Tacrolimus Elimination in Four Patients With a CYP3A5*3/*3 CYP3A4*22/*22 Genotype Combination

Cytochrome P450 3A5 (CYP3A5) and cytochrome P450 3A4 (CYP3A4) are the predominate enzymes responsible for tacrolimus metabolism. The presence of CYP3A4 and CYP3A5 genetic variants significantly affects tacrolimus clearance and dose requirements. CYP3A5*3 is a loss‐of‐function variant resulting in no CYP3A5 enzyme production. CYP3A4*22 is a variant that reduces production of functional CYP3A4 protein. Caucasians commonly carry these variant alleles but are very rarely homozygous for both CYP3A5*3 and CYP3A4*22. This report describes four kidney transplant recipients who carry a rare genotype combination (CYP3A5*3/*3 and CYP3A4*22/*22). These patients were identified from a larger cohort of Caucasian kidney transplant recipients (n=1366). To understand the significance of this genotype combination on tacrolimus troughs and doses, we compared these patients to recipients without this combination. Patients homozygous for both variants are at risk for profound reductions in metabolism of CYP3A substrates. A 342% and a 90.6% increase in the median dose‐normalized trough was observed, when the CYP3A5*3/*3 and CYP3A4*22/*22 genotype combination was compared to the CYP3A5*1/*1 and CYP3A4*1/*1 genotype combination and the CYP3A5*3/*3 and CYP3A4*1/*1 genotype combination, respectively. These four individuals only required on average 2.5 mg/day of tacrolimus. Knowledge of these genotypes would be useful in selecting appropriate tacrolimus doses to avoid overexposure.     

Differences in flurbiprofen pharmacokinetics between CYP2C9*1/*1, *1/*2, and *1/*3 genotypes

OBJECTIVE: This study was conducted to examine differences in flurbiprofen metabolism among individuals with the CYP2C9*1/*1, *1/*2, and *1/*3 genotypes. METHODS: Fifteen individuals with the CYP2C9*1/*1 ( n=5), *1/*2 ( n=5), and *1/*3 ( n=5) genotypes received a single 50-mg oral dose of flurbiprofen. Plasma and urine samples were collected over 24 h, and flurbiprofen and 4'-hydroxyflurbiprofen pharmacokinetic data were compared across genotypes. RESULTS: CYP2C9 genotype was a significant predictor of flurbiprofen metabolism and accounted for 59% of the variability in flurbiprofen AUC(0- infinity ), and approximately 50% of the variability in flurbiprofen oral clearance, formation clearance to 4'-hydroxyflurbiprofen, and the 0 to 24-h urinary metabolic ratio of flurbiprofen to 4'-hydroxyflurbiprofen. Flurbiprofen AUC(0- infinity )was significantly higher and all measures of flurbiprofen clearance were significantly lower in the CYP2C9*1/*3 individuals than in those with *1/*1. Significant differences in these parameters were not detected between *1/*2 subjects and *1/*1 subjects. CONCLUSIONS: CYP2C9 genotype is a significant predictor of flurbiprofen disposition in humans by altering CYP2C9-mediated metabolism and reducing systemic clearance. The effects are most pronounced in individuals carrying the *3 allele.     

CYP3A5 genotype-phenotype analysis in the human kidney reveals a strong site-specific expression of CYP3A5 in the proximal tubule in carriers of the CYP3A5*1 allele

Interindividual variability in the drug-metabolizing activity of the CYP3A5 enzyme is mainly due to a single nucleotide polymorphism in CYP3A5, leading to low expression in homozygous CYP3A5*3/*3 individuals compared with CYP3A5*1 allele carriers. In the human kidney, expression of CYP3A5 has been implicated in blood pressure regulation and calcineurin inhibitor-associated nephrotoxicity. The effect of the CYP3A5*1/*3 polymorphism on the expression level and protein distribution within the human kidney is not well characterized. Therefore, we performed a genotype-phenotype analysis of CYP3A5 mRNA and protein expression in the human kidney. To this end, we analyzed sections of normal kidney tissue obtained from 93 white individuals undergoing nephrectomy by quantitative mRNA expression analysis. Qualitative protein expression analysis of CYP3A5 was performed by immunohistochemistry. Mean renal mRNA expression of carriers of the CYP3A5*1 (n = 12) allele was more than 18-fold higher than that of CYP3A5*3/*3 carriers (n = 81, p < 0.001). Immunohistochemical analysis demonstrated CYP3A5 protein in all epithelia of the nephron in kidney sections with the CYP3A5*3/*3 genotype. In carriers of the CYP3A5*1 allele, a strong increase in protein expression of CYP3A5 was detected, and this was confined to the proximal tubule. This study confirms a significant effect of the CYP3A5*1/*3 polymorphism on CYP3A5 expression in the normal human kidney and reveals a strong nephron segment-specific difference in the CYP3A5 protein expression limited to the proximal tubule.     

