Drugs behave as substrates, inhibitors and inducers of human cytochrome P450 3A4

Human cytochrome P450 (CYP) 3A4 is the most abundant hepatic and intestinal phase I enzyme that metabolizes approximately 50% marketed drugs. The crystal structure of bound and unbound CYP3A4 has been recently constructed, and a small active site and a peripheral binding site are identified. A recent study indicates that CYP3A4 undergoes dramatic conformational changes upon binding to ketoconazole or erythromycin with a differential but substantial (>80%) increase in the active site volume, providing a structural basis for ligand promiscuity of CYP3A4. A number of important drugs have been identified as substrates, inducers and/or inhibitors of CYP3A4. The ability of drugs to act as inducers, inhibitors, or substrates for CYP3A is predictive of whether concurrent administration of these compounds with a known CYP3A substrate might lead to altered drug disposition, efficacy or toxicity. The substrates of CYP3A4 considerably overlap with those of P-glycoprotein (P-gp). To date, the identified clinically important CYP3A4 inhibitors mainly include macrolide antibiotics (e.g., clarithromycin, and erythromycin), anti-HIV agents (e.g., ritonavir and delavirdine), antidepressants (e.g. fluoxetine and fluvoxamine), calcium channel blockers (e.g. verapamil and diltiazem), steroids and their modulators (e.g., gestodene and mifepristone), and several herbal and dietary components. Many of these drugs are also mechanism-based inhibitors of CYP3A4, which involves formation of reactive metabolites, binding to CYP3A4 and irreversible enzyme inactivation. A small number of drugs such as rifampin, phenytoin and ritonavir are identified as inducers of CYP3A4. The orphan nuclear receptor, pregnane X receptor (PXR), have been found to play a critical role in the induction of CYP3A4. The inhibition or induction of CYP3A4 by drugs often causes unfavorable and long-lasting drug-drug interactions and probably fatal toxicity, depending on many factors associated with the enzyme, drugs and the patients. The study of interactions of newly synthesized compounds with CYP3A4 has been incorporated into drug development and detection of possible CYP3A4 inhibitors and inducers during the early stages of drug development is critical in preventing potential drug-drug interactions and side effects. Clinicians are encouraged to have a sound knowledge on drugs that behave as substrates, inhibitors or inducers of CYP3A4, and take proper cautions and close monitoring for potential drug interactions when using drugs that are CYP3A4 inhibitors or inducers.     

Drugs as CYP3A Probes,Inducers, and Inhibitors

Human cytochrome P450 (CYP) 3A subfamily members (mainly CYP3A4 and CYP3A5) mediate the metabolism of approximately half all marketed drugs and thus play a critical role in the drug metabolism. A huge number of studies on CYP3A-mediated drug metabolism in humans have demonstrated that CYP3A activity exhibits marked ethnic and individual variability, in part because of altered levels of CYP3A4 expression by various environmental factors and functionally important polymorphisms present in CYP3A5 gene. Accumulating evidence has revealed that CYP3A4 and CYP3A5 have a significant overlapping in their substrate specificity, inducers and inhibitors. Therefore, it is difficult to define their respective contribution to drug metabolism and drug-drug interactions. Furthermore, P-glycoprotein and CYP3A are frequently co-expressed in the same cells and share a large number of substrates and modulators. The disposition of such drugs is thus affected by both metabolism and transport. In this review, we systematically summarized the frequently used CYP3A probe drugs, inducers and inhibitors, and evaluated their current status in drug development and research.     

