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Kenneth E Thummel

Publications and source records attributed to Kenneth E Thummel.

At least 19 recordsLinked to original sources
PubMed2006

The dietary isothiocyanate sulforaphane is an antagonist of the human steroid and xenobiotic nuclear receptor.

Sulforaphane (SFN) is a biologically active phytochemical found abundantly in broccoli. SFN has been promoted as a putative chemopreventive agent to reduce cancer, and most studies have associated its anti-cancer effects with the induction of phase II xenobiotic metabolism enzymes via activation of the Keap1/Nrf2 antioxidant response pathway. Interestingly, SFN can significantly down-regulate cytochrome P450 3A4 (CYP3A4) expression in human primary hepatocytes. CYP3A4 is responsible for the hepatic and intestinal metabolism of numerous protoxicants, pharmaceutical compounds, and endogenous sterols. Among the most important mediators of CYP3A4 expression is the nuclear hormone receptor, steroid and xenobiotic receptor (SXR; also called "hPXR"). SXR functions as a xenobiotic sensor to coordinately regulate xenobiotic metabolism via transcriptional regulation of xenobiotic-detoxifying enzymes and transporters. Here, we report that SFN is a specific antagonist of human SXR and that it inhibits SXR-mediated induction of drug clearance. SFN can bind directly to SXR, inhibit SXR coactivator recruitment, and efficiently repress SXR activities. Furthermore, SFN inhibited SXR-mediated CYP3A4 expression and CYP3A4-catalyzed midazolam clearance in human primary hepatocytes. Thus, SFN is the first identified naturally occurring antagonist for SXR (hPXR). Because induction of CYP3A4 can result in adverse drug responses (e.g., lack of efficacy), which are a major public health problem, this discovery could lead to the development of important new therapeutic and dietary approaches to reduce the frequency of undesirable inducer-drug interactions.

Administration, Oral↗
PubMed2006

Selective induction of intestinal CYP3A23 by 1alpha,25-dihydroxyvitamin D3 in rats.

Enhancement of CYP3A transcription in both the small intestine and liver of the mouse by activation of a VDR signaling pathway was shown recently by Makishima et al. (Science, 2002). However, in humans and rats, hepatic VDR content is much lower than that found in small intestine, suggesting the possibility of tissue-selective responses to 1,25(OH)(2)D(3). The purpose of this study was to determine the effect of 1,25(OH)(2)D(3) on intestinal and hepatic CYP3A expression in the rat. We found that an acute intraperitoneal treatment (every 48 h) in adult male rats with 1,25(OH)(2)D(3) induced CYP3A transcription selectively in small intestine, but not in liver. At a dose of 100 ng, there was a 6.6-fold increase in intestinal CYP3A23 mRNA after the third treatment (p < 0.05). There were concordant effects of 1,25(OH)(2)D(3) treatment on intestinal CYP3A23 protein levels; 2.2-fold (p < 0.05), 3.5-fold (p < 0.05) and 4.8-fold (p < 0.01) increase following 1-3 doses of 100 ng 1,25(OH)(2)D(3), respectively. In contrast, there was no significant change of CYP3A23 protein content in liver at the 1,25(OH)(2)D(3) doses tested. In support of these findings, there was a 366-fold and 77-fold higher level of VDR mRNA expression in the respective rat and human jejunal mucosa, compared to the liver. These data suggest that the human liver will be less sensitive than the intestine to the transcriptional effects of 1,25(OH)(2)D(3) and that this regulatory pathway may contribute to inter-individual variability in constitutive intestinal CYP3A4 expression.

Animals↗
PubMed2006

Steroid and xenobiotic receptor and vitamin D receptor crosstalk mediates CYP24 expression and drug-induced osteomalacia.

