Selective depletion of HBV-infected hepatocytes by class A capsid assembly modulators requires high levels of intrahepatic HBV core protein.

Capsid assembly mediated by hepatitis B virus (HBV) core protein (HBc) is an essential part of the HBV replication cycle, which is the target for different classes of capsid assembly modulators (CAMs). While both CAM-A ("aberrant") and CAM-E ("empty") disrupt nucleocapsid assembly and reduce extracellular HBV DNA, CAM-As can also reduce extracellular HBV surface antigen (HBsAg) by triggering apoptosis of HBV-infected cells in preclinical mouse models. However, there have not been substantial HBsAg declines in chronic hepatitis B (CHB) patients treated with CAM-As to date. To investigate this disconnect, we characterized the antiviral activity of tool CAM compounds in HBV-infected primary human hepatocytes (PHHs), as well as in HBV-infected human liver chimeric mice and mice transduced with adeno-associated virus-HBV. Mechanistic studies in HBV-infected PHH revealed that CAM-A, but not CAM-E, induced a dose-dependent aggregation of HBc in the nucleus which is negatively regulated by the ubiquitin-binding protein p62. We confirmed that CAM-A, but not CAM-E, induced HBc-positive cell death in both mouse models via induction of apoptotic and inflammatory pathways and demonstrated that the degree of HBV-positive cell loss was positively correlated with intrahepatic HBc levels. Importantly, we determined that there is a significantly lower level of HBc per hepatocyte in CHB patient liver biopsies than in either of the HBV mouse models. Taken together, these data confirm that CAM-As have a unique secondary mechanism with the potential to kill HBc-positive hepatocytes. However, this secondary mechanism appears to require higher intrahepatic HBc levels than is typically observed in CHB patients, thereby limiting the therapeutic potential.

Assessing Pleiotropic Effects of a Mixed-Mode Perpetrator Drug, Rifampicin, by Multiple Endogenous Biomarkers in Dogs.

Rifampicin (RIF) is a mixed-mode perpetrator that produces pleiotropic effects on liver cytochrome P450 enzymes and drug transporters. To assess the complex drug-drug interaction liabilities of RIF in vivo, a known probe substrate, midazolam (MDZ), along with multiple endogenous biomarkers were simultaneously monitored in beagle dogs before and after a 7-day treatment period by RIF at 20 mg/kg per day. Confirmed by the reduced MDZ plasma exposure and elevated 4ß-hydroxycholesterol (4ß-HC, biomarker of CYP3A activities) level, CYP3A was significantly induced after repeated RIF doses, and such induction persisted for 3 days after cessation of the RIF administration. On the other hand, increased plasma levels of coproporphyrin (CP)-I and III [biomarkers of organic anion transporting polypeptides 1b (Oatp1b) activities] were observed after the first dose of RIF. Plasma CPs started to decline as RIF exposure decreased, and they returned to baseline 3 days after cessation of the RIF administration. The data suggested the acute (inhibitory) and chronic (inductive) effects of RIF on Oatp1b and CYP3A enzymes, respectively, and a 3-day washout period is deemed adequate to remove superimposed Oatp1b inhibition from CYP3A induction. In addition, apparent self-induction of RIF was observed as its terminal half-life was significantly altered after multiple doses. Overall, our investigation illustrated the need for appropriate timing of modulator dosing to differentiate between transporter inhibition and enzyme induction. As further indicated by the CP data, induction of Oatp1b activities was not likely after repeated RIF administration. SIGNIFICANCE STATEMENT: This investigation demonstrated the utility of endogenous biomarkers towards complex drug-drug interactions by rifampicin (RIF) and successfully determined the optimal timing to differentiate between transporter inhibition and enzyme induction. Based on experimental evidence, Oatp1b induction following repeated RIF administration was unlikely, and apparent self-induction of RIF elimination was observed.

Lenacapavir: A Novel, Potent, and Selective First-in-Class Inhibitor of HIV-1 Capsid Function Exhibits Optimal Pharmacokinetic Properties for a Long-Acting Injectable Antiretroviral Agent.