Quantitative prediction of hepatic CYP3A activity using endogenous markers in healthy subjects after administration of CYP3A inhibitors or inducers

Accurate prediction of cytochrome P450 (CYP) 3A activity in the early stage of drug development and in clinical practice is important. This study aimed to evaluate the previously constructed CYP3A activity prediction model after administration of CYP3A inhibitors and inducers and to modify the model for better prediction of CYP3A activity. Healthy male subjects received the following study drugs during three study periods: midazolam alone (control phase); midazolam with 200 mg of itraconazole (CYP3A inhibition phase); and midazolam with 150 mg of rifampicin (CYP3A induction phase). We quantified the concentrations of several endogenous CYP3A markers in both urine and plasma using gas chromatography-mass spectrometry. The urinary markers, including 6β-hydroxy (OH)-cortisol/cortisol, 6β-OH-cortisone/cortisone, 16α-OH-dehydroepiandrosterone (DHEA)/DHEA, 16α-OH-androstenedione (A-dione)/A-dione and 7β-OH-DHEA/DHEA, were significantly correlated with midazolam clearance in both the CYP3A inhibition and induction phases. We constructed a statistical prediction model after integrating data from a previous study to predict midazolam clearance as follows: Ln(midazolam clearance) = 2.5545 + 0.3988 × ln(7β-OH-DHEA/DHEA) + 0.1984 × ln(16α-OH-DHEA/DHEA) + 0.5031 × ln(6β-OH-cortisol/cortisol) – 0.1261 [ln(7β-OH-DHEA/DHEA) × ln(6β-OH-cortisol/cortisol)] (r² = 0.75). We suggest that quantitating endogenous markers in vivo coupled with the statistical prediction model may be useful for predicting CYP3A parameters.     

The effect of CYP3A inhibitors and inducers on the pharmacokinetics of telaprevir in healthy volunteers

AIM

To evaluate the effects of ketoconazole, rifampicin and efavirenz on the pharmacokinetics of telaprevir in healthy volunteers.

METHOD

Results from three clinical studies are described. (1) Volunteers received a single 750 mg dose telaprevir with and without a single 400 mg dose ketoconazole. (2) Volunteers received (a) 1250 mg telaprevir followed by three 750 mg doses given every 8 h and (b) four 1250 mg telaprevir doses given every 8 h, with a single 400 mg dose ketoconazole given with the fourth dose of telaprevir. (3) Volunteers received either a single 750 mg dose telaprevir with or without 600 mg once daily rifampicin, or 750 mg every 8 h telaprevir with and without 600 mg once daily efavirenz.

RESULTS

A single 400 mg dose of ketoconazole increased single dose telaprevir exposure: the geometric least-squares mean ratio (GLSMR, with 90% confidence limits) was 1.24 (1.10, 1.41) for C_max and 1.62 (1.45, 1.81) for AUC(0,∞). However, after multiple doses of telaprevir, there was no discernible effect of ketoconazole on telaprevir exposure. Co-administration of rifampicin at steady-state markedly reduced single dose telaprevir exposure with GLSMRs of 0.14 (0.11, 0.18) for C_max and 0.08 (0.07, 0.11) for AUC(0,∞), whereas efavirenz had a smaller effect on telaprevir exposure when both drugs were co-administered at steady-state, with GLSMRs of 0.91 (0.81, 1.02) for C_max, 0.53 (0.44, 0.65) for C_min, and 0.74 (0.65, 0.84) for AUC(0,8 h).

CONCLUSION

CYP3A inducers, rifampicin and efavirenz, can reduce telaprevir exposure to varying degrees based on their potency. The effect of ketoconazole as an inhibitor of telaprevir metabolism is more pronounced after a single dose of telaprevir than after repeated administration.     

细胞色素P450 3A 选择性探针药物的评价

理想的细胞色素P4 5 0 3A(CYP3A)探针药物有助于检测CYP3A活性 ,辅助临床合理用药。本文对CYP3A选择性探针药物进行比较和综合分析 ,评价各种CYP3A选择性探针药物的特点 ,阐明对临床合理用药的指导意义。在已筛选的CYP3A选择性探针药物中 ,咪哒唑仑是一较理想的探药。     

CYP3A4 inhibitors isolated from Licorice

The extract of licorice (Glycyrrhiza uralensis FISHER, Leguminosae) showed CYP3A4 inhibitory activity with the IC50 value of 0.022 mg/ml. Bioassay-guided purification afforded nine compounds, 3-(p-hydroxyphenyl)propionic acid (1), isoliquiritigenin (2), (3R)-vestitol (3), licopyranocoumarin (4), 4-hydroxyguaiacol apioglucoside (5), liquiritin (6), liquiritigenin 7,4'-diglucoside (7), liquiritin apioside (8), and glucoliquiritin apioside (9). Among these compounds, 3, 7, and 5 showed potent CYP3A4 inhibitory activities with IC50 values of 3.6, 17, and 20 microM, respectively. Glycyrrhizin (10), a main constituent of licorice, however, was inactive for CYP3A4 inhibition.     

