Preclinical characterization of the ADME properties of a surrogate anti-IL-36R monoclonal antibody antagonist in mouse serum and tissues.

The decision to pursue a monoclonal antibody (mAb) as a therapeutic for disease intervention requires the assessment of many factors, such as target-biology, including the total target burden and its accessibility at the intended site of action, as well as mAb-specific properties like binding affinity and the pharmacokinetics in serum and tissue. Interleukin-36 receptor (IL-36 R) is a member of the IL-1 family cytokine receptors and an attractive target to treat numerous epithelial-mediated inflammatory conditions, including psoriatic and rheumatoid arthritis, asthma, and chronic obstructive pulmonary disease. However, information concerning the expression profile of IL-36 R at the protein level is minimal, so the feasibility of developing a therapeutic mAb against this target is uncertain. Here, we present a characterization of the properties associated with absorption, distribution, metabolism, and excretion of a high-affinity IL-36 R-targeted surrogate rat (IgG2a) mAb antagonist in preclinical mouse models. The presence of IL-36 R in the periphery was confirmed unequivocally as the driver of non-linear pharmacokinetics in blood/serum, although a predominant site of tissue accumulation was not observed based upon the kinetics of radiotracer. Additionally, the contribution of IL-36 R-mediated catabolism of mAb in kidney was tested in a 5/6 nephrectomized mouse model where minimal effects on serum pharmacokinetics were observed, although analysis of functional mAb in urine suggests that target can influence the amount of mAb excreted. Our data highlight an interesting case of target-mediated drug disposition (TMDD) where low, yet broadly expressed levels of membrane-bound target result in a cumulative effect to drive TMDD behavior typical of a large, saturable target sink. The potential differences between our mouse model and IL-36 R target profile in humans are also presented.

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.

Evaluation of matrix microsampling methods for therapeutic drug candidate quantification in discovery-stage rodent pharmacokinetic studies.

BACKGROUND: AMG 517 or 1-aminobenzotriazole were quantified by LC-MS/MS from low blood/plasma volumes for rat pharmacokinetic (PK) characterization in order to qualify manual/automated dried blood spot (DBS) sampling and plasma separation capillary sampling. In addition, mouse serial automated blood sampling was compared with standard composite sampling. MATERIALS & METHODS: AMG 517 or 1-aminobenzotriazole was administered to rats or mice and multiple microsampling techniques were used to obtain blood or plasma. RESULTS: PK parameters derived from DBS and whole blood-obtained drug concentrations were within 7% for manual DBS and 20% for automated DBS. Plasma PK parameters derived from capillary or standard plasma-obtained drug concentrations differed by 6%. Plasma PK parameters obtained from serial automated blood sampling or manual composite sampling were within 20%. CONCLUSION: Collectively, these results suggest that the microsampling applications that were investigated are attractive approaches for quantifying drug candidates in low matrix volumes that can be successfully employed within discovery-stage rodent PK studies.

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.