Liver Microsomal Cytochrome P-450 Research Articles

Suitable albumin concentrations for enhanced drug oxidation activities mediated by human liver microsomal cytochrome P450 2C9 and other forms predicted with unbound fractions and partition/distribution coefficients of model substrates

Albumin has reportedly enhanced cytochrome P450 (P450)-mediated drug oxidation rates in human liver microsomes. Consequently, measurements of clearances and fractions metabolized could vary depending on the experimental albumin concentrations used.In this study, the oxidation rates of diclofenac and warfarin by human liver microsomes were significantly enhanced in the presence of 0.10% (w/v) bovine serum albumin, whereas those of tolbutamide and phenytoin required 1.0% and 2.0% of albumin for significant enhancement. Values of the fractions metabolized by P450 2C9 for four substrates did not markedly change in the presence of albumin at the above-mentioned concentrations.The oxidation rates of bupropion, omeprazole, chlorzoxazone and phenacetin in human liver microsomes were reportedly enhanced by 0.5%, 1%, 2% and 2% of albumin, respectively. Analysis of reported intrinsic clearance values and suitable albumin concentrations for the currently analyzed substrates and the reported substrates revealed an inverse correlation, with warfarin as an outlier.Suitable albumin concentrations were multivariately correlated with physicochemical properties, that is, the plasma unbound fractions, octanol–water partition coefficient and acid dissociation constant (r = 0.98, p<.0001, n = 10). Therefore, multiple physicochemical properties may be determinants of suitable albumin concentrations for substrate oxidations in human liver microsomes.

Sulfenylation of Human Liver and Kidney Microsomal Cytochromes P450 and Other Drug-Metabolizing Enzymes as a Response to Redox Alteration

The lumen of the endoplasmic reticulum (ER) provides an oxidizing environment to aid in the formation of disulfide bonds, which is tightly regulated by both antioxidant proteins and small molecules. On the cytoplasmic side of the ER, cytochrome P450 (P450) proteins have been identified as a superfamily of enzymes that are important in the formation of endogenous chemicals as well as in the detoxication of xenobiotics. Our previous report described oxidative inhibition of P450 Family 4 enzymes via oxidation of the heme-thiolate cysteine to a sulfenic acid (-SOH) (Albertolle, M. E. et al. (2017) J. Biol. Chem. 292, 11230-11242). Further proteomic analyses of murine kidney and liver microsomes led to the finding that a number of other drug-metabolizing enzymes located in the ER are also redox-regulated in this manner. We expanded our analysis of sulfenylated enzymes to human liver and kidney microsomes. Evaluation of the sulfenylation, catalytic activity, and spectral properties of P450s 1A2, 2C8, 2D6, and 3A4 led to the identification of two classes of redox sensitivity in P450 enzymes: heme-thiolate-sensitive and thiol-insensitive. These findings provide evidence for a mammalian P450 regulatory mechanism, which may also be relevant to other drug-metabolizing enzymes. (Data are available via ProteomeXchange with identifier PXD007913.).

In Vitro Inhibitory Effects of Synthetic Cannabinoid EAM-2201 on Cytochrome P450 and UDP-Glucuronosyltransferase Enzyme Activities in Human Liver Microsomes

