Testosterone 6beta-hydroxylation Research Articles

Beta-caryophyllene oxide and trans-nerolidol affect enzyme activity of CYP3A4 - in vitro and in silico studies.

Evaluation of possible interactions with enzymes of drug metabolism is an important part of studies on safety and, in general, on the properties of any drug or biologically active compound. Here, focus is given on interactions of three sesquiterpenes (beta-caryophyllene oxide (CAO), trans-nerolidol (tNER) and farnesol (FAR)) with CYP3A4. To determine the CYP3A4 activity, specific substrates testosterone (TES) and midazolam (MDZ) were used. In human liver microsomes, the CAO inhibited the MDZ 1´-hydroxylation by mixed type inhibition and K(i) 46.6 microM; TES 6beta-hydroxylation was inhibited more strongly by tNER by the same mechanism and with K(i) of 32.5 microM. Results indicated a possibility of different mode of interaction of both compounds within the active site of CYP3A4 and this was why the molecular docking study was done. The docking experiments showed that the studied sesquiterpenes (CAO and tNER) bound to the CYP3A4 active site cause a significant decrease of binding affinity of substrates tested which corresponded well to the inhibition studies. The inhibition observed, however, most probably does not pose a real harm to microsomal drug metabolism as the levels of sesquiterpenes in plasma (assuming the use of these compounds as spices or flavoring additives) does not usually exceed micromolar range. Hence, the interaction of drugs metabolized by CYP3A4 with sesquiterpenes is less probable.

Effect of Chitosan on Hepatic Drug-Metabolizing Enzymes and Oxidative Stress in Rats Fed Low- and High-Fat Diets

Chitosan is sold worldwide as a lipid-lowering functional food and may be taken with certain medications. To investigate the effect of chitosan on drug-metabolizing enzymes and oxidative stress in the liver, male Wistar rats were fed a low- or high-fat diet with cellulose or chitosan for 4 weeks. A significant decrease in cytochrome P450 (CYP) 3A-catalyzed testosterone 6beta-hydroxylation in liver microsomes was observed in rats fed the chitosan with low- and high-fat diets. The expression of CYP 3A1 and 3A2, however, was suppressed by chitosan in rats fed the low-fat diet only. Furthermore, rats fed the low-fat diet with chitosan had lower hepatic glutathione S-transferase (GST) activity and superoxide dismutase activity and higher total tissue and microsomal lipid hydroperoxides. Hepatic alpha-tocopherol was lower in rats fed the chitosan-containing diet. The results suggest that chitosan is likely to modulate CYP 3A activity and protein expression and GST activity partially in a dietary fat-dependent manner. This change may cause a decrease in the metabolism of drugs catalyzed by these enzymes in liver tissues. Moreover, decrease of alpha-tocopherol level and SOD activity by chitosan partly accounts for the increase of hepatic lipid peroxidation.

Repeated administration of oxycodone modifies the gene expression of several drug metabolising enzymes in the hepatic tissue of male Sprague-Dawley rats, including glutathione S-transferase A-5 (rGSTA5) and CYP3A2†

Clinical use and illicit abuse of the potent opioid agonist oxycodone has dramatically increased over the past decade. Yet oxycodone remains one of the least studied opioids, particularly its interactions on the genomic level. The aim of this study was to examine potential alterations in gene expression of drug metabolising enzymes in the liver tissue of male Sprague-Dawley rats chronically treated with oxycodone. Rats were administered saline or oxycodone 15 mg/kg i.p. twice a day for 8 days. Changes in RNA levels were detected using microarray analysis validated by quantitative real-time PCR; consequent changes in protein expression and functionality were further assessed by Western blotting and activity assays. The expression of several drug metabolising enzymes was modulated by oxycodone treatment: cytochrome P450 (CYP) 2B2, CYP2C13, CYP17A1, epoxide hydrolase 2, carboxylesterase 2, flavin-containing monooxygenase 1, glutathione S-transferase alpha 5 (rGSTA5) and CYP3A2. In particular, the mRNA level of rGSTA5 (formerly GSTYc(2)) was up-regulated by approximately 6.5 fold and CYP3A2 was down-regulated by approximately 7.0 fold. Immunoblotting assays demonstrated a corresponding significant elevation of rGSTA5 protein and repression of CYP3A2 protein. The apparent cytosolic GST activity towards 1-chloro-2,4-dinitrobenzene conjugation and reduction of cumene hydroperoxide were significantly higher in liver from oxycodone-treated rats than that of saline-treated rats. In addition, the microsomal activity of CYP3A2, measured via 6beta-hydroxylation of testosterone, was significantly decreased in oxycodone-treated rats. Repeated oxycodone administration is associated with a significant up-regulation of rGSTA5 and concomitant down-regulation of CYP3A2 mRNA, protein expression and functionality. These results support further in-vivo studies into the clinical impact of our findings.

