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.

Intravenous delivery of GS-441524 is efficacious in the African green monkey model of SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID-19 pandemic, has infected over 260 million people over the past 2 years. Remdesivir (RDV, VEKLURY®) is currently the only antiviral therapy fully approved by the FDA for the treatment of COVID-19. The parent nucleoside of RDV, GS-441524, exhibits antiviral activity against numerous respiratory viruses including SARS-CoV-2, although at reduced in vitro potency compared to RDV in most assays. Here we find in both human alveolar and bronchial primary cells, GS-441524 is metabolized to the pharmacologically active GS-441524 triphosphate (TP) less efficiently than RDV, which correlates with a lower in vitro SARS-CoV-2 antiviral activity. In vivo, African green monkeys (AGM) orally dosed with GS-441524 yielded low plasma levels due to limited oral bioavailability of <10%. When GS-441524 was delivered via intravenous (IV) administration, although plasma concentrations of GS-441524 were significantly higher, lung TP levels were lower than observed from IV RDV. To determine the required systemic exposure of GS-441524 associated with in vivo antiviral efficacy, SARS-CoV-2 infected AGMs were treated with a once-daily IV dose of either 7.5 or 20 mg/kg GS-441524 or IV RDV for 5 days and compared to vehicle control. Despite the reduced lung TP formation compared to IV dosing of RDV, daily treatment with IV GS-441524 resulted in dose-dependent efficacy, with the 20 mg/kg GS-441524 treatment resulting in significant reductions of SARS-CoV-2 replication in the lower respiratory tract of infected animals. These findings demonstrate the in vivo SARS-CoV-2 antiviral efficacy of GS-441524 and support evaluation of its orally bioavailable prodrugs as potential therapies for COVID-19.

Therapeutic treatment with an oral prodrug of the remdesivir parental nucleoside is protective against SARS-CoV-2 pathogenesis in mice.

The coronavirus disease 2019 (COVID-19) pandemic remains uncontrolled despite the rapid rollout of safe and effective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines, underscoring the need to develop highly effective antivirals. In the setting of waning immunity from infection and vaccination, breakthrough infections are becoming increasingly common and treatment options remain limited. In addition, the emergence of SARS-CoV-2 variants of concern, with their potential to escape neutralization by therapeutic monoclonal antibodies, emphasizes the need to develop second-generation oral antivirals targeting highly conserved viral proteins that can be rapidly deployed to outpatients. Here, we demonstrate the in vitro antiviral activity and in vivo therapeutic efficacy of GS-621763, an orally bioavailable prodrug of GS-441524, the parent nucleoside of remdesivir, which targets the highly conserved virus RNA-dependent RNA polymerase. GS-621763 exhibited antiviral activity against SARS-CoV-2 in lung cell lines and two different human primary lung cell culture systems. GS-621763 was also potently antiviral against a genetically unrelated emerging coronavirus, Middle East respiratory syndrome CoV (MERS-CoV). The dose-proportional pharmacokinetic profile observed after oral administration of GS-621763 translated to dose-dependent antiviral activity in mice infected with SARS-CoV-2. Therapeutic GS-621763 administration reduced viral load and lung pathology; treatment also improved pulmonary function in COVID-19 mouse model. A direct comparison of GS-621763 with molnupiravir, an oral nucleoside analog antiviral that has recently received EUA approval, proved both drugs to be similarly efficacious in mice. These data support the exploration of GS-441524 oral prodrugs for the treatment of COVID-19.

Subcutaneous remdesivir administration prevents interstitial pneumonia in rhesus macaques inoculated with SARS-CoV-2.

