Comparison of methods for the prediction of the metabolic sites for CYP3A4-mediated metabolic reactions.

Predictions of the metabolic sites for new chemical entities, synthesized or only virtual, are important in the early phase of drug discovery to guide chemistry efforts in the synthesis of new compounds with reduced metabolic liability. This information can now be obtained from in silico predictions, and therefore, a thorough and unbiased evaluation of the computational techniques available is needed. Several computational methods to predict the metabolic hot spots are emerging. In this study, metabolite identification using MetaSite and a docking methodology, GLUE, were compared. Moreover, the published CYP3A4 crystal structure and computed CYP3A4 homology models were compared for their usefulness in predicting metabolic sites. A total of 227 known CYP3A4 substrates reported to have one or more metabolites adding up to 325 metabolic pathways were analyzed. Distance-based fingerprints and four-point pharmacophore derived from GRID molecular interaction fields were used to characterize the substrate and protein in MetaSite and the docking methodology, respectively. The CYP3A4 crystal structure and homology model with the reactivity factor enabled achieved a similar prediction success (78%) using the MetaSite method. The docking method had a relatively lower prediction success (approximately 57% for the homology model), although it still may provide useful insights for interactions between ligand and protein, especially for uncommon reactions. The MetaSite methodology is automated, rapid, and has relatively accurate predictions compared with the docking methodology used in this study.     

多药耐药蛋白和CYP3A4介导的中草药-西药相互作用

据近年的流行病学报告，国内外越来越多的人终身服用各种中草药，包括各类疾病患者（如：癌症等）和健康人群。随着中草药与处方西药联合应用治疗疾病的日益增多，越来越多的人注意到这两者的同时使用可能引起的中草药-西药相互作用会影响到治疗药物的活性。目前已知P-糖蛋白（P-gp）和CYP3A4一起能构成许多口服吸收药物的高效屏障，然而50％以上的临床用药都会被CYP3A4代谢或被P-gp转运。本文综述了多药耐药蛋白和CYP3A4介导的中草药-西药相互作用，同时也讨论了中草药-西药相互作用的关键因素。     

Comparative metabolic capabilities of CYP3A4, CYP3A5, and CYP3A7.

The human cytochromes P450 (P450) CYP3A contribute to the biotransformation of 50% of oxidatively metabolized drugs. The predominant hepatic form is CYP3A4, but recent evidence indicates that CYP3A5 contributes more significantly to the total liver CYP3A than was originally thought. CYP3A7 is the major fetal form and is rarely expressed in adults. To compare the metabolic capabilities of CYP3A forms for 10 substrates, incubations were performed using a consistent molar ratio (1:7:9) of recombinant CYP3A, P450 reductase, and cytochrome b5. A wide range of substrate concentrations was examined to determine the best fit to kinetic models for metabolite formation. In general, K(m) or S(50) values for the substrates were 3 to 4 times lower for CYP3A4 than for CYP3A5 or CYP3A7. For a more direct comparison of these P450 forms, clearance to the metabolites was determined as a linear relationship of rate of metabolite formation for the lowest substrate concentrations examined. The clearance for 1'-hydroxy midazolam formation at low substrate concentrations was similar for CYP3A4 and CYP3A5. For CYP3A5 versus CYP3A4, clearance values at low substrate concentrations were 2 to 20 times lower for the other biotransformations. The clearance values for CYP3A7-catalyzed metabolite formation at low substrate concentrations were substantially lower than for CYP3A4 or CYP3A5, except for clarithromycin, 4-OH triazolam, and N-desmethyl diltiazem (CYP3A5 - CYP3A7). The CYP3A forms demonstrated regioselective differences in some of the biotransformations. These results demonstrate an equal or reduced metabolic capability for CYP3A5 compared with CYP3A4 and a significantly lower capability for CYP3A7.     

