An efficient screening assay was developed and validated for simultaneous assessment of compound-mediated inhibition of six major human cytochrome P450 （CYP） enzymes. This method employed a cocktail of six probe sub...An efficient screening assay was developed and validated for simultaneous assessment of compound-mediated inhibition of six major human cytochrome P450 （CYP） enzymes. This method employed a cocktail of six probe substrates （i.e., phenacetin, amodiaquine, diclofenac, S-mephenytoin, dextromethorphan and midazolam for CYPIA2, 2C8, 2C9, 2C19, 2D6 and 3A4, respectively） as well as individual prototypical inhibitors of the six CYP enzymes in human liver microsomes under optimized incubation conditions. The corresponding marker metabolites （i.e., acetaminophen, N-desethylamodiaquine, 4-OH-diclofenac, 4-OH-S- mephenytoin, dextrorphan and 1-OH-midazolam） in the incubates were quantified using LC-MS/MS methods either by an internal standard （IS） calibration curve or a simpfified analyte-to-IS peak area ratio approach. The results showed that the IC5o values determined by the cocktail approach were in good agreement with those obtained by the individual substrate approach as well as those reported in the literature. Besides, no remarkable difference was observed between the two quantification approaches. In conclusion, this new cocktail assay can be used for reliable screening of compound-mediated CYP inhibition.展开更多
Uncontrolled cell proliferation is the hall mark of many cancers, and is typically manifested by a deregulation of the cell-division cycle. CDKs play critical roles in regulating cell cycle, apop- tosis and cell diffe...Uncontrolled cell proliferation is the hall mark of many cancers, and is typically manifested by a deregulation of the cell-division cycle. CDKs play critical roles in regulating cell cycle, apop- tosis and cell differentiation. AG-024322 is a multitargeted CDK inhibitor that has been shown to induce cancer cell apoptosis and de- monstrate significant antitumor activity in hu-man tumor xenograft models. This compound is under clinical development as an intravenous anticancer agent. AG-024322 exhibited moder-ate to high systemic clearance across preclini-cal species. In vitro metabolism in human liver microsomes and hepatocytes demonstrates that glucuronidation and oxidation represent the major metabolic pathways of AG-024322. The experiments of chemical inhibition and micro-somes containing individual CYP or UGT iso-forms revealed that CYP3A and UGT1A1 appear to predominantly mediate AG-024322 oxidation and glucuronidation, respectively. Formation kinetics of the two pathways in human liver mi-crosomes suggested that the glucuronidation activity of AG-024322 was approximately 3-fold higher as compared to CYP-mediated oxidation, contributing approximately 37% and 13% of the total clearance, respectively, based on the pro-jected human clearance. UGT1A1 is a poly-morphic isoform involved in glucuronidation of bilirubin. It is of concern if glucuronidation via UGT1A1 plays a major role in the elimination of AG-024322 in humans as competitive inhibition of UGT1A1 has been associated with toxicity (Gilbert and Crigler-Najjar syndromes). There-fore, this information was used to influence the clinical study design to only include subjects having constitutive expression of UGT1A1 in the first human study, thereby decreasing the potential risk of toxicity to patients.展开更多
文摘An efficient screening assay was developed and validated for simultaneous assessment of compound-mediated inhibition of six major human cytochrome P450 （CYP） enzymes. This method employed a cocktail of six probe substrates （i.e., phenacetin, amodiaquine, diclofenac, S-mephenytoin, dextromethorphan and midazolam for CYPIA2, 2C8, 2C9, 2C19, 2D6 and 3A4, respectively） as well as individual prototypical inhibitors of the six CYP enzymes in human liver microsomes under optimized incubation conditions. The corresponding marker metabolites （i.e., acetaminophen, N-desethylamodiaquine, 4-OH-diclofenac, 4-OH-S- mephenytoin, dextrorphan and 1-OH-midazolam） in the incubates were quantified using LC-MS/MS methods either by an internal standard （IS） calibration curve or a simpfified analyte-to-IS peak area ratio approach. The results showed that the IC5o values determined by the cocktail approach were in good agreement with those obtained by the individual substrate approach as well as those reported in the literature. Besides, no remarkable difference was observed between the two quantification approaches. In conclusion, this new cocktail assay can be used for reliable screening of compound-mediated CYP inhibition.
文摘Uncontrolled cell proliferation is the hall mark of many cancers, and is typically manifested by a deregulation of the cell-division cycle. CDKs play critical roles in regulating cell cycle, apop- tosis and cell differentiation. AG-024322 is a multitargeted CDK inhibitor that has been shown to induce cancer cell apoptosis and de- monstrate significant antitumor activity in hu-man tumor xenograft models. This compound is under clinical development as an intravenous anticancer agent. AG-024322 exhibited moder-ate to high systemic clearance across preclini-cal species. In vitro metabolism in human liver microsomes and hepatocytes demonstrates that glucuronidation and oxidation represent the major metabolic pathways of AG-024322. The experiments of chemical inhibition and micro-somes containing individual CYP or UGT iso-forms revealed that CYP3A and UGT1A1 appear to predominantly mediate AG-024322 oxidation and glucuronidation, respectively. Formation kinetics of the two pathways in human liver mi-crosomes suggested that the glucuronidation activity of AG-024322 was approximately 3-fold higher as compared to CYP-mediated oxidation, contributing approximately 37% and 13% of the total clearance, respectively, based on the pro-jected human clearance. UGT1A1 is a poly-morphic isoform involved in glucuronidation of bilirubin. It is of concern if glucuronidation via UGT1A1 plays a major role in the elimination of AG-024322 in humans as competitive inhibition of UGT1A1 has been associated with toxicity (Gilbert and Crigler-Najjar syndromes). There-fore, this information was used to influence the clinical study design to only include subjects having constitutive expression of UGT1A1 in the first human study, thereby decreasing the potential risk of toxicity to patients.
