Intestinal CYP3A4 is not involved in the enantioselective disposition of lansoprazole

The contribution of (S)-lansoprazole to CYP3A4-catalysed sulfoxidation is greater than that of (R)-lansoprazole. The aim was to investigate the effect of grapefruit juice on the enantioselective disposition of lansoprazole among three CYP2C19 genotype groups. Eighteen healthy subjects, consisting of six each of homozygous extensive metabolizers (homEMs), heterozygous extensive metabolizers (hetEMs) and poor metabolizers (PMs), ingested a single oral dose of 60?mg racemic lansoprazole after taking either 200?ml grapefruit juice or water. There was no effect of grapefruit juice on the mean maximum plasma concentrations (C_max) or the elimination half-life for each lansoprazole enantiomer in all three CYP2C19 genotype groups. Similarly, the pharmacokinetic parameters of lansoprazole sulfone remained unaltered by grapefruit juice in all three groups. The CYP3A4-mediated first-pass sulfoxidation of (R)- and (S)-lansoprazole were not influenced by grapefruit juice. In addition, stereoselectivity of the intestinal CYP3A4-catalysed sulfoxidation of (R)- and (S)-lansoprazole was not observed.     

Intestinal CYP3A4 is not involved in the enantioselective disposition of lansoprazole

The contribution of (S)-lansoprazole to CYP3A4-catalysed sulfoxidation is greater than that of (R)-lansoprazole. The aim was to investigate the effect of grapefruit juice on the enantioselective disposition of lansoprazole among three CYP2C19 genotype groups. Eighteen healthy subjects, consisting of six each of homozygous extensive metabolizers (homEMs), heterozygous extensive metabolizers (hetEMs) and poor metabolizers (PMs), ingested a single oral dose of 60 mg racemic lansoprazole after taking either 200 ml grapefruit juice or water. There was no effect of grapefruit juice on the mean maximum plasma concentrations (C(max)) or the elimination half-life for each lansoprazole enantiomer in all three CYP2C19 genotype groups. Similarly, the pharmacokinetic parameters of lansoprazole sulfone remained unaltered by grapefruit juice in all three groups. The CYP3A4-mediated first-pass sulfoxidation of (R)- and (S)-lansoprazole were not influenced by grapefruit juice. In addition, stereoselectivity of the intestinal CYP3A4-catalysed sulfoxidation of (R)- and (S)-lansoprazole was not observed.     

CYP3A4,CYP3A5和MDR1基因多态性对环孢素处置的影响

环孢素是一个广泛用于器官移植患者的免疫抑制剂,具有治疗指数窄,不同个体间药代动力学差异较大的特点。它主要通过肝脏和小肠的CYP3A4和CYP3A5代谢;同时它又是药物转运体的底物。不同个体间药物代谢酶和转运体活性的差异可能是造成不同器官移植患者环孢素药代动力学差异的主要原因。而遗传因素即编码药物代谢酶和转运体基因序列的差异可能是其产生活性差异的分子机制。因此,从编码药物代谢酶和转运体的基因入手,可能会为器官移植患者提供最优的治疗方案。     

Intestinal cell-specific vitamin D receptor (VDR)-mediated transcriptional regulation of CYP3A4 gene

CYP3A4 is the most important drug-metabolizing enzyme that is involved in biotransformation of more than 50% of drugs. Pregnane X receptor (PXR) dominantly controls CYP3A4 inducibility in the liver, whereas vitamin D receptor (VDR) transactivates CYP3A4 in the intestine by secondary bile acids. Four major functional PXR-binding response elements of CYP3A4 have been discovered and their cooperation was found to be crucial for maximal up-regulation of the gene in hepatocytes. VDR and PXR recognize similar response element motifs and share DR3(XREM) and proximal ER6 (prER6) response elements of the CYP3A4 gene.In this work, we tested whether the recently discovered PXR response elements DR4(eNR3A4) in the XREM module and the distal ER6 element in the CLEM4 module (CLEM4-ER6) bind VDR/RXRα heterodimer, whether the elements are involved in the intestinal transactivation, and whether their cooperation with other elements is essential for maximal intestinal expression of CYP3A4.Employing a series of gene reporter plasmids with various combinations of response element mutations transiently transfected into four intestinal cell lines, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP), we found that the CLEM4-ER6 motif interacts with VDR/RXRα heterodimer and partially cooperates with DR3(XREM) and prER6 in both basal and VDR-mediated inducible CYP3A4 regulation in intestinal cells. In contrast, eNR3A4 is involved only in the basal transactivation in intestinal cells and in the PXR-mediated rifampicin-induced transactivation of CYP3A4 in LS174T intestinal cells.We thus describe a specific ligand-induced VDR-mediated transactivation of the CYP3A4 gene in intestinal cells that differs from PXR-mediated CYP3A4 regulation in hepatocytes.     