A model for predicting likely sites of CYP3A4-mediated metabolism on drug-like molecules

We have developed a rapid semiquantitative model for evaluating the relative susceptibilities of different sites on drug molecules to metabolism by cytochrome P450 3A4. The model is based on the energy necessary to remove a hydrogen radical from each site, plus the surface area exposure of the hydrogen atom. The energy of hydrogen radical abstraction is conventionally measured by AM1 semiempirical molecular orbital calculations. AM1 calculations show the following order of radical stabilities for the hydrogen atom abstractions: sp2 centers > heteroatom sp3 centers > carbon sp3 centers. Since AM1 calculations are too time intensive for routine work, we developed a statistical trend vector model, which is used to estimate the AM1 abstraction energy of a hydrogen atom from its local atomic environment. We carried out AM1 and trend vector calculations on 50 CYP3A4 substrates whose major sites of metabolism are known in the literature. A plot of the lowest hydrogen radical formation energy versus its sterically accessible surface area exposure for these 50 substrates shows that only those hydrogen atoms with solvent accessible surface area exposure > or = 8.0 A(2) are susceptible to CYP3A4-mediated metabolism. This approach forms the basis for our general model, which predicts sites on drugs that are susceptible to cytochrome P450 3A4-mediated hydrogen radical abstraction followed by a hydroxylation reaction. This model, in conjunction with specific enzyme site binding requirements, can aid in identifying possible sites of metabolism catalyzed by other cytochrome P450 enzymes.     

CYP3A4-mediated lopinavir bioactivation and its inhibition by ritonavir

The combination of lopinavir (LPV) and ritonavir (RTV) is one of the preferred regimens for the treatment of HIV infection with confirmed efficacy and relatively low toxicity. LPV alone suffers the poor bioavailability due to its rapid and extensive metabolism. RTV boosts the plasma concentration of LPV by suppressing its metabolism and thus increasing LPV efficacy. In the current study, we found that RTV also inhibits LPV bioactivation. LPV bioactivation was investigated in human liver microsomes and cDNA-expressed human cytochromes P450. Twelve GSH-trapped reactive metabolites of LPV were identified by using a metabolomic approach. Semicarbazide-trapped reactive metabolites of LPV were also detected. RTV effectively suppressed all pathways of LPV bioactivation via CYP3A4 inhibition. Our data together with previous reports suggest that LPV plus RTV is an ideal combination because RTV not only boosts LPV plasma concentration, but it decreases LPV bioactivation.     

CYP3A4介导的HIV-1蛋白酶抑制剂的增效作用

HIV蛋白酶抑制剂(PIs)作为治疗艾滋病的有效药物,是肝药酶CYP3A4的底物。它们有生物利用度低、半衰期短、药物在个体之间药动学差异较大等缺点。利托那韦（ritonavir）可以抑制CYP3A4对PIs的代谢作用,因而被用于多种PIs的增效治疗。文中综述了ritonavir的药动学增效作用,重点阐述了ritonavir与各种PIs联合使用在临床上的发展现状、疗效特征以及适应症,并介绍了ritonavir作为增效剂的不足以及未来新的增效剂的发展前景。近几年来关于天然药物对PIs的增效作用也有报道,文中简要介绍了几种具有增效潜力的天然药物。     

CYP3A4 and CYP3A7-mediated carbamazepine 10,11-epoxidation are activated by differential endogenous steroids.

Recently, we reported that several endogenous steroids affect CYP3A4-mediated drug metabolism, using human adult liver microsomes as an enzyme source. Especially, carbamazepine (CBZ) 10,11-epoxidation is activated by androstenedione (AND). In the present studies, we investigated the effects of endogenous steroids on the activity of CBZ 10,11-epoxidation by expressed CYP3A4 and CYP3A7. When expressed CYP3A4 was used as an enzyme source, the addition of AND to the reaction mixture also caused a drastic increase in the activity of CBZ 10,11-epoxidase, and resulted in a change in the kinetics from sigmoid to Michaelis-Menten type. On the other hand, expressed CYP3A7-mediated CBZ 10,11-epoxidation was activated by sulfate conjugate steroids, such as pregnenolone 3-sulfate, 17alpha-hydroxypregnenolone 3-sulfate, and dehydroepiandrosterone 3-sulfate (DHEA-S), whereas the unconjugated form corresponding to these three steroids did not activate the reaction. Especially, DHEA-S was found to be a potent activator of CBZ 10,11-epoxidation by expressed CYP3A7. The kinetic character of CBZ 10,11-epoxidation by CYP3A7 is Michaelis-Menten type regardless of the presence of DHEA-S. The presence of DHEA-S caused a decrease in K(m) and increase in V(max) for CYP3A7-mediated CBZ 10,11-epoxidation, whereas DHEA-S 16alpha-hydroxylation was not affected by the coexistence of CBZ. In conclusion, CYP3A4 and CYP3A7-mediated CBZ 10,11-epoxidations are activated by different types of endogenous steroids. This is the first report regarding CYP3A7 cooperativity.     

