Is the unique benzodiazepine structure interacting with CYP enzymes to affect steroid synthesis in vitro?

The aim of this project was to investigate the endocrine disrupting effects of three γ-aminobutyric acid type A receptor (GABAAR) agonists, diazepam (DZ), oxazepam (OX) and alprazolam (AL) using the steroidogenic in vitro H295R cell line assay, a recombinant CYP17A1 assay, qPCR analysis and computational modelling. Similar effects for DZ and OX on the steroidogenesis were observed in the H295R experiment at therapeutically relevant concentrations. Progestagens and corticosteroids were increased up to 10 fold and androgens were decreased indicating CYP17A1 lyase inhibition. For DZ the inhibition on both the hydroxylase and lyase was confirmed by the recombinant CYP17A1 assay, whereas OX did not appear to directly affect the recombinant CYP17A1 enzyme. Androgens were decreased when exposing the H295R cells to AL, indicating a CYP17A1 lyase inhibition. However, this was not confirmed by the recombinant CYP17A1 assay but a down-regulation in gene expression was observed for StAR and CYP17A1. The present study showed that the three investigated benzodiazepines (BZDs) are rather potent endocrine disruptors in vitro, exerting endocrine effects close the therapeutic Cmax. Both direct and indirect effects on steroidogenesis were observed, but molecular modelling indicated no direct interactions between the heme group in the steroidogenic CYP enzymes and the unique diazepin structure. In contrast, physicochemical properties such as high log P, structure and molecular weight similar to that of steroids appeared to influence the endocrine disrupting abilities of the investigated pharmaceuticals in vitro. Docking of the three BZDs in CYP17A1 and CYP21A2 confirmed that shape complementarity and hydrophobic effects seem to determine the binding modes.

Estimation of the inhibiting impact of abiraterone D4A metabolite on human steroid 21-monooxygenase (CYP21A2).

Abiraterone D4A metabolite, the product of 3ß-hydroxysteroid dehydrogenase activity toward abiraterone, may serve as a potential antitumor agent for the treatment of prostate cancer. The main adverse effect of abiraterone is the disruption of corticosteroid biosynthesis, and the more pharmacologically active abiraterone D4A metabolite may have the same issues. We therefore estimated the inhibiting impact of the abiraterone D4A metabolite on one of the key corticosteroidogenic enzymes - human steroid 21-monooxygenase (CYP21A2). Molecular docking of D4A into the active site of CYP21A2 has been predicted to be similar to abiraterone binding with the enzyme. Abiraterone D4A metabolite, similar to abiraterone, induces type II spectral changes of CYP21A2. The spectral dissociation constant for the abiraterone D4A metabolite-CYP21A2 complex was calculated as 3.4 ±â€¯0.5 µM. Abiraterone D4A metabolite demonstrates competitive/mixed type CYP21A2 inhibition with an inhibitory constant of 1.8 ±â€¯0.8 µM, as obtained by Dixon plot. These results make it possible to predict the adverse effects of the new perspective candidate compound for antitumor therapy.

HIV Drug Efavirenz Inhibits CYP21A2 Activity with Possible Clinical Implications.

