Pharmacokinetics and metabolism of AMG 232, a novel orally bioavailable inhibitor of the MDM2-p53 interaction, in rats, dogs and monkeys: in vitro-in vivo correlation.

1. AMG 232 is a novel inhibitor of the p53-MDM2 protein-protein interaction currently in Phase I clinical trials for multiple tumor indications. The objectives of the investigations reported in this article were to characterize the pharmacokinetic and drug metabolism properties of AMG 232 in pre-clinical species in vivo and in vitro, and in humans in vitro, and to predict its pharmacokinetics in humans through integrating PKDM data. 2. AMG 232 exhibited low clearance (<0.25 × Qh) and moderate to high oral bioavailability in mice, rats and monkeys (>42%), but high clearance (0.74 × Qh) and low oral exposure in dogs (18%). 3. Biotransformation was the major route of elimination of AMG 232 in rats, with only 7% of intravenously administered (14)C-labeled AMG 232 recovered as parent molecule in bile. The major metabolite was an acyl glucuronide as measured by in vivo rat studies and in vitro hepatocyte incubations in multiple species. 4. The in vitro-in vivo correlation of AMG 232 clearance was within 2-fold in pre-clinical species using hepatocytes. AMG 232 was predicted to exhibit low clearance, high volume distribution and long half-life in humans. The predictions are consistent with the preliminary human pharmacokinetic parameters of AMG 232 in clinical trials.

Deletion of Abcg2 has differential effects on excretion and pharmacokinetics of probe substrates in rats.

This study was designed to characterize breast cancer resistance protein (Bcrp) knockout Abcg2(-/-) rats and assess the effect of ATP-binding cassette subfamily G member 2 (Abcg2) deletion on the excretion and pharmacokinetic properties of probe substrates. Deletion of the target gene in the Abcg2(-/-) rats was confirmed, whereas gene expression was unaffected for most of the other transporters and metabolizing enzymes. Biliary excretion of nitrofurantoin, sulfasalazine, and compound A [2-(5-methoxy-2-((2-methyl-1,3-benzothiazol-6-yl)amino)-4-pyridinyl)-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyridin-4-one] accounted for 1.5, 48, and 48% of the dose in the Abcg2(+/+) rats, respectively, whereas it was decreased by 70 to 90% in the Abcg2(-/-) rats. Urinary excretion of nitrofurantoin, a significant elimination pathway, was unaffected in the Abcg2(-/-) rats, whereas renal clearance of sulfasalazine, a minor elimination pathway, was reduced by >90%. Urinary excretion of compound A was minimal. Systemic clearance in the Abcg2(-/-) rats decreased 22, 43 (p<0.05), and 57%, respectively, for nitrofurantoin, sulfasalazine, and compound A administered at 1 mg/kg and 27% for compound A administered at 5 mg/kg. Oral absorption of nitrofurantoin, a compound with high aqueous solubility and good permeability, was not limited by Bcrp. In contrast, the absence of Bcrp led to a 33- and 11-fold increase in oral exposure of sulfasalazine and compound A, respectively. These data show that Bcrp plays a crucial role in biliary excretion of these probe substrates and has differential effects on systemic clearance and oral absorption in rats depending on clearance mechanisms and compound properties. The Abcg2(-/-) rat is a useful model for understanding the role of Bcrp in elimination and oral absorption.

Bioactivation of a novel 2-methylindole-containing dual chemoattractant receptor-homologous molecule expressed on T-helper type-2 cells/D-prostanoid receptor antagonist leads to mechanism-based CYP3A inactivation: glutathione adduct characterization and prediction of in vivo drug-drug interaction.

The 2-methyl substituted indole, 2MI [2-(4-(4-(2,4-dichlorophenylsulfonamido)-2-methyl-1H-indol-5-yloxy)-3-methoxyphenyl)acetic acid] is a potent dual inhibitor of 1) chemoattractant receptor-homologous molecule expressed on T-helper type-2 cells and 2) d-prostanoid receptor. During evaluation as a potential treatment for asthma and allergic rhinitis, 2MI was identified as a mechanism-based inactivator of CYP3A4 in vitro. The inactivation was shown to be irreversible by dialysis and accompanied by an NADPH-dependent increase in 2MI covalent binding to a 55- to 60-kDa microsomal protein, consistent with irreversible binding to CYP3A4. Two glutathione (GSH) adducts, G1 and G2, were identified in vitro, and the more abundant adduct (G1) was unambiguously determined via NMR to be GSH adducted to the 3-position of the 2-methylindole moiety. The potential for a clinical drug-drug interaction arising from mechanism-based inactivation of CYP3A4 by 2MI was predicted using a steady-state model, and a 4.3- to 7.5-fold increase in the exposure of midazolam was predicted at anticipated therapeutic concentrations. To better assess the potential for in vivo drug-drug interactions, the Sprague-Dawley rat was used as an in vivo model. An excellent in vitro-in vivo correlation was observed for the reduction in enzyme steady-state concentration (E'(ss/Ess)) as well as the change in the exposure of a prototypical CYP3A substrate, indinavir (area under the curve (AUC) for indinavir/AUC). In summary, 2MI was identified as a potent mechanism-based inactivator of CYP3A and was predicted to elicit a clinically relevant drug-drug interaction in humans at an anticipated therapeutic concentration.

