Challenges and Opportunities with Non-CYP Enzymes Aldehyde Oxidase, Carboxylesterase, and UDP-Glucuronosyltransferase: Focus on Reaction Phenotyping and Prediction of Human Clearance.

Over the years, significant progress has been made in reducing metabolic instability due to cytochrome P450-mediated oxidation. High-throughput metabolic stability screening has enabled the advancement of compounds with little to no oxidative metabolism. Furthermore, high lipophilicity and low aqueous solubility of presently pursued chemotypes reduces the probability of renal excretion.

Integrated Pharmacokinetic/Pharmacodynamic Analysis for Determining the Minimal Anticipated Biological Effect Level of a Novel Anti-CD28 Receptor Antagonist BMS-931699.

BMS-931699 (lulizumab pegol), a domain antibody (dAb) conjugated with 40-kDa branched polyethylene glycol, is a human anti-CD28 receptor antagonist under development for the treatment of inflammatory and autoimmune diseases. In the present work, the minimal anticipated biologic effect level (MABEL) was determined for BMS-931699 by integrating all the available preclinical data. The relevance of the in vitro mixed lymphocyte reaction (MLR) assay to a whole blood CD28 receptor occupancy (RO) assessment, as well as the relationship between the CD28 RO and the inhibition of T-cell-dependent antibody response to keyhole limpet hemocyanin in vivo, was demonstrated through an integrated pharmacokinetic/pharmacodynamic analysis using anti-hCD28 dAb-001 (differing from BMS-931699 by two additional amino acids at the N-terminus) and a mouse surrogate.

Discovery of ((4-(5-(Cyclopropylcarbamoyl)-2-methylphenylamino)-5-methylpyrrolo[1,2-f][1,2,4]triazine-6-carbonyl)(propyl)carbamoyloxy)methyl-2-(4-(phosphonooxy)phenyl)acetate (BMS-751324), a Clinical Prodrug of p38α MAP Kinase Inhibitor.

In search for prodrugs to address the issue of pH-dependent solubility and exposure associated with 1 (BMS-582949), a previously disclosed phase II clinical p38α MAP kinase inhibitor, a structurally novel clinical prodrug, 2 (BMS-751324), featuring a carbamoylmethylene linked promoiety containing hydroxyphenyl acetic acid (HPA) derived ester and phosphate functionalities, was identified. Prodrug 2 was not only stable but also water-soluble under both acidic and neutral conditions. It was effectively bioconverted into parent drug 1 in vivo by alkaline phosphatase and esterase in a stepwise manner, providing higher exposure of 1 compared to its direct administration, especially within higher dose ranges.

Role of hepatic blood flow and metabolism in the pharmacokinetics of ten drugs in lean, aged and obese rats.

1. The effect of age and obesity on the pharmacokinetics (PK), hepatic blood flow (HBF) and liver metabolism of 10 compounds was determined in rats. The animals fed a high-fat diet were defined as the diet-induced obese (DIO) group, while the animals that were aged similar to the DIO rats but not fed with high-fat diet were called the age-matched (AM) group.

Estimation of the unbound brain concentration of P-glycoprotein substrates or nonsubstrates by a serial cerebrospinal fluid sampling technique in rats.

The unbound concentration in plasma drives the transport of the drug into the brain, and the unbound drug concentration in the central nervous system (CNS) drives the interaction with the target eliciting the pharmacological effect. Delivery of the drug to the CNS is a challenge because of the unique neurovascular unit, which restricts the passage of drugs into the brain. The efflux transporters [especially P-glycoprotein (P-gp)] present at the blood-brain barrier (BBB) act as one of the major detractors for keeping drugs outside the CNS.

Synthesis and evaluation of carbamoylmethylene linked prodrugs of BMS-582949, a clinical p38α inhibitor.

A series of carbamoylmethylene linked prodrugs of 1 (BMS-582949), a clinical p38α inhibitor, were synthesized and evaluated. Though the phosphoryloxymethylene carbamates (3, 4, and 5) and α-aminoacyloxymethylene carbamates (22, 23, and 26) were found unstable at neutral pH values, fumaric acid derived acyloxymethylene carbamates (2, 28, and 31) were highly stable under both acidic and neutral conditions. Prodrugs 2 and 31 were also highly soluble at both acidic and neutral pH values.

Impact of nonlinear midazolam pharmacokinetics on the magnitude of the midazolam-ketoconazole interaction in rats.