Pharmacokinetics of midazolam in CYP3A4- and CYP3A5-genotyped subjects

Objective We investigated whether differences in pharmacokinetics of midazolam, a CYP3A probe, could be demonstrated between subjects with different CYP3A4 and CYP3A5 genotypes.Methods Plasma concentrations of midazolam, and of total (conjugated + unconjugated) 1OH-midazolam, and 4OH-midazolam were measured after the oral administration of 7.5 mg or of 75 µg of midazolam in 21 healthy subjects.Results CYP3A5*7, CYP3A4*1E, CYP3A4*2, CYP3A4*4, CYP3A4*5, CYP3A4*6, CYP3A4*8, CYP3A4*11, CYP3A4*12, CYP3A4*13, CYP3A4*17 and CYP3A4*18 alleles were not identified in the 21 subjects. CYP3A5*3, CYP3A5*6, CYP3A4*1B and CYP3A4*1F alleles were identified in 20, 1, 4 and 2 subjects, respectively. No statistically significant differences were observed for the AUC_inf values between the different genotypes after the 75-µg or the 7.5-mg dose.Conclusion Presently, CYP3A4 and CYP3A5 genotyping methods do not sufficiently reflect the inter-individual variability of CYP3A activity.     

CYP2A6 genetic variation and dexmedetomidine disposition

Purpose

There is a large interindividual variability in dexmedetomidine dose requirements for sedation of patients in intensive care units (ICU). Cytochrome P450 2A6 (CYP2A6) mediates an important route of dexmedetomidine metabolism, and genetic variation in CYP2A6 affects the clearance of other substrate drugs. We examined whether CYP2A6 genotypes affect dexmedetomidine disposition.

Methods

In 43 critically ill ICU patients receiving dexmedetomidine infusions adjusted to achieve the desired level of sedation, we determined a median of five plasma dexmedetomidine concentrations each. Forty subjects were genotyped for five common CYP2A6 alleles and grouped into normal (n?=?33), intermediate (n?=?5), and slow metabolizers (n?=?2).

Results

Using a Bayesian hierarchical nonlinear mixture model, estimated dexmedetomidine clearance was 49.1?L/h (posterior mean; 95% credible interval 41.4–57.6?L/h). There were no significant differences in dexmedetomidine clearance among normal, intermediate, and slow CYP2A6 metabolizer groups.

Conclusion

Genetic variation in CYP2A6 does not appear to be an important determinant of dexmedetomidine clearance in ICU patients.     

Co-regulation of CYP3A4 and CYP3A5 and contribution to hepatic and intestinal midazolam metabolism

We recently demonstrated that a variant allele of CYP3A5 (CYP3A5*3) confers low CYP3A5 expression as a result of improper mRNA splicing. In this study, we further evaluated the regulation of CYP3A5 in liver and jejunal mucosa from white donors. For all tissues, high levels of CYP3A5 protein were strongly concordant with the presence of a wild-type allele of the CYP3A5 gene (CYP3A5*1). CYP3A5 represented greater than 50% of total CYP3A content in nearly all of the livers and jejuna that carried the CYP3A5*1 wild-type allele. Overall, CYP3A5 protein content accounted for 31% of the variability in hepatic midazolam hydroxylation activity. Improperly spliced mRNA (SV1-CYP3A5) was found only in tissues containing a CYP3A5*3 allele. Properly spliced CYP3A5 mRNA (wt-CYP3A5) was detected in all tissues, but the median wt-CYP3A5 mRNA was 4-fold higher in CYP3A5*1/*3 livers compared with CYP3A5*3/*3 livers. Differences in wt-CYP3A5 and CYP3A4 mRNA content explained 53 and 51% of the interliver variability in CYP3A5 and CYP3A4 content, respectively. Hepatic CYP3A4 and CYP3A5 contents were not correlated when all livers were compared. However, for CYP3A5*1/*3 livers, levels of the two proteins were strongly correlated (r = 0.93) as were wt-CYP3A5 and CYP3A4 mRNA (r = 0.76). These findings suggest that CYP3A4 and CYP3A5 genes share a common regulatory pathway for constitutive expression, possibly involving conserved elements in the 5'-flanking region.     