Effect of methylprednisolone on CYP3A4-mediated drug metabolism in vivo

OBJECTIVE: To study the effects of methylprednisolone on the pharmacokinetics and pharmacodynamics of triazolam. METHODS: In this three-phase cross-over study, ten healthy subjects received 0.25 mg oral triazolam on three occasions: on day 1 (no pretreatment, control), on day 8 (1 h after a single dose of 32 mg oral methylprednisolone) and on day 18 (after further treatment with 8 mg oral methylprednisolone daily for 9 days). The plasma concentrations of triazolam were determined up to 10 h, and its effects were measured using four psychomotor tests up to 6 h. RESULTS: The single dose of methylprednisolone showed no significant effects on the pharmacokinetics of triazolam. However, the Digit Symbol Substitution Test result was better (P < 0.05) during the single-dose methylprednisolone phase than during the control phase, the other three tests showing no differences between the phases. The multiple-dose treatment with methylprednisolone reduced the mean peak plasma concentration (Cmax) of triazolam by 30% (P < 0.05) but had no significant effects on the time to Cmax (tmax), elimination half-life (t 1/2), area under the plasma concentration-time curve from 0 h to 10 h (AUC(0-10 h)) and AUC(0-infinity) and did not alter the effects of triazolam. CONCLUSION: A single, relatively high dose of methylprednisolone (32 mg) did not affect cytochrome P450 (CYP)3A4 activity, and treatment with 8 mg methylprednisolone daily for 9 days did not result in clinically significant induction of CYP3A4.     

Expression of CYP3A4 mRNA is correlated with CYP3A4 protein level and metabolic activity in human liver

A relatively large amount of human liver tissue was required to determine the exact activity of human hepatic CYP3A. Although, the quantity of available human liver tissue samples is limited. We measured levels of CYP3A4 mRNA by RT-PCR with a radiolabeled primer specific for CYP3A4 and compared mRNA expression with CYP3A4 protein level and metabolic activity in liver. The level of CYP3A4 mRNA was correlated with the levels of CYP3A4 protein and activity. Our results suggest that CYP3A4 protein and activity levels can be predicted from CYP3A4 mRNA levels determined by RT-PCR and using a very small amount of liver tissue.     

外源化合物对孕烷X受体和组成型雄烷受体介导的细胞色素P4503A4和2B6转录调节共转染体系的优化

目的优化孕烷X受体(hPXR)和组成型雄烷受体(hCAR)介导的细胞色素P450(CPY)3A4和CYP2B6诱导共转染体系,提高检测系统的灵敏度。方法利用invitrogen脂质体2000共同转染表达质粒hPXR/hCAR、报告基因质粒CPY3A4/CYP2B6和内参质粒pRL-TK到HepG-2细胞中。系统以hPXR的激动剂利福平,hCAR的激动剂CITCO为阳性对照组,以二甲基亚砜(DMSO)为溶剂阴性对照组。通过调整3种质粒的转染比例,以利福平/DMSO和CITCO/DMSO的比活值,即阳性药物的诱导倍数作为优化系统灵敏度的指标,分别获得最大比值以表示系统具有最佳灵敏度。结果当共转染体系比例为hPXR/hCAR表达质粒150ng、CPY3A4/CYP2B6报告基因质粒600ng、PLR-TK内参质50ng时,转染体系的检测灵敏度最高。结论针对所使用的转染细胞系和共转染质粒,通过优化质粒的转染比例可提高系统的灵敏度,优化的共转染系统可用于药物代谢酶诱导机制的研究。     

Tamoxifen cytotoxicity in hepatoblastoma cells stably transfected with human CYP3A4

