Pharmacokinetic/pharmacodynamic modeling of crizotinib for anaplastic lymphoma kinase inhibition and antitumor efficacy in human tumor xenograft mouse models.

Crizotinib [Xalkori; PF02341066; (R)-3-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-(1-piperidin-4-yl-1H-pyrazol-4-yl)-pyridin-2-ylamine] is an orally available dual inhibitor of anaplastic lymphoma kinase (ALK) and hepatocyte growth factor receptor. The objectives of the present studies were to characterize: 1) the pharmacokinetic/pharmacodynamic relationship of crizotinib plasma concentrations to the inhibition of ALK phosphorylation in tumors, and 2) the relationship of ALK inhibition to antitumor efficacy in human tumor xenograft models. Crizotinib was orally administered to athymic nu/nu mice implanted with H3122 non-small-cell lung carcinomas or severe combined immunodeficient/beige mice implanted with Karpas299 anaplastic large-cell lymphomas. Plasma concentration-time courses of crizotinib were adequately described by a one-compartment pharmacokinetic model. A pharmacodynamic link model reasonably fit the time courses of ALK inhibition in both H3122 and Karpas299 models with EC(50) values of 233 and 666 ng/ml, respectively. A tumor growth inhibition model also reasonably fit the time course of individual tumor growth curves with EC(50) values of 255 and 875 ng/ml, respectively. Thus, the EC(50) for ALK inhibition approximately corresponded to the EC(50) for tumor growth inhibition in both xenograft models, suggesting that >50% ALK inhibition would be required for significant antitumor efficacy (>50%). Furthermore, based on the observed clinical pharmacokinetic data coupled with the pharmacodynamic parameters obtained from the present nonclinical xenograft mouse model, >70% ALK inhibition was projected in patients with non-small-cell lung cancer who were administered the clinically recommended dosage of crizotinib, twice-daily doses of 250 mg (500 mg/day). The result suggests that crizotinib could sufficiently inhibit ALK phosphorylation for significant antitumor efficacy in patients.

Discovery pharmacokinetic studies in mice using serial microsampling, dried blood spots and microbore LC-MS/MS.

BACKGROUND: Initial pharmacokinetic (PK) studies of discovery compounds are conducted in mice to demonstrate exposure prior to conducting efficacy studies. PK information obtained from a single mouse by serial blood microsampling, dried blood spot collection and analyses using microbore (1 mm internal diameter column) LC-MS/MS is presented. Ex vivo blood to plasma concentration ratios (BPRs) from mouse PK studies were compared with in vitro BPRs for 15 compounds. RESULTS: Two compounds were orally dosed and blood was collected at time points via serial blood sampling. The calculated PK parameters (AUC, T(max) and C(max)) were comparable across liquid blood, dried blood spot and plasma matrices. The BPR results from both methods were comparable. CONCLUSION: Serial blood microsampling has led to reduced animal and compound usage with improved PK data. Ex vivo BPR is suitable in a discovery setting. Microbore LC-MS/MS is well suited in instances where sample volume is limited, and enables faster analyses, reduced solvent use, and less frequent MS source cleaning.

A unique example of drug metabolism: tetra- and penta-oxygenation reactions of capravirine in rats, dogs and humans.

Six tetra- and two penta-oxygenated capravirine metabolites observed in rats, dogs and humans represent the maximum numbers of isomers that can be predicted since oxygenations are restricted at the pyridinyl nitrogen (N-oxidation), sulfur (sulfoxidation), and isopropyl group (hydroxylation), exemplifying a unique case that is very unusual for sequential drug metabolism.

Evaluation of capravirine as a CYP3A probe substrate: in vitro and in vivo metabolism of capravirine in rats and dogs.

Metabolism of [(14)C]capravirine was studied via both in vitro and in vivo means in rats and dogs. Mass balance was achieved in rats and dogs, with mean total recovery of radioactivity >86% for each species. Capravirine was well absorbed in rats but only moderately so in dogs. The very low levels of recovered unchanged capravirine and the large number of metabolites observed in rats and dogs indicate that capravirine was eliminated predominantly by metabolism in both species. Capravirine underwent extensive metabolism via oxygenation reactions (predominant pathways in both species), depicolylation and carboxylation in rats, and decarbamation in dogs. The major circulating metabolites of capravirine were two depicolylated products in rats and three decarbamated products in dogs. However, none of the five metabolites was observed in humans, indicating significant species differences in terms of identities and relative abundances of circulating capravirine metabolites. Because the majority of in vivo oxygenated metabolites of capravirine were observed in liver microsomal incubations, the in vitro models provided good insight into the in vivo oxygenation pathways. In conclusion, the diversity (i.e., hydroxylation, sulfoxidation, sulfone formation, and N-oxidation), multiplicity (i.e., mono-, di-, tri-, and tetraoxygenations), and high enzymatic specificity (>90% contribution by CYP3A4 in humans, CYP3A1/2 in rats, and CYP3A12 in dogs) of the capravirine oxygenation reactions observed in humans, rats, and dogs in vivo and in vitro suggest that capravirine can be a useful CYP3A substrate for probing catalytic mechanisms and kinetics of CYP3A enzymes in humans and animal species.

Metabolism and excretion of capravirine, a new non-nucleoside reverse transcriptase inhibitor, alone and in combination with ritonavir in healthy volunteers.

Metabolism and disposition of capravirine, a new non-nucleoside reverse transcriptase inhibitor, were studied in healthy male volunteers who were randomly divided into two groups (A and B) with five subjects in each group. Group A received a single oral dose of [(14)C]capravirine (1400 mg) and group B received multiple oral doses of ritonavir (100 mg), followed by a single oral dose of [(14)C]capravirine (1400 mg). Mean total recoveries of radioactivity for groups A and B were 86.3% and 79.0%, respectively, with a mean cumulative recovery in urine comparable with that in feces for both groups. Excretion of unchanged capravirine was negligible in urine and low in feces for both groups. The results suggest that capravirine was well absorbed, with metabolism as the principal mechanism of clearance. Capravirine underwent extensive metabolism to a variety of metabolites via oxygenations (mono-, di-, tri-, and tetra-) representing the predominant pathway, glucuronidation, and sulfation in humans. No useful plasma profiles of group A were obtained due to extremely low levels of plasma radioactivity. Analysis of group B plasma indicated that unchanged capravirine was the major radiochemical component, with three monooxygenated products and a glucuronide of capravirine as the major circulating metabolites. Nineteen metabolites were identified using liquid chromatography-multistage ion-trap mass spectrometry methodologies. In summary, coadministration of low-dose ritonavir (a potent CYP3A4 inhibitor) drastically decreased the levels of sequential oxygenated metabolites and markedly increased the levels of the parent drug and primary oxygenated metabolites overall in plasma, urine, and feces.