The utility of remdesivir treatment in COVID-19 patients is currently limited by the necessity to administer this antiviral intravenously, which has generally limited its use to hospitalized patients. Here, we tested a novel, subcutaneous formulation of remdesivir in the rhesus macaque model of SARS-CoV-2 infection that was previously used to establish the efficacy of remdesivir against this virus in vivo. Compared to vehicle-treated animals, macaques treated with subcutaneous remdesivir from 12 h through 6 days post inoculation showed reduced signs of respiratory disease, a reduction of virus replication in the lower respiratory tract, and an absence of interstitial pneumonia. Thus, early subcutaneous administration of remdesivir can protect from lower respiratory tract disease caused by SARS-CoV-2.

Inhaled remdesivir reduces viral burden in a nonhuman primate model of SARS-CoV-2 infection.

Remdesivir (RDV) is a nucleotide analog prodrug with demonstrated clinical benefit in patients with coronavirus disease 2019 (COVID-19). In October 2020, the US FDA approved intravenous (IV) RDV as the first treatment for hospitalized COVID-19 patients. Furthermore, RDV has been approved or authorized for emergency use in more than 50 countries. To make RDV more convenient for non-hospitalized patients earlier in disease, alternative routes of administration are being evaluated. Here, we investigated the pharmacokinetics and efficacy of RDV administered by head dome inhalation in African green monkeys (AGM). Relative to an IV administration of RDV at 10 mg/kg, an approximately 20-fold lower dose administered by inhalation produced comparable concentrations of the pharmacologically active triphosphate in lower respiratory tract tissues. Distribution of the active triphosphate into the upper respiratory tract was also observed following inhaled RDV exposure. Inhalation RDV dosing resulted in lower systemic exposures to RDV and its metabolites as compared with IV RDV dosing. An efficacy study with repeated dosing of inhaled RDV in an AGM model of SARS-CoV-2 infection demonstrated reductions in viral replication in bronchoalveolar lavage fluid and respiratory tract tissues compared with placebo. Efficacy was observed with inhaled RDV administered once daily at a pulmonary deposited dose of 0.35 mg/kg beginning approximately 8 hours post-infection. Moreover, the efficacy of inhaled RDV was similar to that of IV RDV administered once at 10 mg/kg followed by 5 mg/kg daily in the same study. Together, these findings support further clinical development of inhalation RDV.

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.

Prodrugs of a 1'-CN-4-Aza-7,9-dideazaadenosine C-Nucleoside Leading to the Discovery of Remdesivir (GS-5734) as a Potent Inhibitor of Respiratory Syncytial Virus with Efficacy in the African Green Monkey Model of RSV.

A discovery program targeting respiratory syncytial virus (RSV) identified C-nucleoside 4 (RSV A2 EC50 = 530 nM) as a phenotypic screening lead targeting the RSV RNA-dependent RNA polymerase (RdRp). Prodrug exploration resulted in the discovery of remdesivir (1, GS-5734) that is >30-fold more potent than 4 against RSV in HEp-2 and NHBE cells. Metabolism studies in vitro confirmed the rapid formation of the active triphosphate metabolite, 1-NTP, and in vivo studies in cynomolgus and African Green monkeys demonstrated a >10-fold higher lung tissue concentration of 1-NTP following molar normalized IV dosing of 1 compared to that of 4. A once daily 10 mg/kg IV administration of 1 in an African Green monkey RSV model demonstrated a >2-log10 reduction in the peak lung viral load. These early data following the discovery of 1 supported its potential as a novel treatment for RSV prior to its development for Ebola and approval for COVID-19 treatment.

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.

Clinical Pharmacokinetics and Pharmacodynamics of Etelcalcetide, a Novel Calcimimetic for Treatment of Secondary Hyperparathyroidism in Patients With Chronic Kidney Disease on Hemodialysis.