BACKGROUND: The HIV drugs lopinavir and ritonavir have recently been reported to cause transient adrenal insufficiency in preterm newborns. We, therefore, considered HIV drugs as a cause of transiently elevated 17-hydroxyprogesterone (17OHP) levels in a neonatal screening test for congenital adrenal hyperplasia in a preterm girl exposed to zidovudine, efavirenz, tenofovir, and emtricitabine. OBJECTIVE: So far, HIV drugs have not been tested for their effect on steroidogenesis and the steroidogenic enzyme activity of CYP21A2 specifically in an in vitro system. METHODS: We tested the effect of efavirenz, tenofovir, emtricitabine, and zidovudine on steroidogenesis of human adrenal H295R cells. Cells were treated with the drugs at different concentrations including concentrations in therapeutic use. The effect on CYP21A2 activity was assessed by testing the conversion of radiolabeled 17OHP to 11-deoxycortisol. Cell viability was tested by an MTT assay. In addition, recombinant human CYP21A2 protein was used to assess direct drug effects on CYP21A2 activity. RESULTS: We observed significantly decreased CYP21A2 activity in both in vitro testing systems after treatment with efavirenz at therapeutic concentrations. Moreover, efavirenz affected cell viability. By contrast, the other test drugs did not affect steroidogenesis. Follow-up of our patient revealed elevated 17OHP and androgen levels during the first weeks of life, but values normalized spontaneously. Genetic testing for CYP21A2 mutations was negative. Thus, it remains unsettled whether the transient 17OHP elevation in this baby was due to a drug effect. CONCLUSION: The HIV drug efavirenz inhibits CYP21A2 activity in vitro through direct interaction with enzyme catalysis at therapeutic concentrations. This may have clinical implications for HIV treatment in children and adults. However, so far, clinical data are scarce, and further studies are needed to be able to draw clinical conclusions.

Structure-Based Design of Inhibitors with Improved Selectivity for Steroidogenic Cytochrome P450 17A1 over Cytochrome P450 21A2.

Inhibition of androgen biosynthesis is clinically effective for treating androgen-responsive prostate cancer. Abiraterone is a clinical first-in-class inhibitor of cytochrome P450 17A1 (CYP17A1) required for androgen biosynthesis. However, abiraterone also causes hypertension, hypokalemia, and edema, likely due in part to off-target inhibition of another steroidogenic cytochrome P450, CYP21A2. Abiraterone analogs were designed based on structural evidence that B-ring substituents may favorably interact with polar residues in binding CYP17A1 and sterically clash with residues in the CYP21A2 active site. The best analogs increased selectivity of CYP17A1 inhibition up to 84-fold compared with 6.6-fold for abiraterone. Cocrystallization with CYP17A1 validated the intended new contacts with CYP17A1 active site residues. Docking these analogs into CYP21A2 identified steric clashes that likely underlie decreased binding and CYP21A2 inhibition. Overall, these analogs may offer a clinical advantage in the form of reduced side effects.

CYP17A1 inhibitor abiraterone, an anti-prostate cancer drug, also inhibits the 21-hydroxylase activity of CYP21A2.

Abiraterone is an inhibitor of CYP17A1 which is used for the treatment of castration resistant prostate cancer. Abiraterone is known to inhibit several drug metabolizing cytochrome P450 enzymes including CYP1A2, CYP2D6, CYP2C8, CYP2C9, CYP2C19, CYP3A4 and CYP3A5, but its effects on steroid metabolizing P450 enzymes are not clear. In preliminary results, we had observed inhibition of CYP21A2 by 1µM abiraterone. Here we are reporting the effect of abiraterone on activities of CYP21A2 in human adrenal cells as well as with purified recombinant CYP21A2. Cells were treated with varying concentrations of abiraterone for 24h and CYP21A2 activity was measured using [3H] 17-hydroxyprogesterone as substrate. Whole steroid profile changes were determined by gas chromatography-mass spectrometry. Binding of abiraterone to purified CYP21A2 protein was measured spectroscopically. Computational docking was used to study the binding and interaction of abiraterone with CYP21A2. Abiraterone caused significant reduction in CYP21A2 activity in assays with cells and an inhibition of CYP21A2 activity was also observed in experiments using recombinant purified proteins. Abiraterone binds to CYP21A2 with an estimated Kd of 6.3µM. These inhibitory effects of abiraterone are at clinically used concentrations. A loss of CYP21A2 activity in combination with reduction of CYP17A1 activities by abiraterone could result in lower cortisol levels and may require monitoring for any potential adverse effects.

Suppressed Sex Hormone Biosynthesis by Alkylresorcinols: A Possible Link to Chemoprevention.

