Heterogeneity of CYP3A isoforms metabolizing erythromycin and cortisol.

The N-demethylation of erythromycin and 6 beta-hydroxylation of cortisol are both functions of the glucocorticoid-inducible CYP3A in human liver microsomes. To determine whether 6 beta-hydroxylation and erythromycin N-demethylation are catalyzed by similar or distinct CYP3A isoforms, erythromycin N-demethylase activity, as reflected by the recently described 14[C]-erythromycin breath test, was compared with urinary 6 beta-hydroxycortisol/cortisol ratios, a measure of cortisol 6 beta-hydroxylase activity, in nine patients. Erythromycin N-demethylation varied fourfold and 6 beta-hydroxycortisol/cortisol ratios varied sevenfold among the subjects; no correlation was found between these activities (r2 = 0.065). New noninvasive tests of CYP3A strongly suggest cortisol 6 beta-hydroxylation and erythromycin N-demethylation are performed by distinct CYP3A isoforms.     

Influence of CYP3A4, CYP3A5, and ABCB1 genotype and expression on budesonide pharmacokinetics: a possible role of intestinal CYP3A4 expression

Budesonide treatment of chronic inflammatory bowel disease commonly leads to non-response or adverse reactions, possibly because of alterations in efflux transport mediated by the ABCB1 gene product P-glycoprotein or metabolism by CYP3A isoenzymes. Two groups, each consisting of nine healthy volunteers, one with the CYP3A5(*)1/(*)3 genotype (expressors) and the other with the CYP3A5(*)3/(*)3 genotype (non-expressors), were given a single oral dose of 9 mg budesonide. Plasma and urine concentrations of budesonide and its major metabolites were determined using liquid chromatography-tandem mass spectrometry. Subsequently, rectosigmoidal biopsies were taken for analysis of messenger RNA (mRNA) expression. Budesonide pharmacokinetics did not differ between genotype groups. However, intestinal CYP3A4 expression was shown to correlate directly with partial metabolic clearances of 16-hydroxy-prednisolone (r(2) = 0.30; P = 0.010) and 6-hydroxy-budesonide (r(2) = 0.25; P = 0.016), but inversely with budesonide AUC(0-24 h) (r(2) = 0.18; P = 0.040). Interestingly, a strong correlation was found between CYP3A5 and ABCB1 expression in CYP3A5 expressors (r(2) = 0.79; P = 0.001). This study suggests that intestinal CYP3A4 expression has an impact on budesonide pharmacokinetics. Moreover, CYP3A5 and ABCB1 expression appears to be coregulated.     

In Vivo CYP3A4 Activity, CYP3A5 Genotype, and Hematocrit Predict Tacrolimus Dose Requirements and Clearance in Renal Transplant Patients

Tacrolimus is metabolized by CYP3A4 and CYP3A5 and is characterized by a narrow therapeutic index and highly variable pharmacokinetics. This cross-sectional study in 59 renal transplant patients investigated the relationship among in vivo CYP3A4 activity (assessed using midazolam as a drug probe), CYP3A5 genotype on the one hand, and tacrolimus pharmacokinetics on the other hand, taking into account other potential determinants of tacrolimus disposition. In vivo CYP3A4 activity and CYP3A5 genotype explain 56-59% of variability in tacrolimus dose requirements and clearance, contributing ~25 and 30%, respectively. Hematocrit explains an additional 4-14%. These data indicate that CYP3A4- and CYP3A5-mediated tacrolimus metabolisms are major determinants of tacrolimus disposition in vivo and explain a substantial part of the clinically observed high interindividual variability in tacrolimus pharmacokinetics. Furthermore, these data provide a potential basis for a comprehensive approach to predicting tacrolimus dose requirement in individual patients and hence provide a strategy to tailor immunosuppressive therapy in transplant recipients.     