Comparison of inducibility of CYP1A and CYP3A mRNAs by prototypical inducers in primary cultures of human, cynomolgus monkey, and rat hepatocytes

This study was conducted to investigate the effects of treatment with the prototypical inducers rifampicin (Rif), dexamethasone (Dex), and omeprazole (Ome) on the mRNA levels of drug-metabolizing enzymes in primary cultures of cryopreserved human, cynomolgus monkey, and rat hepatocytes. Analysis was performed by quantitative real-time RT-PCR using primers and TaqMan probes. Treatment with Ome substantially increased the mRNA levels of both CYP1A1 and CYP1A2 in human hepatocytes, but increased only the mRNA level of CYP1A1 in monkey hepatocytes, whereas it had no marked effect on the mRNA levels of CYP1A1 or CYP1A2 in rat hepatocytes. Treatment with Rif or Dex did not markedly affect the mRNA level of CYP1A in any of the hepatocyte cultures under the conditions used. All three inducers increased the mRNA level of CYP3A8 in monkey hepatocytes (in the order Rif>Dex>or=Ome), and a similar profile was observed for the mRNA level of CYP3A4 in human hepatocytes, but the potency of induction was markedly attenuated. In contrast, only Dex substantially increased the mRNA level of CYP3A1 in rat hepatocytes, with Rif and Ome showing no effects. These results indicate that the molecular mechanisms responsible for the regulation of CYP1A2 genes differ between humans and cynomolgus monkeys, although the regulatory mechanisms for CYP1A1 and CYP3A genes are similar.     

Proluciferin acetals as bioluminogenic substrates for cytochrome P450 activity and probes for CYP3A inhibition

Cytochrome P450 (P450) assays use probe substrates to interrogate the influence of new chemical entities toward P450 enzymes. We report the synthesis and study of a family of bioluminogenic luciferin acetal substrates that are oxidized by P450 enzymes to form luciferase substrates. The luciferin acetals were screened against a panel of purified P450 enzymes. In particular, one proluciferin acetal has demonstrated sensitive and selective CYP3A4-catalyzed oxidation to a luciferin ester-K(m) and k(cat) are 2.88 μM and 5.87 pmol metabolite · min(-1) · pmol enzyme(-1), respectively. The proluciferin acetal was used as a probe substrate to measure IC(50) values of known inhibitors against recombinant CYP3A4 or human liver microsomes. IC(50) values for the known inhibitors correlate strongly with IC(50) values calculated from the traditional high-performance liquid chromatography-based probe substrate testosterone. Luciferin acetals are rapidly oxidized to unstable hemi-orthoesters by CYP3A resulting in luciferin esters and, therefore, are conducive to simple rapid CYP3A bioluminescent assays.     

Evaluation of 309 molecules as inducers of CYP3A4, CYP2B6, CYP1A2, OATP1B1, OCT1, MDR1, MRP2, MRP3 and BCRP in cryopreserved human hepatocytes in sandwich culture

Abstract

1. Regulation of hepatic metabolism or transport may lead to increase in drug clearance and compromise efficacy or safety. In this study, cryopreserved human hepatocytes were used to assess the effect of 309 compounds on the activity and mRNA expression (using qPCR techniques) of CYP1A2, CYP2B6 and CYP3A4, as well as mRNA expression of six hepatic transport proteins: OATP1B1 (SCLO1B1), OCT1 (SLC22A1), MDR1 (ABCB1), MRP2 (ABCC2), MRP3 (ABCC3) and BCRP (ABCG2).