CYP3A5*3 and CYP3A4*1B allele distribution and genotype combinations: differences between Spaniards and Central Americans

The aim of this study was to detect genotypic differences between three populations of healthy volunteers from Northern Spain (204 subjects), Nicaragua (120 subjects), and El Salvador (112 subjects) regarding CYP3A4*1B and CYP3A5*3 polymorphisms. No significant differences were found by comparing allelic frequencies between the two Central American populations. The CYP3A5*3 allele frequency was significantly different (P < 0.01) between Central Americans (76%) and Spaniards (91%). By contrast, CYP3A4*1B allele was more prevalent among Central Americans (12.5%) than among North Spaniards (4%) (P < 0.01). Analysis of CYP3A4-3A5 genotype combinations revealed that individuals carrying CYP3A4*1B/CYP3A5*1 were more represented in Central Americans (16.9%) than in Spaniards (5.4%), suggesting a marked linkage disequilibrium. These data are compatible with a higher CYP3A enzyme activity in Central Americans as opposed to Spaniards and other white groups, which could imply differences in dose requirements for drugs metabolized by CYP3A and should be considered in allele-disease association studies.     

Effect of CYP3A5 polymorphism on tacrolimus metabolic clearance in vitro.

Previous investigations of solid organ transplant patients treated with tacrolimus showed that individuals carrying a CYP3A5*1 allele have lower dose-adjusted trough blood concentrations compared with homozygous CYP3A5*3 individuals. The objective of this investigation was to quantify the contribution of CYP3A5 to the hepatic and renal metabolic clearance of tacrolimus. Four primary tacrolimus metabolites, 13-O-desmethyl tacrolimus (13-DMT) (major), 15-O-desmethyl tacrolimus, 31-O-desmethyl tacrolimus (31-DMT), and 12-hydroxy tacrolimus (12-HT), were generated by human liver microsomes and heterologously expressed CYP3A4 and CYP3A5. The unbound tacrolimus concentration was low (4-15%) under all incubation conditions. For CYP3A4 and CYP3A5, V(max) was 8.0 and 17.0 nmol/min/nmol enzyme and K(m,u) was 0.21 and 0.21 muM, respectively. The intrinsic clearance of CYP3A5 was twice that of CYP3A4. The formation rates of 13-DMT, 31-DMT, and 12-HT were >or=1.7-fold higher, on average, in human liver microsomes with a CYP3A5*1/*3 genotype compared with those with a homozygous CYP3A5*3/*3 genotype. Tacrolimus disappearance clearances were 15.9 +/- 9.8 ml/min/mg protein and 6.1 +/- 3.6 ml/min/mg protein, respectively, for the two genotypes. In vitro to in vivo scaling using both liver microsomes and recombinant enzymes yielded higher predicted in vivo tacrolimus clearances for patients with a CYP3A5*1/*3 genotype compared with those with a CYP3A5*3/*3 genotype. In addition, formation of 13-DMT was 13.5-fold higher in human kidney microsomes with a CYP3A5*1/*3 genotype compared with those with a CYP3A5*3/*3 genotype. These data suggest that CYP3A5 contributes significantly to the metabolic clearance of tacrolimus in the liver and kidney.     

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.     

CYP3A5*3 and *6 single nucleotide polymorphisms in three distinct Asian populations

OBJECTIVE: To determine the frequencies of two functional single nucleotide polymorphisms, CYP3A5*3 and CYP3A5*6, in the CYP3A5 gene in three distinct Asian ethnic groups, namely, the Chinese, Malays and Indians. METHODS: Single nucleotide polymorphism analyses of CYP3A5*1, *3 and *6 were performed in 296 healthy subjects (108 Chinese, 98 Malays and 90 Indians) using the polymerase chain reaction-restriction fragment length polymorphism method. RESULTS: The *1 allele frequency was 25% in Chinese compared with 40% in Malays and Indians ( P=0.001). The *3 allele frequency was also higher in the Chinese population, being 76% versus 60% in the Malays and Indians ( P=0.001). The Malays and Indians also had allele frequencies significantly different from Caucasian, Japanese and African-American populations (each P相似文献   

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.     