Intestinal first-pass metabolism of CYP3A4 substrates

Cytochrome P450 3A4 (CYP3A4) is present not only in the liver but also in the small intestine, where it functions as a barrier against xenobiotics. Some CYP3A4 substrates exhibit low bioavailability due to intestinal first pass metabolism. The AUCs of such CYP3A4 substrates are remarkably changed by the inhibition, induction, and saturation of CYP3A4 and so prediction of intestinal first-pass metabolism is important. In this article, factors affecting intestinal first-pass metabolism of drugs are reviewed, focusing on the intestinal metabolism by CYP3A. The methods to predict intestinal first-pass metabolism are also reviewed.     

CYP3A4和P糖蛋白与药物的肠道处置

肠CYP3A4介导的生物转化和P糖蛋白介导的药物主动泵出肠细胞是决定口服药物生物利用度的主要因素。有证据显示CYP3A4和P糖蛋白在小肠不是共同调节的,但两者在药物肠道处置中的协同作用已得到体外试验和动物体内试验的证实。进一步了解两者的相互作用有助于改善CYP3A4/P糖蛋白底物的生物利用度。     

Relative roles of CYP2C19 and CYP3A4/5 in midazolam 1'-hydroxylation

1. During the characterization of recombinant CYP2C19, it was observed that this enzyme metabolized midazolam, which is generally regarded as CYP3A4/5 substrate, and we therefore decided to pursue this observation further. 2. CYP2C19 showed a Michaelis-Menten pattern for midazolam 1'-hydroxylation and was inhibited by (+)-N-3-benzylnirvanol and S-mephenytoin, which are a standard potent inhibitor and a substrate of CYP2C19, respectively. 3. The inhibitory potency by CYP3A4/5 inhibitor on the midazolam 1'-hydroxylation in human liver microsomes (HLM) was correlated with the CYP3A4/5 specific catalytic activity, but such correlation was not observed in CYP2C19 enzyme. The in vitro intrinsic clearance value for midazolam 1'-hydroxylation was not changed by the addition of (+)-N-3-benzylnirvanol in four individual HLM preparations. 4. These results indicated that although CYP2C19 is capable of catalyzing midazolam 1'-hydroxylation, CYP3A4/5 play a more important role.     

CYP3A4 gene polymorphisms influence testosterone 6beta-hydroxylation

Three non-synonymous single nucleotide polymorphisms (SNPs) in the CYP3A4 gene were found in 34 cell lines derived from Japanese individuals. These three SNPs (T185S, L293P, and T363M)(1) have been previously reported, but little is known about the effect that these polymorphisms, especially T185S, have on catalytic activity. We measured testosterone hydroxylation in wild-type CYP3A4 and these three variants using a mammalian expression system. Testosterone 6beta-, 2beta-, and 15beta-hydroxylations by the variant CYP3A4 forms T363M (<40%) and T185S (<60%) were reduced as compared with the wild-type in transient expression assays. L293P was similar to the wild-type in testosterone 6beta- and 2beta-hydroxylase activities. Western blot analysis confirmed lower amounts of CYP3A4 protein in the T363M and T185S variants than in the wild-type. Interestingly, Northern blot analysis showed no significant difference among mRNA levels between the wild-type and variants. These results suggest that the T363M and T185S substitutions in CYP3A4 affect either protein expression or stability. These established cell lines provided useful CYP3A4 SNP information in the Japanese.     