CYP3A4、CYP3A5、COMT及OPRM1基因多态性对术后芬太尼镇痛剂量的影响

目的 探讨代谢酶相关基因（CYP3A4*1G、CYP3A5*3、COMT Val158Met）和药物作用靶点基因（OPRM1rs563649）的多态性与芬太尼术后镇痛剂量个体差异的相关性。方法 选择2018年10月至2020年3月复旦大学附属中山医院收治的择期全麻下行腹腔镜辅助结肠癌根治术患者91例，抽取患者外周静脉血，通过Sanger测序技术检测CYP3A4*1G、CYP3A5*3、OPRM1 rs563649及COMT Val158Met各基因多态性，随访患者术后芬太尼镇痛剂量和视觉模拟评分（VAS）分值，分析基因多态性与腹腔镜术后芬太尼镇痛用量的关系。结果 CYP3A4*1G、CYP3A5*3、OPRM1 rs563649及COMT Val158Met各位点在结肠癌患者中突变频率分别为31.87%、65.93%、9.40%、26.92%。携带CYP3A4*1G/*1G突变纯合子基因型的患者术后第1天、第2天芬太尼消耗量减少（P<0.05）。CYP3A5*3、OPRM1rs563649及COMT Val158Met各基因型的芬太尼术后消耗量差异无统计学意义。CYP3A4*1G...     

Association of CYP3A5 polymorphisms with hypertension and antihypertensive response to verapamil

In the CYP3A5 gene, the A>G (*3) and G>A (*6) polymorphisms result in severely decreased expression of CYP3A5 enzyme relative to a normal functional allele (*1). We sought to determine if the CYP3A5 genetic polymorphisms were associated with level of blood pressure (BP), risk of hypertension (HTN), and the antihypertensive response to verapamil. A total of 676 normotensive and hypertensive participants (mean age 49+/-8.2 years) from the Hypertension Genes study and 722 patients (mean age 66+/-9 years) from the International Verapamil/Trandolapril Study Genetic Substudy (INVEST-GENES) were genotyped for CYP3A5 to test for associations with BP, HTN, and in the latter cohort, antihypertensive response to verapamil. CYP3A5 haplotypes were determined using PHASE 2, with any allele containing either (*3) or (*6) designated as non functional. In the HTN genes population, there were no significant differences based on the number of functional CYP3A5 alleles, in systolic blood pressure (SBP) or diastolic blood pressure (DBP) among the normotensive whites or blacks (all P> or =0.70) or in allele frequency between normotensives and hypertensives. In INVEST-GENES, when controlled for baseline BP, race, age, and gender, untreated BP in carriers versus non carriers of a CYP3A5 functional allele was 158.2+/-13.7 and 154.8+/-13.7 (P=0.061), respectively. CYP3A5 functional allele status was marginally associated with the SBP response to verapamil in blacks (P=0.075) and Hispanics (P=0.056), but not in whites (P=0.40), with the effect being largely driven by higher SBP in the carriers of two functional alleles. There was no association with DBP response and CYP3A5 allele status. CYP3A5 genotype does not contribute importantly to BP or risk of HTN, but may influence response to calcium channel blockers in populations in which carrier status of two functional alleles is common.     

Recombinant CYP3A4*17 is defective in metabolizing the hypertensive drug nifedipine, and the CYP3A4*17 allele may occur on the same chromosome as CYP3A5*3, representing a new putative defective CYP3A haplotype

Genetic variation in CYP3A activity may influence the rate of the metabolism and elimination of CYP3A substrates in humans. We previously reported four new CYP3A4 coding variants in three different racial groups. In the present study, we examined metabolism of nifedipine by the recombinant forms of these allelic variants. Metabolism of nifedipine by the L293P (CYP3A4*18), M445T (CYP3A4*3), and P467S (CYP3A4*19) allelic variants was not significantly different from wild-type CYP3A4*1. However, F189S (CYP3A4*17) exhibited a >99% decrease in both V(max) and CL(max) of nifedipine compared with CYP3A4*1. Of 72 racially diverse individuals, CYP3A4*17 was identified in 1 of 24 Caucasian samples [1:5 Eastern European (Adygei ethnic group)]. Genotyping of an extended set of 276 genomic DNAs of Caucasians (100 from the Coriell Repository and an additional 176 from the United States) for CYP3A4*17 detected no additional individuals containing the CYP3A4*17 allele. However, additional genotyping of four more Adygei samples available from Coriell detected an additional individual carrying the CYP3A4*17 allele. New specific polymerase chain reaction-restriction fragment length polymorphism genotyping procedures were developed for the major splice variant of CYP3A5 (CYP3A5*3) and CYP3A4*17. Genotyping revealed that the two individuals carrying CYP3A4*17 were either homozygous or heterozygous for the more frequent CYP3A5*3 allele, suggesting that the two alleles may exist on the same chromosome as a new putative CYP3A poor metabolizer haplotype. We predict that individuals who are homozygous for defective alleles of both of these genes would metabolize CYP3A substrates poorly. The new genetic tests will be useful in future clinical studies to investigate genotype/phenotype associations.     