The balance between bioactivation and degradation of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] is critical for ensuring appropriate biological effects of vitamin D. Cytochrome P450, family 24-mediated (CYP24-mediated) 24-hydroxylation of 1,25(OH)2D3 is an important step in the catabolism of 1,25(OH)2D3. The enzyme is directly regulated by vitamin D receptor (VDR), and it is expressed mainly in the kidney, where VDR is also abundant. A recent report suggests that activation of steroid and xenobiotic receptor (SXR) also enhances the expression of CYP24, providing a new molecular mechanism of drug-induced osteomalacia. However, here we showed that activation of SXR did not induce CYP24 expression in vitro and in vivo, nor did it transactivate the CYP24 promoter. Instead, SXR inhibited VDR-mediated CYP24 promoter activity, and CYP24 expression was very low in tissues containing high levels of SXR, including the small intestine. Moreover, 1,25(OH)2D3-induced CYP24 expression was enhanced in mice lacking the SXR ortholog pregnane X receptor, and treatment of humans with the SXR agonist rifampicin had no effect on intestinal CYP24 expression, despite demonstration of marked CYP3A4 induction. Combined with our previous findings that CYP3A4, not CYP24, plays the dominant role in hydroxylation of 1,25(OH)2D3 in human liver and intestine, our results indicate that SXR has a dual role in mediating vitamin D catabolism and drug-induced osteomalacia.

Animals↗
PubMed2006

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.

Algorithms↗
PubMed2006

MDR1 genotype is associated with hepatic cytochrome P450 3A4 basal and induction phenotype.

OBJECTIVES: Variant cytochrome P450 (CYP) 3A4 alleles cannot explain human variation in CYP3A4 expression. This study investigated whether common single-nucleotide polymorphisms (SNPs) in multidrug resistance 1 (MDR1), encoding P-glycoprotein, or the pregnane X receptor (PXR) were associated with basal or inducible CYP3A4 expression. METHODS: MDR1 G2677T and C3435T SNPs and a PXR 6-base pair (bp) deletion were genotyped in the deoxyribonucleic acid from 144 human livers in 3 cohorts each phenotyped for basal or rifampin (INN, rifampicin)-inducible hepatic CYP3A4 expression (or both) and in 57 human small bowel biopsy specimens from 3 cohorts each phenotyped for either basal or rifampin-induced CYP3A4 expression (or both). RESULTS: Hepatic CYP3A4 expression/function was significantly higher in persons homozygous for the MDR1 2677T (Ser893) allele compared with persons homozygous for 2677G (Ala893) in all 3 hepatic cohorts. For example, homozygous MDR1 2677 TT livers had higher midazolam hydroxylase activity than homozygous 2677 GG livers (1831 +/- 1336 pmol x min(-1) x mg protein(-1) versus 1060 +/- 552 pmol x min(-1) x mg protein(-1), P = .03). In 2 of the 3 groups the association was observed in men but not in women. For example, homozygous MDR1 2677 TT male hepatocytes had significantly higher testosterone 6beta-hydroxylase activity compared with homozygous 2677 GG livers (0.120 +/- 0.06 pmol x min(-1) x mg protein(-1) versus 0.069 +/- 0.04 pmol x min(-1) x mg protein(-1), P = .0002). Conversely, rifampin induction of testosterone 6beta-hydroxylation in primary human hepatocytes was significantly higher in persons homozygous for 2677G (12.0 +/- 5.7-fold) compared with MDR1 homozygous TT carriers (7.3 +/- 4.6-fold) (P = .01). Suggestive evidence for higher CYP3A4 expression in MDR1 2677T carriers was also observed in human intestines. CYP3A4 expression was also related to a 6-bp deletion in PXR in 2 of the liver cohorts. Two-factor ANOVA analysis revealed a significant interaction between the MDR1 2677 SNP and the PXR 6-bp deletion influencing CYP3A4 expression (P = .007). CONCLUSIONS: Individuals homozygous for the MDR1 2677T allele show enhanced constitutive CYP3A4 expression in the liver and intestine, as compared with those homozygous for the MDR1 2677G allele, particularly in men. Conversely, the magnitude of CYP3A4 induction by rifampin is greater in persons with the MDR1 2677G allele and is inversely related to baseline CYP3A4 expression. MDR1 likely influences basal CYP3A4 expression by limiting the intracellular concentration of an endogenous regulator. MDR1 genotype may be a useful predictor of basal CYP3A4 and the extent of some CYP3A4-mediated drug interactions in humans. Moreover, the influence of MDR1 genotype on CYP3A4 expression adds additional complexity to determining the relative contribution of the MDR1 alleles to the disposition of substrates shared by CYP3A4 and MDR1/P-glycoprotein.

Adolescent↗
PubMed2006

Stereochemical aspects of itraconazole metabolism in vitro and in vivo.