EAM-2201, a synthetic cannabinoid, is a potent agonist of the cannabinoid receptors that is widely abused as an illicit recreational drug in combination with other drugs. To evaluate the potential of EAM-2201 as a perpetrator of drug–drug interactions, the inhibitory effects of EAM-2201 on major drug-metabolizing enzymes, cytochrome P450s (CYPs) and uridine 5′-diphospho-glucuronosyltransferases (UGTs) were evaluated in pooled human liver microsomes using liquid chromatography–tandem mass spectrometry (LC-MS/MS). EAM-2201 at doses up to 50 µM negligibly inhibited the activities of eight major human CYPs (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4) and five UGTs (1A1, 1A4, 1A6, 1A9 and 2B7) in human liver microsomes. EAM-2201 exhibited time-dependent inhibition of CYP2C8-catalyzed amodiaquine N-deethylation, CYP2C9-catalyzed diclofenac 4′-hydroxylation, CYP2C19-catalyzed [S]-mephenytoin 4′-hydroxylation and CYP3A4-catalyzed midazolam 1′-hydroxylation with Ki values of 0.54 µM (kinact: 0.0633 min−1), 3.0 µM (kinact: 0.0462 min−1), 3.8 µM (kinact: 0.0264 min−1) and 4.1 µM (kinact: 0.0250 min−1), respectively and competitively inhibited UGT1A3-catalyzed chenodeoxycholic acid 24-acyl-glucuronidation, with a Ki value of 2.4 µM. Based on these in vitro results, we conclude that EAM-2201 has the potential to trigger in vivo pharmacokinetic drug interactions when co-administered with substrates of CYP2C8, CYP2C9, CYP2C19, CYP3A4 and UGT1A3.

In Vivo Modulation of Rat Liver Microsomal Cytochrome P450 Activity by Antimalarial, Anti-HIV, and Antituberculosis Plant Medicines.

Drug interactions are key reasons for adverse drug reactions and attrition from market. Major infectious diseases causing morbidity/mortality in Ghana are malaria, tuberculosis, and HIV/AIDS. In this study, plant medicines commonly used to treat/manage these diseases in Ghana were investigated for their potential to modulate rat cytochrome P450 enzyme activities. Fluorescence and high-performance liquid chromatography–based assays were used to assess effects of antimalarial plant medicines, Fever (FEV), Mal-TF (MAL), and Kantinka terric (KT); anti-TB medicines, Chestico (CHES), CA + ST Pains + HWNT (TF), and Kantinka herbatic (KHB); and anti-HIV/AIDS medicines, Wabco (WAB), AD + T/AD (LIV) and Kantinka BA (KBA) on rat liver microsomal cytochrome P450 enzyme activities. Effects of medicines on rat biochemical and hematological parameters were also assessed. Generally, the medicines altered microsomal CYP1A1/1A2, CYP2B1/2B2, CYP2C9, and CYP2D6 activities. Only KBA elicited an increase (80%) in CYP1A1/1A2 activity. FEV, MAL, CHES, WAB, and LIV strongly inhibited the enzyme activity. All the medicines significantly inhibited CYP2C9 (24%-80%) activity. CYP2D6 activity increased after treatment with MAL, KBA, LIV, and TF. Also, MAL, WAB, LIV, KHB, and CHES increased CYP2B1/2B2 activity, while KT decrease the activity. Generally, the medicines altered liver function in the rats. Cholesterol levels declined after KBA treatment only. White and red blood cell counts, hemoglobin and hematocrit levels were significantly reduced in KT- and KBA-treated rats. Our results suggest that use of the medicines could have implications for drug interactions and safety, particularly if the medicines are administered over prolonged periods. Further investigations are imperative to establish clinical relevance of these results.

In Vitro Antioxidant Potential and Effect of a Glutathione-Enhancer Dietary Supplement on Selected Rat Liver Cytochrome P450 Enzyme Activity.