Simultaneous assessment of CYP3A4 and CYP1A2 activity in vivo with alprazolam and caffeine

Alprazolam (ALP) and caffeine (CAF) were suggested as probe drugs for the activities of CYP3A4 and CYP1A2, respectively. We investigated the disposition of oral ALP (1 mg) and CAF (100 mg) in 17 normal volunteers to establish and validate a procedure for the simultaneous assessment of CYP3A4 and CYP1A2 enzyme activity. Nine received ALP alone, ALP and CAF and CAF alone in an open three-way crossover study to test for pharmacokinetic interaction. Four received ALP after a 2-day pretreatment with ketoconazole, an inhibitor of CYP3A4, and four normal volunteers received ALP after 4 days of rifampin, an inducer of CYP3A4. AUC values of ALP and CAF administered alone were not different from AUC values when both drugs were administered combined, indicating that there is no metabolic interaction. The ratio formed of paraxanthine and CAF correlated significantly with systemic CAF clearance at 3, 4, 6, 8, 10 and 24 h. There was a strong correlation between AUC values of ALP and CAF and the plasma concentration obtained 6, 8, 10, or 24 h after ingestion of the drug. Ketoconazole and rifampin pretreatment significantly changed AUC values of ALP (mean AUC values in microg/l h: ALP = 242.2, ALP + ketoconazole = 426.2, ALP + rifampin = 28.4, ANOVA F = 17.7, P < 0.001). We conclude that ALP and CAF can be administered simultaneously for the assessment of CYP activity. Plasma concentrations 6, 8, 10, and 24 h after drug ingestion reflect AUC of ALP and CAF and therefore in-vivo CYP3A4 and CYP1A2 activity, respectively.     

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.     

Effect of mibefradil on CYP3A4 in vivo

Mibefradil, a calcium channel blocker, was removed from the market because of adverse drug interactions with coadministered CYP3A4 substrates. This study examined the effect of mibefradil on the activity of hepatic and intestinal CYP3A4 in vivo, employing the erythromycin breath test (EBT) and oral midazolam pharmacokinetics. This was a two-period, single-blind, placebo-controlled crossover study in which 8 male volunteers were randomized to the order of receiving placebo and a single 100-mg oral dose of mibefradil. Oral midazolam was coadministered with intravenous [14C N-methyl] erythromycin 1 hour after mibefradil/placebo administration. The EBT was performed 20 minutes following erythromycin administration. Blood and urine were collected during the 36 hours following probe drug administration for analysis of midazolam pharmacokinetics. Coadministration of mibefradil increased the Cmax of midazolam 3-fold, the AUC 8- to 9-fold, and the t1/2 4-fold. Mibefradil coadministration decreased the amount of exhaled 14CO2 in 6 of 8 subjects, with a mean decrease of 25%. It was concluded that a single oral dose of mibefradil significantly inhibits CYP3A4 in intestine and liver. These data support that adverse drug interactions involving mibefradil reflect inhibition of CYP3A4 in intestine and liver. Also, they suggest that the EBT, while a valid probe of in vivo hepatic CYP3A4 activity, is a single time point measurement and may be less sensitive than oral midazolam pharmacokinetics in detecting CYP3A4 inhibition.     

Functionally defective or altered CYP3A4 and CYP3A5 single nucleotide polymorphisms and their detection with genotyping tests

Among the four cytochrome P450 (CYP)3A genes, CYP3A4 and CYP3A5 are the most abundantly expressed in the human liver. Eighty single nucleotide polymorphisms (SNPs) of CYP3A4/5 have been reported to the Human P450 Allele Nomenclature Committee. CYP3A4 alleles with minimal function compared with wild type include the CYP3A4*6 and CYP3A4*17. Alleles with moderately decreased or altered activity include: CYP3A4*2, *8, *11, *12, *13, *16, and *18. CYP3A5 alleles with minimal function include the splice variants CYP3A5*3, *5, *6 and CYP3A5* 10, as well as the null allele CYP3A5*7. Alleles with moderately decreased catalytic activity include CYP3A5*8 and CYP3A5*9. This report reviews the current progress in the functional characterization of CYP3A4 and CYP3A5 SNPs and provides genotyping tests for possible defective variants. A combination of genotyping tests for defective CYP3A4/CYP3A5 haplotypes will be necessary to understand the variations in the metabolism and clinical toxicity of a wide variety of clinical drugs, since these two CYP proteins have overlapping substrate specificities.     