Itraconazole (ITZ) has three chiral centers and is administered clinically as a mixture of four stereoisomers. This study evaluated stereoselectivity in ITZ metabolism. In vitro experiments were carried out using heterologously expressed CYP3A4. Only (2R,4S,2'R)-ITZ and (2R,4S,2'S)-ITZ were metabolized by CYP3A4 to hydroxy-ITZ, keto-ITZ, and N-desalkyl-ITZ. When (2S,4R,2'R)-ITZ or (2S,4R,2'S)-ITZ was incubated with CYP3A4, neither metabolites nor substrate depletion were detected. Despite these differences in metabolism, all four ITZ stereoisomers induced a type II binding spectrum with CYP3A4, characteristic of coordination of the triazole nitrogen to the heme iron (K(s) 2.2-10.6 nM). All four stereoisomers of ITZ inhibited the CYP3A4-catalyzed hydroxylation of midazolam with high affinity (IC(50) 3.7-14.8 nM). Stereochemical aspects of ITZ pharmacokinetics were evaluated in six healthy volunteers after single and multiple oral doses. In vivo, after a single dose, ITZ disposition was stereoselective, with a 3-fold difference in C(max) and a 9-fold difference in C(min) between the (2R,4S)-ITZ and the (2S,4R)-ITZ pairs of diastereomers, with the latter reaching higher concentrations. Secondary and tertiary ITZ metabolites (keto-ITZ and N-desalkyl-ITZ) detected in plasma were of the (2R,4S) stereochemistry. After multiple doses of ITZ, the difference in C(max) and C(min) decreased to 1.5- and 3.8-fold, respectively. The initial difference between the stereoisomeric pairs was most likely due to stereoselective metabolism by CYP3A4, including stereoselective first-pass metabolism as well as stereoselective elimination. However, stereoselective elimination was diminished after multiple dosing, presumably as a result of CYP3A4 autoinhibition. In conclusion, the metabolism of ITZ is highly stereoselective in vitro and in vivo.

Adolescent↗
PubMed2006

Cyclosporin A, tacrolimus and sirolimus are potent inhibitors of the human breast cancer resistance protein (ABCG2) and reverse resistance to mitoxantrone and topotecan.

PURPOSE: Several studies have demonstrated significant interactions between immunosuppressants (e.g., cyclosporin A) and chemotherapeutic drugs that are BCRP substrates (e.g., irinotecan), resulting in increased bioavailability and reduced clearance of these agents. One possible mechanism underlying this observation is that the immunosuppressants modulate the pharmacokinetics of these drugs by inhibiting BCRP. Therefore, the aim of this study was to determine whether the immunosuppressants cyclosporin A, tacrolimus and sirolimus are inhibitors and/or substrates of BCRP. METHODS: First, the effect of the immunosuppressants on BCRP efflux activity in BCRP-expressing HEK cells was measured by flow cytometry. RESULTS: Cyclosporin A, tacrolimus and sirolimus significantly inhibited BCRP-mediated efflux of pheophorbide A, mitoxantrone and BODIPY-prazosin. The EC(50) values of cyclosporin A, tacrolimus and sirolimus for inhibition of BCRP-mediated pheophorbide A efflux were 4.3 +/- 1.9 microM, 3.6 +/- 1.8 microM and 1.9 +/- 0.4 microM, respectively. Cyclosporin A, tacrolimus and sirolimus also effectively reversed resistance of HEK cells to topotecan and mitoxantrone conferred by BCRP. When direct efflux of cyclosporin A, tacrolimus and sirolimus was measured, these compounds were found not to be transported by BCRP. Consistent with this finding, BCRP did not confer resistance to the immunosuppressants in HEK cells. CONCLUSION: These results indicate that cyclosporin A, tacrolimus and sirolimus are effective inhibitors but not substrates of BCRP. These findings could explain the altered pharmacokinetics of BCRP substrate drugs when co-administered with the immunosuppressants and suggest that pharmacokinetic modulation by the immunosuppressants may improve the therapeutic outcome of these drugs.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2006

Adding pharmacogenetics information to drug labels: lessons learned.

