Metabolism and pharmacokinetic study of deuterated osimertinib
Xuyi ZhanShaoyin BaoXumei LiShaojun ZhouMaha Raja DaharNengming LinXiugui ChenChengshan NiuKaige JiYusheng WuKui ZengZhihua TangLushan Yu
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Osimertinib is a highly selective third-generation irreversible inhibitor of epidermal growth factor receptor mutant, which can be utilized to treat non-small cell lung cancer. As the substrate of cytochrome P450 enzyme, it is mainly metabolized by the CYP3A enzyme in humans. Among the metabolites produced by osimertinib, AZ5104, and AZ7550, which are demethylated that is most vital. Nowadays, deuteration is a new design approach for several drugs. This popular strategy is deemed to improve the pharmacokinetic characteristics of the original drugs. Therefore, in this study the metabolism profiles of osimertinib and its deuterated compound (osimertinib-d3) in liver microsomes and human recombinant cytochrome P450 isoenzymes and the pharmacokinetics in rats and humans were compared. After deuteration, its kinetic isotope effect greatly inhibited the metabolic pathway that produces AZ5104. The plasma concentration of the key metabolite AZ5104 of osimertinib-d3 in rats and humans decreased significantly compared with that of the osimertinib. This phenomenon was consistent with the results of the metabolism studies in vitro. In addition, the in vivo results indicated that osimertinib-d3 had higher systemic exposure (AUC) and peak concentration (Cmax ) compared with the osimertinib in rats and human body.Keywords:
Osimertinib
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Human cytochrome P450 3A7 (CYP3A7) and cytochrome P450 3A4 (CYP3A4) are hepatic metabolising enzymes which participates in the biotransformation of endo- and exogenous substances in foetuses and neonates respectively. These CYP3A enzymes display an inverse relationship: CYP3A7 is the dominant enzyme in the foetal liver, whereas the expression of CYP3A4 is low. After parturition there is a shift in the expression, thus CYP3A7 is down regulated, while the level of CYP3A4 gradually increases and becomes the dominant metabolising CYP3A enzyme in the adult. The minipig is increasingly being used as a model for humans in biomedical studies, because of its many similarities with the human physiology and anatomy. The aim of this study was to examine whether, as in humans, a shift is seen in the hepatic expression of a CYP3A7- like enzyme to cytochrome P450 3A29 (CYP3A29) (an orthologue to the human CYP3A4) in minipigs. This was elucidated by examining the hepatic mRNA expression of CYP3A7 and CYP3A29 in 39 foetuses and newborn Göttingen minipigs using quantitative real time polymerase chain reaction (qPCR). Furthermore the immunochemical level of CYP3A7-LE and CYP3A29 was measured in liver microsomes using western blotting. The expression of CYP3A29 was approximately 9- fold greater in neonates compared to foetuses, and a similar difference was reflected on the immunochemical level. It was not possible to detect a significant level of foetal CYP3A7 mRNA, but immunoblotting showed a visible difference depending on age. This study demonstrates an increase in the expression of CYP3A29, the CYP3A4 orthologue in perinatal minipigs as in humans, which suggests that the minipig could be a good model when testing for human foetal toxicity towards CYP3A4 substrates.
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1. The expression of small intestinal cytochromes P450 (P450s) has not been systematically measured in cynomolgus monkeys, which are widely used in preclinical drug studies to predict pharmacokinetics and toxicity in humans: therefore, P450 content of small intestine was quantified in 35 cynomolgus monkeys by immunoblotting using 11 selective antibodies.2. CYP2D, CYP2J2, CYP3A4 and CYP3A5 were detected in all 35 animals, while CYP1A and CYP2C9/19 were detected in 31 and 17 animals, respectively. CYP2C9 and CYP2C19 were detected with the same antibody. CYP1D, CYP2A, CYP2B6, CYP2C76 and CYP2E1 were not detected in any of the 35 animals examined.3. On analysis of pooled microsomes (35 animals), CYP3A (3A4 + 3A5) was most abundant (79% of total immunoquantified CYP1-3 proteins), followed by CYP2J2 (13%), CYP2C9/19 (4%), CYP1A (3%) and CYP2D (0.4%). On the analysis of individual microsome samples, each P450 content varied 2-to-6-fold between animals, and no sex differences were observed in any P450 content.4. These findings should help to increase the understanding of drug metabolism, especially the first-pass effect, in cynomolgus monkey small intestines.