Background There is considerable evidence that many people take dietary supplements including those of herbal origin as an alternative therapy to improve their health. One such supplement, with an amalgam of constituents, is CellGevity®. However, the effect of this dietary supplement on drug-metabolizing enzymes is poorly understood, as it has not been studied extensively. Therefore, we investigated the effect of CellGevity dietary supplement on selected rat liver microsomal cytochrome P450 (CYP) enzymes, the most common drug-metabolizing enzymes. We also determined the total antioxidant potential of this dietary supplement in vitro. Methods To determine the antioxidant potential of CellGevity dietary supplement, 2,2-diphenyl-2-picryl-hydrazyl (DPPH), total phenolic, and flavonoid assays were used after initial preparation of a solution form of the supplement (low dose, LD; 4 mg/kg and high dose, HD; 8 mg/kg). Rats received oral administration of these doses of the supplement for 7 days, after which the effect of the supplement on selected liver CYP enzymes was assessed using probe substrates and spectroscopic and high-performance liquid chromatographic methods. Rats which received daily administration of 80 mg/kg of phenobarbitone and distilled water served as positive and negative controls, respectively. Results The IC50 value of the supplement 0.34 ± 0.07 mg/ml compared to 0.076 ± 0.03 mg/ml of the BHT (positive control). The total phenolic content of the supplement at a concentration of 2.5 mg/ml was 34.97 g gallic acid equivalent (GAE)/100 g while its total flavonoid content at a concentration of 2.5 mg/ml was 6 g quercetin equivalent (QE)/100 g. The supplement significantly inhibited rat CYP2B1/2B2 (LDT 92.4%; HDT 100%), CYP3A4 (LDT 81.2%; HDT 71.7%), and CYP2C9 (LDT 21.7%; HDT 28.5%) while it had no significant inhibitory effect on CYPs 1A1/1A2, CYP1A2, and CYP2D6. Conclusion CellGevity dietary supplement possesses moderate antioxidant activity in vitro and has an inhibitory effect on selected rat liver CYP enzymes, suggesting its potential interaction with drugs metabolized by CYP enzymes.

Influence of Derivatives of Resveratrol on Enzyme Activities in Human Liver Microsomal Cytochromes P450

Influence of Derivatives of Resveratrol on Enzyme Activities in Human Liver Microsomal Cytochromes P450

Fatty Acid Oxidation in Zebrafish Adipose Tissue Is Promoted by 1α,25(OH)2D3

1α,25(OH)2D3 (vitamin D3) is crucial for mineral homeostasis in mammals, but the precise effects of 1α,25(OH)2D3 in adipose tissue remain to be clarified invivo. The initial 25-hydroxylation is catalyzed by liver microsomal cytochrome P450 2R1 (CYP2R1), which is conserved in vertebrates. To probe the physiological function(s) of 1α,25(OH)2D3 in teleosts, we generated two independent cyp2r1-deficient zebrafish lines. These mutants exhibit retarded growth and increased obesity, especially in the visceral adipose tissue (VAT). These defects could be rescuedwith 25(OH)D3 treatments. ChIP-PCR analyses demonstrated that pgc1a is the target of the vitamin D receptor in the liver and VAT of zebrafish. Significantly decreased protein levels of Pgc1a, impaired mitochondrial biogenesis, and free fatty acid oxidation are also observed in the cyp2r1 mutant VAT. Our results demonstrate that regulation of 1α,25(OH)2D3 during lipid metabolism occurs through the regulation of Pgc1a for mitochondrial biogenesis and oxidative metabolism within zebrafish VAT.

An integrated strategy for the quantitative analysis of endogenous proteins: A case of gender-dependent expression of P450 enzymes in rat liver microsome

Liquid chromatography mass spectrometry based methods provide powerful tools for protein analysis. Cytochromes P450 (CYPs), the most important drug metabolic enzymes, always exhibit sex-dependent expression patterns and metabolic activities. To date, analysis of CYPs based on mass spectrometry is still facing critical technical challenges due to the complexity and diversity of CYP isoforms besides lack of corresponding standards. The aim of present work consisted in developing a label-free qualitative and quantitative strategy for endogenous proteins, and then applying to the gender-difference study for CYPs in rat liver microsomes (RLMs). Initially, trypsin digested RLM specimens were analyzed by the nanoLC-LTQ-Orbitrap MS/MS. Skyline, an open source and freely available software for targeted proteomics research, was then used to screen the main CYP isoforms in RLMs under a series of criteria automatically, and a total of 40 and 39 CYP isoforms were identified in male and female RLMs, respectively. More importantly, a robust quantitative method in a tandem mass spectrometry-multiple reaction mode (MS/MS-MRM) was built and optimized under the help of Skyline, and successfully applied into the CYP gender difference study in RLMs. In this process, a simple and accurate approach named ‘Standard Curve Slope” (SCS) was established based on the difference of standard curve slopes of CYPs between female and male RLMs in order to assess the gender difference of CYPs in RLMs. This presently developed methodology and approach could be widely used in the protein regulation study during drug pharmacological mechanism research.

