Cytochrome P450 Complex Research Articles

Quantitative Determination of Oxalic and Phytic Acids of Lippia Multiflora Moldenke (Verbenaceae) Leaves and In Silico Study of Their Interaction with Haemoglobin S and 2,3-DPG-Mutase

The aim of this study is to measure oxalic and phytic acids from the leaves of Lippia multiflora and to evaluate their anti-sickle cell activity in silico. The histological characterization of the leaves of L. multiflora was performed by optical micrography. Theseanti-nutritional factors oxalic acid and phytic acid were determined by the standard method. Discovery Studio, ChemDraw and Autodock Vina software were used in molecular modeling. The pharmacokinetic and toxicological profile was evaluated using the bioinformatics descriptor SWISSADME. Histological analysis shows that fibres, sclerites and suber fragments are characteristic tissues of L. multiflora leaves. These leaves contain oxalic acid (insoluble oxalates: 51.457±27.653 mg/100 g DM; total oxalates: 59.483±0.704 mg/100 g MS; calcium oxalates: 0.216±0.0021 mg/100 g MS) and phytic acid (18.400±5.062 mg/100 g MS). Oxalic acid interacts more effectively with 2,3-diphospgoglycerate mutase (ΔG=-5.30±0.03 kcal/mol) while phytic acid interacts more strongly with deoxyhemoglobin S (ΔG=-9.30±0.01 kcal/mol) in silico. Phytic acid interacts with deoxyhemoglobin S by two hydrogen bonds (Arginine 31 and Lysine 120) while oxalic acid forms five hydrogen bonds with 2,3-DPG mutase (Arginine 10, Asparagine 17; Arginine 62; Histidine 188 and Glycine 189). The pharmacopharmacokinetic profile shows that these compounds do not inhibit cytochrome P450 complex enzymes and are not toxic. Both compounds represent added value for L. multiflora in the symptomatic treatment of sickle cell disease. In addition to attenuating hypersideremia, they can inhibit erythrocyte sickle formation via interaction with both protein receptors.

Allelic and Genotype Frequencies of CYP2B6∗2 (64C > T) and CYP2B6∗3 (777C > A) in Three Dominant Ethnicities of the Iranian Population.

Cytochrome P450 complex plays a key role in drug metabolism. CYP2B6 has an essential part in Cytochrome P450 complex metabolism. This study aims to determine the allelic distribution of CYP2B6∗2 and CYP2B6∗3 in three main Iranian ethnicities: Fars, Turk, and Kurd. The study was conducted on 174 unrelated healthy volunteers from three main Iranian ethnicities. After DNA extraction from peripheral blood samples, genotyping of CYP2B6∗2 and ∗3 was performed using tetra ARMS and ARMS PCR, respectively. The average age of 174 cases was 40.69 ± 11.87 (mean ± SD) and 39.06 ± 11.63 (mean ± SD) for males and females. In the CYP2B6∗2 variant, the genotyping frequency of wild type (C/C), heterozygous (C/T), and homozygous mutant (T/T) was 8.7%, 86%, and 5.2%, respectively. The CYP2B6∗2 (c.64C > T) allele frequency was 48.2% (95% CI: (37.8-58.6)). In the CYP2B6∗3 variant, the frequency of wild type (C/C), heterozygous (C/T), and homozygous mutant (T/T) was 75.3%, 11%, and 13.6%, respectively. The CYP2B6∗3 (c.777C > A) allelic frequency was 19.1% (95% CI: (17.5-20.7)). Allelic distribution in three main Iranian ethnicities, i.e., Turk, Kurd, and Fars, is remarkably higher than that in other populations, even that in Southern Iran. High frequencies of CYP2B6∗2 and ∗3 in the Iranian population highly affect drug responsiveness. Understanding such variability could help to increase drug efficacy and reduce its toxicity.

<em>In silico</em> pharmacokinetic and toxicological study of Flavone analogues

Flavone analogs are natural compounds of the flavonoid class that have a wide range of biological activities. The present study aimed to predict, with the aid of in silico methodologies, the oral bioavailability and pharmacokinetic and toxicological analyzes for three flavone analogues (apigenin, chrysin and luteonlin). The study revealed that the analogues have good oral availability, favorable pharmacokinetic and toxicological parameters. The Virtual Screening performed to predict oral bioavailability revealed that all analogues did not violate Lipinski's Rule. The in silico pharmacokinetic study revealed that all analogues have high intestinal absorption, do not cross the blood-brain barrier, are permeable by Caco-2 cells and do not inhibit P-glycoprotein. The in silico ADME study showed that all analogues inhibit the enzymes of the cytochrome P450 complex (CYP4501A2, CYP4502C9, CYP4502C19, CYP4503A4) and not only the CYP4502D6 enzyme. The in silico Toxicology study indicated that the analogues do not show toxicity by the AMES Test and are not carcinogenic. Apigenin and chrysin have low toxicity, while luteolin has moderate toxicity.