Electron transfer properties and catalytic competence of cytochrome b 5 in the fusion protein Hmwb 5-EGFP in reactions catalyzed by cytochrome P450 3A4

In the present paper we describe studies on molecular mechanisms of protein-protein interactions between cytochrome P450 3A4 (CYP3A4) and cytochrome b(5), the latter being incorporated into the artificial recombinant protein Hmwb(5)-EGFP containing full-length cytochrome b(5) (functional module) and a mutant form of the green fluorescent protein EGFP (signal module) fused into a single polypeptide chain. It is shown that cytochrome b(5) within the fusion protein Hmwb(5)-EGFP can be reduced by NADPH-cytochrome P450 reductase in the presence of NADPH, the rate of reduction being dependent on solution ionic strength, indicating that the signal module does not prevent the interaction of the flavo- and hemeproteins. Interaction of cytochrome P450 3A4 and Hmwb(5)-EGFP was estimated based on spin equilibrium shift of cytochrome P450 3A4 to high-spin state in the presence of Hmwb(5)-EGFP, as well as based on steady-state fluorescence anisotropy of the EGFP component of the fusion protein in the presence of CYP3A4. The engineering of chimeric protein Hmwb(5)-EGFP gives an independent method to determine dissociation constant for the complex of cytochrome P450 and cytochrome b(5) that is less sensitive to environmental factors compared to spectrophotometric titration used before. Reconstitution of catalytic activity of cytochrome P450 3A4 in the reaction of testosterone 6beta-hydroxylation in the presence of Hmwb(5)-EGFP indicates that cytochrome b(5) in the fusion protein is able to stimulate the hydroxylation reaction. Using other fusion proteins containing either cytochrome b(5) or its hydrophilic domain to reconstitute catalytic activity of cytochrome P450 3A4 showed that the hydrophobic domain of cytochrome b(5) participates not only in hemeprotein interaction, but also in electron transfer from cytochrome b(5) to cytochrome P450.

In Vitro Inhibition and Induction of Human Liver Cytochrome P450 Enzymes by Milnacipran

Milnacipran (Savella) inhibits both norepinephrine and serotonin reuptake and is distinguished by a nearly 3-fold greater potency in inhibiting norepinephrine reuptake in vitro compared with serotonin. We evaluated the ability of milnacipran to inhibit and induce human cytochrome P450 enzymes in vitro. In human liver microsomes, milnacipran did not inhibit CYP1A2, 2B6, 2C8, 2C9, 2C19, or 2D6 (IC(50) >or= 100 microM); whereas, a comparator with dual reuptake properties [duloxetine (Cymbalta)] inhibited CYP2D6 (IC(50) = 7 microM) and CYP2B6 (IC(50) = 15 microM) with a relatively high potency. Milnacipran inhibited CYP3A4/5 in a substrate-dependent manner (i.e., midazolam 1'-hydroxylation IC(50) approximately 30 microM; testosterone 6beta-hydroxylation IC(50) approximately 100 microM); whereas, duloxetine inhibited both CYP3A4/5 activities with equal potency (IC(50) = 37 and 38 microM, respectively). Milnacipran produced no time-dependent inhibition (<10%) of P450 activity, whereas duloxetine produced time-dependent inhibition of CYP1A2, 2B6, 2C19, and 3A4/5. To evaluate P450 induction, freshly isolated human hepatocytes (n = 3) were cultured and treated once daily for 3 days with milnacipran (3, 10, and 30 microM), after which microsomal P450 activities were measured. Whereas positive controls (omeprazole, phenobarbital, and rifampin) caused anticipated P450 induction, milnacipran had minimal effect on CYP1A2, 2C8, 2C9, or 2C19 activity. The highest concentration of milnacipran (30 microM; >10 times plasma C(max)) produced 2.6- and 2.2-fold increases in CYP2B6 and CYP3A4/5 activity (making it 26 and 34% as effective as phenobarbital and rifampin, respectively). Given these results, milnacipran is not expected to cause clinically significant P450 inhibition or induction.