In vitro activity of LY281389 and comparison with erythromycin and oral beta-lactams.

LY281389 is a new 14-member ring macrolide which is presently being developed for possible clinical use against bacterial infections. We compared the in vitro activity of LY281389 with erythromycin, ampicillin, augmentin and cephalexin against 610 clinical isolates. The new drug inhibited 97 and 11% of methicillin-sensitive and methicillin-resistant Staphylococcus aureus isolates, respectively, 59% of coagulase-negative staphylococci, 63% of enterococci and 74% of Haemophilus influenzae. All the 171 isolates of Streptococcus Lancefield group A, group B and Streptococcus pneumoniae were susceptible to LY281389 at MIC values ranging between 0.03 and 0.24 micrograms/ml. In vitro activity of LY281389 against the bacteria tested was comparable to that of erythromycin.     

Pharmacogenetics of acenocoumarol: CYP2C9, CYP2C19, CYP1A2, CYP3A4, CYP3A5 and ABCB1 gene polymorphisms and dose requirements

BACKGROUND AND OBJECTIVE: Acenocoumarol (AC) is a coumarin derivative, vitamin K antagonist anticoagulant drug. It has a narrow therapeutic index and shows large pharmacokinetic and pharmacodynamic interindividual variability. Our objective was to investigate the association between AC dose requirements to achieve a target level of anticoagulation and genetic polymorphisms of genes possibly associated with its metabolism (CYP1A2, CYP2C9, CYP2C19, CYP3A4, CYP3A5) and transport (ABCB1). METHODS: Ninety-six Bulgarian patients treated orally with AC for at least 3 months were included. They were separated into three groups according to their AC dose requirement, i.e. low, medium and high. RESULTS AND DISCUSSION: CYP2C9*1/*3 (associated with an intermediate CYP2C9 activity), CYP2C9*2/*2, and CYP2C9*2/*3 genotypes (associated with a low CYP2C9 activity) were more prevalent in the group with low dose requirement of AC compared with the other two groups (P = 0.003). The frequency of CYP2C9*1/*1 genotype, which is associated with an extensive CYP2C9 activity, was higher in the group of patients with high dose requirements (79%), compared with the groups of the medium and low dose requirements (67% and 21% respectively). In addition, the ABCB1 2677GG/3435CC haplotype was associated with use of lower AC dose, whereas the 2677TT/3435TT and 2677GT/3435TT haplotypes were associated with use of higher AC dose (P = 0.03). The distribution of polymorphisms of other genes did not show significant differences between the three groups. CONCLUSION: In vivo, cytochromes P450 isoforms other than CYP2C9 [corrected] were not significantly associated with dose requirement of AC. In our Bulgarian patients, the presence of CYP2C9*2 or/and CYP2C9*3 alleles, as well as the ABCB1 2677GG/3435CC haplotype were associated with low dose requirement of AC.     

Identification and characterization of CYP3A4*20, a novel rare CYP3A4 allele without functional activity