The US Food and Drug Administration approved a revised package insert for two cancer drugs to include information about the increased risk of severe adverse events owing to enzyme deficiencies caused by genetic variants. The label revisions stopped short of recommending or requiring pharmacogenetic testing prior to or following an adverse event. Despite (or because of) the lack of specific recommendations, we believe the actions taken by US Food and Drug Administration will have implications for pharmacogenetics research, clinical integration, and other policy considerations. We review the reasons behind the cautious label changes and discuss some of the lessons that can be learned from these experiences.

Antineoplastic Agents↗
PubMed2006

Mutual repression between steroid and xenobiotic receptor and NF-kappaB signaling pathways links xenobiotic metabolism and inflammation.

While it has long been known that inflammation and infection reduce expression of hepatic cytochrome P450 (CYP) genes involved in xenobiotic metabolism and that exposure to xenobiotic chemicals can impair immune function, the molecular mechanisms underlying both of these phenomena have remained largely unknown. Here we show that activation of the nuclear steroid and xenobiotic receptor (SXR) by commonly used drugs in humans inhibits the activity of NF-kappaB, a key regulator of inflammation and the immune response. NF-kappaB target genes are upregulated and small bowel inflammation is significantly increased in mice lacking the SXR ortholog pregnane X receptor (PXR), thereby demonstrating a direct link between SXR and drug-mediated antagonism of NF-kappaB. Interestingly, NF-kappaB activation reciprocally inhibits SXR and its target genes whereas inhibition of NF-kappaB enhances SXR activity. This SXR/PXR-NF-kappaB axis provides a molecular explanation for the suppression of hepatic CYP mRNAs by inflammatory stimuli as well as the immunosuppressant effects of xenobiotics and SXR-responsive drugs. This mechanistic relationship has clinical consequences for individuals undergoing therapeutic exposure to the wide variety of drugs that are also SXR agonists.

Carcinoma, Hepatocellular↗
PubMed2005

Intestinal and hepatic CYP3A4 catalyze hydroxylation of 1alpha,25-dihydroxyvitamin D(3): implications for drug-induced osteomalacia.

The decline in bone mineral density that occurs after long-term treatment with some antiepileptic drugs is thought to be mediated by increased vitamin D(3) metabolism. In this study, we show that the inducible enzyme CYP3A4 is a major source of oxidative metabolism of 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] in human liver and small intestine and could contribute to this adverse effect. Heterologously-expressed CYP3A4 catalyzed the 23- and 24-hydroxylation of 1,25(OH)(2)D(3). No human microsomal cytochrome P450 enzyme tested, other than CYP3A5, supported these reactions. CYP3A4 exhibited opposite product stereochemical preference compared with that of CYP24A1, a known 1,25(OH)(2)D(3) hydroxylase. The three major metabolites generated by CYP3A4 were 1,23R,25(OH)(3)D(3), 1,24S,25(OH)(3)D(3), and 1,23S,25(OH)(3)D(3). Although the metabolic clearance of CYP3A4 was less than that of CYP24A1, comparison of metabolite profiles and experiments using CYP3A-specific inhibitors indicated that CYP3A4 was the dominant source of 1,25(OH)(2)D(3) 23- and 24-hydroxylase activity in both human small intestine and liver. Consistent with this observation, analysis of mRNA isolated from human intestine and liver (including samples from donors treated with phenytoin) revealed a general absence of CYP24A1 mRNA. In addition, expression of CYP3A4 mRNA in a panel of duodenal samples was significantly correlated with the mRNA level of a known vitamin D receptor gene target, calbindin-D9K. These and other data suggest that induction of CYP3A4-dependent 1,25(OH)(2)D(3) metabolism by antiepileptic drugs and other PXR ligands may diminish intestinal effects of the hormone and contribute to osteomalacia.

Base Sequence↗
PubMed2005

Effect of VKORC1 haplotypes on transcriptional regulation and warfarin dose.