Lenacapavir (LEN) is a picomolar first-in-class capsid inhibitor of human immunodeficiency virus type 1 (HIV-1) with a multistage mechanism of action and no known cross resistance to other existing antiretroviral (ARV) drug classes. LEN exhibits a low aqueous solubility and exceptionally low systemic clearance following intravenous (IV) administration in nonclinical species and humans. LEN formulated in an aqueous suspension or a PEG/water solution formulation showed sustained plasma exposure levels with no unintended rapid drug release following subcutaneous (SC) administration to rats and dogs. A high total fraction dose release was observed with both formulations. The long-acting pharmacokinetics (PK) were recapitulated in humans following SC administration of both formulations. The SC PK profiles displayed two-phase absorption kinetics in both animals and humans with an initial fast-release absorption phase, followed by a slow-release absorption phase. Noncompartmental and compartmental analyses informed the LEN systemic input rate from the SC depot and exit rate from the body. Modeling-enabled deconvolution of the input rates from two processes: absorption of the soluble fraction (minor) from a direct fast-release process leading to the early PK phase and absorption of the precipitated fraction (major) from an indirect slow-release process leading to the later PK phase. LEN SC PK showed flip-flop kinetics due to the input rate being substantially slower than the systemic exit rate. LEN input rates via the slow-release process in humans were slower than those in both rats and dogs. Overall, the combination of high potency, exceptional stability, and optimal release rate from the injection depot make LEN well suited for a parenteral long-acting formulation that can be administered once up to every 6 months in humans for the prevention and treatment of HIV-1.

18O-Enabled High-Throughput Acyl Glucuronide Stability Assay.

Acyl glucuronides (AGs) are common metabolites of carboxylic acid-containing compounds. In some circumstances, AGs are suspected to be involved in drug toxicity due to formation of acyl migration products that bind covalently to cellular components. The risk of this adverse effect has been found to be correlated with the chemical stability of the AG, and assays have been described that monitor acyl migration by liquid chromatography coupled with mass spectrometry (LC-MS). This analysis can be challenging as it requires baseline chromatographic separation of the unmigrated 1-ß-acyl glucuronide from the migrated isomers and thus needs to be individually optimized for each aglycone. Therefore, a high-throughput assay that eliminates LC method development is desirable. Herein, we report an improved acyl glucuronide stability assay based on the rate of 18O-incorporation from [18O] water, which is compatible with high-throughput bioanalytical LC-MS workflows. Synthetic AGs with shorter migration half-lives showed faster incorporation of 18O. The level of differential incorporation of 18O following a 24 h incubation correlates well with the migration tendency of AGs. This assay was developed further, exploring in situ generation of AGs by human hepatic microsomal fraction. The results from 18 in situ-formed acyl glucuronides were similar to those obtained using authentic reference standards. In this format, this new 18O-labeling method offers a simplified workflow, requires no LC method development or AG reference standard, and thus facilitates AG liability assessment in early drug discovery.

Human and nonclinical disposition of [14C]bictegravir, a potent integrase strand-transfer inhibitor for the treatment of HIV-1 infection.

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 absorption, metabolism, distribution, and elimination (ADME) characteristics of BIC were determined through in vivo nonclinical and clinical studies (IND 121318).[14C]BIC was rapidly absorbed orally in mice, rats, monkeys and human. The cumulative dose recovery was high in nonclinical species (>80%) and humans (95.3%), with most of the excreted dose recovered in faeces. Quantifiable radioactivity with declining concentration was observed in rat tissues suggesting reversible binding. Unchanged BIC was the most abundant circulating component in all species along with two notable metabolites M20 (a sulphate conjugate of hydroxylated BIC) and M15 (a glucuronide conjugate of BIC). BIC was primarily eliminated by hepatic metabolism followed by excretion of the biotransformed products into faeces. In vitro drug-drug interaction (DDI) studies with M15 and M20 demonstrated that no clinically relevant interactions were expected.Overall, BIC is a novel and potent INSTI with a favourable resistance, PK, and ADME profile that provides important improvements over other currently available INSTIs for the treatment of HIV-1.

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.

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.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.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. .

Key Metabolic Enzymes Involved in Remdesivir Activation in Human Lung Cells.