Inhibitory Effects of 12 Ginsenosides on the Activities of Seven Cytochromes P450 in Human Liver Microsomes

Ginseng, a traditional herbal drug, has been used in Eastern Asia for more than 2000 years. Various ginsenosides, which are the major bioactive components of ginseng products, have been shown to exert numerous beneficial effects on the human body when co-administered with drugs. However, this may give rise to ginsenoside-drug interactions, which is an important research consideration. In this study, acassette assay was performed the inhibitory effects of 12 ginsenosides on seven cytochrome P450 (CYP) isoforms in human liver microsomes (HLMs) using LC-MS/MS to predict the herb-drug interaction. After incubation of the 12 ginsenosides with seven cocktail CYP probes, the generated specific metabolites were quantified by LC-MS/MS to determine their activities. Ginsenoside Rb1 and F2 showed strong selective inhibitory effect on CYP2C9-catalyzed diclofenac 4′-hydroxylation and CYP2B6-catalyzed bupropion hydroxylation, respectively. Ginsenosides Rd showed weak inhibitory effect on the activities of CYP2B6, 2C9, 2C19, 2D6, 3A4, and compound K, while ginsenoside Rg3 showed weak inhibitory effects on CYP2B6. Other ginsenosides, Rc, Rf, Rg1, Rh1, Rf, and Re did not show significant inhibitory effects on the activities of the seven CYPs in HLM. Owing to the poor absorption of ginsenosides after oral administration in vivo, ginsenosides may not have significant side effects caused by interaction with other drugs.

Inhibition of cytochrome P450 3A enzyme by Millettia aboensis: its effect on the pharmacokinetic properties of efavirenz and nevirapine

The chronic and comorbid nature of HIV infection necessitate the use of multiple drugs including herbs to relieve symptoms with a possible increase in herb–drug interaction cases. This study was designed to evaluate the effect of Millettia aboensis (Hook. f.) Baker, Fabaceae, on cytochrome P450 3A isoenzyme and the influence of this effect on the bioavailability of two antiretroviral agents. In vitro effect of ethanol extract of M. aboensis on intestinal and liver microsomes extracted from female rats was assessed using erythromycin-N-demethylation assay method while in vivo effects were determined by estimating simvastatin plasma concentrations in rats. The effect of the extract on pharmacokinetic parameters of orally administered efavirenz (25mg/kg) and nevirapine (20mg/kg) was determined in rats divided into groups (n=5). Plasma drug concentrations were assayed using HPLC and pharmacokinetic parameters determined through a non-compartmental analysis as implemented in WinNonlin pharmacokinetic program. The extract inhibited both intestinal and liver microsomal cytochrome P450 3A isoenzyme activities in vitro and enhanced simvastatin absorption in vivo with possible inhibition of metabolizing enzymes as indicated by significant (p<0.05) increase in maximal concentration, area under curve and mean resident time of the drug. However, further in vivo interaction studies in animal model did not produce significant (p>0.05) changes in the pharmacokinetic parameters of efavirenz and nevirapine. HPLC fingerprinting indicated the presence of quercetin and kaempferol in the extract. These findings revealed M. aboensis as an inhibitor of cytochrome P450 3A enzyme but, with no significant effect on the bioavailability of orally administered nevirapine and efavirenz.

Oxidation of R- and S-omeprazole stereoselectively mediated by liver microsomal cytochrome P450 2C19 enzymes from cynomolgus monkeys and common marmosets