Tamoxifen can exert its effects through the competitive inhibition of estrogen receptors or other mechanisms. HepG2 cells lacking estrogen receptors and engineered to overexpress CYP3A4, the most important CYP to metabolize the drug, appear to be a good model to study the effects of tamoxifen metabolites. Tamoxifen altered cell cycle of transduced HepG2 cells, decreased G0/G1 cell numbers, diminished proliferation index and induced cell death mostly in cells overexpressing CYP3A4 but was without significant effect on cytotoxicity or proliferation of cells engineered to overexpress CYP2E1 or on empty vector transfected cells. Tamoxifen did not change MDR1 levels irrespectively on CYP450s expression, but inhibited by approximately 50% p-gp functions in all cell types. Drug treatment significantly increased dehydroepiandrosterone sulfotransferase activity and sulfotransferase inhibition significantly decreased tamoxifen cytotoxicity. Our results support the view that metabolic activation of tamoxifen in liver cells may proceed via CYP450-mediated metabolism and subsequent sulfotransferase-mediated activation and point to the role of CYP3A4 and dehydroepiandrosterone sulfotransferase in adverse tamoxifen effects.     

Development of template systems for ligand interactions of CYP3A5 and CYP3A7 and their distinctions from CYP3A4 template

Starting from established CYP3A4 Template (DMPK. 2019, and 2020), CYP3A5 and CYP3A7 Templates have been constructed to be reliable tools for verification of their distinct catalytic properties. A distinct occupancy was observed on CYP3A4-selective ligands, but not on the non-selective ligands, in simulation experiments. These ligands often invade into Bay-1 region during the migration from Entrance to Site of oxidation in simulation experiments. These results offered an idea of the distinct localization of Bay-1 residue on CYP3A5 Template, in which the Bay-1 residue stayed closely to Template border. The idea also accounted for the higher oxidation rates of CYP3A5, than of CYP3A4, of noscapine and schisantherin E through their enhanced sitting-stabilization. Typical CYP3A7 substrates such as zonisamide and retinoic acids took their placements without occupying a left side region of Template for their metabolisms. In turn, the occupancies of the left-side region were inevitably observed among poor ligands of CYP3A7. Altered extent of IJK-Interaction or localization of a specific residue at the left-side would thus explain distinct catalytic properties of CYP3A7 on Template. These data suggest the alteration of each one of Template region, from CYP3A4 Template, led to the distinct catalytic properties of CYP3A5 and CYP3A7 forms.     

A literature review of enzyme kinetic parameters for CYP3A4-mediated metabolic reactions of 113 drugs in human liver microsomes: structure-kinetics relationship assessment

Cytochrome P450 (CYP) enzymes represent a superfamily of hemoproteins that are involved in the metabolism of a wide variety of endogenous and exogenous compounds. For a given CYP enzyme, kinetic properties of a substrate are usually related to substrate lipophilicity (log P or log D(7.4)). In this review, enzyme kinetic parameters (K(m), V(max), and V(max)/K(m)) of 215 CYP3A4-mediated metabolic reactions of 113 drugs in human liver microsomes were obtained from the literature, and lipophilicity values of the 113 drugs were calculated using the ACD/Labs 8.0 program. A low degree of K(m)- or (V(max)/K(m))-lipophilicity correlation, but no V(max)-lipophilicity correlation, is exhibited for the CYP3A4-mediated reactions. Overall, K(m) decreases, but V(max)/K(m) increases, with increasing substrate lipophilicity, and V(max) appears to be independent of substrate lipophilicity. In other words, a low K(m) generally confers a high V(max)/K(m) ratio for a substrate. The degree of lipophilicity-kinetics correlations is related to both reaction types (or reaction mechanisms) and regiochemical positions (or physicochemical properties) of the reaction groups of the substrates. Among the categorized CYP3A4-mediated reactions, the best lipophilicity-kinetics correlation is achieved for carbon hydroxylation, followed by N-dealkylation. No or little lipophilicity-kinetics correlations are seen for N, S-oxidation and other reactions. Within the hydroxylation group, aliphatic hydroxylation shows the best lipophilicity-kinetics correlation while hydroxylation on a carbon atom adjacent to an aromatic ring does not show any lipophilicity-kinetics correlation. The detailed structural and kinetic data sets of the human liver microsomal CYP3A4-mediated reactions represent a specialized database useful for researchers working in the area of structure-metabolism relationship modeling and analysis.     