Alkylresorcinols (ARs, 5-n-alkylresorcinols) are amphiphilic phenolic lipids in whole grain rye and wheat, with a long odd-numbered carbon chain. A preventive effect of whole grain diet on sex hormone-dependent cancers has been recognized, but the active component(s) or mechanisms are not known. We have investigated the effects of the ARs C15:0, C19:0, and C21:0, individually and in combination, on steroid hormone production by using the human adrenocortical cell line H295R. Decreased synthesis of dehydroepiandrosterone (DHEA), testosterone, and estradiol was demonstrated at low concentrations of C15:0 and C19:0. There were no indications of additive effects on steroid secretion from the combined treatment with equimolar concentrations of the three ARs. Gene expressions of CYP21A2, HSD3B2, and CYP19A1 were downregulated and CYP11A1 was upregulated by the ARs. The results on gene expression could not explain the effects on steroidogenesis, which may be due to direct effects on enzyme activities, such as inhibition of CYP17A1. Our results demonstrate suppressed synthesis of testosterone and estradiol by ARs suggesting a novel mechanism for ARs in the chemoprevention of prostate and breast cancer.

Novel functionalized 5-(phenoxymethyl)-1,3-dioxane analogs exhibiting cytochrome P450 inhibition: a patent evaluation WO2015048311 (A1).

Cytochrome P450's (CYP's) constitute a diverse group of over 500 monooxygenase hemoproteins, catalyzing transformations that involve xenobiotic metabolism, steroidogenesis and other metabolic processes. Over-production of the steroid hormone cortisol is implicated in the progression of diseases such as diabetes, heart failure and hypertension, stroke, Cushing's syndrome, obesity and renal failure, among others. The biosynthesis of cortisol involves a cascade of cholesterol metabolizing reactions regulated through three major CYP proteins: 17α-hydroxylase-C17/20-lyase (CYP17), 21-hydroxylase (CYP21), and 11ß-hydroxylase (CYP11B1). Excess activities of these enzymes are linked to the progression of malignancies including prostate, breast, ovarian, and uterine cancers. A series of novel functionalized dioxane analogs have been developed and recently patented as CYP17, CYP21, and CYP11B1 inhibitors, which lead to the modulation of cortisol production as a method for treating, delaying, slowing, and inhibiting the implicated diseases. The findings disclosed in this patent have been analyzed and compared with the literature data on inhibitors of CYP17, CYP21, and CYP11B1. The compiled data provide insight into the novel functionality of the compounds described in the patent. In this regard, an objective opinion on the effectiveness and novel biochemistry of these compounds in comparison to current CYP inhibitors used in the treatment of cortisol-related diseases is presented in this paper.

Inhibitory effect of Aristolochia fruit on Cytochrome P450 isozymes in vitro and in vivo.

The mature fruits of Aristolochia debilis, known in China by the name, "Madouling" has been popularly prescribed in Asia, particularly in China, to treat a range of conditions including gynaecological problems, arthritis and wound healing. This study was aimed to evaluate the potential effect of Madouling on the cytochrome P450 (CYP) isozymes in vitro in microsomal fractions and in vivo in rats. The influence of Madouling on CYPs activity was first explored by an in vitro method of estimating levels of four respective metabolites in rat liver microsomes. The results were re-examined in vivo in rats by using a cocktail approach involving the probe drugs theophylline, tolbutamide, chlorzoxazone and dapsone. Pharmacokinetics of the four substrates was used to analyze the activities of the targeting isozymes. In vitro study revealed that Madouling decreased the activity of CYP1A2, 3A1 and 2E1. However, no significant influence on CYP2C6 was found. These results coincided with those of in vivo study to a great degree except that in vivo estimation the herb didn't inhibit CYP1A2 significantly. From the data obtained, Madouling is suggested as a candidate for clinically significant CYP interactions. Drug co-administrated with Madouling may need dose adjustment.

Assessment of the potential of polyphenols as a CYP17 inhibitor free of adverse corticosteroid elevation.