Racemic omeprazole has been used for clinically treating gastric acid-related diseases and also as a typical human cytochrome P450 (P450) 2C19 probe substrate in preclinical studies. S-Omeprazole has been developed as a single enantiomer medicine, which has been reported not to be associated with polymorphic human P450 2C19 phenotypes. In this study, 5-hydroxylation and sulfoxidation activities, with respect to stereoselective R- and S-omeprazole oxidations by liver microsomes from experimental animals including non-human primates and humans, were investigated in vitro. Liver microsomes from humans, cynomolgus monkeys, and mice preferentially mediated R-omeprazole 5-hydroxylations, however those from marmosets, minipigs, dogs, and rats preferentially mediated S-omeprazole 5-hydroxylations. High catalytic activities were observed for recombinant human P450 2C19 in R-omeprazole 5-hydroxlations, cynomolgus monkey P450 2C19 in both R- and S-omeprazole 5-hydroxlations, and marmoset P450 2C19 in S-omeprazole 5-hydroxlations. On the other hand, human, cynomolgus monkey, and marmoset P450 3A enzymes preferentially mediated S-omeprazole sulfoxidations. Correlation and kinetic analyses revealed a high affinity of polymorphic cynomolgus monkey and marmoset liver microsomal P450 2C19 enzymes with respect to R- and S-omeprazole 5-hydroxylations, respectively, and a high capacity of cynomolgus monkey and marmoset liver microsomal P450 3A4 for omeprazole 5-hydroxylations and sulfoxidations. R-and S-omeprazole 5-hydroxylation activities in cynomolgus monkey and marmoset liver microsomes were significantly different among wild-type, heterozygous, and homozygous animals genotyped for cynomolgus monkey P450 2C19 p.[(Phe100Asn; Ala103Val; Ile112Leu)] and for marmoset P450 2C19 p.[(Phe7Leu; Ser254Leu; Ile469Thr)], respectively. The results of this study demonstrate polymorphic cynomolgus monkey and marmoset P450 2C19-dependent omeprazole oxidation activities with individual variations similar to humans.

Comprehensive evaluation of liver microsomal cytochrome P450 3A (CYP3A) inhibition: comparison of cynomolgus monkey and human

1. Members of the cytochrome P450 3A (CYP3A) subfamily metabolize numerous compounds and serve as the loci of drug–drug interactions (DDIs). Because of high amino acid sequence identity with human CYP3A, the cynomolgus monkey has been proposed as a model species to support DDI risk assessment.2. Therefore, the objective of this study was to evaluate 35 known inhibitors of human CYP3A using human (HLM) and cynomolgus monkey (CLM) liver microsomes. Midazolam was employed as substrate to generate IC50 values (concentration of inhibitor rendering 50% inhibition) in the absence and presence of a preincubation (30 mins) with NADPH.3. In the absence of preincubation, the IC50 values generated with CLM were similar to those obtained with HLM (86% within 2-fold; 100% within 3-fold difference). However, significant differences (up to 48-fold) in preincubation IC50 were observed with 17% of the compounds (raloxifene, bergamottin, nicardipine, mibefradil, ritonavir, and diltiazem).4. Our results indicate that in most cases the cynomolgus monkey can be a viable DDI model. However, significant species differences in time-dependent CYP3A inhibition can be observed for some compounds. In the case of raloxifene, such a difference can be ascribed to a specific CYP3A4 amino acid residue.

Interaction of isoflavonoids with human liver microsomal cytochromes P450: inhibition of CYP enzyme activities

1. The possibility of interaction of isoflavonoids with concomitantly taken drugs to determined isoflavonoids safety was studied. Inhibition of nine forms of cytochrome P450 (CYP3A4, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2C9, CYP2D6 and CYP2E1) by 12 isoflavonoids (daidzein, genistein, biochanin A, formononetin, glycitein, equol and six glucosides, daidzin, puerarin, genistin, sissotrin, ononin and glycitin) was studied systematically.2. The most potent inhibitors were genistein and daidzein inhibiting noncompetitively the CYP2C9 with Ki of 35.95 ± 6.96 and 60.56 ± 3.53 μmol/l and CYP3A4 (inhibited by genistein with Ki of 23.25 ± 5.85 μmol/l also by a noncompetitive mechanism). Potent inhibition of CYP3A4 was observed also with biochanin A (Ki of 57.69 ± 2.36 μmol/l) and equol (Ki of 38.47 ± 2.32 μmol/l).3. Genistein and daidzein inhibit noncompetitively CYP3A4 and CYP2C9. With plasma levels in micromolar range, a clinically important interaction with concomitantly taken drugs does not seem to be probable.