CYP3 phylogenomics: evidence for positive selection of CYP3A4 and CYP3A7

OBJECTIVE: CYP3A metabolizes 50% of currently prescribed drugs and is frequently involved in clinically relevant drug interactions. The understanding of roles and regulations of the individual CYP3A genes in pharmacology and physiology is incomplete. METHODS: Using genomic sequences from 16 species we investigated the evolution of CYP3 genomic loci over a period of 450 million years. RESULTS: CYP3A genes in amniota evolved from two ancestral CYP3A genes. Upon the emergence of eutherian mammals, one of them was lost, whereas, the other acquired a novel genomic environment owing to translocation. In primates, CYP3A underwent rapid evolutionary changes involving multiple gene duplications, deletions, pseudogenizations, and gene conversions. The expansion of CYP3A in catarrhines (Old World monkeys, great apes, and humans) differed substantially from New World primates (e.g. common marmoset) and strepsirrhines (e.g. galago). We detected two recent episodes of particularly strong positive selection acting on primate CYP3A protein-coding sequence: (i) on CYP3A7 early in hominoid evolution, which was accompanied by a restriction of its hepatic expression to fetal period and (ii) on human CYP3A4 following the split of the chimpanzee and human lineages. In agreement with these findings, three out of four positively selected amino acids investigated in previous biochemical studies of CYP3A affect the activity and regioselectivity. CONCLUSIONS: CYP3A7 and CYP3A4 may have acquired catalytic functions especially important for the evolution of hominoids and humans, respectively.     

Cytotoxicity of safrole in HepaRG cells: studies on the role of CYP1A2-mediated ortho-quinone metabolic activation

1. Safrole is a natural compound categorized as a group 2B carcinogen extracted from sassafras oil or certain other essential oils. The hepatotoxicity of safrole has always been highly concerned. So, the purpose of this study was to evaluate the role of cytochrome P450 (CYP450)-mediated reactive metabolites (RMs) formation and its induced cytotoxicity in HepaRG cells.

2. Safrole belongs to the methylenedioxyphenyl structure which could be activated to RMs. Two metabolites (M1, M2) and three new glutathione conjugates (M3–M5) of safrole ortho-oquinone RMs were found in HepaRG cells. Using human recombinant CYP450 enzymes and chemical inhibitor method, the metabolism of safrole RMs was predominantly carried out through the CYP1A2 with minor contributions by CYP2E1.

3. Induction of CYP1A2 by omeprazole (OME) enhanced safrole-induced cytotoxicity, compared with treatment with safrole alone, whereas inhibition of CYP1A2 by alpha-naphthoflavone (α-NAP) decreased the cytotoxicity. The cytotoxicity of cell induced by safrole was related to the amount of RMs formation. Besides, pretreatment with L-buthionine sulfoximine (BSO) to deplete intracellular GSH markedly enhanced safrole-induced cytotoxicity. OME induced the safrole-induced GSH exhaustion, and GSH depletion by safrole was not via oxidation of GSH and occurred prior to the increase in ROS. Furthermore, mitochondrial membrane potential (ΔΨ_m) could be aggravated by the inducer of CYP1A2 together with safrole. Collectively, these data suggest that the ortho-quinone RM may mediate safrole hepatotoxicity, and CYP1A2 was the core enzyme in ortho-quinone RMs-mediated safrole hepatotoxicity.     

Cyclic AMP-independent activation of CYP3A4 gene expression by forskolin

Forskolin and cAMP have been shown to have paradoxical effects in the regulation of expression levels of mRNA of cytochrome P450 3A (CYP3A) family members. We demonstrate in this study that forskolin upregulated the promoter for CYP3A4 independent of its ability to increase cAMP levels. This activity was explained showing forskolin directly activated the pregnane-X-receptor, a known regulator of CYP3A genes.     