Numerous groups have described the rat as an in vivo model for the assessment and prediction of drug-drug interactions (DDIs) in humans involving the inhibition of cytochrome P450 3A forms. Even for a well-established substrate-inhibitor pair like midazolam-ketoconazole, however, the magnitude of the DDI in rats (e.g.

Discovery of pyrrolo[2,1-f][1,2,4]triazine C6-ketones as potent, orally active p38α MAP kinase inhibitors.

Pyrrolo[2,1-f][1,2,4]triazine based inhibitors of p38α have been prepared exploring functional group modifications at the C6 position. Incorporation of aryl and heteroaryl ketones at this position led to potent inhibitors with efficacy in in vivo models of acute and chronic inflammation.

Discovery of 4-(5-(cyclopropylcarbamoyl)-2-methylphenylamino)-5-methyl-N-propylpyrrolo[1,2-f][1,2,4]triazine-6-carboxamide (BMS-582949), a clinical p38α MAP kinase inhibitor for the treatment of inflammatory diseases.

The discovery and characterization of 7k (BMS-582949), a highly selective p38α MAP kinase inhibitor that is currently in phase II clinical trials for the treatment of rheumatoid arthritis, is described. A key to the discovery was the rational substitution of N-cyclopropyl for N-methoxy in 1a, a previously reported clinical candidate p38α inhibitor. Unlike alkyl and other cycloalkyls, the sp(2) character of the cyclopropyl group can confer improved H-bonding characteristics to the directly substituted amide NH.

Examination of CYP3A and P-glycoprotein-mediated drug-drug interactions using animal models.

With the advent of polytherapy for cancer treatment it has become prudent to minimize, as much as possible, the potential for drug-drug interactions (DDI). Toward this end, the metabolic and transporter pathways involved in the disposition of a drug candidate (phenotyping) and potential for inhibition and induction of drug-metabolizing enzymes and transporters are evaluated in vitro. Such in vitro human data can be made available prior to human dosing and enable in vitro to in vivo-based predictions of clinical outcomes.

Preclinical pharmacokinetics and in vitro metabolism of brivanib (BMS-540215), a potent VEGFR2 inhibitor and its alanine ester prodrug brivanib alaninate.

Purpose: Brivanib alaninate is a prodrug of brivanib (BMS-540215), a potent oral VEGFR-2 inhibitor and is currently in development for the treatment of hepatocellular and colon carcinomas. In vitro and in vivo studies were conducted to characterize the preclinical pharmacokinetics and disposition of brivanib and brivanib alaninate, and antitumor efficacy in mice bearing human xenografts.

Methods: In vitro studies were conducted in liver and intestinal fractions, plasma and Caco-2 cells to assess the metabolic stability.

Novel tricyclic inhibitors of IkappaB kinase.

The design and synthesis of a novel series of oxazole-, thiazole-, and imidazole-based inhibitors of IkappaB kinase (IKK) are reported. Biological activity was improved compared to the pyrazolopurine lead, and the expedient synthesis of the new tricyclic systems allowed for efficient exploration of structure-activity relationships. This, combined with an iterative rat cassette dosing strategy, was used to identify compounds with improved pharmacokinetic (PK) profiles to advance for in vivo evaluation.

The discovery of (R)-2-(sec-butylamino)-N-(2-methyl-5-(methylcarbamoyl)phenyl) thiazole-5-carboxamide (BMS-640994)-A potent and efficacious p38alpha MAP kinase inhibitor.

A novel structural class of p38alpha MAP kinase inhibitors has been identified via iterative SAR studies of a focused deck screen hit. Optimization of the lead series generated 6e, BMS-640994, a potent and selective p38alpha inhibitor that is orally efficacious in rodent models of acute and chronic inflammation.

Benzothiazole based inhibitors of p38alpha MAP kinase.

Rational design, synthesis, and SAR studies of a novel class of benzothiazole based inhibitors of p38alpha MAP kinase are described. The issue of metabolic instability associated with vicinal phenyl, benzo[d]thiazol-6-yl oxazoles/imidazoles was addressed by the replacement of the central oxazole or imidazole ring with an aminopyrazole system. The proposed binding mode of this new class of p38alpha inhibitors was confirmed by X-ray crystallographic studies of a representative inhibitor (6a) bound to the p38alpha enzyme.

Design, synthesis, and anti-inflammatory properties of orally active 4-(phenylamino)-pyrrolo[2,1-f][1,2,4]triazine p38alpha mitogen-activated protein kinase inhibitors.