In vivo inhibition of CYP3A-mediated midazolam metabolism by anchusan in rats

Cytochrome P450 (CYP)-mediated drug interactions caused by Kampo medicine have not been investigated sufficiently. The current study was conducted to reveal the effect of anchusan, a commonly used Kampo formula for gastrointestinal disease, on CYP3A-mediated drug metabolism in rats. The pharmacokinetics of midazolam (MDZ) was investigated after the single or one-week administration of anchusan (500 mg/kg) to evaluate its inhibitory and inducible effect on CYP3A, respectively. MDZ was administrated 16 h after the last anchsan treatment in the multiple dose study, while their intervals were 2 or 16 h in the single dose study. Unexpectedly, the multiple-pretreatment of anchusan increased the AUC of MDZ by 2.4-fold rather than decreasing it, and the CYP3A contents and activities were unchanged in hepatic and intestinal microsomes of these rats. In contrast, no significant inhibitory effects on MDZ metabolism were observed by the single anchusan pretreatment. In vitro study showed that the preincubation of anchusan and some of its component extracts with rat liver microsomes reduced CYP3A activity in a time- and NADPH-dependent manner. These results suggested that anchusan increased the serum MDZ concentration in rats, at least in part, by the time-dependent inhibition of CYP3A.     

Flavonoids diosmetin and luteolin inhibit midazolam metabolism by human liver microsomes and recombinant CYP 3A4 and CYP3A5 enzymes

We evaluated the effects of increasing concentrations of the flavonoids salvigenin, diosmetin and luteolin on the in vitro metabolism of midazolam (MDZ), a probe substrate for cytochrome P450 (CYP) 3A enzymes, which is converted into 1'-hydroxy-midazolam (1'-OH-MDZ) and 4-hydroxy-midazolam (4-OH-MDZ) by human liver microsomes. Salvigenin had only a modest effect on MDZ metabolism, whereas diosmetin and luteolin inhibited in a concentration-dependent manner the formation of both 1'-OH-MDZ and 4-OH-MDZ, with apparent K(i) values in the 30-50mumol range. Both diosmetin and luteolin decreased 1'-OH-MDZ formation by human recombinant CYP3A4, but not CYP3A5, whereas they decreased 4-OH-MDZ formation by both recombinant enzymes. To assess whether any relationship exists between the physico-chemical characteristics of flavones and their effects on MDZ metabolism, we tested the effects of three other flavones (flavone, tangeretin, chrysin) on MDZ metabolism by human liver microsomes. Whereas flavones possessing more than two hydroxyl groups (luteolin, diosmetin) inhibited MDZ biotransformation, flavones lacking hydroxyl groups in their A and B rings (flavone, tangeretin) stimulated MDZ metabolism. We also found close relationships between the maximum stimulatory or inhibitory effects of flavones on 1'-OH-MDZ and 4-OH-MDZ formation rates and their log of octanol/water partition coefficients (logP) or their total number of hydroxyl groups. The results of the study may be of clinical relevance since they suggest that luteolin and diosmetin may cause pharmacokinetic interactions with co-administered drugs metabolized via CYP3A.     

Bufalin inhibits CYP3A4 activity in vitro and in vivo

Aim： To investigate the inhibitory interactions of bufalin and CYP3A4. Methods： Recombinant human CYP3A4 was incubated with bufalin in vitro. Bufalin was administered ig and iv to Wistar rats to further estimate its impact on CYP3A4, and midazolam was given to index the activity of CYP3A4. Results： The IC50 of bufalin was 14.52 μmol/L. Bufalin affected CYP3A4 activity with increases in AUC0-t and t1/2, and decreases in CL and the formation of 1-hydroxy-midazolam after ig or iv administration of midazolam （P〈0.05）. An increase in Cmax after ig bufalin administration （P〈0.05） was observed. Conclusion： Bufalin showed a modest but significant inhibition of CYP3A4 both in vitro and in vivo. The likelihood of an interaction between bufalin and the CYP3A4-metabolized drugs in human might not be negated.