Mechanism-based inactivation of human cytochrome P450 1A2 and 3A4 isoenzymes by anti-tumor triazoloacridinone C-1305

1. 5-Dimethylaminopropylamino-8-hydroxytriazoloacridinone, C-1305, is a promising anti-tumor therapeutic agent with high activity against several experimental tumors.2. It was determined to be a potent and selective inhibitor of liver microsomal and human recombinant cytochrome P450 (CYP) 1A2 and 3A4 isoenzymes. Therefore, C-1305 might modulate the effectiveness of other drugs used in multidrug therapy.3. The objective of this study was to investigate the mechanism of the observed C-1305-mediated inactivation of CYP1A2 and CYP3A4.4. Our findings indicated that C-1305 produced a time- and concentration-dependent decrease in 7-ethoxycoumarin O-deethylation (CYP1A2, KI = 10.8 ± 2.14 μM) and testosterone 6β-hydroxylation (CYP3A4, KI = 9.1 ± 2.82 μM). The inactivation required the presence of NADPH, was unaffected by a nucleophilic trapping agent (glutathione) and a reactive oxygen species scavenger (catalase), attenuated by a CYP-specific substrate (7-ethoxycoumarin or testosterone), and was not reversed by potassium ferricyanide. The estimated partition ratios of 1086 and 197 were calculated for the inactivation of CYP1A2 and CYP3A4, respectively.5. In conclusion, C-1305 inhibited human recombinant CYP1A2 and CYP3A4 isoenzymes by mechanism-based inactivation. The obtained knowledge about specific interactions between C-1305 and/or its metabolites, and CYP isoforms would be useful for predicting the possible drug–drug interactions in potent multidrug therapy.

Trans-Resveratrol Enhances the Anticoagulant Activity of Warfarin in a Mouse Model.

Aim: Resveratrol is a popular ingredient in dietary supplements. Some patients concomitantly use dietary supplements and medicines in Japan. In the present study, we determined whether trans-resveratrol and melinjo (Gnetum gnemon L.) seed extract (MSE), which contains resveratrol dimers, interacted with drugs using a mouse model.Methods: Male C57BL/6J mice were fed experimental diets containing 0.005%, 0.05%, or 0.5% (w/w) trans-resveratrol or MSE for 1 or 12 weeks. The expression of liver cytochrome P-450 (CYP) mRNA and activity of liver microsomal CYP were measured. To determine the influence of resveratrol or MSE on drug efficacy, the anticoagulant activity of warfarin was examined in mice that were fed diets containing trans-resveratrol or MSE for 12 weeks.Results: When the mice were fed experimental diets for 1 week, none of the doses of trans-resveratrol and MSE affected body weight, liver weight, or plasma AST and ALT levels. Trans-resveratrol also did not affect CYP1A1, CYP1A2, CYP2C, or CYP3A activities. In contrast, 0.5% MSE slightly increased CYP1A1 activity. When the mice were fed experimental diets for 12 weeks, 0.05% trans-resveratrol increased CYP1A1, CYP2C, and CYP3A activities, whereas 0.5% MSE suppressed CYP3A activity. Under these conditions, 0.5% trans-resveratrol enhanced the anticoagulant activity of warfarin, although CYP2C activity increased. However, MSE did not affect the anticoagulant activity of warfarin.Conclusion: The 0.05% trans-resveratrol did not interact with warfarin in a mouse model, whereas 0.5% trans-resveratrol may have enhanced the anticoagulant activity of warfarin.

