in nonclinical species and consistent with the tissue distribution observed by whole-body autoradiography in rats. LEN does not distribute to brain, consistent with being a P-glycoprotein (P-gp) substrate. Mechanistic drug disposition studies with [14C]LEN in intravenously dosed bile duct-cannulated rats and dogs showed a substantial amount of unchanged LEN (31%–60% of dose) excreted in feces, indicating that intestinal excretion (IE) was a major clearance pathway for LEN in both species. Coadministration of oral elacridar, a P-gp inhibitor, in rats decreased CL and IE of LEN. Renal excretion was < 1% of dose in both species. In plasma, almost all radioactivity was unchanged LEN. Low levels of metabolites in excreta included LEN conjugates with glutathione, pentose, and glucuronic acid, which were consistent with metabolites formed in vitro in Hμrel hepatocyte cocultures and those observed in human. Our studies highlight the importance of IE for efflux substrates that are highly metabolically stable compounds with slow elimination rates.
SIGNIFICANCE STATEMENT LEN is a long-acting injectable that exists as conformationally stable atropisomers. Due to an atropisomeric interconversion rate that significantly exceeds the in vivo elimination rate, the atropisomer ratio of LEN remains constant in circulation. The disposition of LEN highlights that intestinal excretion has a substantial part in the elimination of compounds that are metabolically highly stable and efflux transporter substrates.
Introduction
Lenacapavir (LEN; Fig. 1) is a subcutaneously administered human immunodeficiency virus (HIV) therapeutic with properties that are well suited for a long-acting injectable (LAI; Subramanian et al., 2023). LEN is a picomolar inhibitor of human immunodeficiency virus type 1 (HIV-1) capsid protein with no known cross resistance to other current antiretroviral drug classes (Yant et al., 2019; Link et al., 2020; Margot et al., 2021). LEN displays low aqueous solubility and a remarkably low systemic clearance (CL) in nonclinical species and humans (Subramanian et al., 2023; Weber et al., 2024). LEN, in either a suspension or a solution formulation, forms a drug depot after subcutaneous (SC) administration in rats and dogs. The LEN release rate from the SC depot is much slower than its systemic elimination rate (Subramanian et al., 2023). Consequently, the apparent terminal half-life (t1/2) of LEN after SC dosing is notably higher than after intravenous (IV) dosing (flip-flop kinetics). The combined characteristics of LEN—slow and continuous release from the SC depot, a low systemic exit rate, and an exceptional potency—facilitated the clinical development of a LAI at a dose-volume suitable for SC administration (Sager et al., 2019; Begley et al., 2020; Subramanian et al., 2023). In humans, target concentrations of LEN in plasma were maintained for at least 6 months after a single 927 mg LEN SC dose (946.2 mg as LEN sodium) of the polyethylene glycol (PEG)/water solution formulation (Begley et al., 2020; Dvory-Sobol et al., 2022). LEN (Sunlenca) administered SC twice yearly, in combination with other antiretrovirals, was recently approved for heavily treatment-experienced adults with HIV-1 infection (Segal-Maurer et al., 2022; Sunlenca, 2023a, 2023b; Ogbuagu et al., 2023). LEN LAI formulations are also being investigated in clinical studies for HIV preexposure prophylaxis and, in combination with other antiretrovirals in virologically suppressed people with HIV-1.
LEN exists as a mixture of two interconvertible atropisomers that arise due to restricted rotation about the biaryl carbon-carbon bond in the molecule, denoted as LEN.1 and LEN.2. LEN.1 and LEN.2 are assigned as Sa and Ra, respectively, according to International Union of Pure and Applied Chemistry nomenclature following the Cahn-Ingold-Prelog system.
LEN exists as a mixture of two atropisomers in dynamic equilibrium (Fig. 1) that arise due to restricted rotation about the biaryl carbon-carbon bond in the molecule. LEN contains multiple fixed chiral centers, and the restricted rotation results in two interconvertible atropodiastereomers (Bringmann et al., 2005). Henceforth, these are denoted as LEN.1 and LEN.2 and referred to as atropisomers. LEN.1 and LEN.2 are assigned as Sa and Ra, respectively, according to International Union of Pure and Applied Chemistry nomenclature following the Cahn-Ingold-Prelog system. LEN.2 is the dominant species at equilibrium. Disposition studies with radiolabeled drugs quantitatively assess routes of elimination, total drug pharmacokinetics (PK), and levels of parent drug and metabolites in excreta and circulation (Penner et al., 2012). In this article, we describe the characterization of LEN atropisomer interconversion along with nonclinical PK and disposition of LEN in vitro and in vivo following IV administration in rat and dog. The results presented herein provide an understanding of the LEN disposition in nonclinical species and human.
Materials and Methods
Reagents and Compounds
LEN (Fig. 1) free acid, LEN sodium salt, LEN.2 (purified lot), and internal standards for mass spectrometry-based analyses were synthesized by Gilead Sciences (Foster City, CA). [3H]LEN (5.8 Ci/mmol; ≥ 99% radiopurity) and [14C]LEN (113 mCi/mmol; ≥ 99.2% radiopurity) incorporating the radiolabels in specific locations of the molecule (Supplemental Fig. 1) were synthesized by Vitrax (Placentia, CA) and Moravek Biochemicals (Brea, CA), respectively. All other chemicals or reagents, unless otherwise specified, were purchased from BD Biosciences (Woburn, MA), Sigma-Aldrich (St. Louis, MO; Gillingham, UK), VWR (West Chester, PA), Thermo Fisher Scientific (Carlsbad, CA), or Toronto Research Chemicals (North York, ON, Canada).
In Vitro Studies
LEN concentrations in the in vitro experiments were chosen fit-for-purpose in the context of each experiment and appropriate for the analytical sensitivity to accurately determine the property.