Remdesivir (RDV; GS-5734, Veklury), the first FDA-approved antiviral to treat COVID-19, is a single-diastereomer monophosphoramidate prodrug of an adenosine analogue. RDV is taken up in the target cells and metabolized in multiple steps to form the active nucleoside triphosphate (TP) (GS-443902), which, in turn, acts as a potent and selective inhibitor of multiple viral RNA polymerases. In this report, we profiled the key enzymes involved in the RDV metabolic pathway with multiple parallel approaches: (i) bioinformatic analysis of nucleoside/nucleotide metabolic enzyme mRNA expression using public human tissue and lung single-cell bulk mRNA sequence (RNA-seq) data sets, (ii) protein and mRNA quantification of enzymes in human lung tissue and primary lung cells, (iii) biochemical studies on the catalytic rate of key enzymes, (iv) effects of specific enzyme inhibitors on the GS-443902 formation, and (v) the effects of these inhibitors on RDV antiviral activity against SARS-CoV-2 in cell culture. Our data collectively demonstrated that carboxylesterase 1 (CES1) and cathepsin A (CatA) are enzymes involved in hydrolyzing RDV to its alanine intermediate MetX, which is further hydrolyzed to the monophosphate form by histidine triad nucleotide-binding protein 1 (HINT1). The monophosphate is then consecutively phosphorylated to diphosphate and triphosphate by cellular phosphotransferases. Our data support the hypothesis that the unique properties of RDV prodrug not only allow lung-specific accumulation critical for the treatment of respiratory viral infection such as COVID-19 but also enable efficient intracellular metabolism of RDV and its MetX to monophosphate and successive phosphorylation to form the active TP in disease-relevant cells.

Characterization of Seasonal Pharmacokinetic Variability in Woodchucks.

The eastern woodchuck (Marmota monax) is a hibernating species extensively used as an in vivo efficacy model for chronic human hepatitis B virus infection. Under laboratory conditions, woodchucks develop a pseudohibernation condition; thus, the pharmacokinetics (PK) of small-molecule therapeutics may be affected by the seasonal change. The seasonal PK of four probe compounds were characterized over 12 months in seven male and nine female laboratory-maintained woodchucks. These compounds were selected to study changes in oxidative metabolism [antipyrine (AP)], glucuronidation [raltegravir (RTG)], renal clearance [lamivudine (3TC)], and hepatic function [indocyanine green (ICG)]. Seasonal changes in physiologic parameters and PK were determined. Seasonal body weight increases were ≥30%. Seasonal changes in body temperature and heart rate were <10%. The mean AP exposure remained unchanged from April to August 2017, followed by a significant increase (≥1.0-fold) from August to December and subsequent decrease to baseline at the end of study. A similar trend was observed in RTG and 3TC exposures. The ICG exposure remained unchanged. No significant sex difference in PK was observed, although female woodchucks appeared to be less susceptible to seasonal PK and body weight changes. Significant seasonal PK changes for AP, RTG, and 3TC indicate decreases in oxidative metabolism, phase II glucuronidation, and renal clearance during pseudohibernation. The lack of seasonal change in ICG exposure suggests there are no significant changes in hepatic function. This information can be used to optimize the scheduling of woodchuck studies to avoid seasonally driven variation in drug PK. SIGNIFICANCE STATEMENT: Woodchuck is a hibernating species and is commonly used as a nonclinical model of hepatitis B infection. Investigation of seasonal PK changes is perhaps of greater interest to pharmaceutical industry scientists, who use the woodchuck model to optimize the scheduling of woodchuck studies to avoid seasonally driven variation in drug PK and/or toxicity. This information is also valuable to drug metabolism and veterinary scientists in understanding woodchuck's seasonal metabolism and behavior under the pseudohibernation condition.

Organic Anion-Transporting Polypeptide Genes Are Not Induced by the Pregnane X Receptor Activator Rifampin: Studies in Hepatocytes In Vitro and in Monkeys In Vivo.

