To assess the effects of Ile359 to Leu359 change on CYP2C9-mediated metabolism, we performed site-directed mutagenesis and cDNA expression in yeast for CYP2C9 and examined in detail the kinetics of seven metabolic reactions by wild-type CYP2C9 (Ile359) and its Leu359 variant. For the metabolism of all the substrates studied, the Leu359 variant exhibited smaller Vmax/Km values than did the wild-type. The differences in the Vmax/Km values between the wild-type and the Leu359 variant varied from 3.4-fold to 26.9-fold. The Leu359 variant had higher Km values than did the wild-type for all the reactions studied. Among the seven reactions studied, the greatest difference in the Vmax values between the wild-type and the Leu359 variant was for piroxicam 5'-hydroxylation (408 versus 19 pmol/min/nmol P450), whereas there were no differences in the Vmax values between the wild-type and the Leu359 variant for diclofenac 4'-hydroxylation and tolbutamide methylhydroxylation. These results indicate that the Ile359 to Leu359 change significantly decreases the catalytic activity of all the CYP2C9-mediated metabolisms studied, whereas the extent of the reduction in activity and changes of the kinetic parameters varies between substrates. Moreover, the amino acid substitution decreased the enantiomeric excess in the formation of 5-(4-hydroxyphenyl)-5-phenylhydantoin from phenytoin.

We established intracranial tumor implantation models of EML4-ALK-positive NSCLC. Alectinib showed potent efficacy against intracranial EML4-ALK-positive tumor. These results demonstrated that alectinib might provide therapeutic opportunities for crizotinib-treated patients with brain metastases.

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9-Substituted 6,6-dimethyl-11-oxo-6,11-dihydro-5H-benzo[b]carbazoles were discovered as highly selective and potent anaplastic lymphoma kinase (ALK) inhibitors by structure-based drug design. The high target selectivity was achieved by introducing a substituent close to the E(0) region of the ATP binding site, which has a unique amino acid sequence. Among the identified inhibitors, compound 13d showed highly selective and potent inhibitory activity against ALK with an IC(50) value of 2.9 nM and strong antiproliferative activity against KARPAS-299 with an IC(50) value of 12.8 nM. The compound also displayed significant antitumor efficacy in an established ALK fusion gene-positive anaplastic large-cell lymphoma (ALCL) xenograft model in mice without body weight loss.

A method of assessing the risk of drug-drug interaction (DDI) caused by mechanism-based inhibition (MBI) was developed for early-stage drug development using cytochrome P450 (CYP) 3A4 inhibition screening data. CYP3A4 inhibition was evaluated using a fluorescent substrate with or without preincubation containing an inhibitor. The results showed that five well-known mechanism-based inhibitors, but not the competitive inhibitor ketoconazole, had lower IC(50) after preincubation, suggesting the utility of the IC(50) shift by preincubation to discern mechanism-based inhibitors. A method to approximately predict the change in the area under the concentration-time curve (AUC) of a co-administered drug by MBI was found using IC(50) shift data and the unbound mean plasma concentration of the inhibitor. From our predictions of change in the AUC for 38 drugs using this method, all mechanism-based inhibitors causing change in the AUC of more than 200% were predicted to be high risk. In conclusion, our method provides a simple assessment of the risk of DDI from mechanism-based inhibitors, especially in the early stages of drug development.

1. The in vitro metabolism of alectinib, a potent and highly selective oral anaplastic lymphoma kinase inhibitor, was investigated. 2. The main metabolite (M4) in primary human hepatocytes was identified, which is produced by deethylation at the morpholine ring. Three minor metabolites (M6, M1a, and M1b) were also identified, and a minor peak of hydroxylated alectinib (M5) was detected as a possible precursor of M4, M1a, and M1b. 3. M4, an important active major metabolite, was produced and further metabolized to M6 by CYP3A, indicating that CYP3A enzymes were the principal contributors to this route. M5 is possibly produced by CYP3A and other isoforms as the primary step in metabolism, followed by oxidation to M4 mainly by CYP3A. Alternatively, M5 could be oxidized to M1a and M1b via an NAD-dependent process. None of the non-CYP3A-mediated metabolism appeared to be major. 4. In conclusion, this study suggests that involvement of multiple enzymes in the metabolism of alectinib reduces its potential for drug-drug interactions.

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