Inhibition of 17α-hydroxylase/17,20-lyase (CYP17), which dictates the proceeding of androgen biosynthesis, is recommended as an effective treatment for androgen-dependent diseases. However, androgen depletion by selective CYP17 inhibition is accompanied with corticosteroid elevation, which increases risk of cardiovascular diseases. In this study, we evaluated the likelihood of polyphenols as a CYP17 inhibitor without cardiovascular complications. All examined polyphenols significantly inhibited CYP17 in human adrenocortical H295R cells, but their effects on androgen and cortisol biosynthesis were diverse. Resveratrol was the most potent CYP17 inhibitor with an approximate IC50 of 4 µM, and the inhibition might weigh on the 17α-hydroxylase activity more than the 17,20-lyase activity. Resveratrol also inhibited 21α-hydroxylase (CYP21) essential for corticosteroid biosynthesis but to a lesser extent, thus preventing the occurrence of cortisol elevation following CYP17 blockade. Although transcriptional down-regulation was important for α-naphthoflavone-mediated CYP17 inhibition, resveratrol inhibited CYP17 and CYP21 mainly at the level of enzyme activity rather than enzyme abundance and cytochrome P450 electron transfer. Daidzein also inhibited CYP17 and CYP21 although less potent than resveratrol. Daidzein was the only polyphenol showing inhibition of 3ß-hydroxysteroid dehydrogenase type II (3ßHSD2). The exceptional 3ßHSD2 inhibition led to dehydroepiandrosterone accumulation alongside daidzein-caused androgen biosynthetic impairment. In contrast, androgen and cortisol secretion was increased or remained normal under α-naphthoflavone and ß-naphthoflavone treatments, suggesting that CYP17 inhibition was counteracted by increased substrate generation. α-naphthoflavone and ß-naphthoflavone also enhanced the formation of cortisol from 17-hydroxyprogesterone and testosterone from androstenedione. Our findings suggest a potential application of resveratrol in androgen deprivation therapy.

Evidence that adrenal hexose-6-phosphate dehydrogenase can effect microsomal P450 cytochrome steroidogenic enzymes.

The role of adrenal hexose-6-phosphate dehydrogenase in providing reducing equivalents to P450 cytochrome steroidogenic enzymes in the endoplasmic reticulum is uncertain. Hexose-6-phosphate dehydrogenase resides in the endoplasmic reticulum lumen and co-localizes with the bidirectional enzyme 11ß-hydroxysteroid dehydrogenase 1. Hexose-6-phosphate dehydrogenase likely provides 11ß-hydroxysteroid dehydrogenase 1 with NADPH electrons via channeling. Intracellularly, two compartmentalized reactions generate NADPH upon oxidation of glucose-6-phosphate: cytosolic glucose-6-phosphate dehydrogenase and microsomal hexose-6-phosphate dehydrogenase. Because some endoplasmic reticulum enzymes require an electron donor (NADPH), it is conceivable that hexose-6-phosphate dehydrogenase serves in this capacity for these pathways. Besides 11ß-hydroxysteroid dehydrogenase 1, we examined whether hexose-6-phosphate dehydrogenase generates reduced pyridine nucleotide for pivotal adrenal microsomal P450 enzymes. 21-hydroxylase activity was increased with glucose-6-phosphate and, also, glucose and glucosamine-6-phosphate. The latter two substrates are only metabolized by hexose-6-phosphate dehydrogenase, indicating that requisite NADPH for 21-hydroxylase activity was not via glucose-6-phosphate dehydrogenase. Moreover, dihydroepiandrostenedione, a non-competitive inhibitor of glucose-6-phosphate dehydrogenase, but not hexose-6-phosphate dehydrogenase, did not curtail activation by glucose-6-phosphate. Finally, the most compelling observation was that the microsomal glucose-6-phosphate transport inhibitor, chlorogenic acid, blunted the activation by glucose-6-phosphate of both 21-hydroxylase and 17-hydroxylase indicating that luminal hexose-6-phosphate dehydrogenase can supply NADPH for these enzymes. Analogous kinetic observations were found with microsomal 17-hydroxylase. These findings indicate that hexose-6-phosphate dehydrogenase can be a source, but not exclusively so, of NADPH for several adrenal P450 enzymes in the steroid pathway. Although the reduced pyridine nucleotides are produced intra-luminally, these compounds may also slowly transverse the endoplasmic reticulum membrane by unknown mechanisms.