Effects of CYP3A4*1G and CYP3A5*3 polymorphisms on pharmacokinetics of tylerdipine hydrochloride in healthy Chinese subjects

1. The aim of this analysis was to explore the influence of CYP3A4*1G and CYP3A5*3 polymorphisms on the pharmacokinetics of tylerdipine in healthy Chinese subjects.

2. A total of 64 and 63 healthy Chinese subjects were included and identified as the genotypes of CYP3A4*1G and CYP3A5*3, respectively. Plasma samples were collected for up to 120?h post-dose to characterize the pharmacokinetic profile following single oral dose of the drug (5, 15, 20, 25 and 30?mg). Plasma levels were measured by a high-performance liquid chromatography-mass spectrometry (LC-MS/MS). The pharmacokinetic parameters were calculated using non-compartmental method. The maximum concentration (C_max) and the area under the curve (AUC_0–24?h) were all corrected by the dose given.

3. In the wild-type group, the mean dose-corrected AUC_0–24?h was 1.35-fold larger than in CYP3A4*1G carriers (p?=?.018). Among the three CYP3A5 genotypes, there showed significantly difference (p?=?.008) in the t_1/2, but no significant difference was observed for the AUC_0–24?h and C_max. In subjects with the CYP3A5*3/*3 genotype, the mean t_1/2 was 1.35-fold higher than in CYP3A5*1/*1 group (p?=?.007). And the t_1/2 in CYP3A5*3 carriers also was 1.32-fold higher than in the wild-type group (p?=?.004).

4. CYP3A4*1G and CYP3A5*3 polymorphisms may influence tylerdipine pharmacokinetic in healthy Chinese subjects.

     

No ethnic difference between Caucasian and Japanese hepatic samples in the expression frequency of CYP3A5 and CYP3A7 proteins

Ethnic differences in the pharmacokinetics of nifedipine, a substrate of CYP3A, and in CYP3A7 expression have been reported. The aim of the present study was to measure the protein levels of CYP3A4, CYP3A5, and CYP3A7 and nifedipine oxidation activity in hepatic microsomes from 15 Caucasian and 15 Japanese patients for comparison between the two ethnic groups. Nifedipine oxidation activity and CYP3A4 protein level were well correlated. No significant difference between Caucasian and Japanese microsomal samples was found in nifedipine oxidation activity or in the CYP3A4 protein level. CYP3A5 was detected in 6 of 15 Caucasian samples and in 5 of 15 Japanese samples, but no ethnic difference was found in either the frequency of expression or its protein level. CYP3A7 was found in 10 of 15 Caucasian samples and in 14 of 15 Japanese samples. Although the estimated CYP3A7 protein level was higher in the Japanese than in the Caucasian samples, its protein level was much lower than that of CYP3A4. These results imply that the contribution of CYP3A5 or CYP3A7 to the purported Caucasian-Japanese ethnic difference in the overall CYP3A activity seems to be small.     

Genetic signature consistent with selection against the CYP3A4*1B allele in non-African populations

Cytochrome P450 3A enzymes (CYP3A) play a major role in the metabolism of steroid hormones, drugs and other chemicals, including many carcinogens. The individually variable CYP3A expression, which remains mostly unexplained, has been suggested to affect clinical phenotypes. We investigated the CYP3A locus in five ethnic groups. The degree of linkage disequilibrium (LD) differed among ethnic groups, but the most common alleles of the conserved LD regions were remarkably similar. Non-African haplotypes are few; for example, only four haplotypes account for 80% of common European Caucasian alleles. Large LD blocks of high frequencies were suggestive of selection. Accordingly, European Caucasian and Asian cohorts each contained a block of single nucleotide polymorphism (SNPs) with very high P excess values. The overlap between these blocks in these two groups contained only two of the investigated 26 SNPs and one of them was the CYP3A4*1B allele. The region centromeric of CYP3A4*1B exhibited high haplotype homozygosity in European Caucasians as opposed to African-Americans. CYP3A4*1B showed a moderate effect on CYP3A4 mRNA and protein expression, as well as on CYP3A activity assessed as Vmax of testosterone 6beta-hydroxylation in a liver bank. Our data are consistent with a functional relevance of CYP3A4*1B and with selection against this allele in non-African populations. The elimination of CYP3A4*1B involved different parts of the CYP3A locus in European Caucasians and Asians. Because CYP3A4 is involved in the vitamin D metabolism, rickets may have been the underlying selecting factor.     