Evaluation of possible inhibition of human liver drug metabolizing cytochromes P450 by two new acetylcholinesterase oxime-type reactivators

Two non-symmetric bispyridine oxime – based reactivators of acetylcholinesterase enzyme (AChE), labeled as K027 (1-(4-carbamoylpyridinium)-3-(4-hydroxyiminomethylpyridinium)-propane dibromide) and K203 ((E)-1-(4- carbamoylpyridinium)-4-(4-hydroxyiminomethylpyridinium)-but-2-ene dibromide) were tested for their potential to inhibit activities of human liver microsomal cytochromes P450 (CYP). Both oximes are very potent reactivators of organophosphate-inhibited AChE.An interaction of both compounds with CYP in human liver microsomal preparation was detected using difference spectroscopy. The compounds were shown to bind to CYP enzymes with spectral binding constants of 5.04 ± 1.79 nM (K027) and 5.2 ± 2.6 nM (K203). Enzymology studies were subsequently performed aimed at determining which of the nine most important CYP involved in drug is affected by this interaction. The results have shown no prominent inhibition of individual CYP activities with either compounds except in the case of CYP2E1 and K203. Diagnostic Dixon plot revealed that K203 acted as an uncompetitive inhibitor of CYP2E1. Inhibition of this activity however is not as prominent as to make a potent drug interaction likely. Hence, the interaction of K027 and K203 oxime-type AChE reactivators with human liver microsomal CYP enzymes does not seem to be of prominent clinical importance and both compounds could be safely used in this respect as antidotes with low risk of drug interactions.

A Focal Adhesion Kinase-Derived Peptide Binds the Src SH3 Domain in Two Orientations, As Demonstrated Using Paramagnetic Nuclear Magnetic Resonance.

SH3 binding peptides contain polyproline helices and are classified according to their binding orientations as N-to-C-terminal or C-to-N-terminal. We have tested the hypothesis that such a peptide binds in both orientations but with different populations. A focal adhesion kinase (FAK)-derived peptide was tested for its binding orientation on the Src SH3 domain. Paramagnetic tags were introduced at several positions on the SH3 domain, and on the basis of the paramagnetic relaxation enhancements (PREs) of the amide protons, the positions of the paramagnetic centers were determined. Two peptides were synthesized with (13)C-enriched Ala or Pro, at the N-terminal or C-terminal side of the peptide, and the intermolecular PREs were measured. The results provide compelling evidence that the FAK-derived peptide binds the SH3 domain in two orientations. In the major state, the SH3 domain binds the peptide in the N-C orientation, whereas 20% of the time, the peptide binds in the C-N orientation. We conclude that the distinction between N-C and C-N orientations, which is based on crystal structures, might be artificial. The pseudosymmetric nature of the polyproline helix might allow for binding in both orientations in the solution state.

Caffeine 7-N-demethylation and C-8-oxidation mediated by liver microsomal cytochrome P450 enzymes in common marmosets

1. 3-N-Demethylation of caffeine (1,3,7-trimethylxanthine) is mediated by human cytochrome P450 1A2, whereas 7-N-demethylation and C-8-hydroxylation are reportedly catalyzed by monkey P450 2C9 and rat P450 1A2, respectively.2. Roles of marmoset P450 enzymes in caffeine oxidation were investigated using nine marmoset liver microsomes and 14 recombinantly expressed marmoset P450 enzymes.3. Predominant caffeine 7-N-demethylation and C-8-hydroxylation activities in marmoset liver microsomes were moderately (r = 0.78, p < 0.05) and highly (r = 0.82, p < 0.01) correlated with midazolam 1′-hydroxylation activities, respectively, while the former was not strongly affected by ketoconazole or α-naphthoflavone.4. Caffeine C-8-hydroxylation in liver microsomes was inhibited by ketoconazole and activated by α-naphthoflavone, suggesting main involvements of P450 3As.5. Recombinant marmoset P450 3As had high Vmax/Km values for C-8-hydroxylation, comparable to Km values for marmoset liver microsomes. Marmoset P450 1As efficiently mediated caffeine 3-N-demethylation and C-8-hydroxylation with apparently lower Km values than those of liver microsomes.6. These results collectively suggest highly active marmoset P450 3A enzymes toward caffeine 8-hydorxylaiton and involvement of multiple P450 isoforms including P450 1A in caffeine 7-N- and 3-N-demethylations in marmoset livers. Marmoset P450s have slightly different properties to human or monkey P450s regarding caffeine metabolic pathways.