Induction potentials of the pregnane X receptor (PXR) activator rifampin (RIF) on transporter genes [e.g., organic anion-transporting polypeptides (OATPs)] are still in its infancy or remain controversial in the field. The present investigations characterized changes in transporter gene expression by RIF in sandwich-cultured hepatocytes from multiple donors of human and cynomolgus monkey using real-time quantitative reverse transcription polymerase chain reaction method. Three-day treatment of RIF significantly induced CYP3A4 (∼60-fold induction), but not CYP1A2 and CYP2D6 genes. SLC51B was the most highly induced uptake transporter gene (>10-fold) in both human and monkey hepatocytes. A greater induction of CYP2C9 was observed in monkey hepatocytes than that in humans. ATP-binding cassette (ABC)B1 and ABCC2 were induced slightly above 2-fold in human and monkey hepatocytes and appeared to be dose-dependent. The induction of OATP and other transporter genes was generally less than 2-fold and considered not clinically relevant. SLCO2B1 was not detectable in monkey hepatocytes. To investigate in vivo OATP induction, RIF (18 mg/kg per day) was orally dosed to cynomolgus monkeys for 7 days. Pitavastatin and antipyrine were intravenously dosed before and after RIF treatment as exogenous probes of OATP and CYP activities, respectively. Plasma coproporphyrin-I (CP-I) and coproporphyrin-III (CP-III) were measured as OATP endogenous biomarkers. Although a significant increase of antipyrine clearance (CL) was observed after RIF treatment, the plasma exposures of pitavastatin, CP-I, and CP-III remained unchanged, suggesting that OATP function was not significantly altered. The results suggested that OATP transporters were not significantly induced by PXR ligand RIF. The data are consistent with current regulatory guidances that the in vitro characterization of transporter induction during drug development is not required. SIGNIFICANCE STATEMENT: Organic anion-transporting polypeptide (OATP) genes were not induced by rifampin in sandwich-cultured human and monkey hepatocytes OATP functions measured by OATP probe pitavastatin and endogenous marker coproporphyrins were not altered in monkeys in vivo by 7-day rifampin treatment. The data suggested that OATP transporters are unlikely induced by the pregnane X receptor ligand rifampin, which are consistent with current regulatory guidances that the in vitro characterization of OATP1B induction during drug development is not required.

Pharmacokinetics and Disposition of Momelotinib Revealed a Disproportionate Human Metabolite-Resolution for Clinical Development.

Momelotinib (MMB), a small-molecule inhibitor of Janus kinase (JAK)1/2 and of activin A receptor type 1 (ACVR1), is in clinical development for the treatment of myeloproliferative neoplasms. The pharmacokinetics and disposition of [14C]MMB were characterized in a single-dose, human mass-balance study. Metabolism and the pharmacologic activity of key metabolites were elucidated in multiple in vitro and in vivo experiments. MMB was rapidly absorbed following oral dosing with approximately 97% of the radioactivity recovered, primarily in feces with urine as a secondary route. Mean blood-to-plasma [14C] area under the plasma concentration-time curve ratio was 0.72, suggesting low association of MMB and metabolites with blood cells. [14C]MMB-derived radioactivity was detectable in blood for ≤48 hours, suggesting no irreversible binding of MMB or its metabolites. The major circulating human metabolite, M21 (a morpholino lactam), is a potent inhibitor of JAK1/2 and ACVR1 in vitro. Estimation of pharmacological activity index suggests M21 contributes significantly to the pharmacological activity of MMB for the inhibition of both JAK1/2 and ACVR1. M21 was observed in disproportionately higher amounts in human plasma than in rat or dog, the rodent and nonrodent species used for the general nonclinical safety assessment of this molecule. This discrepancy was resolved with additional nonclinical studies wherein the circulating metabolites and drug-drug interactions were further characterized. The human metabolism of MMB was mediated primarily by multiple cytochrome P450 enzymes, whereas M21 formation involved initial P450 oxidation of the morpholine ring followed by metabolism via aldehyde oxidase.

Selective ORAI1 Inhibition Ameliorates Autoimmune Central Nervous System Inflammation by Suppressing Effector but Not Regulatory T Cell Function.