2. The results showed that 6% of compounds induced CYP1A2 activity (1.5-fold increase); 30% induced CYP2B6 while 23% induced CYP3A4. qPCR data identified 16, 33 or 32% inducers of CYP1A2, CYP2B6 or CYP3A4, respectively. MRP2 was induced by 27 compounds followed by MDR1 (16)?>?BCRP (9)?>?OCT1 (8)?>?OATP1B1 (5)?>?MRP3 (2).

3. CYP3A4 appeared to be down-regulated (≥2-fold decrease in mRNA expression) by 53 compounds, 10 for CYP2B6, 6 for OCT1, 4 for BCRP, 2 for CYP1A2 and OATP1B1 and 1 for MDR1 and MRP2.

4. Structure–activity relationship analysis showed that CYP2B6 and CYP3A4 inducers are bulky lipophilic molecules with a higher number of heavy atoms and a lower number of hydrogen bond donors. Finally, a strategy for testing CYP inducers in drug discovery is proposed.     

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.     

Development of novel furanocoumarin dimers as potent and selective inhibitors of CYP3A4.

Grapefruit juice has been found to cause an increase in the oral bioavailability of many therapeutic agents. Such interactions are believed to result from the mechanism-based inhibition of CYP3A4 activity in the intestine. Furanocoumarin dimers present in the juice have been found to be extremely potent inhibitors of CYP3A4 activity. The aim of this work was to synthesize and test a series of dimers with a view to defining the relationship between structure and inhibitory activity and establish whether they might make suitable probes of CYP3A4 activity. Eleven furanocoumarin dimers were synthesized and evaluated as inhibitors of CYP3A4 using human liver microsomes, with testosterone as the marker substrate. Four of the most potent dimers were also investigated for their effects on CYP3A4 activity in the human intestine and on five additional hepatic cytochrome P450 isoforms. The dimers showed potent dose-dependent inhibition of CYP3A4 activity in both liver and intestine; IC50 values ranged from 0.021 +/- 0.002 to 0.146 +/- 0.041 microM (mean +/- S.D. n = 3). Of the four dimers evaluated further, all showed time-dependent inhibition of CYP3A4 activity. 88Prop showed moderate inhibition of both CYP2C19 and CYP1A2 with IC50 values of 4.42 +/- 0.01 and 1.98 +/- 0.34 microM, 88Octa was found to inhibit CYP2C19 (IC50 = 3.16 +/- 0.01 microM) and 58Prop to inhibit CYP1A2 (IC50 = 2.39 +/- 0.77 microM). Minimal inhibition of CYP2D6 and CYP2C9 was observed (IC50 > 10 microM). In conclusion, all the dimers tested were extremely potent inhibitors of CYP3A4 activity. In particular, dimer 55EE was highly selective toward the enzyme, suggesting that this compound is a suitable probe for determining the contribution of CYP3A4 to drug metabolism.     

人细胞色素P450 3A4突变体CYP3A4.3,CYP3A4.4,CYP3A4.5和CYP3A4.18重组酶的异源表达与活性

目的重组表达人细胞色素P450(CYP)3A4突变体CYP3A4.3,CYP3A4.4,CYP3A4.5和CYP3A4.18蛋白,为CYP3A4代谢活性的体外研究提供单一酶源。方法应用杆状病毒表达系统构建含有上述各CYP3A4突变体基因序列的重组病毒,将其连同含人源还原型烟酰胺腺嘌呤二核苷磷酸-P450氧化还原酶(POR)和细胞色素b5基因的重组病毒共同感染昆虫草地夜蛾细胞Sf9得到CYP3A4突变体与POR和细胞色素b5共表达的重组蛋白,分别以高效液相色谱法和荧光分析法测定各重组酶对睾酮和7-甲氧基-4-三氟甲基香豆素的代谢活性。结果在mRNA分子水平上验证了CYP3A4突变体CYP3A4*3,CYP3A4*4,CYP3A4*5和CYP3A4*18基因在Sf9细胞中的转录。感染了各重组病毒的Sf9细胞裂解液对睾酮和7-苄氧基-4-三氟甲基香豆素有明显代谢。结论应用杆状病毒-昆虫细胞表达系统在体外成功表达了具有催化活性的人CYP3A4突变体CYP3A4.3,CYP3A4.4,CYP3A4.5和CYP3A4.18蛋白,其中CYP3A4.5活性显著低于野生型蛋白,CYP3A4.18活性显著高于野生型蛋白,而CYP3A4.3和CYP3A4.4与野生型蛋白活性近似。     