Up-Regulation of Transporters and Enzymes by the Vitamin D Receptor Ligands, 1α,25-Dihydroxyvitamin D3and Vitamin D Analogs, in the Caco-2 Cell Monolayer

The effects of 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] on gene expression and function were studied in Caco-2 cells. Microarray analyses, real-time quantitative polymerase chain reactions, and Western blotting were used to determine the mRNA and protein expression of transporters and enzymes after 1,25(OH)(2)D(3) or vehicle (0.1% ethanol) treatment for 1, 3, 6, and 10 days. The mRNA and protein expressions of the apical sodium-dependent bile acid transporter, oligopeptide transporter 1, multidrug resistance-associated protein (MRP) 3, and sulfotransferase 1E1 remained unchanged with 1,25(OH)(2)D(3) treatment, whereas those for CYP3A4, multidrug resistance protein 1, and MRP2 were significantly increased (P < 0.05). 1,25(OH)(2)D(3) treatment significantly enhanced MRP4 protein expression by increasing protein stability without affecting mRNA expression, as confirmed in cycloheximide experiments. Marked increase in 6beta-hydroxylation of testosterone by CYP3A4 was also observed in the 6-day 1,25(OH)(2)D(3)-treated (100 nM) cell lysate. The transport of [(3)H]digoxin, the P-glycoprotein (P-gp) substrate, after treatment with 100 nM 1,25(OH)(2)D(3) for 3 days revealed a higher apparent permeability (P(app)) value in the basal (B)-to-apical (A) direction over that of vehicle treatment (15.1 +/- 0.53 x 10(-6) versus 11.8 +/- 0.58 x 10(-6) cm/s; P < 0.05), whereas the P(app) in the A-to-B direction was unchanged; the efflux ratio was increased (from 5.8 to 8.0). Reduced cellular retention of 5-(and-6)-carboxy-2',7'-dichlorofluorescein, suggestive of higher MRP2 activity, was observed in the 3-day 100 nM 1,25(OH)(2)D(3)-treated cells over controls. Higher protein expression of CYP3A4, MRP2, P-gp, and MRP4 was also observed after a 6-day treatment with other vitamin D analogs (100 nM 1alpha-hydroxyvitamin D(3),1alpha-hydroxyvitamin D(2) or Hectorol, and 25-hydroxyvitamin D(3)) in Caco-2 cells, suggesting a role of 1,25(OH)(2)D(3) and analogs in the activation of enzymes and transporters via the vitamin D receptor.

Potential of Lansoprazole as a Novel Probe for Cytochrome P450 3A Activity by Measuring Lansoprazole Sulfone in Human Liver Microsomes

Cytochrome P450 (CYP) 3A enzymes are responsible for the metabolism of many drugs. It is useful to know CYP3A activity in individual patients undergoing drug therapy so as to predict the efficacies or adverse events. Lansoprazole is metabolized to Lansoprazole sulfone (LS) by CYP3A, while to 5-hydroxylansoprasole by CYP2C19. The aim of this study was to evaluate whether lansoprazole can be used to assess CYP 3A activity in human liver. Lansoprazole sulfoxidation activity in 14 human liver microsomes was determined as the ratio of lansoprazole/LS, measuring these parameters by high-performance liquid chromatography. Testosterone 6beta-hydroxylation (T6beta-OH) activity, a known marker for CYP3A activity was also measured together with lansoprazole sulfoxidation activity. Lansoprazole sulfoxidation activity was also analyzed in microsomes preincubat-ed with anti-CYP2C19 antibody. Interindividual variation was observed in lansoprazole sulfoxidation activity and T6beta-OH activities of those microsomes, respectively. Lansoprazole sulfoxidation activity was significantly correlated with T6beta-OH activity and CYP3A protein level. Lansoprazole sulfoxidation activity in microsomes with anti-CYP2C19 antibody was closely correlated with T6beta-OH activity. In contrast, lansoprazole 5-hydroxylation activity was correlated with the CYP2C19 activity. These results suggest that metabolism of lansoprazole to LS by CYP3A occurs independently of metabolism by CYP2C19. LS can be used as a new marker of CYP3A activity.