The influence of rosuvastatin on liver microsomal CYP2C6 in hereditary hypertriglyceridemic rat.

OBJECTIVES: The aim of this study was to investigate whether rosuvastatin affects expression and activity of rat CYP2C6. This cytochrome P450 is considered to be a counterpart of human CYP2C9, which metabolizes many drugs, including diclofenac, ibuprofen or warfarin. DESIGN: Male hereditary hypertriglyceridemic (HHTg) rats were fed standard laboratory diet (STD) or high cholesterol diet (HCD: STD + 1% of cholesterol w/w + 10% of lard fat w/w) for 21 days. A third group of rats were fed high a cholesterol diet with rosuvastatin added (0.03% w/w). Expression of CYP2C6 was measured in liver samples using real-time PCR (mRNA level) and Western blotting (protein level). Formation of diclofenac metabolites (typical enzyme activity of CYP2C6) was analyzed using HPLC with UV detection. RESULTS: Administration of rosuvastatin to HHTg rats resulted in significantly increased mRNA expression and enzyme activity in HCD-fed animals; changes of CYP2C6 protein were non-significant. These results suggest that CYP2C6 expression and activity are positively affected by rosuvastatin in hereditary hypertriglyceridemic rats after intake of HCD. CONCLUSION: The results presented open the possibility that in humans, rosuvastatin may affect the metabolism of many drugs by influencing expression and activity of CYP2C6 (counterpart of human CYP2C9). Further studies are needed to elucidate the effects of this statin on CYP2C9 in humans.

The influence of Aspalathus linearis (Rooibos) and dihydrochalcones on adrenal steroidogenesis: quantification of steroid intermediates and end products in H295R cells.

The steroid hormone output of the adrenal gland is crucial in the maintenance of hormonal homeostasis, with hormonal imbalances being associated with numerous clinical conditions which include, amongst others, hypertension, metabolic syndrome, cardiovascular disease, insulin resistance and type 2 diabetes. Aspalathus linearis (Rooibos), which has been reported to aid stress-related symptoms linked to metabolic diseases, contains a wide spectrum of bioactive phenolic compounds of which aspalathin is unique. In this study the inhibitory effects of Rooibos and the dihydrochalcones, aspalathin and nothofagin, were investigated on adrenal steroidogenesis. The activities of both cytochrome P450 17α-hydroxylase/17,20 lyase and cytochrome P450 21-hydroxylase were significantly inhibited in COS-1 cells. In order to study the effect of these compounds in H295R cells, a human adrenal carcinoma cell line, a novel UPLC-MS/MS method was developed for the detection and quantification of twenty-one steroid metabolites using a single chromatographic separation. Under both basal and forskolin-stimulated conditions, the total amount of steroids produced in H295R cells significantly decreased in the presence of Rooibos, aspalathin and nothofagin. Under stimulated conditions, Rooibos decreased the total steroid output 4-fold and resulted in a significant reduction of aldosterone and cortisol precursors. Dehydroepiandrosterone-sulfate levels were unchanged, while the levels of androstenedione (A4) and 11ß-hydroxyandrostenedione (11ßOH-A4) were inhibited 5.5 and 2.3-fold, respectively. Quantification of 11ßOH-A4 showed this metabolite to be a major product of steroidogenesis in H295R cells and we confirm, for the first time, that this steroid metabolite is the product of the hydroxylation of A4 by human cytochrome P450 11ß-hydroxylase. Taken together our results demonstrate that Rooibos, aspalathin and nothofagin influence steroid hormone biosynthesis and the flux through the mineralocorticoid, glucocorticoid and androgen pathways, thus possibly contributing to the alleviation of negative effects arising from elevated glucocorticoid levels.

[Metabolism of nicousamide in rat and human liver in vitro].