BACKGROUND: The management of warfarin therapy is complicated by a wide variation among patients in drug response. Variants in the gene encoding vitamin K epoxide reductase complex 1 (VKORC1) may affect the response to warfarin. METHODS: We conducted a retrospective study of European-American patients receiving long-term warfarin maintenance therapy. Multiple linear-regression analysis was used to determine the effect of VKORC1 haplotypes on the warfarin dose. We determined VKORC1 haplotype frequencies in African-American, European-American, and Asian-American populations and VKORC1 messenger RNA (mRNA) expression in human liver samples. RESULTS: We identified 10 common noncoding VKORC1 single-nucleotide polymorphisms and inferred five major haplotypes. We identified a low-dose haplotype group (A) and a high-dose haplotype group (B). The mean (+/-SE) maintenance dose of warfarin differed significantly among the three haplotype group combinations, at 2.7+/-0.2 mg per day for A/A, 4.9+/-0.2 mg per day for A/B, and 6.2+/-0.3 mg per day for B/B (P<0.001). VKORC1 haplotype groups A and B explained approximately 25 percent of the variance in dose. Asian Americans had a higher proportion of group A haplotypes and African Americans a higher proportion of group B haplotypes. VKORC1 mRNA levels varied according to the haplotype combination. CONCLUSIONS: VKORC1 haplotypes can be used to stratify patients into low-, intermediate-, and high-dose warfarin groups and may explain differences in dose requirements among patients of different ancestries. The molecular mechanism of this warfarin dose response appears to be regulated at the transcriptional level.

Anticoagulants↗
PubMed2005

Contribution of CYP3A5 to hepatic and renal ifosfamide N-dechloroethylation.

Ifosfamide nephrotoxicity is attributed to the formation of a toxic metabolite, chloroacetaldehyde, via N-dechloroethylation, a reaction that is purportedly catalyzed by CYP3A and CYP2B6. Because allelic variants of CYP3A5 are associated with polymorphic expression of microsomal CYP3A5 in human liver and kidneys, we hypothesized that ifosfamide N-dechloroethylation depends on CYP3A5 genotype. We compared ifosfamide N-dechloroethylation activity in cDNA-expressed CYP3A4 and CYP3A5. Ifosfamide N-dechloroethylation was also assessed in liver (N = 20) and kidney (N = 21) microsomes from human donors with different CYP3A5 genotypes. Ifosfamide N-dechloroethylation was catalyzed by recombinant CYP3A5 at a rate comparable with recombinant CYP3A4. In human liver microsomes matched for CYP3A4 protein content, N-dechloroethylation was more than 2-fold higher in that from donors carrying CYP3A5*1 allele that express CYP3A5 relative to that from donors homozygous for the mutant CYP3A5*3. Correlation analysis revealed that ifosfamide N-dechloroethylation was significantly associated with CYP3A4 and CYP3A5 protein concentration but not with age, sex, or CYP2B6 protein concentration. In hepatic microsomes not expressing CYP3A5 protein, ifosfamide N-dechloroethylation was inhibited 53 to 61% and 0 to 3% by monoclonal antibodies specific for CYP3A4/5 or CYP2B6, respectively. Ifosfamide N-dechloroethylation was not detected in renal microsomes obtained from CYP3A5*3/*3 donors. In contrast, it was readily measurable in microsomes isolated from four kidneys of CYP3A5*1 carriers, which was almost completely inhibited by the CYP3A inhibitor ketoconazole. CYP2B6 protein could not be detected in this panel of human renal microsomes. In conclusion, CYP3A5*1 genotype is associated with higher rates of ifosfamide N-dechloroethylation in human liver and kidneys.

Antineoplastic Agents, Alkylating↗
PubMed2005

Pharmacogenetic determinants of human liver microsomal alfentanil metabolism and the role of cytochrome P450 3A5.