The function of CD4(+) T cells is dependent on Ca(2+) influx through Ca(2+) release-activated Ca(2+) (CRAC) channels formed by ORAI proteins. To investigate the role of ORAI1 in proinflammatory Th1 and Th17 cells and autoimmune diseases, we genetically and pharmacologically modulated ORAI1 function. Immunization of mice lacking Orai1 in T cells with MOG peptide resulted in attenuated severity of experimental autoimmune encephalomyelitis (EAE). The numbers of T cells and innate immune cells in the CNS of ORAI1-deficient animals were strongly reduced along with almost completely abolished production of IL-17A, IFN-γ, and GM-CSF despite only partially reduced Ca(2+) influx. In Th1 and Th17 cells differentiated in vitro, ORAI1 was required for cytokine production but not the expression of Th1- and Th17-specific transcription factors T-bet and RORγt. The differentiation and function of induced regulatory T cells, by contrast, was independent of ORAI1. Importantly, induced genetic deletion of Orai1 in adoptively transferred, MOG-specific T cells was able to halt EAE progression after disease onset. Likewise, treatment of wild-type mice with a selective CRAC channel inhibitor after EAE onset ameliorated disease. Genetic deletion of Orai1 and pharmacological ORAI1 inhibition reduced the leukocyte numbers in the CNS and attenuated Th1/Th17 cell-mediated cytokine production. In human CD4(+) T cells, CRAC channel inhibition reduced the expression of IL-17A, IFN-γ, and other cytokines in a dose-dependent manner. Taken together, these findings support the conclusion that Th1 and Th17 cell function is particularly dependent on CRAC channels, which could be exploited as a therapeutic approach to T cell-mediated autoimmune diseases.

Drug disposition model of radiolabeled etelcalcetide in patients with chronic kidney disease and secondary hyperparathyroidism on hemodialysis.

Etelcalcetide (AMG 416) is an allosteric activator of the calcium-sensing receptor for treatment of secondary hyperparathyroidism in patients with chronic kidney disease (CKD) on hemodialysis. To characterize the time course of etelcalcetide in different matrices (plasma, dialysate, urine, and feces), a drug disposition model was developed. Nonlinear mixed-effect modeling was used to describe data from six adults with CKD on hemodialysis who received a single intravenous dose of [14C]etelcalcetide (10 mg; 710 nCi) after hemodialysis (study NCT02054572). A three-compartment model with the following attributes adequately described the observed concentration-time profiles of etelcalcetide in the different matrices: biotransformation in the central compartment; elimination in dialysate, urine, and feces; and a nonspecific elimination process. The terminal half-life of total C-14 in plasma was approximately 56 days. The ratio of conjugation-deconjugation rate constants between etelcalcetide and biotransformed products was 11.3. Simulations showed that three hemodialysis sessions per week for 52 weeks would contribute to 60.1% of the total clearance of etelcalcetide following single-dose intravenous etelcalcetide administration. Minimal amounts were eliminated in urine (2.5%) and feces (5.7%), whereas nonspecific elimination accounted for 31.2% of total elimination. In addition to removal of etelcalcetide, ~10% of small-molecular weight biotransformed products was estimated to have been removed through hemodialysis and in urine. This model provided a quantitative approach to describe biotransformation, distribution, and elimination of etelcalcetide, a unique synthetic D-amino acid peptide, in the relevant patient population.

Nonclinical Pharmacokinetics, Disposition, and Drug-Drug Interaction Potential of a Novel d-Amino Acid Peptide Agonist of the Calcium-Sensing Receptor AMG 416 (Etelcalcetide).