Drug Interactions of Thalidomide with Midazolam and Cyclosporine A: Heterotropic Cooperativity of Human Cytochrome P450 3A5

There is growing clinical interest of thalidomide because of its immunomodulatory and antiangiogenic properties, despite its teratogenicity. However, little information about thalidomide has been reported regarding its precise effects on drug-metabolizing enzymes. We investigated the effects of thalidomide on cytochrome P450 (P450) enzymes in human liver microsomes to clarify the potential for possible drug interactions. Thalidomide inhibited S-mephenytoin 4'-hydroxylation activities of recombinant P450 2C19 and human liver microsomes: the apparent concentration of thalidomide producing 50% inhibition was approximately 270 microM for P450 2C19. Midazolam 4-hydroxylation activities were suppressed by thalidomide, but activities of 1'-hydroxylation and total midazolam oxidation and testosterone 6beta-hydroxylation were enhanced in the presence of thalidomide. Recombinant P450 3A5 was found to have altered kinetics at clinically relevant concentrations of thalidomide (10-30 microM). P450 3A4 was also affected, but only at higher thalidomide concentrations. Enhanced midazolam hydroxylation by thalidomide was also seen in liver microsomal samples harboring the CYP3A5*1 allele. Similarly enhanced rates of cyclosporine A clearance were observed in P450 3A5 and liver microsomes expressing P450 3A5 in the presence of thalidomide. A proposed effector constant for thalidomide corresponded roughly to its clinical plasma levels. Docking studies with a P450 3A5 homology model, based on the published structure of P450 3A4, revealed close interaction between thalidomide and the heme of P450 3A5. The present results suggest that total midazolam metabolism or cyclosporine A clearance may be increased by thalidomide in a dose-dependent manner. Unexpected drug interactions involving thalidomide might occur via heterotropic cooperativity of polymorphic P450 3A5.

A gel‐free MS‐based quantitative proteomic approach accurately measures cytochrome P450 protein concentrations in human liver microsomes

The human cytochrome P450 (P450) superfamily consists of membrane-bound proteins that metabolize a myriad of xenobiotics and endogenous compounds. Quantification of P450 expression in various tissues under normal and induced conditions has an important role in drug safety and efficacy. Conventional immunoquantification methods have poor dynamic range, low throughput, and a limited number of specific antibodies. Recent advances in MS-based quantitative proteomics enable absolute protein quantification in a complex biological mixture. We have developed a gel-free MS-based protein quantification strategy to quantify CYP3A enzymes in human liver microsomes (HLM). Recombinant protein-derived proteotypic peptides and synthetic stable isotope-labeled proteotypic peptides were used as calibration standards and internal standards, respectively. The lower limit of quantification was approximately 20 fmol P450. In two separate panels of HLM examined (n = 11 and n = 22), CYP3A, CYP3A4 and CYP3A5 concentrations were determined reproducibly (CV <or=27%). The MS-based method strongly correlated with the immunoquantification method (r(2)>or=0.87) and marker activities (r(2)>or=0.88), including testosterone 6beta-hydroxylation (CYP3A), midazolam 1'-hydroxylation (CYP3A), itraconazole 6-hydroxylation (CYP3A4) and CYP3A5-mediated vincristine M1 formation (CYP3A5). Taken together, our MS-based method provides a specific, sensitive and reliable means of P450 protein quantification and should facilitate P450 characterization during drug development, especially when specific substrates and/or antibodies are unavailable.

A Comprehensive in Vitro and in Silico Analysis of Antibiotics That Activate Pregnane X Receptor and Induce CYP3A4 in Liver and Intestine

We have investigated several in silico and in vitro methods to improve our ability to predict potential drug interactions of antibiotics. Our focus was to identify those antibiotics that activate pregnane X receptor (PXR) and induce CYP3A4 in human hepatocytes and intestinal cells. Human PXR activation was screened using reporter assays in HepG2 cells, kinetic measurements of PXR activation were made in DPX-2 cells, and induction of CYP3A4 expression and activity was verified by quantitative polymerase chain reaction, immunoblotting, and testosterone 6beta-hydroxylation in primary human hepatocytes and LS180 cells. We found that in HepG2 cells CYP3A4 transcription was activated strongly (> 10-fold) by rifampin and troleandomycin; moderately (> or = 7-fold) by dicloxacillin, tetracycline, clindamycin, griseofulvin, and (> or = 4-fold) erythromycin; and weakly (> 2.4-fold) by nafcillin, cefaclor, sulfisoxazole, and (> 2-fold) cefadroxil and penicillin V. Similar although not identical results were obtained in DPX-2 cells. CYP3A4 mRNA and protein expression were induced by these antibiotics to differing extents in both liver and intestinal cells. CYP3A4 activity was significantly increased by rifampin (9.7-fold), nafcillin and dicloxacillin (5.9-fold), and weakly induced (2-fold) by tetracycline, sufisoxazole, troleandomycin, and clindamycin. Multiple pharmacophore models and docking indicated a good fit for dicloxacillin and nafcillin in PXR. These results suggest that in vitro and in silico methods can help to prioritize and identify antibiotics that are most likely to reduce exposures of medications (such as oral contraceptive agents) which interact with enzymes and transporters regulated by PXR. In summary, nafcillin, dicloxacillin, cephradine, tetracycline, sulfixoxazole, erythromycin, clindamycin, and griseofulvin exhibit a clear propensity to induce CYP3A4 and warrant further clinical investigation.