Protein Binding and Blood to Plasma Ratio Assessments.
The extent of binding of LEN (2 μM) in plasma, purified human serum albumin (HSA; 40 mg/mL), and human alpha-1-acid glycoprotein (hAAG; 0.8 mg/mL) were determined by equilibrium dialysis with a 24-hour incubation at 37°C. The free fraction in human hepatic microsomal fraction (0.5 mg protein/mL) was assessed at an initial LEN concentration of 2 μM with a 3-hour incubation by equilibrium dialysis at 37°C. The whole blood to plasma ratios (BPR) were determined following LEN (0.5 μM) incubation in whole blood from nonclinical species and human at 37°C for 1 hour.
Permeability, Efflux, and Uptake Transporter Assessment.
The bidirectional membrane permeability of LEN (1 μM) was assessed in human colorectal adenocarcinoma (Caco-2) cell monolayers. LEN (0.1 μM) was assessed as a substrate for efflux transporters in transwell assays using Madin Darby canine kidney cell monolayers overexpressing human multidrug resistance 1 [P-glycoprotein (P-gp)] or breast cancer resistance protein (BCRP). Transporter expression-dependent changes in the bidirectional permeability assay were confirmed using control inhibitors valspodar tested at 1 μM (P-gp) or Ko143 tested at 0.5 μM (BCRP). Additional details are provided in the Supplemental Methods.
LEN (0.025 μM) was also evaluated as a potential substrate for uptake transporters organic anion transporting polypeptide (OATP)1B1 and OATP1B3 using CHO cells transfected with the individual transporters. The uptake rate of LEN in OATP1B1- and OATP1B3-overexpressing cells was determined in the absence or presence of a control inhibitor rifampicin (40 μM; Vavricka et al., 2002). Hepatic uptake of LEN was also assayed in cryopreserved human hepatocytes in the absence or presence of a control inhibitor rifamycin (100 μM; Vavricka et al., 2002) as the inhibitor of hepatic uptake by OATP transporters. Additional details are provided in the Supplemental Methods.
Metabolic Stability and Profiling.
Metabolic stability was determined by measuring the rates of appearance of radiolabeled metabolites from the [3H]LEN (0.25 μM) substrate following incubation (2, 12, 25, 45, and 65 minutes) in the pooled hepatic microsomal fractions in the presence of a cofactor mixture that consisted of reduced nicotinamide adenine dinucleotide phosphate, uridine 5′-diphospho-glucuronic acid, and incubation (0, 1, 3, and 6 hours) in cryopreserved hepatocytes from Sprague–Dawley rat, beagle dog, and human (mixed sex). [3H]LEN with a higher specific activity (5.8 Ci/mmol) was used for a quantitative assessment of metabolite formation across species at a low substrate concentration and with minimal sample processing. The rates of metabolite formation were determined following liquid chromatography (LC) separation followed by online radioflow detection. Formation of metabolites from [3H]LEN were quantified using peak area of radioactivity. The predicted hepatic clearance was calculated using the determined in vitro t1/2 values without correcting for plasma or liver microsomal protein binding (Obach et al., 1997). The predicted hepatic extraction was computed by comparing the predicted hepatic clearance to the hepatic blood flow.
Metabolism of [14C]LEN (10 μM) was determined following incubation (2 hours) in pooled hepatic microsomes in the presence of nicotinamide adenine dinucleotide phosphate, uridine 5′-diphospho-glucuronic acid, and glutathione from Wistar Han (WH) rat, beagle dog, cynomolgus and rhesus monkey, and human. The rates of metabolism and the metabolite profiles of LEN were also assessed using Hμrel rat, dog, and human hepatic cocultures (mixed sex). Hμrel hepatic cocultures were incubated with [14C]LEN (10 μM) for 7 days with sample collection at 0, 72, and 168 hours. Metabolite formation and identification were determined following LC separation by offline radio-detection and simultaneous inline high-resolution mass spectrometry (HRMS). Additional details are provided in the Supplemental Methods, Supplemental Table 3, and Supplemental Table 4.
Atropisomer Interconversion Rate and Stability in Solutions, Serum, and Plasma
Crystallization of LEN sodium salt yields material highly enriched in LEN.2 (∼98% pure). A sample of this LEN.2 enriched crystalline material was added to human serum containing 1.5% Kolliphor (w/v), which was added to improve the low aqueous solubility of LEN. The sample was sonicated for approximately 5 minutes and then filtered to remove undissolved material. D2O was added (15% of total volume) and 19F nuclear magnetic resonance (NMR) spectra were collected approximately every 7 minutes at 37°C on an Agilent 400-MR DD2 NMR spectrometer operating at 376 MHz for 19F. Trifluoroethyl peaks corresponding to LEN.1 and LEN.2 were well resolved, and the relative concentrations of LEN.1 and LEN.2 were determined by integration. The sample was allowed to equilibrate for approximately 25 hours at 37°C before acquiring 19F NMR spectra in triplicate to determine the atropisomer ratio at equilibrium. The rate constants were determined from a plot of -ln(([A] – [A]eq)/([A]0 – [A]eq)) versus time, where [A] represents the relative concentration of LEN.2 and [A]0 and [A]eq refer to the relative concentration of LEN.2 over the sum of LEN.1 and LEN.2 concentrations at t = 0 and at equilibrium, respectively. The rotational energy barrier was computed from the rate constants using the Eyring equation. Additional details are provided in the Supplemental Methods. The interconversion rate of the atropisomers was also assessed in methanol and an aqueous ethanolic solution (10 mM K3PO4:ethanol 1:1 v/v) at pH 2, 7, 12 and at 5°C, 25°C, 37°C.
