Impact of hydrolysis-mediated clearance on the pharmacokinetics of novel anaplastic lymphoma kinase inhibitors.

Compound 1 [(E)-4-fluoro-N-(6-((4-(2-hydroxypropan-2-yl)piperidin-1-yl)methyl)-1-((1S,4S)-4-(isopropylcarbamoyl)cyclohexyl)-1H-benzo[d]imidazol-2(3H)-ylidene)benzamide], a new, potent, selective anaplastic lymphoma kinase (ALK) inhibitor with potential application for the treatment of cancer, was selected as candidate to advance into efficacy studies in mice. However, the compound underwent mouse-specific enzymatic hydrolysis in plasma to a primary amine product (M1). Subsequent i.v. pharmacokinetics studies in mice showed that compound 1 had high clearance (CL) and a short half-life. Oral dose escalation studies in mice indicated that elimination of compound 1 was saturable, with higher doses achieving sufficient exposures above in vitro IC(50). Chemistry efforts to minimize hydrolysis resulted in the discovery of several analogs that were stable in mouse plasma. Three were taken in vivo into mice and showed decreased CL corresponding to increased in vitro stability in plasma. However, the more stable compounds also showed reduced potency against ALK. Kinetic studies in NADPH-fortified and unfortified microsomes and plasma produced submicromolar K(m) values and could help explain the saturation of elimination observed in vivo. Predictions of CL based on kinetics from hydrolysis and NADPH-dependent pathways produced predicted hepatic CL values of 3.8, 3.0, 1.6, and 1.2 l/h⋅kg for compound 1, compound 2 [(E)-3,5-difluoro-N-(6-((4-(2-hydroxypropan-2-yl)piperidin-1-yl)methyl)-1-((1s,4s)-4-(isopropylcarbamoyl)cyclohexyl)-1H-benzo[d]imidazol-2(3H)-ylidene)benzamide], compound 3 [(E)-3-chloro-5-fluoro-N-(6-((4-(2-hydroxypropan-2-yl)piperidin-1-yl)methyl)-1-((1s,4s)-4-(isopropylcarbamoyl)cyclohexyl)-1H-benzo[d]imidazol-2(3H)-ylidene)benzamide], and compound 4 [(E)-N-(6-((4-(2-hydroxypropan-2-yl)piperidin-1-yl)methyl)-1-((1s,4s)-4-(isopropylcarbamoyl)cyclohexyl)-1H-benzo[d]imidazol-2(3H)-ylidene)-3-(trifluoromethyl)benzamide], respectively. The in vivo observed CLs for compounds 1, 2, 3, and 4 were 5.52, 3.51, 2.14, and 2.66 l/h⋅kg, respectively. These results indicate that in vitro metabolism kinetic data, incorporating contributions from both hydrolysis and NADPH-dependent metabolism, could be used to predict the systemic CL of compounds cleared via hydrolytic pathways provided that the in vitro assays thoroughly investigate the processes, including the contribution of other metabolic pathways and the possibility of saturation kinetics.

LC-MS/MS bioanalytical method development for AMG 900: resolution of an isobaric interference in rodent in vivo studies.

AMG 900 is an orally available small molecule that is a highly potent and selective pan-aurora kinase inhibitor currently in development for the treatment of advanced human cancers. A co-eluting, isobaric interference was discovered in preliminary LC-MS/MS analyses of rodent in vivo pharmacokinetic samples during preclinical evaluation of AMG 900. The interference was identified as a major circulating N-oxide metabolite which partially converted to an [M+H-O](+) ion under the conditions of atmospheric pressure chemical ionization. A selective liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of AMG 900 and its N-oxide metabolite in plasma was developed and successfully applied for the bioanalysis of discovery stage preclinical rodent pharmacokinetic studies.

The discovery and optimization of a novel class of potent, selective, and orally bioavailable anaplastic lymphoma kinase (ALK) inhibitors with potential utility for the treatment of cancer.

A class of 2-acyliminobenzimidazoles has been developed as potent and selective inhibitors of anaplastic lymphoma kinase (ALK). Structure based design facilitated the rapid development of structure-activity relationships (SAR) and the optimization of kinase selectivity. Introduction of an optimally placed polar substituent was key to solving issues of metabolic stability and led to the development of potent, selective, orally bioavailable ALK inhibitors. Compound 49 achieved substantial tumor regression in an NPM-ALK driven murine tumor xenograft model when dosed qd. Compounds 36 and 49 show favorable potency and PK characteristics in preclinical species indicative of suitability for further development.

In vitro and in vivo pharmacokinetic characterizations of AMG 900, an orally bioavailable small molecule inhibitor of aurora kinases.

AMG 900 is a small molecule being developed as an orally administered, highly potent, and selective pan-aurora kinase inhibitor. The aim of the investigations was to characterize in vitro and in vivo pharmacokinetic (PK) properties of AMG 900 in preclinical species. AMG 900 was rapidly metabolized in liver microsomes and highly bound to plasma proteins in the species tested. It was a weak Pgp substrate with good passive permeability. AMG 900 exhibited a low-to-moderate clearance and a small volume of distribution. Its terminal elimination half-life ranged from 0.6 to 2.4 h. AMG 900 was well-absorbed in fasted animals with an oral bioavailability of 31% to 107%. Food intake had an effect on rate (rats) or extent (dogs) of AMG 900 oral absorption. The clearance and volume of distribution at steady state in humans were predicted to be 27.3 mL/h/kg and 93.9 mL/kg, respectively. AMG 900 exhibited acceptable PK properties in preclinical species and was predicted to have low clearance in humans. AMG 900 is currently in Phase I clinical testing as a treatment for solid tumours. Preliminary human PK results appear to be consistent with the predictions.