BACKGROUND: There is considerable unexplained interindividual variability in the clearance of alfentanil. Alfentanil undergoes extensive metabolism by cytochrome P4503A4 (CYP3A4). CYP3A5 is structurally similar to CYP3A4 and metabolizes most CYP3A4 substrates but is polymorphically expressed. Livers with the CYP3A5*1 allele contain higher amounts of the native CYP3A5 protein than livers homozygous for the mutant CYP3A5*3 allele. This investigation tested the hypothesis that alfentanil is a substrate for CYP3A5 and that CYP3A5 pharmacogenetic variability influences human liver alfentanil metabolism. METHODS: Alfentanil metabolism to noralfentanil and N-phenylpropionamide was determined in microsomes from two groups of human livers, characterized for CYP3A4 and CYP3A5 protein content: low CYP3A5 (2.0-5.2% of total CYP3A, n = 10) and high CYP3A5 (46-76% of total CYP3A, n = 10). Mean CYP3A4 content was the same in both groups. The effects of the CYP3A inhibitors troleandomycin and ketoconazole, the latter being more potent toward CYP3A4, on alfentanil metabolism were also determined. RESULTS: In the low versus high CYP3A5 livers, respectively, noralfentanil formation was 77 +/- 31 versus 255 +/- 170 pmol . min . mg, N-phenylpropionamide formation was 8.0 +/- 3.1 versus 20.5 +/- 14.0 pmol . min . mg, and the metabolite ratio was 9.5 +/- 0.4 versus 12.7 +/- 1.4 (P < 0.05 for all). There was a poor correlation between alfentanil metabolism and CYP3A4 content but an excellent correlation when CYP3A5 (i.e., total CYP3A content) was considered (r = 0.81, P < 0.0001). Troleandomycin inhibited alfentanil metabolism similarly in the low and high CYP3A5 livers; ketoconazole inhibition was less in the high CYP3A5 livers. CONCLUSION: In microsomes from human livers expressing the CYP3A5*1 allele and containing higher amounts of CYP3A5 protein, compared with those with the CYP3A5*3 allele and little CYP3A5, there was greater alfentanil metabolism, metabolite ratios more closely resembled those for expressed CYP3A5, and inhibitors with differing CYP3A4 and CYP3A5 selectivities had effects resembling those for expressed CYP3A5. Therefore, alfentanil is metabolized by human liver microsomal CYP3A5 in addition to CYP3A4, and pharmacogenetic variability in CYP3A5 expression significantly influences human liver alfentanil metabolism in vitro. Further investigation is warranted to assess whether the CYP3A5 polymorphism is a factor in the interindividual variability of alfentanil metabolism and clearance in vivo.

Alfentanil↗
PubMed2005

CYP3A probes can quantitatively predict the in vivo kinetics of other CYP3A substrates and can accurately assess CYP3A induction and inhibition.

Because the rates at which therapeutics are cleared from the body can affect their effectiveness, knowing and accounting for the variables that contribute to drug clearance is of utmost importance when designing a drug dosage regimen for patients. The activities of the manifold cytochrome P450 enzymes [(CYPs), the most clinically important of which is often CYP3A] must be considered as they are essential for modification and metabolism of compounds (i.e., therapeutic, xenobiotic, etc.) prior to their excretion. To this end, much research has been expended on trying to identify and develop drug probes that accurately predict the metabolism of CYP3A substrates in individuals. Recently, Benet has written on the futility of such an enterprise; however, other researchers believe the identification of valuable predictive probes is not only possible but crucial.

Alfentanil↗
PubMed2004

In vitro metabolism of cyclosporine A by human kidney CYP3A5.

The objectives of this study were to characterize and compare the metabolic profile of cyclosporine A (CsA) catalyzed by CYP3A4, CYP3A5 and human kidney and liver microsomes, and to evaluate the impact of the CYP3A5 polymorphism on product formation from parent drug and its primary metabolites. Three primary CsA metabolites (AM1, AM9 and AM4N) were produced by heterologously expressed CYP3A4. In contrast, only AM9 was formed by CYP3A5. Substrate inhibition was observed for the formation of AM1 and AM9 by CYP3A4, and for the formation of AM9 by CYP3A5. Microsomes isolated from human kidney produced only AM9 and the rate of product formation (2 and 20 microM CsA) was positively associated with the detection of CYP3A5 protein and presence of the CYP3A5*1 allele in 4 of the 20 kidneys tested. A kinetic experiment with the most active CYP3A5*1-positive renal microsomal preparation yielded an apparent Km (15.5 microM) similar to that of CYP3A5 (11.3 microM). Ketoconazole (200 nM) inhibited renal AM9 formation by 22-55% over a CsA concentration range of 2-45 microM. Using liver microsomes paired with similar CYP3A4 content and different CYP3A5 genotypes, the formation of AM9 was two-fold higher in CYP3A5*1/*3 livers, compared to CYP3A5*3/*3 livers. AM19 and AM1c9, two of the major secondary metabolites of CsA, were produced by CsA, AM1 and AM1c when incubated with CYP3A4, CYP3A5, kidney microsomes from CYP3A5*1/*3 donors and all liver microsomes. Also, the formation of AM19 and AM1c9 was higher from incubations with liver and kidney microsomes with a CYP3A5*1/*3 genotype, compared to those with a CYP3A5*3/*3 genotype. Together, the data demonstrate that CYP3A5 may contribute to the formation of primary and secondary metabolites of CsA, particularly in kidneys carrying the wild-type CYP3A5*1 allele.