The potential for changes in the proportion of the two atropisomers was assessed following incubation of LEN (0.1 or 1.0 μM) at 37°C for 24 hours in PBS, 1:1 acetonitrile/water, or in plasma (Sprague–Dawley rat, beagle dog, and human). The incubation samples were quenched with acetonitrile-containing internal standard and centrifuged, and supernatants were quantified by LC-HRMS based on chromatographic peak areas of the individual isomers. Additional details are provided in the Supplemental Methods.
In Vivo Studies
All nonclinical studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the US National Institutes of Health and were approved by the institution’s Animal Care and Use Committee. All radiolabeled studies were managed using Debra software (Lablogic Systems, VA). An overview of the nonclinical PK and absorption, distribution, metabolism, and excretion (ADME) studies is shown in Table 1.
In vivo study design to evaluate the disposition of LEN following a single IV dose of LEN
LEN PK in Nonclinical Species.
The PK of LEN was assessed following a single 30-minute IV infusion of LEN at 1 mg/kg in intact male Sprague–Dawley rat, beagle dog, cynomolgus monkey, and rhesus monkey. The dose was formulated as a solution in 5% ethanol, 20% propylene glycol, 45% PEG 300, and 30% 10 mM phosphate buffer (v/v/v/v). Serial blood samples were collected following a 30-minute IV infusion, and LEN plasma concentrations were quantified using a liquid chromatograhy-tandem mass spectrometry (LC-MS/MS method). Plasma PK parameters for total LEN (i.e., LEN.1 + LEN.2 combined peaks) were estimated using noncompartmental analysis by Phoenix WinNonlin software, version 6.4 (Pharsight Corporation, Mountain View, CA).
LEN Disposition in Rat.
The PK, mass balance, metabolism, and excretion of [14C]LEN were assessed following a single IV bolus dose in intact and bile duct-cannulated (BDC) male WH rats (G1, G2, G5; Table 1). The tissue distribution of [14C]LEN was assessed following a single IV bolus dose in pigmented (Long-Evans) and nonpigmented (WH) intact rats (G3, G4; Table 1). BDC WH rats (RccHan:WIST) and intact LE rats (HsdBlu:LE) aged 7 to 10 weeks (Envigo RMS, Inc., Indianapolis, IN) were assigned into groups. Blood was collected via exsanguination (cardiac puncture) under isoflurane anesthesia into tubes containing K2EDTA at designated time points postdose from two animals/time point. Blood was placed on wet ice until aliquoted for radioanalysis and centrifuged to obtain plasma. Tissues from nonpigmented and pigmented rats were collected at designated time points for analysis by quantitative whole-body autoradiography (QWBA) and liquid scintillation counting (LSC). Urine, feces, bile, residual carcasses (rats only), and nonbiological samples (cage rinses, cage wash, cage wipe, cage debris, bile cannula rinse, and jacket rinse, as applicable) were collected at designated time points after dosing for analysis of excretion of radioactivity and mass balance.
The excretion of LEN was assessed in bile duct-intact and BDC male WH rats following a single IV dose of LEN without and with elacridar (ELA; a P-gp and BCRP inhibitor; Dash et al., 2017; Ward and Smith, 2004) treatment (G6 to G9; Table 1). Rats received oral doses of ELA (30 mg/kg) twice daily (approximately 12 hours apart) for 4 days (total of 7 doses). The first ELA dose was administered approximately 15 minutes prior to the LEN infusion dose on day 1. ELA was formulated as a solution in 10% N-methyl-2-pyrrolidone, 20% PEG 300, and 70% water (v/v/v). PK and excretion of unchanged LEN in bile, feces, and urine were quantified by an LC-MS/MS method.
LEN Disposition in Dog.
The PK, mass balance, metabolism, and excretion of [14C]LEN were assessed following a single IV bolus dose in intact and BDC male beagle dogs (G10, G11; Table 1). Beagle dogs aged 8 to 11 months (Covance Research Products, Cumberland, VA) were assigned into groups and administered [14C]LEN IV at a dose of 1 mg/kg (15 μCi/kg). The dosing vehicle was 5% ethanol, 20% propylene glycol, 45% PEG 300, and 30% water, pH 2 (v/v/v/v). Animals dosed intravenously were not fasted. Animals dosed orally were fasted overnight through approximately 4 hours postdose.
Blood was collected via a jugular vein into tubes containing K2EDTA from each animal at designated time points postdose, maintained in chilled cryoracks, then centrifuged to obtain plasma. Urine, feces, bile, and nonbiological samples (cage rinses, cage wash, cage wipe, cage debris, bile cannula rinse, and jacket rinse, as applicable) were collected at designated time points after dosing for analysis of excretion of radioactivity and mass balance.
Quantification of 14C Radioactivity.
The amounts of 14C radioactivity present in samples from rats and dogs were quantified by LSC using Model 2900TR or 2910TR liquid scintillation counters (Packard Instrument Company, Fallbrook, MA). Blood samples were treated with a commercial solubilizing agent, incubated for ≥ 1 hour at approximately 60°C, and mixed with disodium EDTA (0.1 M) and 30% hydrogen peroxide. After foaming had subsided, the blood samples were mixed with scintillation cocktail and analyzed by LSC. Plasma, urine, or bile samples were mixed with Ultima Gold XR scintillation cocktail (PerkinElmer, Waltham, MA) and analyzed by LSC. Feces samples were homogenized in ethanol/water (20:80, v/v), and weighed duplicate aliquots were digested in 1 N sodium hydroxide and analyzed by LSC. Residual rat carcasses were digested in 1 N sodium hydroxide until dissolved, homogenized in ethanol, and analyzed as duplicate aliquots by LSC. Blood and plasma PK parameters for total radioactivity were estimated using noncompartmental analysis by Phoenix WinNonlin software, version 6.4. The amounts of radioactivity in excreta were reported as the percentage of the 14C recovered dose.