A novel structural class of p38 mitogen-activated protein (MAP) kinase inhibitors consisting of substituted 4-(phenylamino)-pyrrolo[2,1- f][1,2,4]triazines has been discovered. An initial subdeck screen revealed that the oxindole-pyrrolo[2,1- f][1,2,4]triazine lead 2a displayed potent enzyme inhibition (IC 50 60 nM) and was active in a cell-based TNFalpha biosynthesis inhibition assay (IC 50 210 nM). Replacement of the C4 oxindole with 2-methyl-5- N-methoxybenzamide aniline 9 gave a compound with superior p38 kinase inhibition (IC 50 10 nM) and moderately improved functional inhibition in THP-1 cells.

Effect of commonly used organic solvents on the kinetics of cytochrome P450 2B6- and 2C8-dependent activity in human liver microsomes.

The effect of common organic solvents on the activities of various human cytochromes P450 has been reported. However, very little is known about their influence on CYP2B6 and CYP2C8 enzymes. The purpose of this study was to investigate the effect of solvents on the kinetics of representative CYP2B6 (bupropion hydroxylase) and CYP2C8 (paclitaxel hydroxylase) reactions in human liver microsomes.

Preclinical pharmacokinetics and in vitro metabolism of dasatinib (BMS-354825): a potent oral multi-targeted kinase inhibitor against SRC and BCR-ABL.

Purpose: Dasatinib (BMS-354825), a potent oral multi-targeted kinase inhibitor against SRC and BCR-ABL, has recently been approved for the treatment of chronic myelogenous leukaemia (CML) in imatinib-acquired resistance and intolerance. In vitro and in vivo studies were conducted to characterize the pharmacokinetics and metabolism of dasatinib in mouse, rat, dog, and monkey. Possible mechanisms contributing to the incomplete oral bioavailability of dasatinib in animals were investigated.

Development and validation of a preclinical food effect model.

A preclinical canine model capable of predicting a compound's potential for a human food effect was developed. The beagle dog was chosen as the in vivo model. A validation set of compounds with known propensities for human food effect was studied.

In vivo animal models for investigating potential CYP3A- and Pgp-mediated drug-drug interactions.

With the advent of polytherapy it has become prudent to minimize, as much as possible, the potential for drug-drug interactions. Towards this end, the metabolic and transporter pathways involved in the disposition of a drug candidate (phenotyping) are evaluated in vitro employing available human tissue and specific reagents. Likewise, in vitro screening for inhibition and induction of drug-metabolizing enzymes and transporters is conducted also.

Novel fluoro-substituted camptothecins: in vivo antitumor activity, reduced gastrointestinal toxicity and pharmacokinetic characterization.

Purpose: The novel fluoro-substituted camptothecin analog, BMS-286309, and its prodrug, BMS-422461, were evaluated for their pharmacologic, toxicologic, metabolic and pharmacokinetic developmental potential.

Methods: In vitro and in vivo assays were used to assess the compounds for topoisomerase I activity, antitumor activity, gastrointestinal (GI) toxicity, and pharmacokinetic parameters.

Results: BMS-286309-induced topoisomerase I-mediated DNA breaks in vitro and was similar in potency to camptothecin.

Preclinical pharmacokinetics and oral bioavailability of BMS-310705, a novel epothilone B analog.

Purpose: BMS-310705, a novel semisynthetic derivative of epothilone B, is a tubulin-polymerization agent currently in phase I clinical trials for anticancer therapy. The in vitro and in vivo pharmacokinetics and oral bioavailability of BMS-310705 were investigated in mice, rats, and dogs. In addition, comparison of the pharmacokinetics of BMS-310705 using various formulations was conducted in rats.

P-glycoprotein plays a role in the oral absorption of BMS-387032, a potent cyclin-dependent kinase 2 inhibitor, in rats.

Purpose: BMS-387032, a novel cyclin-dependent kinase 2 inhibitor, is currently in phase I clinical trials for anticancer therapy. The oral bioavailability of BMS-387032 has been found to be about 31% in rats. Absorption and first-pass metabolism were evaluated as possible reasons for the incomplete oral bioavailability in rats.

Selective Itk inhibitors block T-cell activation and murine lung inflammation.

Nonreceptor protein tyrosine kinases including Lck, ZAP-70, and Itk play essential roles in T-cell receptor (TCR) signaling. Gene knockout studies have revealed that mice lacking these individual kinases exhibit various degrees of immunodeficiency; however, highly selective small molecule inhibitors of these kinases as potential immunosuppressive agents have not been identified. Here we discovered two novel compounds, BMS-488516 and BMS-509744, that potently and selectively inhibit Itk kinase activity.