Potent N-(1,3-thiazol-2-yl)pyridin-2-amine vascular endothelial growth factor receptor tyrosine kinase inhibitors with excellent pharmacokinetics and low affinity for the hERG ion channel.

A series of N-(1,3-thiazol-2-yl)pyridin-2-amine KDR kinase inhibitors have been developed that possess optimal properties. Compounds have been discovered that exhibit excellent in vivo potency. The particular challenges of overcoming hERG binding activity and QTc increases in vivo in addition to achieving good pharmacokinetics have been acomplished by discovering a unique class of amine substituents. These compounds have a favorable kinase selectivity profile that can be accentuated with appropriate substitution.

Optimization of the indolyl quinolinone class of KDR (VEGFR-2) kinase inhibitors: effects of 5-amido- and 5-sulphonamido-indolyl groups on pharmacokinetics and hERG binding.

Modifications to the basic side-chain of early lead structures of the indolyl quinolinone class of KDR kinase inhibitors resulted in improved pharmacokinetic and ancillary profiles. Specifically, compounds bearing 5-amido- and 5-sulphonamido-indolyl substituents exhibited lower plasma clearance and weaker binding affinity for the I(Kr) potassium channel hERG.

Metabolic activation of a pyrazinone-containing thrombin inhibitor. Evidence for novel biotransformation involving pyrazinone ring oxidation, rearrangement, and covalent binding to proteins.

Compound I, (2-[3-[(2,2-difluoro-2(2-pyridyl)ethyl)amino]-6-methyl-2-oxohydropyrazinyl]-N-[(3-fluoro(2-pyridyl))methyl]acetamide, is a potent competitive inhibitor of thrombin that reacts stoichiometrically with the protease. Compounds of this class possess therapeutic potential as anticoagulation agents. During the metabolic characterization of compound I, evidence was obtained for extensive metabolic activation of the pyrazinone ring system. Following administration of (14)C-labeled I to rats, significant levels of irreversibly bound radioactivity to proteins were detected in rat plasma and liver. LC/MS/MS analysis of metabolites formed in rat and human liver microsomes fortified with glutathione (GSH) revealed the presence of two structurally distinct GSH adducts. It is proposed that the first of these GSH conjugates derives from a two electron oxidation of the 6-methyl-2-oxo-3-aminopyrazinone moiety to afford an electrophilic imine-methide intermediate, while the second is formed by addition of GSH to an epoxide formed by P-450-mediated oxidation of the double bond at the 5-6 position of the pyrazinone ring. The addition of GSH to the proposed epoxide facilitates opening of the pyrazinone ring and a rearrangement to afford a stable, rearranged imidazole-containing metabolite. Elucidation of the metabolic activation pathways of I provides structural guidance for the design of thrombin inhibitors with decreased potential for the generation of chemically reactive intermediates.

Design and synthesis of a pro-drug of vinblastine targeted at treatment of prostate cancer with enhanced efficacy and reduced systemic toxicity.

Chemotherapy of prostate cancer with antimitotic agents such as vinblastine and doxorubicin is only marginally effective, due to dose-limiting systemic toxicity. Herein we report the development of peptidyl conjugate 5 of the cytotoxic agent vinblastine (1), along with the results of its in vitro and in vivo evaluation as a pro-drug targeted at prostate cancer cells. Prostate-derived tumors are known to produce significant amounts of prostate specific antigen (PSA), a serine protease with chymotrypsin-like properties. Earlier work in these laboratories established that an appropriately engineered peptidyl pro-drug will release active cytotoxic agent strictly within the microenvironment of the tumor tissue (Garsky, V. M., et al. J. Med.Chem. 2001, 44, 4216-4224). Conjugate 5, which features an octapeptide segment attached by an ester linkage at the 4-position of vinblastine (1), undergoes rapid cleavage by PSA (T(1/2) = 12 min) between the Gln and Ser residues. In nude mouse xenograft studies, 5 reduced circulating PSA levels by 99% and tumor weight by 85% at a dose just below its MTD. By contrast, the putative end-point metabolite, the cytotoxic agent des-acetyl vinblastine (1b), was ineffective in reducing PSA levels and tumor burden at its maximum tolerated doses. Additional data from metabolism studies on 5 support the supervention of a novel in vivo processing mechanism, the spontaneous release of 1b from a dipeptidyl intermediate driven by favorable diketopiperazine formation.