The amount of 14C radioactivity was quantified by QWBA in nonpigmented and pigmented male rats in 52 tissues. Sagittal sections (40-μm thickness) were collected on adhesive tape in a CM3600 cryomicrotome (Leica Biosystems, Buffalo Grove, IL) and dried at −20°C. The mounted sections were exposed to phosphorimaging screens along with fortified blood standards for 4 days, and the screens were scanned using a Typhoon scanner. MCID analysis software (InterFocus Imaging Ltd., Cambridge, UK) was used to generate calibrated standard curves from the autoradiographic standard image data. Concentrations of 14C in tissues were determined from each standard curve as nanocuries/g, then converted to ng equivalents/g based on [14C]LEN specific activity.
Metabolite Profiling and Identification.
Radiochromatograms of plasma, bile, and feces obtained following administration of [14C]LEN from rats and dogs were generated by LC with fraction collection followed by offline radio detection, and the identity of the radiopeaks was determined by simultaneous inline HRMS. A detailed description of sample preparation and analytical methods for metabolite profiling and identification is provided in the Supplemental Methods. In brief, plasma samples were pooled by time point and were further combined to generate a single across intact rats (0.083–168 hours) and dogs (0.083–72 hours) area under the concentration-time curve (AUC) pooled sample by using a time-weighted pooling method (Hop et al., 1998). Bile and feces samples were prepared by weight-proportional pooling across time intervals, and the prepared plasma and excreta samples were analyzed. Urine was not profiled because of low radioactivity (< 1% of dose) in both species.
The 14C radioactivity associated with unchanged LEN and each identifiable metabolite was determined. Quantification of [14C]LEN and its metabolites in pooled plasma, bile, and feces samples in rats and dogs was based on integration of the respective metabolite profile peaks in the radiochromatograms and the radioactive concentration or dose recovered in the corresponding sample. The limit of quantitation (LOQ) for radioactivity in plasma, bile, and feces from rats and dogs was established at 1% of each chromatographic analysis (run) and a peak height of 10 cpm (limit of detection). For a given metabolite and matrix, values below the LOQ were only reported if at least one time point or interval had a value above the LOQ. For plasma, levels of LEN and its metabolites were determined as a percentage in the AUC-pool radiochromatogram. Plasma samples pooled by time point were also analyzed to determine the LEN atropisomer ratio. For excreta, levels of LEN and its metabolites were determined as a percentage of the administered radiodose.
Data Statistics.
All data, where feasible, are reported as mean and S.D. along with replicate values. No other statistical tests were performed.
Results
In Vitro Assessments
A summary of the in vitro properties of LEN is presented in Table 2. LEN has a high molecular weight (968 Da) for a small molecule drug and is strongly lipophilic with a high log D7.4 value (3.7). LEN showed low forward (absorptive) permeability and high reverse permeability through Caco-2 monolayers resulting in a high efflux ratio. LEN was found to be a substrate for human P-gp, but not human BCRP, based on the observed changes in its bidirectional permeability in P-gp and BCRP overexpressing cells in the absence and presence of inhibitors (Supplemental Table 1). Consistent with a substrate of P-gp, the efflux of LEN decreased from 35 to 1.1 with the addition of P-gp inhibitor valspodar in P-gp overexpressing cells. BCRP inhibitor Ko143 had no effect on the efflux of LEN in BCRP overexpressing cells. LEN was not a substrate for human OATP1B1 and OATP1B3 hepatic uptake transporters because the uptake rates of LEN were comparable in the presence or absence of rifampicin (Supplemental Table 2). LEN uptake into cryopreserved human hepatocytes at 37°C was not inhibited by rifamycin (100 mM), further confirming that LEN was not a substrate of hepatic OATP transporters, and there was no evidence for human hepatic uptake of LEN in vitro.
In vitro properties of LEN
LEN BPR was ≤ 0.67 and similar across species. LEN was modestly excluded from the cellular fraction of blood. The binding of LEN to plasma proteins was high, with less than 1.5% free in all tested species. The highest free fraction of 1.46% was observed in human plasma. LEN demonstrated very high binding to HSA with 0.01% free in a physiologically relevant concentration (40 mg/mL) of HSA. LEN bound moderately to hAAG with a mean of 7.0% free at a typical level of 0.8 mg/mL hAAG. LEN is expected to be bound primarily to albumin in human plasma. LEN binding to human hepatic microsomes was high (free fraction of 10.8%).
[3H]LEN exhibited low turnover and showed high metabolic stability across all species in both microsomes and hepatocytes (Table 2). The predicted hepatic extraction ranged from 1% to 6% of the hepatic blood flow across the nonclinical species. LEN was also predicted to have a low hepatic metabolic clearance in humans.
Interconversion and Stability of Atropisomers
The 19F NMR spectrum of the trifluoroethyl moiety at equilibrium in human serum at 37°C is shown in Fig. 2A. The atropisomer ratio did not significantly change between the 7-hour and 25-hour time points, and the ratio of [A]eq/[B]eq, where A and B represent LEN.2 and LEN.1, respectively, was determined to be 4.43. The rate of change of LEN.2 to LEN.1 in human serum is shown in Fig. 2B. Peaks for LEN.2 and LEN.1 in the 19F NMR spectra were integrated to determine [A] at each time point (Fig. 2B), and a plot of -ln(([A] – [A]eq)/([A]0 – [A]eq)) versus time was constructed (Fig. 2C). The slope of this plot is equal to the sum of the rate constants (k1 + k2). At equilibrium, k1[A]eq = k2[B]eq and since [A]eq/[B]eq = 4.43, k1 and k2 were calculated to be 9.63 × 10−5 s−1 and 4.26 × 10−4 s−1, respectively. The energy barrier to rotation about the LEN biaryl bond was determined to be 23.87 kcal/mol for LEN.2 → LEN.1 and 22.95 kcal/mol for LEN.1 → LEN.2, with half-lives of 0.45 hours and 2.00 hours for LEN.1 and LEN.2, respectively. The energy barriers and half-lives of hours are consistent with class 2 atropisomers (LaPlante et al., 2011).