BACKGROUND: The major drug-metabolizing enzyme cytochrome P450 (CYP) 3A4 is genetically conserved. One outlier of Brazilian descent was found in a clinical pharmacokinetic trial exhibiting a 6-fold higher exposure than expected to an investigational drug, shown to be a CYP3A4 substrate. We aimed to investigate the genetic background of this finding. METHODS: The allelic variant of the CYP3A4 gene present in the outlier was sequenced, and the corresponding complementary deoxyribonucleic acid was expressed in yeast and human embryonic kidney cells. The outlier was phenotyped by use of intravenous administration of 1 mg midazolam. Analysis of phenotype and genotype correlation was carried out. The prevalence of the new allele was screened for in a white population. RESULTS: We identified a subject who heterozygously carried a novel CYP3A4 allele, named CYP3A4*20, with a premature stop codon yielding a truncated protein. Heterologous expression revealed that the CYP3A4.20 enzyme does not incorporate heme and thus is devoid of catalytic activity. CYP3A phenotyping in vivo showed that CYP3A4*20 exhibits a clear genotype-phenotype correlation, demonstrated by the subject's low systemic midazolam clearance (2.99 mL x min(-1) x kg(-1)). Genotyping of a white German population (n = 428) and relatives of the subject, as well as a review of published CYP3A4 sequencing data, suggests that CYP3A4*20 is a rare variant allele (<0.06% in white subjects). CONCLUSIONS: CYP3A4*20 represents the first CYP3A4 allele to be identified that has been shown to be devoid of functional activity. It causes an intermediate CYP3A4 metabolizer phenotype in a heterozygous carrier. Subjects of this genotype might be susceptible to side effects during drug therapy with substrates or inhibitors of CYP3A4.     

Influence of solute carriers on the pharmacokinetics of CYP3A4 probes

We hypothesized that the assessment of baseline CYP3A4 activity is influenced by probe-specific differences in hepatocellular uptake mechanisms. There was no significant correlation between the erythromycin breath test (ERMBT) parameters and midazolam clearance in 30 cancer patients (R(2) < 0.01), regardless of their CYP3A5 genotype status. In cellular models overexpressing 10 different solute carriers, erythromycin uptake was significantly increased by OATP1A2 (P < 0.005) and OATP1B3 (P < 0.01). Midazolam was not a substrate for any of the tested transporters. In a separate cohort of 119 patients, 6 nonsynonymous variants in the OATP1B3 gene SLCO1B3 were identified. Individuals carrying two copies of the T allele at the 334 locus had a 2.4-fold lower value for ERMBT 1/T(max) (P = 0.001), a measure reflecting more rapid hepatic uptake. These findings suggest that differential affinities for solute carriers should be considered when selecting an appropriate phenotypic probe to allow tailored dosing of pharmaceuticals that are CYP3A4 substrates.     

St John's Wort: effect on CYP3A4 activity

BACKGROUND: St John's Wort is a widely used herbal product. Information regarding its potential for drug interactions is required for responsible treatment of patients using St John's Wort. CYP3A4 is a metabolic enzyme implicated in most clinically significant drug-drug interactions. OBJECTIVE: To determine the in vivo effect of reagent-grade St John's Wort extract on CYP3A4 activity through evaluation of urinary 6-beta-hydroxycortisol/cortisol ratios. METHODS: Thirteen subjects ranging in age from 18 to 25 years participated in this unblinded, multiple-dose, single-treatment before-after trial conducted in a university-based pharmacokinetics and biopharmaceutics laboratory. Each subject ingested a 300-mg tablet of reagent-grade St John's Wort extract standardized to 0.3% hypericin three times a day for 14 days. Baseline and posttreatment CYP3A4 activity was assessed with the urinary 6-beta-hydroxycortisol/cortisol ratio after a 24-hour urine collection. RESULTS: The mean +/- SD urinary 6-beta-hydroxycortisol/cortisol ratio significantly increased (P = .003) from a baseline value of 7.1 +/- 4.5 to 13 +/- 4.9. The mean +/- SD percentage increase was 114% +/- 95%, with a range from -25% to 259%. All but one subject had an increase in the ratio. CONCLUSIONS: Treatment with St John's Wort for 14 days resulted in significant increases in the urinary 6-beta-hydroxycortisol/cortisol ratio. This finding suggests that St John's Wort is an inducer of CYP3A4.     