Delineation of the interactions between the chemotherapeutic agent eribulin mesylate (E7389) and human CYP3A4

Eribulin mesylate (E7389), a structurally simplified, synthetic analog of the marine natural product halichondrin B, acts by inhibiting microtubule dynamics via mechanisms distinct from those of other tubulin-targeted agents. Eribulin is currently in Phase III clinical trials for the treatment of metastatic breast cancer. Since drug-induced modulation of cytochrome P450 enzymes, particularly CYP3A4, is a frequent cause of drug-drug interactions, we examined the effects of eribulin on the activity and expression of hepatic and recombinant CYP3A4 (rCYP3A4) in vitro. Identification of the enzyme(s) responsible for eribulin metabolism was based on compound depletion and metabolite formation in reaction mixtures containing subcellular liver fractions or primary human hepatocytes, plus recombinant Phases I and II metabolic enzymes. The role of the enzyme(s) identified was confirmed using enzyme-selective inhibitors and the correlation with prototypic enzyme activity. The effect of eribulin on enzymatic activity was characterized using both microsomal preparations and recombinant enzymes, while the possible modulation of protein expression was evaluated in primary cultures of human hepatocytes. Eribulin was primarily metabolized by CYP3A4, resulting in the formation of at least four monooxygenated metabolites. In human liver microsomal preparations, eribulin suppressed the activities of CYP3A4-mediated testosterone and midazolam hydroxylation with an apparent K (i) of approximately 20 microM. Eribulin competitively inhibited the testosterone 6beta-hydroxylation, nifedipine dehydration, and R-warfarin 10-hydroxylation activities of rCYP3A4, with an average apparent K (i) of approximately 10 microM. These inhibitions were reversible, with no apparent mechanism-based inactivation. Eribulin did not induce the expression or activities of CYP1A and CYP3A enzymes in human primary hepatocytes, and clinically relevant concentrations of eribulin did not inhibit CYP3A4-mediated metabolism of various therapeutic agents, including carbamazepine, diazepam, paclitaxel, midazolam, tamoxifen, or terfenadine. Eribulin was predominantly metabolized by CYP3A4. Although eribulin competitively inhibited the testosterone 6beta-hydroxylation, nifedipine dehydration, and R-warfarin 10-hydroxylation activities of rCYP3A4, it did not induce or inhibit hepatic CYP3A4 activity at clinically relevant concentrations. As eribulin does not appear to affect the metabolism of other therapeutic agents by CYP3A4, our data suggest that eribulin would not be expected to inhibit the metabolism of concurrently administered drugs that are metabolized by CYP3A4, suggesting a minimal risk of drug-drug interactions in the clinical setting.

Identification of Human Liver Cytochrome P450 Enzymes Involved in Biotransformation of Vicriviroc, a CCR5 Receptor Antagonist

Vicriviroc (SCH 417690), a CCR5 receptor antagonist, is currently under investigation for the treatment of human immunodeficiency virus infection. The objective of this study was to identify human liver cytochrome P450 enzyme(s) responsible for the metabolism of vicriviroc. Human liver microsomes metabolized vicriviroc via N-oxidation (M2/M3), O-demethylation (M15), N,N-dealkylation (M16), N-dealkylation (M41), and oxidation to a carboxylic acid metabolite (M35b/M37a). Recombinant human CYP3A4 catalyzed the formation of all these metabolites, whereas CYP3A5 catalyzed the formation of M2/M3 and M41. CYP2C9 only catalyzed the formation of M15. There was a high correlation between the rates of formation of M2/M3, M15, and M41, which was determined using 10 human liver microsomal samples and testosterone 6beta-hydroxylation catalyzed by CYP3A4/5 (r > or = 0.91). Ketoconazole and azamulin (inhibitors of CYP3A4) were potent inhibitors of the formation of M2/M3, M15, M41, and M35b/M37a from human liver microsomes. A CYP3A4/5-specific monoclonal antibody (1 microg/microg of protein) inhibited the formation of all metabolites from human liver microsomes by 86 to 100%. The results of this study suggest that formation of the major vicriviroc metabolites in human liver microsomes is primarily mediated via CYP3A4. CYP2C9 and CYP3A5 most likely play a minor role in the biotransformation of this compound. These enzymology data will provide guidance to design clinical studies to address any potential drug-drug interactions.