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We verified a physiologically-based pharmacokinetic (PBPK) model to predict cytochrome P450 3A4/5-mediated drug-drug interactions (DDIs). A midazolam (MDZ)-ketoconazole (KTZ) interaction study in 24 subjects selected by CYP3A5 genotype, and liquid chromatography and mass spectroscopy quantification of CYP3A4/5 abundance from independently acquired and genotyped human liver (n = 136) and small intestinal (N = 12) samples, were conducted. The observed CYP3A5 genetic effect on MDZ systemic and oral clearance was successfully replicated by a mechanistic framework incorporating the proteomics-informed CYP3A abundance and optimized small intestinal CYP3A4 abundance based on MDZ intestinal availability (FG ) of 0.44. Furthermore, combined with a modified KTZ PBPK model, this framework recapitulated the observed geometric mean ratio of MDZ area under the curve (AUCR) following 200 or 400 mg KTZ, which was, respectively, 2.7-3.4 and 3.9-4.7-fold in intravenous administration and 11.4-13.4 and 17.0-19.7-fold in oral administration, with AUCR numerically lower (P > 0.05) in CYP3A5 expressers than nonexpressers. In conclusion, the developed mechanistic framework supports dynamic prediction of CYP3A-mediated DDIs in study planning by bridging DDIs between CYP3A5 expressers and nonexpressers.
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Gender-related differences in drug pharmacokinetics have frequently been considered as potentially important determinants for the clinical effectiveness of drug therapy. Major molecular factors involved in drug disposition include drug-metabolising enzymes and drug transporters. Oxidative drug metabolism by cytochrome P450 (CYP) enzymes is a major pathway for drug elimination. CYP3A4,the major human drug-metabolizing CYP enzymes,has repeatedly been suggested higher metabolic activity in women than that in men,which sex-dependent secretory patterns of growth hormone that may be responsible for.
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Background: Cytochrome P450 3A4 (CYP3A4) is an important drug-metabolizing enzyme that is expressed in the liver and small intestine of humans. Various factors influence the expression of CYP3A4, but gender difference in CYP3A4 expression remains debatable. Objective: To clarify gender difference of hepatic and intestinal CYP3A4 in CYP3A-humanized mice generated by a human artificial chromosome (HAC) vector system. The CYP3A-humanized (CYP3AHAC) mice have essential regulatory regions, including promoters and enhancers, and unknown elements affecting the expression of CYP3A4. Methods: We examined the expression and activity of hepatic and intestinal CYP3A4 in male and female CYP3A-HAC mice. CYP3A activity was determined as α- and 4-hydroxylation activity of triazolam in liver and intestinal microsomes. Expression level of CYP3A protein was determined by Western blot analysis. Expression level of CYP3A4 mRNA was measured by quantitative real-time PCR. Results: The results showed that triazolam hydroxylation activities and protein levels of CYP3A in the liver were significantly higher in female than in male CYP3A-HAC mice, whereas those in the intestine were not significantly different between the genders. In addition, the expression of CYP3A4 mRNA showed a tendency similar to that found for the activity and expression of CYP3A protein in the liver and intestine of CYP3A-HAC mice. Conclusion: These findings suggest that the expression and activity levels of CYP3A4 in the liver are higher in females than in males, whereas there is no gender difference in the levels in the intestine of CYP3A-HAC mice. Keywords: CYP3A4, liver, intestine, sex, humanized mice, CYP3A-humanized mice.