Influence of ABCB1, CYP3A4*18B and CYP3A5*3 polymorphisms on cyclosporine A pharmacokinetics in bone marrow transplant recipients

The aim of this study was to retrospectively evaluate the effect of polymorphisms in the CYP3A4, CYP3A5 and ABCB1 genes on the dose-adjusted concentration and dose requirement of cyclosporine A (CsA) in Chinese recipients during the early period after bone marrow or hematopoietic stem cell transplantation. Ninety-one bone marrow transplant recipients were genotyped by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay or by direct sequencing for the C1236T, G2677T/A and C3435T polymorphisms in CYP3A4*18B, CYP3A5*3, and ABCB1, respectively. The concentration at zero before administration (C₀) and concentration at 2 h after administration (C₂) of whole blood CsA were measured by fluorescence polarization immunoassay. Dose-adjusted C₀ and C₂ were determined and compared among groups with different genotypes. Compared with CYP3A5*3/*3 individuals, CYP3A5*1/*1 subjects have a significantly lower dose-adjusted C₀ and C₂ at days 1–10 and a higher dose requirement for CsA at days 16–30 (p < 0.05). In addition, homozygotes for the ABCB1 3435T mutant have a significantly higher dose-adjusted C₀ and C₂ and a lower dose requirement compared with wildtype (p < 0.05). Similar results were also derived for carriers of the T-G-C haplotype in CYP3A5 producers compared with non-carriers (p < 0.05 and p < 0.01, respectively). In summary, the ABCB1 3435T SNP, T-G-C haplotype in CYP3A5 producers, and CYP3A5*3 SNP are all associated with differences in CsA pharmacokinetics and dose requirements during the first month after bone marrow or hematopoietic stem cell transplantation. Genetic testing can therefore help to determine initial dosage and individualize immunosuppressive therapy.     

Effects of CYP2C9*1/*3 genotype on the pharmacokinetics of flurbiprofen in Korean subjects

Archives of Pharmacal Research - The aim of this study was to investigate the impact of CYP2C9*1/*3 genotype on the pharmacokinetics of flurbiprofen and its metabolite. The CYP2C9 genotypes were...     

A urine metabolic ratio of dextromethorphan and 3-methoxymorphinan as a probe for CYP3A activity and prediction of cyclosporine clearance in healthy volunteers.

Dextromethorphan (DM) is metabolized in the body to dextrophan (DT) and 3-methoxymorphinan (3-MM) by cytochrome P450 (CYP) 2D6 and 3A4, respectively, and cyclosporine (CsA) is a known substrate of CYP3A4. We attempted to determine if the urine metabolic ratio of DM:3-MM at various time intervals during 24 hours is predictive of CsA clearance in 11 healthy volunteers. Each subject took DM 30 mg orally, and serial urine samples were collected at 0-4, 4, and 4-24, and 0-24 hours. Subjects then were randomly assigned to receive either oral microemulsion CsA 5 mg/kg or intravenous CsA 1.5 mg/kg in a crossover fashion in a two-sequence pharmacokinetic study with a wash-out period of at least 7 days. A total of 17 blood samples were collected from each subject in the CsA pharmacokinetic study over 24 hours. Urinary DM, DT, and 3-MM were quantified by high-performance liquid chromatography (HPLC) with a fluorescence detector, and blood CsA concentrations were analyzed by HPLC with ultraviolet detection. All subjects were extensive metabolizers of CYP2D6 as determined by metabolic ratios of DM:DT (mean+/-SD 0.0255+/-0.048). There was no correlation between CYP2D6 and CYP3A4 (p=0.38). The metabolic ratios of DM:3-MM in any urine samples during the 24-hour collection period did not predict CsA pharmacokinetics, although the 0-24 hour sample had an unexpected positive correlation with CsA clearance (r2 = 0.38, p<0.0001). The correlation was similar for metabolic ratios of DM:3-MM with intravenous CsA clearance (r2 = 0.5, p<0.0001). Metabolic ratios of DM:3-MM based on 24-hour cumulative urine collection did not appear to have clinical utility in predicting CYP3A activity measured by CsA clearance.     