This paper is aimed to study the metabolic kinetics of nicousamide in rat liver microsomes and cytosol and to identify the major metabolite and drug metabolizing enzymes involved in the metabolism of nicousamide in rat and human liver microsomes by selective inhibitors in vitro. The concentration of nicousamide was determined by HPLC-UV method. The metabolite of nicousamide in rat and human liver microsomes was isolated and identified by LC-MS/MS. The major metabolite of nicousamide in rat and human liver microsomes was identified to be 3-(3'-carboxy-4'-hydroxy-anilino-carbo-)-6-amino-7-hydroxy-8-methyl-coumarin (M1). The metabolite of nicousamide in rat plasma, urine, bile and liver was consistent with M1. The metabolism of nicousamide can be catalyzed by several reductases, including CYP450 reductases, cytochrome b5 reductases and CYP2C6 in rat liver microsomes, as well as xanthine oxidase and DT-diaphorase in rat liver cytosol.

Differential inhibition of CYP17A1 and CYP21A2 activities by the P450 oxidoreductase mutant A287P.

P450 oxidoreductase (POR) has a pivotal role in facilitating electron transfer from nicotinamide adenine dinucleotide phosphate to microsomal cytochrome P450 (CYP) enzymes, including the steroidogenic enzymes CYP17A1 and CYP21A2. Mutations in POR have been shown recently to cause congenital adrenal hyperplasia with apparent combined CYP17A1 and CYP21A2 deficiency that comprises a variable clinical phenotype, including glucocorticoid deficiency, ambiguous genitalia, and craniofacial malformations. To dissect structure-function relationships potentially explaining this phenotypic diversity, we investigated whether specific POR mutations have differential effects on CYP17A1 and CYP21A2. We compared the impact of missense mutations encoding for single amino acid changes in three distinct regions of the POR molecule: 1), Y181D and H628P close to the central electron transfer area, 2) S244C located within the hinge close to the flavin adenine dinucleotide and flavin mononucleotide domains of POR, and 3) A287P that is clearly distant from the two other regions. Functional analysis using a yeast microsomal assay with coexpression of human CYP17A1 or CYP21A2 with wild-type or mutant human POR revealed equivalent decreases in CYP17A1 and CYP21A2 activities by Y181D, H628P, and S244C. In contrast, A287P had a differential inhibitory effect, with decreased catalytic efficiency (Vmax/Km) for CYP17A1, whereas CYP21A2 retained near normal activity. In vivo analysis of urinary steroid excretion by gas chromatography/mass spectrometry in 11 patients with POR mutations showed that A287P homozygous patients had the highest corticosterone/cortisol metabolite ratios, further indicative of preferential inhibition of CYP17A1. These findings provide novel mechanistic insights into the redox regulation of human steroidogenesis. Differential interaction of POR with electron-accepting CYP enzymes may explain the phenotypic variability in POR deficiency, with additional implications for hepatic drug metabolism by POR-dependant CYP enzymes.

Interactions between neuroleptics and CYP2C6 in rat liver--in vitro and ex vivo study.