Enantiospecific effects of chiral drugs on cytochrome P450 inhibition in vitro

1. The aim of this work was to examine the differences in the inhibitory potency of individual enantiomers and racemic mixtures of selected chiral drugs on human liver microsomal cytochromes P450.2. The interaction of enantiomeric forms of six drugs (tamsulosin, tolterodine, citalopram, modafinil, zopiclone, ketoconazole) with nine cytochromes P450 (CYP3A4, CYP2E1, CYP2D6, CYP2C19, CYP2C9, CYP2C8, CYP2B6, CYP2A6, CYP1A2) was examined. HPLC methods were used to estimate the extent of the inhibition of specific activity in vitro.3. Tamsulosin (TAM) and tolterodine (TOL) inhibited CYP3A4 activity with an enantiospecific pattern. The inhibition of CYP3A4 activity differed for R-TAM (Ki 2.88 ± 0.12 µM) and S-TAM (Ki 14.22 ± 0.53 µM) as well as for S-TOL (Ki 1.71 ± 0.03 µM) and R-TOL (Ki 4.78 ± 0.17 µM). Also, the inhibition of CYP2C19 by ketoconazole (KET) cis-enantiomers exhibited enantioselective behavior: the (+)-KET (IC50 23.64 ± 6.25 µM) was more potent than (−)-KET (IC50 66.12 ± 12.6 µM). The inhibition of CYP2C19 by modafinil (MOD) enantiomers (R-MOD IC50 = 51.79 ± 8.58 µM, S-MOD IC50 = 48.62 ± 9.74 µM) and the inhibition of CYP2D6 by citalopram (CIT) enantiomers (R-CIT IC50 = 68.17 ± 5.70 µM, S-CIT IC50 = 62.63 ± 7.89 µM) was not enantiospecific.4. Although enantiospecific interactions were found (TAM, TOL, KET), they are probably not clinically relevant as the plasma levels are generally lower than the drug concentration needed for prominent inhibition (at least 50% of CYP activity).

Further Characterization of the Metabolism of Desloratadine and Its Cytochrome P450 and UDP-glucuronosyltransferase Inhibition Potential: Identification of Desloratadine as a Relatively Selective UGT2B10 Inhibitor.

Desloratadine (Clarinex), the major active metabolite of loratadine (Claritin), is a nonsedating antihistamine used for the treatment of seasonal allergies and hives. Previously we reported that the formation of 3-hydroxydesloratadine, the major human metabolite of desloratadine, involves three sequential reactions, namely N-glucuronidation by UGT2B10 followed by 3-hydroxylation by CYP2C8 followed by deconjugation (rapid, nonenzymatic hydrolysis of the N-glucuronide). In this study we assessed the perpetrator potential of desloratadine based on in vitro studies of its inhibitory effects on cytochrome P450 and UDP-glucuronosyltransferase (UGT) enzymes in human liver microsomes (HLM). Desloratadine (10 µM) caused no inhibition (<15%) of CYP1A2, CYP2C8, CYP2C9, or CYP2C19 and weak inhibition (32-48%) of CYP2B6, CYP2D6, and CYP3A4/5. In cryopreserved human hepatocytes (CHH), which can form the CYP2C8 substrate desloratadine N-glucuronide, desloratadine did not inhibit the CYP2C8-dependent metabolism of paclitaxel or amodiaquine. Assessment of UGT inhibition identified desloratadine as a potent and relatively selective competitive inhibitor of UGT2B10 (Ki value of 1.3 μM). Chemical inhibition of UGT enzymes in HLM demonstrated that nicotine (UGT2B10 inhibitor) but not hecogenin (UGT1A4 inhibitor) completely inhibited the conversion of desloratadine (1 µM) to 3-hydroxydesloratadine in HLM fortified with both NADPH and UDP-glucuronic acid. 3-Hydroxydesloratadine formation correlated well with levomedetomidine glucuronidation (UGT2B10 marker activity) with a panel of individual CHH (r(2) = 0.72). Overall, the results of this study confirm the role of UGT2B10 in 3-hydroxydesloratadine formation and identify desloratadine as a relatively selective in vitro inhibitor of UGT2B10.