Biotransformation↗
PubMed2004

Evidence of significant contribution from CYP3A5 to hepatic drug metabolism.

CYP3A4 and CYP3A5 exhibit significant overlap in substrate specificity but can differ in product regioselectivity and formation activity. To further explore this issue, we compared the kinetics of product formation for eight different substrates, using heterologously expressed CYP3A4 and CYP3A5 and phenotyped human liver microsomes. Both enzymes displayed allosteric behavior toward six of the substrates. When it occurred, the "maximal" intrinsic clearance was used for quantitative comparisons. Based on this parameter, CYP3A5 was more active than CYP3A4 in catalyzing total midazolam hydroxylation (3-fold) and lidocaine demethylation (1.4-fold). CYP3A5 exhibited comparable metabolic activity as CYP3A4 (90-110%) toward dextromethorphan N-demethylation and carbamazepine epoxidation. CYP3A5-catalyzed erythromycin N-demethylation, total flunitrazepam hydroxylation, testosterone 6beta-hydroxylation, and terfenadine alcohol formation occurred with an intrinsic clearance that was less than 65% that of CYP3A4. Using two sets of human liver microsomes with equivalent CYP3A4-specific content but markedly different CYP3A5 content (group 1, predominantly CYP3A4; group 2, CYP3A4 + CYP3A5), we assessed the contribution of CYP3A5 to product formation rates determined at low substrate concentrations (< or = Km). Mean product formation rates for group 2 microsomes were 1.4- to 2.2-fold higher than those of group 1 (p < 0.05 for 5 of 8 substrates). After adjusting for CYP3A4 activity (itraconazole hydroxylation), mean product formation rates for group 2 microsomes were still significantly higher than those of group 1 (p < 0.05 for 3 substrates). We suggest that, under conditions when CYP3A5 content represents a significant fraction of the total hepatic CYP3A pool, the contribution of CYP3A5 to the clearance of some drugs may be an important source of interindividual variability.

Animals↗
PubMed2004

Role of itraconazole metabolites in CYP3A4 inhibition.

Itraconazole (ITZ) is a potent inhibitor of CYP3A in vivo. However, unbound plasma concentrations of ITZ are much lower than its reported in vitro Ki, and no clinically significant interactions would be expected based on a reversible mechanism of inhibition. The purpose of this study was to evaluate the reasons for the in vitro-in vivo discrepancy. The metabolism of ITZ by CYP3A4 was studied. Three metabolites were detected: hydroxy-itraconazole (OH-ITZ), a known in vivo metabolite of ITZ, and two new metabolites: keto-itraconazole (keto-ITZ) and N-desalkyl-itraconazole (ND-ITZ). OHITZ and keto-ITZ were also substrates of CYP3A4. Using a substrate depletion kinetic approach for parameter determination, ITZ exhibited an unbound K(m) of 3.9 nM and an intrinsic clearance (CLint) of 69.3 ml.min(-1).nmol CYP3A4(-1). The respective unbound Km values for OH-ITZ and keto-ITZ were 27 nM and 1.4 nM and the CLint values were 19.8 and 62.5 ml.min(-1).nmol CYP3A4(-1). Inhibition of CYP3A4 by ITZ, OH-ITZ, keto-ITZ, and ND-ITZ was evaluated using hydroxylation of midazolam as a probe reaction. Both ITZ and OH-ITZ were competitive inhibitors of CYP3A4, with unbound Ki (1.3 nM for ITZ and 14.4 nM for OH-ITZ) close to their respective Km. ITZ, OH-ITZ, keto-ITZ and ND-ITZ exhibited unbound IC50 values of 6.1 nM, 4.6 nM, 7.0 nM, and 0.4 nM, respectively, when coincubated with human liver microsomes and midazolam (substrate concentration < Km). These findings demonstrate that ITZ metabolites are as potent as or more potent CYP3A4 inhibitors than ITZ itself, and thus may contribute to the inhibition of CYP3A4 observed in vivo after ITZ dosing.

Cytochrome P-450 CYP3A↗