The aim of the present study was to investigate the influence of classic and atypical neuroleptics on the activity of rat CYP2C6 measured as a rate of warfarin 7-hydroxylation. The reaction was studied in control liver microsomes in the presence of neuroleptics, as well as in microsomes of rats treated intraperitoneally for one day or two weeks (twice a day) with pharmacological doses (mg/kg) of the drugs (promazine, levomepromazine, thioridazine, perazine 10, chlorpromazine, haloperidol 0.3, risperidone 0.1, sertindole 0.05), in the absence of the neuroleptics in vitro. Some of the neuroleptics added in vitro to control liver microsomes decreased the activity of CYP2C6. Sertindole and levomepromazine (Ki = 25 and 31 microM, respectively) were the most potent inhibitors of the rat CYP2C6 among the drugs studied. Their effects were more pronounced than those of the other phenothiazines tested: thioridazine and chlorpromazine (Ki = 88 and 91 microM, respectively), promazine and perazine (Ki = 322 and 341 microM, respectively), risperidone (Ki = 414 microM) or haloperidol (Ki = 606 microM). The investigated neuroleptics--when given to rats in vivo for one day or two weeks--did not produce any indirect effect on CYP2C6 via other mechanisms, except for levomepromazine, which increased the activity of the enzyme after 24-h exposure. Therefore, the direct inhibitory effect of levomepromazine on CYP2C6 may be attenuated by an indirect mechanism at the beginning of the neuroleptic therapy. In summary, the obtained results show direct inhibitory effects of some phenothiazine neuroleptics and sertindole on the activity of CYP2C6 in vitro in rat liver microsomes. Considering relatively high pharmacological doses and therapeutic concentrations of phenothiazines, it seems that the inhibitory effect of levomepromazine (and other phenothiazines with Ki values below 100 microM) found in vitro may be of physiological and pharmacological importance in vivo.

Phytoestrogen resveratrol suppresses steroidogenesis by rat adrenocortical cells by inhibiting cytochrome P450 c21-hydroxylase.

BACKGROUND AND AIM: The phytoestrogen resveratrol is found in grapes, mulberries and peanuts, all of which are consumed regularly by humans. Resveratrol is also used in chemotherapy against cancer and aging and as a cardioprotectant. The aim of the present study was to characterize the effects of resveratrol on rat adrenal steroidogenesis and to study the underlying mechanism. METHODS: Adrenocortical cells were isolated from the adrenal glands of normal male rats (in vitro) and from male rats administered resveratrol in their diet for 12 weeks (ex vivo). Cells from resveratrol-treated and non-treated rats were tested ex vivo for responsiveness to ACTH and cells from normal rats were tested in vitro for responsiveness to ACTH in the presence and absence of resveratrol. Corticosterone and progesterone production were measured by RIA and expression of steroidogenic enzymes analyzed by PAGE/Western blotting. RESULTS: Corticosterone production was inhibited 47% by 50 microM resveratrol in vitro and 20% ex vivo, while progesterone production was elevated to 400% of the control value in in vitro experiments. Resveratrol treatment decreased adrenal cytochrome P450 c21-hydroxylase expression in vivo and cell culture conditions. No changes in cell viability or morphology were caused by exposure to resveratrol in both ex vivo and in vitro experiments. CONCLUSION: Resveratrol suppresses corticosterone production by primary rat adrenocortical cell cultures in vitro and ex vivo by inhibiting cytochrome P450 c21-hydroxylase.

Cytochrome p450 2C inhibition reduces post-ischemic vascular dysfunction.

Cytochrome p450 (CYP) inhibitors provide protection against myocardial infarction following both global and focal cardiac ischemia and reperfusion (I/R). We hypothesized that sulfaphenazole, an inhibitor of CYP2C6 and 9, also attenuates post-ischemic endothelial dysfunction by reducing CYP-mediated superoxide generation (which scavenges nitric oxide (NO)), thereby restoring NO bioavailability and vascular tone. Rat hearts were perfused in the Langendorff mode for 20 min in the presence, or absence, of sulfaphenazole and then subjected to 30 min global no-flow ischemia followed by 15 min reperfusion. Septal coronary resistance arteries were isolated and mounted on glass cannulae for measurements of luminal diameter. Preconstricted arteries were exposed to acetylcholine to elicit endothelium-dependent, NO-mediated vasodilation. Acetylcholine caused near maximal dilation in control tissues not subjected to I/R. Following I/R, endothelium-dependent vasodilation was reduced. Pretreatment with sulfaphenazole restored endothelial sensitivity to acetylcholine. Vasoresponsiveness to endothelium-independent vasodilators, sodium nitroprusside and isoproterenol, were also reduced following I/R. However, sensitivity to endothelium-independent vasodilators was not restored by pretreatment with sulfaphenazole. I/R-induced superoxide production was assessed by dihydroethidium staining of flash frozen hearts. Sulfaphenazole treatment significantly reduced superoxide production in arterial walls following I/R injury. We conclude that sulfaphenazole restores post-ischemic endothelium-dependent, NO-mediated vasodilation by reducing superoxide production, suggesting that CYP2C9 plays a key role in post-ischemic vascular dysfunction.