Modulation of CYP2D6 and CYP3A4 metabolic activities by Ferula asafetida resin

Present study investigated the potential effects of Ferula asafetida resin on metabolic activities of human drug metabolizing enzymes: CYP2D6 and CYP3A4. Dextromethorphan (DEX) was used as a marker to assess metabolic activities of these enzymes, based on its CYP2D6 and CYP3A4 mediated metabolism to dextrorphan (DOR) and 3-methoxymorphinan (3-MM), respectively. In vitro study was conducted by incubating DEX with human liver microsomes and NADPH in the presence or absence of Asafetida alcoholic extract. For clinical study, healthy human volunteers received a single dose of DEX alone (phase-I) and repeated the same dose after a washout period and four-day Asafetida treatment (phase-II). Asafetida showed a concentration dependent inhibition on DOR formation (in vitro) and a 33% increase in DEX/DOR urinary metabolic ratio in clinical study. For CYP3A4, formation of 3-MM in microsomes was increased at low Asafetida concentrations (10, 25 and 50 μg/ml) but slightly inhibited at the concentration of 100 μg/ml. On the other hand, in vivo observations revealed that Asafetida significantly increased DEX/3-MM urinary metabolic ratio. The findings of this study suggest that Asafetida may have a significant effect on CYP3A4 metabolic activity. Therefore, using Ferula asafetida with CYP3A4 drug substrates should be cautioned especially those with narrow therapeutic index such as cyclosporine, tacrolimus and carbamazepine.     

Establishment of knockdown of superoxide dismutase 2 and expression of CYP3A4 cell system to evaluate drug-induced cytotoxicity

Drug-induced hepatotoxicity is a major problem in drug development, and oxidative stress is known as one of the causes. Superoxide dismutases (SODs) are important antioxidant enzymes against reactive oxygen species (ROS). Mitochondria are the major source of superoxide production, and SOD2 is mainly localized in mitochondria and, with other SODs, plays an important role in scavenging superoxide. In this study, we established SOD2-knockdown cells. An adenovirus vector with short hairpin RNA against rat SOD2 (AdSOD2-shRNA) was constructed, and infection of AdSOD2-shRNA to rat hepatic BRL3A cells resulted in significant decreases of SOD2 mRNA and protein by 60%, and SOD2 activity by 50% after 3 days infection. We previously constructed an adenovirus expressing cytochrome P450 3A4 (AdCYP3A4). Co-infection of AdSOD2-shRNA and AdCYP3A4 to BRL3A cells was carried out to evaluate the superoxide- and CYP3A4-mediated formation of active metabolites, and mitochondrial toxicity, ROS and superoxide radical production and lipid peroxidation were selected to assess the cell viability. Albendazole, carbamazepine, dapsone, flutamide, isoniazid, nifedipine, sulfamethoxazole, trazodone, troglitazone, and zidovudine demonstrated significant increases of SOD2- and CYP3A4-mediated cytotoxicity. In conclusion, we constructed a highly sensitive cell system to evaluate oxidative stress and CYP3A4 mediated cytotoxicity that could be useful in preclinical drug development.     

Different effects of proton pump inhibitors and famotidine on the clopidogrel metabolic activation by recombinant CYP2B6, CYP2C19 and CYP3A4

Inhibitory potential of proton pump inhibitors (PPIs) and famotidine, an H(2) receptor antagonist, on the metabolic activation of clopidogrel was evaluated using recombinant CYP2B6, CYP2C19 and CYP3A4. Formation of the active metabolite from an intermediate metabolite, 2-oxo-clopidogrel, was investigated by liquid chromatography-tandem mass spectrometry and three peaks corresponding to the pharmacologically active metabolite and its stereoisomers were detected. Omeprazole potently inhibited clopidogrel activation by CYP2C19 with an IC(50) of 12.8 μmol/L and more weakly inhibited that by CYP2B6 and CYP3A4. IC(50) of omeprazole for CYP2C19 and CYP3A4 was decreased about two- and three-fold, respectively, by 30-min preincubation with NADPH. Lansoprazole, esomeprazole, pantoprazole, rabeprazole and rabeprazole thioether, a major metabolite, also inhibited metabolic activation by CYP2C19, with an IC(50) of 4.3, 8.9, 48.3, 36.2 and 30.5 μmol/L, respectively. In contrast, famotidine showed no more than 20% inhibition of clopidogrel activation by CYP2B6, CYP2C19 and CYP3A4 at up to 100 μmol/L and had no time-dependent CYP2C19 and CYP3A4 inhibition. These results provide direct evidence that PPIs inhibit clopidogrel metabolic activation and suggest that CYP2C19 inhibition is the main cause of drug-drug interaction between clopidogrel and omeprazole. Famotidine is considered as a safe anti-acid agent for patients taking clopidogrel.