Etelcalcetide, a d-amino acid peptide, is an intravenous calcimimetic approved for the treatment of secondary hyperparathyroidism. Etelcalcetide binds the calcium-sensing receptor and increases its sensitivity to extracellular calcium, thereby decreasing secretion of parathyroid hormone (PTH) by chief cells. Etelcalcetide and its low-molecular-weight transformation products are rapidly cleared by renal excretion in healthy subjects, but clearance is substantially reduced and dependent on hemodialysis in end-stage renal disease. The effective half-life is 3-5 days in patients undergoing hemodialysis 3 times a week. A clinical study using a single microtracer intravenous dose of [14 C]etelcalcetide indicated that 60% of the administered dose was eliminated in dialysate. Etelcalcetide undergoes reversible disulfide exchange with serum albumin to form a serum albumin peptide conjugate that is too large (67 kDa) to be dialyzed, until a subsequent exchange forms etelcalcetide or a low-molecular-weight transformation product. This exchange from albumin is apparent after hemodialysis, when it partially restores etelcalcetide concentrations in plasma. Etelcalcetide has no known risks for drug-drug interactions. In phase 3 studies, 74%-75% of hemodialysis patients with secondary hyperparathyroidism who received etelcalcetide achieved a >30% PTH reduction from baseline versus 8%-10% of patients who received placebo. The pharmacokinetics and pharmacodynamics of etelcalcetide in hemodialysis patients supports a 5-mg starting dose administered after hemodialysis and uptitration in 2.5- or 5-mg increments every 4 weeks to a maximum dose of 15 mg 3 times a week.

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.

Clinical immunogenicity of the d-amino acid peptide therapeutic etelcalcetide: Method development challenges and anti-drug antibody clinical impact assessments.

The immunogenicity risk assessment and bioanalytical strategy for novel therapeutics should account for both unique biophysical properties and potential consequences of immunogenicity. When assessing the immunogenicity risk of etelcalcetide, a peptide agonist of the calcium-sensing receptor, we considered the potential that the d-amino acid 'backbone' and biotransformation of etelcalcetide could allow the drug to act as a hapten. As a consequence, we validated and implemented a surface plasmon resonance immunoassay platform with both etelcalcetide and etelcalcetide-'carrier' surfaces to detect anti-drug antibodies (ADA). No evidence of in-vitro neutralizing activity with surrogate controls was detected despite multiple immunization approaches and a sensitive cell-based activity assay. Therefore, a neutralizing assay was not implemented for clinical support. We conducted an integrated analysis of immunogenicity data pooled from two pivotal placebo-controlled trials to define the clinical impact of anti-etelcalcetide antibodies. While both pre-existing and developing anti-etelcalcetide antibodies were detected, we show here that they have no consequences for clinical exposure, efficacy, or safety of etelcalcetide.

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.

Pharmacokinetics, Biotransformation, and Excretion of [14C]Etelcalcetide (AMG 416) Following a Single Microtracer Intravenous Dose in Patients with Chronic Kidney Disease on Hemodialysis.

Etelcalcetide (AMG 416) is a novel synthetic peptide calcium-sensing receptor activator in clinical development as an intravenous calcimimetic for the treatment of secondary hyperparathyroidism in patients with chronic kidney disease (CKD) on hemodialysis. Etelcalcetide is composed of seven D-aminoacids with an L-cysteine linked to a D-cysteine by a disulfide bond. A single intravenous dose of [14C]etelcalcetide (10 mg; 26.3 kBq; 710 nCi) was administered to patients with CKD on hemodialysis to elucidate the pharmacokinetics, biotransformation, and excretion of etelcalcetide in this setting. Blood, dialysate, urine, and feces were collected to characterize the pharmacokinetics, biotransformation product profiles, mass balance, and formation of anti-etelcalcetide antibodies. Accelerator mass spectrometry was necessary to measure the microtracer quantities of C-14 excreted in the large volumes of dialysate and other biomatrices. An estimated 67 % of the [14C]etelcalcetide dose was recovered in dialysate, urine, and feces 176 days after dose administration. Etelcalcetide was primarily cleared by hemodialysis, with approximately 60 % of the administered dose eliminated in dialysate. Minor excretion was observed in urine and feces. Biotransformation resulted from disulfide exchange with endogenous thiols, and preserved the etelcalcetide D-amino acid backbone. Drug-related radioactivity circulated primarily as serum albumin peptide conjugate (SAPC). Following removal of plasma etelcalcetide by hemodialysis, re-equilibration occurred between SAPC and L-cysteine present in blood to partially restore the etelcalcetide plasma concentrations between dialysis sessions. No unanticipated safety signals or anti-etelcalcetide or anti-SAPC antibodies were detected.