Molecular Characterization of Specifically Active Recombinant Fused Enzymes Consisting of CYP3A4, NADPH-Cytochrome P450 Oxidoreductase, and Cytochrome b5

Microsomal cytochrome P450 3A4 (CYP3A4) catalyzes monooxygenase reactions toward a diverse group of exogenous and endogenous substrates and requires cytochrome b5 (b5) in the oxidation of the typical substrate testosterone. To analyze the molecular interaction among CYP3A4, NADPH-cytochrome P450 oxidoreductase (P450 reductase), and b5, we constructed several fused enzyme genes and expressed them in Saccharomyces cerevisiae. The recombinant fused enzymes CYP3A4-truncated (t)-P450 reductase-t-b5 (3RB) and CYP3A4-t-b5-t-P450 reductase (3BR) in yeast microsomes showed a higher specific activity in 6beta-hydroxylation of testosterone than did the reconstitution premixes of CYP3A4, P450 reductase, and b5. The purified fused enzymes exhibited lower Km values and substantially increased Vmax values in 6beta-hydroxylation of testosterone and oxidation of nifedipine. Moreover, the fused enzymes showed significantly higher activities in cytochrome c reduction than the reconstitution premixes. Although the affinity of 3RB toward cytochrome c was twice as high as that of 3BR, 3BR and 3RB showed nearly the same affinity toward NADPH/NADH. In addition, the heme of the CYP3A4 moiety of 3RB was reduced preferentially and more rapidly than that of 3BR, whereas the heme of the b5 moiety of 3BR was selectively reduced compared with that of 3RB. These results suggest that the conformation of the 3RB molecule was the most suitable for high activity because of appropriate ordering of the CYP3A4, P450 reductase, and b5 moieties for efficient electron flow. Thus, we believe that the b5 moiety plays an important role in the efficient transfer of the second electron in the vicinity of the CYP3A4 moiety.

Different Roles of Pummelo Furanocoumarin and Cytochrome P450 3A5*3 Polymorphism in the Fate and Action of Felodipine

Herein we aim to test if pummelo furanocoumarins can inhibit cytochrome P450 (CYP) 3A both in vitro and in vivo, and to explore the influence of CYP3A5*3 (GenBank AC005020: A22893-->G) polymorphism in the pharmacokinetics and pharmacological response to felodipine. Fruit juices of pummelo grapefruit (Citrus paradisi Macf., G), 'Guanximiyou' (C. grandis Osbeck vs. Guanxi, P) and 'Changshanhuyou' (C. changshanhuyou Y.B. Chang, H) were selected by screening Citrus fruit juices for their furanocoumarin contents and their inhibition of testosterone 6beta-hydroxylation in human liver microsomes. Twelve healthy male Chinese were administered 250 mL G, P, H or water (W) alternatively with 26-mumol (10-mg) plain tablet felodipine, and were observed for 12 h. G had more furanocoumarins and at higher levels than P while H had none, and their potencies for in vitro CYP3A inhibition were in the order as G > P > H. The geometric mean and 90% confidence intervals of pharmacokinetic parameters for human oral felodipine with G, P, H and W were respectively as follows: peak plasma concentration (nmol.L(-1)), 37 (32-44), 25 (21-29), 19 (16-22) and 18 (15-21); area under the plasma concentration-time curve (nmol.h.L(-1)), 118 (103-136), 84 (73-97), 64 (56-74) and 59 (51-68). Subjects showed higher heart rates with G than with H or W. CYP3A5*3 polymorphism showed no significant effect on felodipine pharmacokinetics and related hemodynamic changes. This work supports the hypothesis that CYP3A inhibition by furanocoumarins caused pummelo fruit juice-drug interaction; while the role of CYP3A5 in the population pharmacokinetics of felodipine and blood pressure response appear to be limited.