LEN atropisomer characterization in human serum at 37°C by 19F NMR. (A) 19F NMR spectrum of the trifluoroethyl moiety in LEN at equilibrium. (B) % LEN.2 (major isomer) change over time. (C) Transformed plot to compute the sum of rate constants (see method section for details).
The half-life for interconversion between LEN.1 and LEN.2 was less than 1 hour in an aqueous ethanolic solution at pH 7 and 37°C. Following incubation of LEN containing both LEN.1 and LEN.2 at its natural equilibrium in plasma from Sprague–Dawley rats (LaPlante et al., 2011), beagle dogs, and humans, or in PBS and water acetonitrile buffers for 24 hours, the ratio of LEN.1 and LEN.2 remained constant (Table 3). The results showed that the balance between the two atropisomers is stable in aqueous media with time and unaffected by binding proteins or enzymes in plasma.
Atropisomer stability of LEN in plasma and buffer
In Vivo Studies
PK of LEN in Plasma.
The plasma LEN PK parameters following a single IV infusion administration to preclinical species are shown in Fig. 3. The in vivo PK parameters and the difference between the in vitro predicted CL and in vivo observed CL are presented in Tables 4 and 5, respectively. The average observed plasma CL of LEN was low across all tested species (1% to 12% of hepatic blood flow). The in vivo observed CL values were higher than the in vitro predicted CL values even with predicted clearance calculated without any correction for binding using the well-stirred model. The difference between the observed and predicted CL values were more pronounced when comparing the CL and the difference between the in vivo measured and in vitro predicted CL values (ΔCL) as percent of liver blood flow. The average volume of distribution at steady state (Vss) in all species was larger than that of total body water. The elimination t1/2 ranged from 14.6 to 38.1 hours across species.
Plasma concentration-time profiles of LEN following single IV 30-minute infusion administration of LEN at 1 mg/kg to Sprague–Dawley rat, beagle dog, cynomolgus, and rhesus monkeys (n = 6 or 9; mean ± S.D.).
In vivo plasma LEN PK parameters following a single IV 30-min infusion at 1 mg/kg to nonclinical species (n = 6 or 9; mean ± S.D.)
LEN in vivo observed CL was higher than in vitro predicted CL in all species
Pharmacokinetics of Total Radioactivity in Blood and Plasma.
The blood and plasma PK profiles for total radioactivity following a single IV administration of [14C]LEN in rats and dogs are shown in Supplemental Fig. 3 and Supplemental Fig. 4. Following IV administration to WH rats, the mean Cmax of radioactivity in blood and plasma were 1400 and 2380 ng equivalents [14C]LEN/g, respectively, observed at the first collection time point (0.083 hours postdose). The mean concentrations of radioactivity in blood and plasma then declined through 840 and 672 hours, respectively, and were below the limit of quantitation thereafter through 1680 hours postdose. Mean AUC extrapolated to time infinity values for total radioactivity were 53,000 and 90,900 ng equivalents [14C]LEN·hours/g, respectively.
Following IV administration of [14C]LEN to intact dogs, the mean Cmax of radioactivity in blood and plasma were 765 and 1290 ng equivalents [14C]LEN/g, respectively, observed at the first collection time point (0.083 hours). The mean concentrations of radioactivity in blood and plasma then declined to 9.33 and 8.09 ng equivalents [14C]LEN/g, respectively, by 336 hours and were below the limit of quantitation thereafter through 672 hours. AUClast values for total radioactivity in blood and plasma were 16,500 and 26,700 ng equivalents [14C]LEN·hours/g, respectively.
Tissue Distribution.
The blood-to-plasma AUC ratio of total radioactivity was 0.583 for rats and 0.618 for dogs and was similar to in vitro-derived values (Table 2). These data suggest a limited association of LEN-derived radioactivity with blood cells. Concentrations of total radioactivity in blood and representative tissues determined by QWBA following a single IV infusion of [14C]LEN in nonpigmented and pigmented rats are presented in Table 6. [14C]LEN-derived radioactivity was detected in most tissues by the first collection time point (0.5 hours postdose). Distribution of radioactivity was similar in both nonpigmented and pigmented rats. Most of the tissues reached maximum concentration by 0.5 hours postdose in nonpigmented rats and by 0.5 or 2 hours postdose in pigmented rats. Generally, the radioactivity was preferentially distributed into organs of elimination with the liver containing the highest concentration of radioactivity of the tissues sampled. No quantifiable or low levels of radioactivity were detected in brain and testes, respectively, suggesting the distribution of [14C]LEN-derived radioactivity was restricted by the blood-to-brain and blood-to-testes barriers. Radioactivity was cleared from all tissues in both strains except liver by 672 hours (28 days) postdose and was cleared from liver by 1344 hours (56 days) postdose. No significant melanin binding, e.g., in pigmented uveal tract and pigmented skin, was observed. The extensive tissue distribution was consistent with the Vss measured in the rat from noncompartmental analysis PK analysis.
Concentrations of total radioactivity (ng Eq/g) in blood and selected tissues determined by QWBA following a single IV [14C]LEN dose to nonpigmented and pigmented rats
Excretion.
The cumulative excretion of radioactivity over a period of up to 34 days following a single IV dose of [14C]LEN in intact and BDC rats and in intact and BDC dogs is summarized in Table 7, and the cumulative dose-recovery profiles are shown in Supplemental Fig. 3 and Supplemental Fig. 4. The mean cumulative overall recovery of dosed radioactivity was > 87% in both species. The excretion routes in intact animals were consistent across species, with the majority of the excreted dose recovered in feces (> 86% of [14C] dose in intact animals) and minor amounts recovered in urine (< 1.0% of [14C] dose). In BDC rats and dogs, a substantial amount of the dose was excreted in bile (rat, 41.7%; dog, 32.4%), and approximately 35% to 63% was excreted in feces (rat, 35.2%; dog, 63.0%).