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In humans, 75% of all drugs are metabolized by the cytochrome P450-dependent monooxygenase system. Enzymes encoded by the CYP2C, CYP2D, and CYP3A gene clusters account for ∼80% of this activity. There are profound species differences in the multiplicity of cytochrome P450 enzymes, and the use of mouse models to predict pathways of drug metabolism is further complicated by overlapping substrate specificity between enzymes from different gene families. To establish the role of the hepatic and extrahepatic P450 system in drug and foreign chemical disposition, drug efficacy, and toxicity, we created a unique mouse model in which 30 cytochrome P450 genes from the Cyp2c, Cyp2d, and Cyp3a gene clusters have been deleted. Remarkably, despite a wide range of putative important endogenous functions, Cyp2c/2d/3a KO mice were viable and fertile, demonstrating that these genes have evolved primarily as detoxification enzymes. Although there was no overt phenotype, detailed examination showed Cyp2c/2d/3a KO mice had a smaller body size (15%) and larger livers (20%). Changes in hepatic morphology and a decreased blood glucose (30%) were also noted. A five-drug cocktail of cytochrome P450 isozyme probe substrates were used to evaluate changes in drug pharmacokinetics; marked changes were observed in either the pharmacokinetics or metabolites formed from Cyp2c, Cyp2d, and Cyp3a substrates, whereas the metabolism of the Cyp1a substrate caffeine was unchanged. Thus, Cyp2c/2d/3a KO mice provide a powerful model to study the in vivo role of the P450 system in drug metabolism and efficacy, as well as in chemical toxicity.
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Aim To investigate whether Rhizoma curcumaecould induce pregnane X receptor(PXR)-mediated transcriptional expression of CYP3A4 and study the modulatory effects on the enzyme activity and mRNA expression of CYP3A in the rat liver.Methods Transient cotransfection reporter gene assays were performed in HepG2 cells;the rat liver microsomal cytochrome P450 and CYP3A isoenzyme-erythromycin N-demethylase(ERD)activities were determined by UV chromatography;the mRNA expression level of CYP3A was detected by reverse transcriptase-polymerase chain reaction(RT-PCR).Results In vitroinvestigation showed Rhizoma curcumaeand its four selected constituents could induce the CYP3A4 transcriptional expression by activating PXR.In vivoinvestigation showed the CYP450 content of liver microsomes and enzyme activity of CYP3A were markedly increased and induced by Rhizoma curcumaeextract;at the mRNA level, the expression of CYP3A1 and CYP3A2 gene were markedly induced by Rhizoma curcumaeextract.Conclusions Rhizoma curcumaeand its four selected constituents could induce the expression of the CYP3A4 gene transcriptional expression through activating PXR;Rhizoma curcumaeextract could increase and induce the enzyme activity and mRNA expression of CYP3A.
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Drug metabolism is an enzymatic biotransformation of drugs. The early stage of drug metabolism generally consists of phase I reactions such as oxidation, reduction, and hydrolysis, effected by introducing a polar group into the parent molecule. The phase I reactions are followed by conjugations with hydrophilic compounds such as glucuronic acid and glutathione, to yield a more hydrophilic metabolite (phase II reactions). Cytochrome P450 (P450 or CYP) represents the enzyme that metabolizes drugs with various manners of oxidation as the phase I reaction. P450 is comprised of a large superfamily of heme-containing membrane-binding proteins that are classified into families and subfamilies. Most of the P450 related to drug metabolisms belong to CYP1, CYP2, or CYP3 families that are known as "drug metabolizing enzymes." Two or more P450 isoforms are frequently involved in the metabolism of the same drug, suggesting broad substrate specificity. P450 exists mainly in the liver, but may also exist in various organs such as the brain, lung, gastrointestinal tract, kidneys, and gonads. CYP3A4 is the most abundant isoform, occupying approx 30% of the total P450 amount in the human liver (1). Because many therapeutic drugs are metabolized by CYP3A4, drug interactions related to the isoform occur frequently and interindividual variation of CYP3A4 activity is sometimes clinically significant via altering pharmacokinetics and pharmacodynamic actions. Furthermore, when a drug substrate of CYP3A4 is administered orally, the CYP3A4 activity in the intestine has a clinically significant effect on the bioavailability of the drug.
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