Inhibitory Effects of Saturated and Polyunsaturated Fatty Acids on the Cytochrome P450 3A Activity in Rat Liver Microsomes

This study was conducted to investigate the inhibitory effects of saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and polyunsaturated fatty acids (PUFAs) on cytochrome P450 3A (CYP3A). The inhibition of 6beta-hydroxy testosterone formation from testosterone in rat liver microsomes was used as an index of CYP3A activity. In the present study, among the three types of fatty acids, the rank order of inhibitory effects of fatty acids was SFAs<MUFAs<PUFAs. Among the four PUFAs examined (linoleic acid, gamma-linolenic acid, retinoic acid, and docosahexaenoic acid; DHA), the highest inhibitory effect of CYP3A-catalyzed testosterone metabolism was observed with DHA, which inhibited testosterone metabolism by 94%. The percentage inhibition (I) of fatty acids on testosterone 6beta-hydroxylation was predicted from the number of double bonds and the carbon chain length of the fatty acids. The correlation between the predicted and observed I values yielded the r(2) values of 0.759 and 0.907 using the linear and non-linear equations, respectively. Moreover, the goodness-of-fit assessed using the mean absolute error (MAE) was 17.6 with the linear model and 11.3 with the non-linear model. These observations suggested that fatty acids showed the variable inhibitory effects on CYP3A-mediated testosterone oxidation and that the number of double bonds, as well as the carbon chain lengths of fatty acids, may be important in the inhibition of CYP3A.

Regulation of Constitutive Mouse Hepatic Cytochromes P450 and Growth Hormone Signaling Components by 3-Methylcholanthrene

3-Methylcholanthrene (MC) activates the aryl hydrocarbon receptor and increases expression of cytochrome P450 (P450) enzymes such as CYP1A1. MC also decreases expression of CYP2C11, the major hepatic P450 in male rats that is regulated by pulsatile growth hormone (GH) secretion via a pathway partially dependent on signal transducer and activator of transcription 5b (STAT5b). If disruption of this GH signaling pathway is important for MC's ability to suppress CYP2C11 transcription, we hypothesize that MC suppresses other male-specific genes (e.g., mouse Cyp2d9) regulated by pulsatile GH with STAT5b dependence. We examined the time course of MC's effects on hepatic P450s and GH signaling components in male C57BL/6 mice. P450 content, heme content, and NADPH P450 oxidoreductase activity were induced 2.3-, 1.8-, and 1.3-fold, respectively, by MC. MC dramatically induced CYP1A1 mRNA, protein, and catalytic activity. MC caused a 42% decrease in CYP2D9 protein, a 28% decrease in CYP2D9 mRNA, and a 27% decrease in testosterone 16alpha-hydroxylation activity. MC caused a pronounced decrease in CYP3A protein; however, there was no apparent change in testosterone 6beta-hydroxylation activity, and changes in mRNA levels for CYP3A forms were relatively small. Expression of GH receptor and major urinary protein 2, a gene regulated by GH with STAT5b dependence, was decreased by MC at the mRNA level. These results show that MC suppresses mouse Cyp2d9, a pulsatile GH- and STAT5b-dependent male-specific gene, via a pretranslational mechanism that may involve disrupted GH signaling. Mouse CYP3A protein levels are dramatically decreased by MC via a mechanism that is not yet understood.

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

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. 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). 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). 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.

AUTOINDUCTION OF MKC-963 [(R)-1-(1-CYCLOHEXYLETHYLAMINO)-4-PHENYLPHTHALAZINE] METABOLISM IN HEALTHY VOLUNTEERS AND ITS RETROSPECTIVE EVALUATION USING PRIMARY HUMAN HEPATOCYTES AND CDNA-EXPRESSED ENZYMES