Cumulative dose recovery following a single IV [14C]LEN dose to rats and dogs (mean ± S.D.)
Pharmacokinetics and Excretion of LEN in the Absence and Presence of ELA in Rats.
Plasma PK and fecal excretion profiles following a single IV dose without or with oral ELA pretreatment to intact and BDC rats are shown in Fig. 4, and a summary of the respective PK parameters are presented in Table 8. The systemic exposure (AUC) was increased 1.7- to 1.8-fold with ELA administration with a commensurate decrease in LEN CL. The LEN recovery was also assessed in feces, and recovery of intact LEN increased approximately 3-fold and 2.1-fold in intact and BDC animals, respectively. The excretion in bile and urine, i.e., minimal amounts of intact LEN, was unchanged and was consistent with the 14C data (Table 7).
LEN PK and fecal excretion profiles following a single LEN IV administration with and without ELA oral pretreatment. (A) LEN at 1.0 mg/kg in intact male WH rats (mean ± S.D., n = 3/4) and (B) LEN at 10 mg/kg in BDC male WH rats (mean ± S.D., n = 4/6). LEN levels in urine and bile from BDC animals (B) were negligible (< 0.1% and < 0.5% of the dose, respectively).
Pharmacokinetic and excretion of LEN following IV administration of LEN without or with ELA pretreatment in intact or BDC rats (mean ± S.D.)
Metabolite Profiles
In Vitro.
The turnover of [14C]LEN was extremely low across all in vitro systems and species with no turnover observed in human hepatic microsomes or hepatocytes. The turnover of [14C]LEN was further investigated in Hμrel hepatic cocultures from rat, dog, and human with a 7-day incubation period; results are presented in Table 9. [14C]LEN was metabolized via phase 2 conjugation with hexose and glucuronic acid and via oxidation followed by glutathione conjugation. No direct oxidation metabolites were detected. Also, the ratio of LEN.1 and LEN.2 ranged from 0.19 to 0.21 in Hμrel incubates across all species and remained stable in the sampling period. These ratios were consistent with those observed in plasma and buffer in vitro.
Metabolism of [14C]LEN in Hμrel hepatic cocultures
In Vivo.
Chromatograms of LEN and its metabolites observed in AUC-pooled plasma, bile, and feces from rats and dogs are shown in Fig. 5, and data are provided in Tables 10 and 11, respectively. A summary of representative LC-HRMS data for LEN and its biotransformation products is provided in Supplemental Table 5. A scheme of the proposed in vivo biotransformation pathways is shown in Fig. 6.
Radiochromatograms from pooled plasma, feces, and bile samples after a single IV dose of [14C]LEN to male WH rats and beagle dogs. (A) Rat AUC0.083-168 h plasma. (B) Rat feces. (C) Rat bile. (D) Dog AUC0.083-72 h plasma. (E) Dog feces. (F) Dog bile. Plasma and feces profiles are from intact animals. LEN and the three most abundant metabolites each exist as atropisomers (designated as .1 and .2). The observation of metabolite atropisomers is likely a function of their structure, abundance, and limit of detection.
Plasma profiles following IV administration of [14C]LEN
Summary of [14C]LEN derived biotransformation products in excreta following IV administration of [14C]LEN
Proposed biotransformation pathways of LEN following a single IV administration of [14C]LEN in rat, dog, and human (Weber et al., 2024). LEN and the three most abundant metabolites each exist as atropisomers (designated as .1 and .2). The observation of metabolite atropisomers is likely a function of their structure, abundance, and limit of detection.
In rat and dog plasma, unchanged LEN represented > 99% of radioactivity in AUC-pooled plasma. No LEN-derived metabolite was identified in plasma in rat and dog. The LEN.1 to LEN.2 ratio determined in individual time-point plasma pools showed the atropisomer ratio was 0.10 to 0.12 in rat and 0.17 to 0.20 in dog; the atropisomer ratio remained unchanged over time (0.083 to 168 hours) in both species.
In rat excreta, unchanged LEN was the major component recovered in feces from both intact and BDC rats accounting for 64.2% and 30.5% of the dose, respectively. Excreted metabolites comprised glucuronide conjugates and oxidative metabolites. Biliary excretion represented approximately 42% of the administered dose recovered in bile. Less than 0.1% of the dose represented unchanged LEN in bile. Glutathione conjugates (five metabolites: M4, M8, M9, M10, M11; Table 11) and a cysteine conjugate (M1) formed upon oxidation followed by an initial glutathione addition were the most abundant metabolites (total ∼26.2%) in rat bile. LEN-glucuronide (M13; ∼5.7%) was a smaller component in rat bile.
In dog excreta, unchanged LEN was the major component in radioactivity recovered in feces from both intact and BDC dogs and accounted for 65.1% and 59.6% of the dose, respectively. Excreted metabolites included phase 2 conjugates (pentose, hexose, and glucuronic acid) and oxidative metabolites. Biliary excretion represented approximately 31% of the administered dose in bile through 96 hours postdose. Most of the radioactivity recovered in bile was unchanged LEN and accounted for approximately 21.4% of the dose. LEN-glucuronide (M13) and oxy-LEN (M19) accounted for 4.2% and 4.8% of the dose in bile, respectively.