Inhibition of rat liver estrogen 2/4-hydroxylase activity by troleandomycin: comparison with erythromycin and roxithromycin

Administration of troleandomycin (0.5 mmol.kg-1 p.o. daily for 5 days) decreased by 61% and 36%, respectively, the estradiol and ethinylestradiol 2/4-hydroxylase activities of hepatic microsomes from male Sprague-Dawley rats killed 2 hr after the last dose. This decrease did not appear to be due to the in vivo formation of the inactive cytochrome P-450 p Fe(II)-metabolite complex, since disruption of this complex with potassium ferricyanide did not increase estrogen hydroxylase activities. Troleandomycin administration, however, essentially suppressed cytochrome P-450 UT-A (one of the P-450 forms involved in the hydroxylation of estrogens) and resulted in the appearance of cytochrome P-450 forms whose estradiol hydroxylase activity was inhibitable by troleandomycin in vitro. Similarly, troleandomycin (2 mM) inhibited by 60% estradiol and ethinylestradiol 2/4-hydroxylase activities in microsomes from dexamethasone-treated rats, although it had no inhibitory effect in microsomes from control rats. In contrast, erythromycin and roxithromycin (2 mM) exerted no inhibitory effect, even in microsomes from dexamethasone-treated rats. In vivo, these macrolides (0.5 mmol.kg-1 p.o. daily for 5 days) decreased moderately cytochrome P-450 UT-A levels and estradiol 2/4-hydroxylase activity, and did not modify ethinylestradiol 2/4-hydroxylase activity. We conclude that the administration of troleandomycin, but not that of erythromycin or roxithromycin, decreases ethinylestradiol 2/4-hydroxylase activity in male rat liver microsomes, as a possible consequence of decreased cytochrome P-450 UT-A levels and of the induction of glucocorticoid-responsive P-450 forms whose ethinylestradiol hydroxylase activity is inhibitable by troleandomycin.     

CYP3A5*3 and CYP3A4*18 single nucleotide polymorphisms in a Chinese population

BACKGROUND: Human cytochrome P450 3A evolved to catalyze the metabolism of numerous common therapy drugs and endogenous molecules. Members of the CYP3A are the majority expressed in human liver and intestine, and there are marked interindividual differences in their protein expression and activity. The activity of CYP3A enzyme in Chinese is highly variable, exceeding 14-fold, and contributes greatly to variation in oral bioavailability and systemic clearance of CYP3A substrates. The genetic factors play an important role in the interindividual variability in CYP3A activity. Detection of CYP3A5 and CYP3A4 variant alleles and knowledge about their allelic frequency in specific ethnic groups are important to lead to individualized drug dosing and improved therapeutics. METHODS: We determined the allelic frequency of the CYP3A5*3 and CYP3A4*18 in a group of 302 Chinese subjects by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays. RESULTS: In the group of 302 unrelated individuals, the frequency of the CYP3A5*3 and CYP3A4*18 variant allele in Chinese population were 0.778 (95% CI: 0.754, 0.802) and 0.01 (95% CI: 0, 0.02), respectively. CONCLUSIONS: We developed a simple assay for the detection of the CYP3A4*18 allele and showed that in a Chinese population, CYP3A4*18 and CYP3A5*3 allelic frequencies are similar to that reported previously in Chinese resident in Taiwan. The frequency of the CYP3A5*3 allele in Chinese population is similar to the Japanese but lower than Caucasians. Meanwhile, our findings suggest that an approximate 62% of the Chinese population carrying CYP3A5*3/*3 genotype may appear not to express CYP3A5 protein.     

In vivo CYP3A activity is significantly lower in cyclosporine-treated as compared with tacrolimus-treated renal allograft recipients

In vitro studies have identified cyclosporine and tacrolimus as CYP3A inhibitors. In the current study in renal allograft recipients, we used intravenously and orally administered midazolam as a drug probe to assess whether the study drugs at doses that are generally used in clinical practice have differential effects on in vivo hepatic and first-pass CYP3A activities. Systemic and apparent oral midazolam clearance were 24% (269 ± 73 vs. 354 ± 102?ml/min, P = 0.022) and 31% (479 ± 190 vs. 688 ± 265?ml/min, P = 0.013), respectively, lower in cyclosporine-treated patients (n = 20) than in matched tacrolimus-treated patients (n = 20). The latter displayed midazolam clearances similar to those in two larger cohorts of nonmatched tacrolimus-treated patients (n = 58 and n = 80) and to those receiving a calcineurin inhibitor-free regimen (n = 6). This implies that in vivo hepatic and first-pass CYP3A activities are significantly lower in patients receiving cyclosporine than in those receiving tacrolimus, indicating that, at the doses generally used in clinical practice, cyclosporine is the stronger of the two with respect to CYP3A inhibition. This observation has important implications in the context of drug-drug interactions in transplant recipients.     