On the mechanism of hepatocarcinogenesis of benzodiazepines: evidence that diazepam and oxazepam are CYP2B inducers in rats, and both CYP2B and CYP4A inducers in mice

The aim of this study was to evaluate diazepam and oxazepam as cytochrome P450 inducers at doses previously shown to cause liver tumors in mice but not rats. In rats, diazepam and oxazepam induced CYP2B, and were as effective as phenobarbital despite lacking phenobarbital's tumor-promoting effect in rats. In mice, diazepam and oxazepam induced both CYP2B and CYP4A at dietary doses associated with liver tumor formation. It remains to be determined why diazepam and oxazepam induce CYP4A in mice but not rats and whether this difference accounts for the apparent species difference in the tumor-promoting activity of diazepam and oxazepam.     

Genetic polymorphisms of the CYP3A4, CYP3A5, CYP3A7 and CYP1A2 among the Jordanian population

Cytochromes P450 (CYP450) plays an extremely vital role in oxidation, reduction, and peroxidation of numerous endogenous and exogenous compounds, like drugs and procarcinogens. Mainly, expression occurs in the liver, in varying polymorphic forms. Therefore, proposed as biomarkers of susceptibility to carcinogenicity and toxicity. The objective of this study was to find the allelic frequencies of CYP3A5*2,*3,*4,*5,*6,*7, CYP3A4*1B, CYP3A7*1C and CYP1A2*1C, *1D, *1E, *1F enzymes in Jordanians, and to compare them with other ethnic groups. We used polymerase chain reaction-restriction fragment length (PCR-RFLP) to genotype alleles, and we calculated frequencies using Hardy Weinberg's equation (HWE). Allelic frequencies results were: CYP3A5*2 (0.2%), CYP3A5*3 (86.6%), CYP3A5*6 (1.7%), CYP*3A5*4,*5*7 not detected, CYP3A4*1B (11.7%), CYP3A7*1C (1.7%). Finally 6.5%, 18.2%, 6.0%, 67.3% were the results of CYP1A2*1C, 1D, 1E and 1F, respectively. In conclusion, genotyping method and results of this study can be adopted or used in pharmacotherapy, toxicity and carcinogenic studies in Jordan.     

Spin probes as mechanistic inhibitors and active site probes of thymidylate synthetase

C-4- and C-5-substituted analogues of dUMP were examined as inhibitors of thymidylate synthetase and as topographical probes of its active site by electron spin resonance (ESR). The C-5-substituted spin-labeled analogues pDUAP (2) and a pDUTT (3) as well as the unlabeled AAdUMP (1) were competitive inhibitors with Ki's of 9.2, 89, and 7.9 microM, respectively. The C-4-spin-labeled pls4dU (4) displayed no inhibition activity. Scatchard plots as determined by ESR gave similar association constants for 2 (Kassoc = 1.9 X 10(5) M-1) and for 3 (Kassoc = 2.4 X 10(5) M-1). Both of these values are similar to the Kassoc of FdUMP indicating that the bulky substituent in position 5 does not interfere with the formation of the binary complex. The enzyme-C-5-spin-labeled nucleotide complexes indicate the presence of similarly immobilized spin labels by ESR, whereas no binding and immobilization were noticed with the C-4-spin-labeled nucleotide. A model for the active-site geometry of the enzyme was derived which suggests that the C-5 substituents point toward the opening of the binding cavity whose depth is at least 12 A. Also, the approximate 10-fold increased inhibitory activity of 2 as compared to that of 3 may be attributed to the significant electron withdrawing properties of the C-5 substituent in 2. Finally, the set of probes used for the binding and inhibition of thymidylate synthetase gives direct experimental evidence that an electron-withdrawing C-5 substituent primarily affects the formation of the ternary complex and will not substantially influence the stability of the binary complex.