Discussion
LEN is a multistage HIV-1 capsid inhibitor and is approved for SC administration every 6 months in heavily treatment-experienced people with HIV (Daar et al., 2019; Yant et al., 2019; Bester et al., 2020; Sunlenca, 2023a, 2023b). In this work, we present the in vitro, nonclinical PK and disposition properties of LEN that identified very low systemic clearance, which was a favorable component for development as a LAI. LEN is one of a few Food and Drug Administration-approved drugs that exhibit atropisomerism (Glunz, 2018; Basilaia et al., 2022; Perreault et al., 2022; McVicker and O’Boyle, 2024; https://drughunter.com/articles/synthetic-access-to-stable-atropisomers-in-drug-discovery-via-catalysis/). LEN is a class 2 atropisomer, and surprisingly, there are even fewer approved drug examples of class 2 atropisomers (Basilaia et al., 2022; Glunz, 2018; McVicker and O’Boyle, 2024; Toenjes and Gustafson, 2018). The LEN.1 to LEN.2 ratio for LEN injected at 309 mg/mL as a solution formulation is approximately 1:3 as measured by 19F NMR (Gilead internal data). Class 2 atropisomers are typically developed as a mixture based on the influence of the interconversion rates on the timespan of drug shelf-life and its in vivo t1/2. We sought to determine the interconversion rates between LEN.1 and LEN.2 to understand the contribution toward PK and disposition of LEN. The interconversion half-life between the two LEN atropisomers, LEN.1 (minor) and LEN.2 (major), is less than 2 hours in solution across all studied conditions. In examples where the atropisomeric interconversion is markedly faster than the t1/2 of the in vivo elimination, systemic exposure is controlled by the fast interconversion rate. The LEN plasma t1/2 of approximately 24 hours in rats and dogs (Table 4) and 274 hours in human (Weber et al., 2024) substantially exceeds the t1/2 of atropisomer interconversion in solution (≤2 hours). Thus, the rotational isomers are expected to remain in equilibrium in vivo. In vitro, LEN showed no detectable change in relative distribution between the two atropisomers over 24 hours in human plasma (Table 3) and in Hμrel incubates across all species. The findings are consistent with the lack of atropisomer ratio changes in vivo in rat and dog plasma dosed with [14C]LEN (Table 3) and indicate that the balance between the two atropisomers is stable at a ratio of approximately 1:4 over time, consistent with the free energy of interconversion, and unaffected by enzymes or binding to proteins in plasma.
During the discovery of LEN, considerable challenges were overcome to identify a molecule that effectively inhibited the functions of HIV capsid, had high metabolic stability, and was amenable to LAI administration in vivo (Subramanian et al., 2023). LEN is very different from the typical drug-like small molecule agent with a high molecular weight (968 g/mol), low clearance, and high picomolar potency (Link et al., 2020; Weber et al., 2024). Taken together, the properties of LEN were highly promising for consideration as a LAI candidate (Subramanian et al., 2023).
The PK and ADME of LEN were assessed following single-dose IV dose of [14C]LEN in rats and dogs. Mean BPR concentration ratios of total radioactivity after dosing were < 1 in both species, indicating that LEN was predominantly distributed to plasma rather than the cellular components of blood. Following an IV [14C]LEN dose in rats, quantifiable radioactivity was observed in tissues at 168 hours postdose and through 672 hours in the liver, although declining concentrations indicated reversible binding. High liver distribution was not attributable to hepatic OATP transporter-mediated uptake (Table 2), and the mechanism for high liver to plasma ratio is not presently identified. Distribution of [14C]LEN to brain was almost negligible, consistent with LEN being a P-gp substrate. Distribution patterns of radioactivity to the uveal tract of the eye and pigmented skin suggested that [14C]LEN-related radioactivity was not selectively associated with melanin-containing tissues. The extensive distribution of LEN to most tissues was consistent with the Vss in rats, which also indicated significant extravascular distribution. The tissue distribution study also informed the human dosimetry calculations and enabled administration of a 200-μCi [14C]LEN IV dose to characterize the disposition of [14C]LEN in healthy human volunteers (Weber et al., 2024). The LEN Vss was moderate in nonclinical species (1.6 to 5.2 L/kg; Table 4) and substantially higher in humans (∼23.8 L/kg; Weber et al., 2024). These differences are not accounted for by the plasma free-fraction differences across species, and the mechanism of higher Vss in humans compared with nonclinical species is not presently understood.
The observed CL of LEN was low in nonclinical species (Table 4) and humans (Weber et al., 2024). However, the in vivo observed CL values were higher than the in vitro metabolic predicted CL values even when no binding correction was used in the well-stirred model. The difference between the observed and predicted CL values was more pronounced when comparing the CL and ΔCL values as percent of liver blood flow (Table 5). For example, the hepatocyte-predicted CL was approximately one-third and approximately one-sixth of the observed CL in rat and human, respectively.
[14C]LEN metabolism was investigated in Hμrel hepatic cocultures, which offered an in vitro system to characterize low-turnover drugs (Burton et al., 2018). Following a 7-day incubation period in the Hμrel system, [14C]LEN was found to be metabolized to phase 2 conjugates (with hexose and glucuronic acid) and oxidative metabolites leading to glutathione and cysteine conjugates (Table 9). In vivo, similar phase 2 conjugates (plus a pentose conjugate) and oxidative metabolites were observed in bile and feces with an overall abundance that was higher in rat compared with dog and human (Table 11).
