Distribution, metabolism, and excretion of the anti-angiogenic compound SU5416.

SU5416, 3-(3,5-dimethyl-1H-pyrrol-2-ylmethylene)-1,3-dihydro-indol-2-one, is a potent inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinase, Flk-1/KDR (fetal liver kinase 1/kinase insert domain-containing receptor), also known as VEGF receptor 2 (VEGFR2). It was the first VEGFR2 inhibitor to enter clinical trials for the treatment of colorectal and non-small cell lung cancers. Pre-clinical evaluation of SU5416 included studies related to the distribution, metabolism and excretion of this compound. These studies have provided information useful in understanding the disposition and metabolism of the indolinone class of chemicals, which has not been studied previously with therapeutic intent. The lessons we learned from SU5416 have been successfully applied in developing next generation indolinone compounds targeting tumor angiogenesis.

Simultaneous determination of Z-SU5416 and its interconvertible geometric E-isomer in rat plasma by LC/MS/MS.

SU5416 is a selective inhibitor of vascular endothelial growth factor (VEGF) receptor, which plays a major role in vascular angiogenesis. SU5416 exists as the thermodynamically stable and pharmacologically active cis isomer (Z-isomer) in the solid state. In light-exposed solutions the unstable trans isomer (E-isomer) is formed. The E-isomer is unstable for synthesis and isolation and the analytical standard of the E-isomer is unavailable. A new, simple, fast and reliable LC/MS/MS method was developed to quantify both isomers simultaneously in rat plasma samples in order to support the study of disposition kinetics of Z- and E-SU5416. This method is sensitive (LOQ = 0.5 ng/ml), reproducible, and has a wide linear range (0.5-2500 ng/ml).

Application of LC/MS/MS in the quantitation of SU101 and SU0020 uptake by 3T3/PDGFr cells.

SU101 or leflunomide, has been studied extensively because of its anti-cancer and immunomodulating properties. The parent isoxazole compound is converted in vitro and metabolized in vivo to an open ring isomeric form, SU0020. Several pharmacological activities have been reported for the parent and metabolite compounds including inhibition of platelet-derived growth factor (PDGF)-mediated signaling for the parent compound and inhibition of de novo pyrimidine biosynthesis for the metabolite. The inhibition of PDGF-mediated signaling and the anti-tumor properties have been ascribed to the parent compound. In spite of its short plasma half-life of the parent molecule, SU101 can be administered intermittently in animal tumor models and retain efficacy. Therefore, the relationship between plasma levels of SU101 and its efficacy in tumor-implanted immuno-compromised mice is not well established. This study was conducted to assess the concentration of SU101 in 3T3/PDGFr alpha and beta cells (NIH3T3 mouse fibroblasts engineered to overexpress human PDGFr alpha or beta) to better understand the cellular levels of SU101 and SU0020. Two strains of 3T3/PDGFr cells (alpha and beta) were incubated with 1, 25, and 100 microM concentrations of SU101 for 1, 6, 24, and 48 hours. Quantitation of SU101 and SU0020 in these cell lines was achieved by a specific and sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) method. Interestingly, in both alpha and beta cell lysates SU101 was much more concentrated than SU0020. The greater concentration of SU101 versus SU0020 that was observed may be due to the preferential partitioning of SU101 into the cells and this shows that significant levels of the parent drug can reach the pharmacological site of action for inhibition of PDGF receptors. The data suggest that the conversion of SU101 to SU0020 is much slower in these cells than in the incubation media.

Single- and multiple-dose disposition kinetics of sunitinib malate, a multitargeted receptor tyrosine kinase inhibitor: comparative plasma kinetics in non-clinical species.

PURPOSE: The purpose of these extensive non-clinical studies was to assess pharmacokinetics and dispositional properties of sunitinib and its primary active metabolite (SU12662). METHODS: Sunitinib was administered in single and repeat oral doses in mice, rats, and monkeys. Assessments were made using liquid-chromatography-tandem mass spectrometric methods, radioactive assays, and quantitative whole body autoradiography. RESULTS: Sunitinib was readily absorbed with good oral bioavailability and linear kinetics at clinically-relevant doses. SU12662 plasma levels were less than those of sunitinib in mice and monkeys, but greater in rats. Sunitinib was extensively distributed with moderate-to-high systemic clearance and eliminated primarily into feces. Single- and repeat-dosing kinetics were similar. A prolonged half-life allowed once-daily dosing, enabling adequate systemic exposure with limited-to-moderate accumulation. In multiple-dose studies with cyclic dosing, drug plasma concentrations cleared from one cycle to the next. CONCLUSIONS: Sunitinib exhibited advantageous pharmacokinetic and dispositional properties in non-clinical species, translating into favorable properties in humans.

Effects of prototypical microsomal enzyme inducers on cytochrome P450 expression in cultured human hepatocytes.

Cultured human hepatocytes are a valuable in vitro system for evaluating new molecular entities as inducers of cytochrome P450 (P450) enzymes. The present study summarizes data obtained from 62 preparations of cultured human hepatocytes that were treated with vehicles (saline or dimethylsulfoxide, 0.1%), beta-naphthoflavone (33 microM), phenobarbital (100 or 250 microM), isoniazid (100 microM) and/or rifampin (20 or 50 microM), and examined for the expression of P450 enzymes based on microsomal activity toward marker substrates, or in the case of CYP2C8, the level of immunoreactive protein. The results show that CYP1A2 activity was markedly induced by beta-naphthoflavone (on average 13-fold, n = 28 preparations), and weakly induced by phenobarbital (1.9-fold, n = 25) and rifampin (2.3-fold, n = 22); CYP2A6 activity tended to be increased with phenobarbital (n = 7) and rifampin (n = 3) treatments, but the effects were not statistically significant; CYP2B6 was induced by phenobarbital (6.5-fold, n = 13) and rifampin (13-fold, n = 14); CYP2C8 was induced by phenobarbital (4.0-fold, n = 4) and rifampin (5.2-fold, n = 4); CYP2C9 was induced by phenobarbital (1.8-fold, n = 14) and rifampin (3.5-fold, n = 10); CYP2C19 was markedly induced by rifampin (37-fold, n = 10), but relatively modestly by phenobarbital (7-fold, n = 9); CYP2D6 was not significantly induced by phenobarbital (n = 5) or rifampin (n = 5); CYP2E1 was induced by phenobarbital (1.7-fold, n = 5), rifampin (2.2-fold, n = 5), and isoniazid (2.3-fold, n = 5); and, CYP3A4 was induced by phenobarbital (3.3-fold, n = 42) and rifampin (10-fold, n = 61), but not by beta-naphthoflavone. Based on these observations, we generalize that beta-naphthoflavone induces CYP1A2 and isoniazid induces CYP2E1, whereas rifampin and, to a lesser extent phenobarbital, tend to significantly and consistently induce enzymes of the CYP2A, CYP2B, CYP2C, CYP2E, and CYP3A subfamilies but not the 2D subfamily.