MKC-963, (R)-1-(1-cyclohexylethylamino)-4-phenylphthalazine, a potent inhibitor of platelet aggregation, was synthesized and used in clinical trials in the 1990s. In the process of clinical study, it was found that urinary excretion ratios for 6beta-hydroxycortisol and free cortisol increased significantly in parallel with decreases in the plasma concentrations of MKC-963 after repeated oral administration of the compound to healthy volunteers. These findings suggested that MKC-963 caused autoinduction (defined as the ability of a drug to induce enzymes that enhance its own metabolism, resulting in dispositional tolerance) in humans, and clinical studies using the compound were stopped. This experience prompted us to reevaluate the effects of this compound on CYP3A4 using primary human hepatocytes and cDNA-expressed human cytochrome P450 (P450) enzymes to determine whether the autoinduction of MKC-963 metabolism in humans could have been predicted if these in vitro systems had been used for the evaluation of MKC-963 in the preclinical study. The results of in vitro study showed that MKC-963 increased CYP3A4 mRNA expression level and activity of testosterone 6beta-hydroxylation to extents similar to those observed with rifampicin in primary human hepatocytes. In addition, approximately 90% of the MKC-963 metabolism in human liver microsomes was estimated to be attributable to CYP3A4. These in vitro findings are in good agreement with the results of clinical study, suggesting that studies using human hepatocytes and cDNA-expressed human P450s are useful for assessing the autoinductive nature of compounds under development before starting clinical studies.

Quantitative prediction of macrolide drug-drug interaction potential from in vitro studies using testosterone as the human cytochrome P4503A substrate

Macrolide antibiotics are mechanism-based inactivators of CYP3A enzymes that exhibit varying degrees of inhibitory potency. Our aim was to predict quantitatively the drug-drug interaction (DDI) potential of five macrolides from in vitro studies using testosterone as the CYP3A substrate, and to compare the predictions generated from human liver microsomal and recombinant CYP3A4 data. The in vitro kinetic constants of CYP3A inactivation (K (I) and k (inact)) were estimated by varying the time of pre-incubation and the concentration of troleandomycin, erythromycin, clarithromycin, roxithromycin or azithromycin. CYP3A activity was determined from the measurement of testosterone 6beta-hydroxylation with human liver microsomes (HLM) and recombinant CYP3A4 as the enzyme sources. The mechanism-based pharmacokinetic model was fitted with inactivation data to predict the increase in oral area under the plasma concentration-time curve (AUC) for midazolam. All five macrolides inactivated testosterone 6beta-hydroxylation by HLM and recombinant CYP3A4 with k (inact) values in the range of 0.023 to 0.058 min(-1). The potency of inactivation (K (I)) was higher using recombinant CYP3A4 as the enzyme source. The oral AUCs for midazolam were predicted from HLM data to increase 16.6, 5.3, 4.6, 1.6 and 1.2-fold due to the inhibition of metabolic clearance by troleandomycin, erythromycin, clarithromycin, roxithromycin and azithromycin, respectively. These results are within the range of the AUC ratios reported for clinical DDI studies. The predicted AUC increases generated using recombinant CYP3A4 overestimated the magnitude of the DDIs. The DDI potential of five macrolide antibiotics was quantitatively predicted from in vitro studies using testosterone as the CYP3A substrate with HLM as the enzyme source.

Differential Inhibition of Cytochrome P450 3A4, 3A5 and 3A7 by Five Human Immunodeficiency Virus (HIV) Protease Inhibitors in vitro

The effects of five HIV protease inhibitors (amprenavir, indinavir, nelfinavir, ritonavir and saquinavir) on cytochrome P450 (CYP) 3A4, 3A5 and 3A7 activities were studied in vitro using testosterone 6beta-hydroxylation in recombinant CYP3A4, CYP3A5 and CYP3A7 enzymes. The protease inhibitors showed differential inhibitory effects on the three CYP3A forms. Ritonavir and saquinavir were non-selective and preferential inhibitors of CYP3A4 and CYP3A5 (K(i) 0.03 microM and 0.6-0.8 microM for ritonavir and saquinavir, respectively), and weaker inhibitors of CYP3A7 (K(i) 0.6 microM and 1.8 microM, respectively). Nelfinavir was a potent and non-selective inhibitor of all three CYP3A forms (K(i) 0.3-0.4 microM). Amprenavir and indinavir preferentially inhibited CYP3A4 (K(i) 0.1 microM and 0.2 microM, respectively), with weaker inhibitory effects on CYP3A5 (K(i) 0.5 microM and 2.2 microM, respectively) and CYP3A7 (K(i) 2.1 microM and 10.6 microM, respectively). In conclusion, significant differences exist in the inhibitory potency of protease inhibitors for different CYP3A forms. Ritonavir, nelfinavir, saquinavir and amprenavir seem to be prone to drug-drug interactions by inhibiting both CYP3A4 and CYP3A5. Especially nelfinavir and ritonavir also have a potential to inhibit foetal CYP3A7-mediated drug metabolism and some endogenous pathways that may be crucial to normal foetal development, while indinavir has the lowest potential to inhibit CYP3A5 and CYP3A7.