Mechanistic ADME studies following LEN IV administration in rats and dogs aided in understanding the disconnect between the in vivo observed CL and the in vitro predicted CL. In both species, the cumulative recovery of dosed radioactivity was high at > 87%. Biliary excretion represented approximately 42% of the administered dose recovered in bile in rats and 31% in dogs, while renal excretion was minimal in both species (< 1% of dose). Unchanged LEN was the major component recovered in feces from both intact and BDC animals, accounting for 64.2% and 30.5% of the dose, respectively, in rats and 65.1% and 59.6% of the dose, respectively, in dogs (Table 7). Recovery of unchanged LEN in feces after IV administration in BDC animals indicated that IE was a significant elimination pathway for LEN in rat and dog in addition to biliary excretion, while metabolic clearance played a lesser role. In a mechanistic study designed to understand the excretion of unchanged LEN in feces observed in the rat [14C]LEN ADME study, LEN was administered IV without or with pretreatment of ELA, a P-gp and BCRP inhibitor, to bile duct-intact rats or BDC rats. LEN IE decreased by two- to threefold in the presence of ELA while no effect was observed in the negligible excretion of LEN in bile or urine (Table 8, Fig. 4). The systemic exposure (AUC) commensurately increased with ELA administration, although the overall impact on LEN CL was more modest (an increase of 1.7- to 1.8-fold).
Results from [14C]LEN nonclinical and human mass balance and disposition studies were similar. Unchanged [14C]LEN was the only component identified in rat and dog plasma. In human plasma, LEN was also the predominant component (68.8% of radioactivity exposure; Weber et al., 2024), and no single circulating metabolites exceeded 10% of the total plasma radioactivity. Unchanged LEN was the most abundant component in human feces, accounting for a mean of 32.9% of the administered IV dose (Weber et al., 2024). In vitro, LEN is a substrate for human P-gp but not BCRP. Collectively, the data show that IE of LEN is mediated by P-gp regulating LEN fecal excretion and a significant clearance mechanism of LEN in rat, dog, and likely human. Taken together, the similar low in vitro metabolic clearance, low systemic clearance, and parent drug excretion in the feces of humans after IV dosing strongly suggest that IE is a significant clearance mechanism for LEN in humans.
IE is an underappreciated disposition mechanism for small molecule drugs because it requires ADME studies to be conducted following IV administration in intact and BDC animals (Zhang et al., 2021). Often, radiolabeled ADME studies are performed following oral administration to support oral drug development, and any parent drug recovered in feces is assumed to be an unabsorbed drug. Thus, oral ADME studies can be inadequate for identification of IE as a possible clearance mechanism. IE has a major role in a molecule such as LEN because it shows low permeability, prominent P-gp-mediated efflux, and very low total CL. Overall, these studies demonstrate that the atropisomer mixture of LEN maintains a constant ratio of approximately 1:4 in vivo and that IE is a major route of LEN elimination. The results highlight the importance of IE as a mechanism for highly metabolically stable compounds with slow elimination rates. The tools of modern drug discovery have increased the number of molecules with low metabolic clearance, and IE is likely to become a more important pathway for small molecule drugs.
Acknowledgments
The authors thank CRL Laboratories (Legacy Agilux Laboratories, Worcester), and Labcorp Laboratories (Legacy Covance Laboratories, Madison) for conducting the nonclinical in vivo PK studies. The authors thank the Labcorp ADME team, Jessica M. Henderson, and Sara Leitz for conducting the radiolabeled in vivo studies. The authors also thank Sibylle Wilbert for editorial assistance with this manuscript.
Data Availability
The authors declare that all the data supporting the findings of this study are available within the paper and its Supplemental Materials.
Authorship Contributions
Participated in research design: Zheng, Murray, Bashir, Tse, Link, Yoon, Chiu, Rowe, Smith, Subramanian.
Conducted experiments: Lu, Carr, Mwangi, Wang, Hao, Staiger, Kozon, Gohdes.
Contributed new reagents or analytic tools: Lu, Carr, Mwangi, Schroeder, Graupe, Rowe.
Performed data analysis: Zheng, Lu, Carr, Mwangi, Wang, Gohdes.
Wrote or contributed to the writing of the manuscript: Lu, Carr, Murray, Link, Yoon, Chiu, Smith, Subramanian.
Footnotes
- Received May 13, 2024.
- Accepted July 31, 2024.
This work was supported by Gilead Sciences, Inc.
All authors, apart from M.B. and M.G., were employees of Gilead Sciences for the body of work reported in this manuscript.
Primary laboratory of origin: Gilead Sciences, Inc. (Foster City, CA).
Parts of this work were previously disclosed as poster presentations: 1. Lu B, et al.; A robust LC-MS/MS method demonstrated free interconversions between the two lenacapavir atropisomers; 2021, ASMS poster #FP126. 2. Zheng J, et al.; Intestinal excretion is a major disposition pathway of lenacapavir in rat and dog; 2021, FASEB 35(S1); ASPET poster #R423.
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This article has supplemental material available at jpet.aspetjournals.org.
Abbreviations
- ADME
- absorption, distribution, metabolism, and excretion
- AUC
- area under the concentration-time curve
- BDC
- bile duct-cannulated
- BCRP
- breast cancer resistance protein
- BPR
- whole blood to plasma concentration ratio
- CL
- clearance
- hAAG
- human alpha-1-acid glycoprotein
- HIV
- human immunodeficiency virus
- HIV-1
- human immunodeficiency virus type 1
- HRMS
- high-resolution mass spectrometry
- HSA
- human serum albumin
- IV
- intravenous
- LAI
- long-acting injectable
- LC
- liquid chromatography
- LC-MS/MS
- liquid chromatography-tandem mass spectrometry
- LEN
- lenacapavir (also known as GS-6207)
- LOQ
- limit of quantitation
- LSC
- liquid scintillation counting
- NMR
- nuclear magnetic resonance
- PEG
- polyethylene glycol
- P-gp
- P-glycoprotein
- PK
- pharmacokinetic(s)
- QWBA
- quantitative whole-body autoradiography
- SC
- subcutaneous
- t1/2
- half-life
- Vss
- volume of distribution at steady state
- WH
- Wistar Han
- Copyright © 2024 by The Author(s)
This is an open access article distributed under the CC BY Attribution 4.0 International license.