In vivo CYP3A4 heteroactivation is a possible mechanism for the drug interaction between felbamate and carbamazepine

Atypical (non-Michaelis-Menten) kinetics are commonly observed with CYP3A4 substrates in vitro. If relevant in vivo, cytochrome P450 heteroactivation could give rise to increased drug clearance. To test the possible in vivo relevance of atypical cytochrome P450 kinetics, we investigated the role of heteroactivation in the therapeutically relevant drug interaction between the anti-epileptics felbamate and carbamazepine. Felbamate heteroactivates CYP3A4-mediated formation of carbamazepine-10,11-epoxide (carbamazepine-ep), the major metabolite of carbamazepine, in human liver microsomes and recombinant CYP3A4 at relevant in vivo concentrations of both drugs (maximum activation 98% at 10 microM carbamazepine, 1 mM felbamate). Felbamate (50-500 microM) did not induce CYP3A4, as based on mRNA measurements in human liver slices. The further metabolism of carbamazepine-ep was inhibited (38% by 500 microM felbamate) in human liver slices. We propose a methodology to predict changes in steady-state plasma concentrations (Css) of parent drug and metabolite from in vitro heteroactivation and inhibition data, including prediction of the increase in fraction metabolized. A meta-analysis of reported in vivo effects of felbamate on Csscarbamazepine was performed to allow evaluation of this approach. The predicted effect of in vitro heteroactivation on Csscarbamazepine corresponds well to that observed in vivo. Combining the effect of heteroactivation on the fraction metabolized to carbamazepine-ep, and inhibition of its further metabolism, predicts a change in Csscarbamazepine-ep that falls within the range observed in vivo. Our results strongly suggest that in vivo heteroactivation of CYP3A4 is a possible mechanism of the clinically observed drug interaction between felbamate and carbamazepine.     

CYP3A activity in African American and European American men: population differences and functional effect of the CYP3A4*1B5'-promoter region polymorphism

OBJECTIVE: Cytochrome P4503A (CYP3A) activity exhibits considerable interindividual variability. Possible differences in CYP3A activity were investigated in European American and African American men with the use of midazolam as an in vivo probe. METHODS: Midazolam was simultaneously administered intravenously (1 mg, [15N3]-labeled) and orally (2 mg, unlabeled in capsule form) to 15 young healthy European American men and a similar group of men of African American descent. Plasma concentration-time curves were measured. The subjects were subsequently genotyped with respect to the CYP3A4*B1 polymorphism (A-290G) in the 5'-promoter (nifedipine-specific element) region. RESULTS: The oral bioavailability of midazolam was about equally determined by intestinal and hepatic extraction with CYP3A activity at the former site exhibiting greater variability. Oral bioavailability was related to intestinal metabolism (r = 0.98), whereas hepatic CYP3A activity contributed little to the interindividual variability (r = 0.03). A lower systemic clearance (265+/-54 versus 310+/-56 mL/min; P = .04), but not oral clearance, was observed in African Americans. With one exception, the African Americans possessed a variant CYP3A4*1B allele (4 heterozygotes A/G and 10 homozygote G/G), whereas all of the European Americans were wild-type homozygotes (A/A). Hepatic CYP3A activity and the systemic clearance of midazolam were about 30% lower in G/G homozygotes than in A/A homozygotes (252+/-53 versus 310+/-54 mL/min; P = .02), and a gene-dose effect was present (P = .01). There was no genotype/phenotype relationship with respect to the oral clearance of midazolam. CONCLUSION: Comparison of CYP3A activity between populations is complicated by frequency distribution differences in the regulatory CYP3A4*1B polymorphism and lower hepatic CYP3A activity associated with the variant allele. However, this reduction is modest; therefore no major and clinically important difference in CYP3A activity is present between Americans of African or European descent.