Mechanism of inhibition of cytochrome P450 C21 enzyme activity by autoantibodies from patients with Addison's disease.

OBJECTIVE: To study possible mechanisms for the inhibition of cytochrome P450 C21 (steroid 21-hydroxylase) enzyme activity by P450 C21 autoantibodies (Abs) in vitro. DESIGN: Two possible mechanisms for the inhibition of P450 C21 enzyme activity by P450 C21 Abs were studied: (a) conformational changes in the P450 C21 molecule induced by Ab binding and (b) the effects of Ab binding to P450 C21 on the electron transfer from the nicotinamide adenine dinucleotide phosphate reduced (NADPH) cytochrome P450 reductase (CPR) to P450 C21. METHODS: The effect of P450 C21 Ab binding on the conformation of recombinant P450 C21 in yeast microsomes was studied using an analysis of the dithionite-reduced CO difference spectra. The effect of P450 C21 Abs on electron transfer was assessed by analysis of reduction of P450 C21 in the microsomes in the presence of CO after addition of NADPH. RESULTS: Our studies confirmed the inhibiting effect of P450 C21 Abs on P450 C21 enzyme activity. Binding of the Abs did not induce significant change in the P450 C21 peak at 450nm (native form) and did not produce a detectable peak at 420 nm (denatured form) in the dithionite-reduced CO difference spectra. This indicated that conformation of P450 C21 around the heme was not altered compared with the native structure. However, incubation of the P450 C21 in yeast microsomes with P450 C21 Ab inhibited the fast phase electron transfer from the CPR to P450 C21. CONCLUSIONS: Our observations suggested that the mechanism by which P450 C21 Abs inhibit P450 C21 enzyme activity most likely involves inhibition of the interaction between the CPR and P450 C21.

Greater than the sum of its parts: combining models for useful ADMET prediction.

In silico ADMET (absorption, distribution, metabolism, excretion, and toxicity) models are important tools in combating late-stage attrition in the drug discovery process. This work shows how ADMET models can be combined to tailor predictions depending on one's needs. We demonstrate how the judicious use of data and considered combination of predictions can produce models that provide truly useful answers. This approach is illustrated with the prediction of hERG channel blocking and cytochrome P450 2D6 inhibition, where combination of two predictive models (with >80% of compounds correctly predicted) resulted in models with even better predictive values (with >90% of compounds correctly predicted for those classes of interest).

Selectivities of human cytochrome P450 inhibitors toward rat P450 isoforms: study with cDNA-expressed systems of the rat.

The aim of this study was to determine the selectivities of chemical inhibitors for human cytochrome P450 (P450) isoforms toward the corresponding rat P450 isoforms by using cDNA-expressed rat P450s (CYP1A2, CYP2A1, CYP2C6, CYP2C11, CYP2D2, CYP2E1, CYP3A1, and CYP3A2). Among the inhibitor probes for human P450s used in this study, only sulfaphenazole showed a selective inhibitory effect on the activity of the corresponding rat P450 isoform (CYP2C6). Furafylline also preferentially inhibited the activity of rat CYP1A2. However, methoxalen and ketoconazole more strongly inhibited the activities of other P450 isoforms than those of the corresponding rat P450 isoforms, CYP2A1 and CYP3A1/2, respectively. On the other hand, quinidine and aniline had little effect on the activities of the corresponding rat P450 isoforms, CYP2D2, and rat CYP2E1, respectively. These results suggest that chemical probes that have been used for human P450 isoforms do not always exhibit the same selectivity for the corresponding rat P450 isoforms. However, it appears that sulfaphenazole can be used as a selective inhibitor for rat CYP2C6. In addition, furafylline may also be a relatively selective inhibitor for rat CYP1A2.