Association of MDR1, CYP3A4*18B, and CYP3A5*3 polymorphisms with cyclosporine pharmacokinetics in Chinese renal transplant recipients

Objective  The objective of this study was to retrospectively evaluate the effects of MDR1, CYP3A4*18B, and CYP3A5*3 genetic polymorphisms on cyclosporine A (CsA) pharmacokinetics in Chinese renal transplant patients during the first month after transplantation. Methods  A total of 103 renal transplant recipients receiving CsA were genotyped for MDR1 (C1236T, G2677T/A, and C3435T), CYP3A4*18B, and CYP3A5*3. The predose and 2-h postdose concentrations of CsA (C₀ and C₂, respectively) were determined by fluorescence polarization immunoassay, and their relationships with corresponding genotypes and haplotypes were investigated. Results  Patients with a CYP3A4*1/*1 genotype were found to have a higher dose-adjusted concentration compared with those with CYP3A4*18B/*18B, as follows: for C₂, 19.3% (P = 0.008) during days 8-15, 35.2% (P = 0.008) during days 16–30, and for C₀, 39.7% (P = 0.012) during days 16–30. The dose-adjusted C₀ was higher in patients with MDR1 1236CC compared with those with 1236TT in the first month postoperation. The dose-adjusted C₀ in patients with the CYP3A5*3/*3 genotype was 25.5% and 30.7% higher than those with the wild-type genotype during days 8–15 (P = 0.011) and days 16–30 (P = 0.015), respectively. Haplotype analysis revealed that the dose-adjusted C₀ was higher in the first month following surgery in carriers of haplotype MDR1 CAC than in noncarriers. Polymorphisms of MDR1 and CYP3A5*3 did not affect dose-adjusted C_2. Conclusion  The data suggests that the CYP3A4*18B genotype affects CsA pharmacokinetics during the first month following surgery in Chinese renal transplant recipients. Patients with CYP3A4*18B alleles may require higher doses of CsA to reach the target levels. Large prospective studies may be needed to further explore the impact of MDR1 and CYP3A5*3 polymorphisms on CsA pharmacokinetics in renal transplant recipients.     

CYP3A5~*3和CYP3A4~*18B基因多态性对肾移植患者环孢素药代动力学的影响

目的回顾性研究肾脏移植后1mon,CYP3A5*3和CYP3A4*18B基因多态性对CsA药代动力学参数的影响。方法采用PCR-RFLP方法分析了63名肾脏移植患者CYP3A5*3和CYP3A4*18B基因型;荧光偏正免疫法用于检测肾移植患者静脉全血中的CsA浓度。结果在63名肾移植患者中,CYP3A5*3和CYP3A4*18B突变等位基因发生频率分别为0.770(95CI:0.767～0.773),0.235(95CI:0.235～0.241),而且这些等位基因表现出完全连锁不平衡。在移植术后1mon内,携带CYP3A4*1/*1野生型纯合子患者的C0以及剂量校正谷血浓度(C0/D)均明显高于携带CYP3A4*1/*18B杂合子或CYP3A4*18B/*18B突变型纯合子患者(P<0.05,Mann-WhitneyUtest);CYP3A5*1/*1基因型组的给药剂量明显高于CYP3A5*1/*3或CYP3A5*3/*3基因型组(P=0.004<0.01,Kruakal-Wallistest);CYP34*18B和CYP3A5*3联合考虑,对于CYP3A5表达组,同样发现C0、C0/D在CYP3A4*1/*1组C0以及C0/D均明显高于CYP3A4*1/*18B或CYP3A4*18B/*18B组(P<0.05,Mann-WhitneyUtest);而其他药动学参数在CYP3A5*3及CYP3A4*18B各组间相比差异则没有统计学意义。结论CYP3A5*3和(或)CYP3A4*18B基因多态性对肾移植后1monCsA药代动力学有一定影响,移植前CYP3A5*3基因型的分析仍需进一步研究。