AMG 416 (etelcalcetide) is a novel synthetic peptide agonist of the calcium-sensing receptor composed of a linear chain of seven d-amino acids (referred to as the d-amino acid backbone) with a d-cysteine linked to an l-cysteine via a disulfide bond. AMG 416 contains four basic d-arginine residues and is a +4 charged peptide at physiologic pH with a mol. wt. of 1048.3 Da. The pharmacokinetics (PK), disposition, and potential of AMG 416 to cause drug-drug interaction were investigated in nonclinical studies with two single (14)C-labels placed either at a potentially metabolically labile acetyl position or on the d-alanine next to d-cysteine in the interior of the d-amino acid backbone. After i.v. dosing, the PK and disposition of AMG 416 were similar in male and female rats. Radioactivity rapidly distributed to most tissues in rats with intact kidneys, and renal elimination was the predominant clearance pathway. No strain-dependent differences were observed. In bilaterally nephrectomized rats, minimal radioactivity (1.2%) was excreted via nonrenal pathways. Biotransformation occurred primarily via disulfide exchange with endogenous thiol-containing molecules in whole blood rather than metabolism by enzymes, such as proteases or cytochrome P450s; the d-amino acid backbone remained unaltered. A substantial proportion of the plasma radioactivity was covalently conjugated to albumin. AMG 416 presents a low risk for P450 or transporter-mediated drug-drug interactions because it showed no interactions in vitro. These studies demonstrated a (14)C label on either the acetyl or the d-alanine in the d-amino acid backbone would be appropriate for clinical studies.

Nonclinical Safety Profile of Etelcalcetide, a Novel Peptide Calcimimetic for the Treatment of Secondary Hyperparathyroidism.

Etelcalcetide is a novel d-amino acid peptide that functions as an allosteric activator of the calcium-sensing receptor and is being developed as an intravenous calcimimetic for the treatment of secondary hyperparathyroidism in patients with chronic kidney disease on hemodialysis. To support clinical development and marketing authorization, a comprehensive nonclinical safety package was generated. Primary adverse effects included hypocalcemia, tremoring, and convulsions. Other adverse effects were considered sequelae of stress associated with hypocalcemia. Cardiovascular safety evaluations in the dog revealed an anticipated prolongation of the corrected QT interval that was related to reductions in serum calcium. Etelcalcetide did not affect the human ether-a-go-go gene ion channel current. Etelcalcetide was mutagenic in some strains of Salmonella, however, based on the negative results in 2 in vitro and 2 in vivo mammalian genotoxicity assays, including a 28-day Muta mouse study, etelcalcetide is considered nongenotoxic. Further support for a lack of genotoxicity was provided due to the fact that etelcalcetide was not carcinogenic in a 6-month transgenic rasH2 mouse model or a 2-year study in rats. There were no effects on fertility, embryo-fetal development, and prenatal and postnatal development. All of the adverse effects observed in both rat and dog were considered directly or secondarily related to the pharmacologic activity of etelcalcetide and the expected sequelae associated with dose-related reductions in serum calcium due to suppression of parathyroid hormone secretion. These nonclinical data indicate no safety signal of concern for human risk beyond that associated with hypocalcemia and associated QT prolongation.

Critical Cysteine Residues in Both the Calcium-Sensing Receptor and the Allosteric Activator AMG 416 Underlie the Mechanism of Action.

AMG 416 is a novel D-amino acid-containing peptide agonist of the calcium-sensing receptor (CaSR) that is being evaluated for the treatment of secondary hyperparathyroidism in chronic kidney disease patients receiving hemodialysis. The principal amino acid residues and their location in the CaSR that accommodate AMG 416 binding and mode of action have not previously been reported. Herein we establish the importance of a pair of cysteine residues, one from AMG 416 and the other from the CaSR at position 482 (Cys482), and correlate the degree of disulfide bond formation between these residues with the pharmacological activity of AMG 416. KP-2067, a form of the CaSR agonist peptide, was included to establish the role of cysteine in vivo and in disulfide exchange. Studies conducted with AMG 416 in pigs showed a complete lack of pharmacodynamic effect and provided a foundation for determining the peptide agonist interaction site within the human CaSR. Inactivity of AMG 416 on the pig CaSR resulted from a naturally occurring mutation encoding tyrosine for cysteine (Cys) at position 482 in the pig CaSR. Replacing Cys482 in the human CaSR with serine or tyrosine ablated AMG 416 activity. Decidedly, a single substitution of cysteine for tyrosine at position 482 in the native pig CaSR provided a complete gain of activity by the peptide agonist. Direct evidence for this disulfide bond formation between the peptide and receptor was demonstrated using a mass spectrometry assay. The extent of disulfide bond formation was found to correlate with the extent of receptor activation. Notwithstanding the covalent basis of this disulfide bond, the observed in vivo pharmacology of AMG 416 showed readily reversible pharmacodynamics.