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.

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.

Determination of Etelcalcetide Biotransformation and Hemodialysis Kinetics to Guide the Timing of Its Dosing.

INTRODUCTION: Etelcalcetide, a novel calcimimetic agonist of the calcium-sensing receptor for treatment of secondary hyperparathyroidism in chronic kidney disease patients on hemodialysis, is a d-amino acid linear heptapeptide with a d-cysteine that is linked to an l-cysteine by a disulfide bond. In addition to binding to the calcium-sensing receptor, etelcalcetide is biotransformed by disulfide exchange in whole blood to predominantly form a covalent serum albumin peptide conjugate (SAPC). Key factors anticipated to affect the pharmacokinetics and disposition of etelcalcetide in chronic kidney disease patients on hemodialysis are the drug's intrinsic dialytic properties and biotransformation kinetics. METHODS: These factors were investigated using in vitro methods, and the findings were modeled to derive corresponding kinetic rate constants. RESULTS: Biotransformation was reversible after incubation of etelcalcetide or SAPC in human whole blood. The rate of SAPC formation from etelcalcetide was 18-fold faster than the reverse process. Clearance of etelcalcetide by hemodialysis was rapid in the absence of blood and when hemodialysis was initiated immediately after addition of etelcalcetide to blood. Preincubation of etelcalcetide in blood for 3 hours before hemodialysis resulted in formation of SAPC and decreased its clearance due to the slow rate of etelcalcetide formation from SAPC. Etelcalcetide hemodialysis clearance was >16-fold faster than its biotransformation. DISCUSSION: These results indicate that etelcalcetide should be administered after hemodialysis to avoid elimination of a significant fraction of the dose.

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.

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.

P450-mediated O-demethylated metabolite is responsible for rat hepatobiliary toxicity of pyridyltriazine-containing PI3K inhibitors.

The dysregulation of phosphatidylinositol 3-kinase (PI3K)-dependent pathways is implicated in several human cancers making it an attractive target for small molecule PI3K inhibitors. A series of potent pyridyltriazine-containing inhibitors of class Ia PI3Ks were synthesized and a subset of compounds was evaluated in exploratory repeat-dose rat toxicology studies. Daily oral dosing of compound 1: in Sprague Dawley rats for four consecutive days was associated with hepatobiliary toxicity that included biliary epithelial hyperplasia and hypertrophy, periductular edema, biliary stasis, and acute peribiliary inflammatory infiltrates. These histological changes were associated with clinical pathology changes that included increased serum liver enzymes, total bile acids, and bilirubin. The predominant clearance pathway of 1: was shown in vitro and in a bile-duct cannulated rat (14)C-ADME study to be P450-mediated oxidative metabolism. An O-demethylated pyridine metabolite, M3: , was identified as a candidate proximal metabolite that caused the hepatotoxicity. Co-administration of the pan-P450 inhibitor 1-aminobenzotriazole with 1: to rats significantly reduced the formation of M3: and prevented liver toxicity, whereas direct administration of M3: reproduced the toxicity. Structural changes were introduced to 1: to make the methoxypyridine ring less susceptible to P450 oxidation (compound 2: ), and addition of a methyl group to the benzylic carbon (compound 3: ) improved the pharmacokinetic profile. These changes culminated in the successful design of a clinical candidate 3: (AMG 511) that was devoid of liver toxicity in a 14-day rat toxicity study. Herein, we describe how a metabolism-based structure-activity relationship analysis allowed for the successful identification of a PI3K inhibitor devoid of off-target toxicity.