The imidazo[1,2-a]pyridine ring system as a scaffold for potent dual phosphoinositide-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitors.

Based on lead compound 1, which was discovered from a high-throughput screen, a series of PI3Kα/mTOR inhibitors were evaluated that contained an imidazo[1,2-a]pyridine as a core replacement for the benzimidazole contained in 1. By exploring various ring systems that occupy the affinity pocket, two fragments containing a methoxypyridine were identified that gave <100 nM potency toward PI3Kα in enzyme and cellular assays with moderate stability in rat and human liver microsomes. With the two methoxypyridine groups selected to occupy the affinity pocket, analogs were prepared with various fragments intended to occupy the ribose pocket of PI3Kα and mTOR. From these analogs, tertiary alcohol 18 was chosen for in vivo pharmacodynamic evaluation based on its potency in the PI3Kα cellular assay, microsomal stability, and in vivo pharmacokinetic properties. In a mouse liver pharmacodynamic assay, compound 18 showed 56% inhibition of HFG-induced AKT (Ser473) phosphorylation at a 30 mg/kg dose.

Comparison of information-dependent acquisition, SWATH, and MS(All) techniques in metabolite identification study employing ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry.

Sensitive and selective liquid chromatography-mass spectrometry (LC-MS) analysis is a powerful and essential tool for metabolite identification in drug discovery and development. An MS(2) (or tandem, MS/MS) mass spectrum is acquired from the fragmentation of a precursor ion by multiple methods including information-dependent acquisition (IDA), SWATH (sequential window acquisition of all theoretical fragment-ion spectra), and MS(All) (also called MS(E)) techniques. We compared these three techniques in their capabilities to produce comprehensive MS(2) data by assessing both metabolite MS(2) acquisition hit rate and the quality of MS(2) spectra. Rat liver microsomal incubations from eight test compounds were analyzed with four methods (IDA, MMDF (multiple mass defect filters)-IDA, SWATH, or MS(All)) using an ultrahigh-performance liquid chromatography-qudrupole time-of-flight mass spectrometry (UHPLC-Q-TOF MS) platform. A combined total of 227 drug-related materials (DRM) were detected from all eight test article incubations, and among those, 5% and 4% of DRM were not triggered for MS(2) acquisition with IDA and MMDF-IDA methods, respectively. When the same samples were spiked to an equal volume of blank rat urine (urine sample), the DRM without MS(2) acquisition increased to 29% and 18%, correspondingly. In contrast, 100% of DRM in both matrixes were subjected to MS(2) acquisition with either the SWATH or MS(All) method. However, the quality of the acquired MS(2) spectra decreased in the order of IDA, SWATH, and MS(All) methods. An average of 10, 9, and 6 out of 10 most abundant ions in MS(2) spectra were the real product ions of DRM detected in microsomal samples from IDA, SWATH, and MS(All) methods, respectively. The corresponding numbers declined to 9, 6, and 3 in the urine samples. Overall, IDA-based methods acquired qualitatively better MS(2) spectra but with a lower MS(2) acquisition hit rate than the other two methods. SWATH outperformed the MS(All) method given its better quality of MS(2) spectra with an identical MS(2) acquisition hit rate.

Phosphoinositide-3-kinase inhibitors: evaluation of substituted alcohols as replacements for the piperazine sulfonamide portion of AMG 511.

Replacement of the piperazine sulfonamide portion of the PI3Kα inhibitor AMG 511 (1) with a range of aliphatic alcohols led to the identification of a truncated gem-dimethylbenzylic alcohol analog, 2-(5-(4-amino-6-methyl-1,3,5-triazin-2-yl)-6-((5-fluoro-6-methoxypyridin-3-yl)amino)pyridin-3-yl)propan-2-ol (7). This compound possessed good in vitro efficacy and pharmacokinetic parameters and demonstrated an EC50 of 239 ng/mL in a mouse liver pharmacodynamic model measuring the inhibition of hepatocyte growth factor (HGF)-induced Akt Ser473 phosphorylation in CD1 nude mice 6 h post-oral dosing.

Accelerated and Biopredictive In Vitro Release Testing Strategy for Single Agent and Combination Long-Acting Injectables.

The purpose of this study was to develop an in vitro release testing (IVRT) strategy to predict the pre-clinical performance of single agent and combination long acting injectable (LAI) suspension products. Two accelerated IVRT methods were developed using USP apparatus 2 to characterize initial, intermediate, and terminal phases of drug release. Initial and intermediate phases were captured using a suspension cup with moderate agitation to ensure a constant, low surface area exposure of the LAI suspension to the release media. The terminal phase was obtained by exposing the LAI suspension to a high initial paddle speed. This resulted in smaller suspension particulates with high cumulative surface area that were dispersed throughout the release media, enabling rapid drug release. The in vitro release profiles obtained with these two methods in 48 h or less were independently time scaled to reflect the in vivo time scale of approximately 1800 h. Level-A in vitro in vivo correlations (IVIVCs) were separately developed for each method and active pharmaceutical ingredient (API) using in vivo absorption profiles obtained by deconvolution of rat plasma concentration-time profiles. The IVIVCs were successfully validated for each API. This work provides a framework for evaluating individual phases of drug release of complex LAIs to ultimately predict their in vivo performance.

Pharmacokinetics, Disposition, and Biotransformation of [14C]Lenacapavir, a Novel, First-in-Class, Selective Inhibitor of HIV-1 Capsid Function, in Healthy Participants Following a Single Intravenous Infusion.

BACKGROUND AND OBJECTIVE: Lenacapavir (LEN) is a novel, first-in-class, multistage, selective inhibitor of human immunodeficiency virus type 1 (HIV-1) capsid function recently approved for the treatment of HIV-1 infection in heavily treatment-experienced adults with multidrug-resistant HIV-1 infection. The purpose of this multicohort study was to evaluate the pharmacokinetics, metabolism, excretion, safety, and tolerability of LEN following a single intravenous (IV) infusion of 10 mg LEN or 20 mg [14C]LEN in healthy participants. METHODS: Twenty-one healthy adult participants were enrolled into the study and received either a single IV dose of 10 mg LEN (n = 8 active, n = 3 placebo; cohort 1) or a single IV dose of 20 mg [14C]LEN containing 200 µCi (n = 10; cohort 2). Blood, urine, and feces samples (when applicable) were collected after dosing, and radioactivity (cohort 2) was assessed using liquid scintillation counting in both plasma and excreta. LEN in plasma was quantified by liquid chromatography (LC) tandem mass spectroscopy (MS/MS) method bioanalysis. Metabolite profiling in plasma and excreta were performed using LC-fraction collect (FC)-high-resolution MS and LC-FC-accelerator mass spectrometry in plasma. RESULTS: Between the 10 mg and 20 mg doses of LEN, the observed plasma exposure of LEN doubled, while the elimination half-life was similar. Following administration of 20 mg [14C]LEN (200 µCi), the mean cumulative recovery of [14C] radioactivity was 75.9% and 0.24% from feces and urine, respectively. The mean whole [14C] blood-to-plasma concentration ratio was 0.5-0.7, which showed a low distribution of LEN to red blood cells. Intact LEN was the predominant circulating species in plasma (representing 68.8% of circulating radioactivity), and no single metabolite contributed to > 10% of total radioactivity exposure through 1176 h postdose. Similarly, intact LEN was the most abundant component (32.9% of administered dose; 75.9% of recovered dose) measured in feces, with metabolites accounting for trace amounts. These results suggest metabolism of LEN is not a primary pathway of elimination. Of the metabolites observed in the feces, the three most abundant metabolites were direct phase 2 conjugates (glucuronide, hexose, and pentose conjugates), with additional metabolites formed to a lesser extent via other pathways. The administered LEN IV doses were generally safe and well-tolerated across participants in this study. CONCLUSIONS: The results of this mass balance study indicated that LEN was majorly eliminated as intact LEN via the feces. The renal pathway played a minor role in LEN elimination (0.24%). In addition, no major circulating metabolites in plasma or feces were found, indicating minimal metabolism of LEN.