<em>In silico</em> pharmacokinetic and toxicological study of Cinnamic Acid analogues

Cinnamic acid analogs are natural phenolic compounds that have a wide range of biological and therapeutic activities. The present work aimed to predict, through in silico methodologies, the oral bioavailability and pharmacokinetic and toxicological analyzes for four cinnamic acid analogues (caffeic acid, ferulic acid, p-coumaric acid and synaptic acid). The study revealed that the analogues have good oral bioavailability, favorable pharmacokinetic and toxicological parameters. The Virtual Screening performed to predict oral bioavailability indicated that all analogues do not violate Lipinski's Rule. The in silico ADME study of pharmacokinetic parameters showed that all derivatives have high intestinal absorption, are permeable by Caco-2 cells, do not cross the blood-brain barrier, do not inhibit P-glycoprotein. There will be no inhibition of the cytochrome P450 complex isoenzymes (CYP450). The in silico Toxicological study revealed that the analogues do not have toxicity by the AMES Test, are not carcinogenic and do not present acute oral toxicity.

Evaluation of parental and transgenerational effects of clotrimazole in Daphnia magna — A multi-parametric approach

Azole antifungals inhibit the cytochrome P450 complex, decreasing the production of ergosterol in fungi, and compromising the biosynthesis of ecdysteroids in crustaceans, which are hormones regulating reproduction and ecdysis. The azole antifungal clotrimazole (CLO) raises environmental concerns due its toxicity. This work evaluated the effects on the number of moults, feeding rate, growth, reproduction, transgenerational reproductive effects on two different generations (F0, parental generation; and F1, organisms born from F0), and energetic balance in Daphnia magna. Neonates (<24 h) were exposed to sublethal concentrations (0, 2.7, and 3.4 mg/L) of CLO, to assess its effects on the moulting process. Neonates were also exposed to environmentally realistic concentrations of CLO (0, 30, 150, 750, and 3750 ng/L) for 24 and 96 h, to assess adverse effects on their feeding behaviour. Effects on energy reserves (fatty acids, glycogen, and protein levels) were also measured in animals exposed to CLO. A reproduction test was carried out to evaluate the amount and size of neonates from F0 and F1 generations. CLO exposure decreased the number of moults, and the size of organisms, but did not alter the feeding pattern of 5 days old individuals. However, neonates (<24 h) exposed to CLO had a significant decrease in their feeding pattern. CLO decreased the fatty acids content in exposed animals, but did not change glycogen and protein. CLO also decreased the size of adult daphnids from the third brood, born from animals exposed in F0; in F1 animals, the size of neonates from the third brood was decreased. This study evidenced the toxic effects caused by CLO on growth, feeding and reproduction of D. magna. Nevertheless, it is not possible to conclude whether the effects are due to the inhibition of cytochrome P450 enzymes, or to unspecific effects caused by general toxic stress and decreased nutrition.

Elucidating the biodegradation pathway and catabolic genes of benzophenone-3 in Rhodococcus sp. S2-17

A new bacterium, Rhodococcus sp. S2-17, which could completely degrade an emerging organic pollutant, benzophenone-3 (BP-3), was isolated from contaminated sediment through an enrichment procedure, and its BP-3 catabolic pathway and genes were identified through metabolic intermediate and transcriptomic analyses and biochemical and genetic studies. Metabolic intermediate analysis suggested that strain S2-17 may degrade BP-3 using a catabolic pathway progressing via the intermediates BP-1, 2,4,5-trihydroxy-benzophenone, 3-hydroxy-4-benzoyl-2,4-hexadienedioic acid, 4-benzoyl-3-oxoadipic acid, 3-oxoadipic acid, and benzoic acid. A putative BP-3 catabolic gene cluster including cytochrome P450, flavin-dependent oxidoreductase, hydroxyquinol 1,2-dioxygenase, maleylacetate reductase, and α/β hydrolase genes was identified through genomic and transcriptomic analyses. Genes encoding the cytochrome P450 complex that demethylates BP-3 to BP-1 were functionally verified through protein expression, and the functions of the other genes were also verified through knockout mutant construction and intermediate analysis. This study suggested that strain S2-17 might have acquired the ability to catabolize BP-3 by recruiting the cytochrome P450 complex and α/β hydrolase, which hydrolyzes 4-benzoyl-3-oxoadipic acid to benzoic acid and 3-oxoadipic acid, genes, providing insights into the recruitment of genes of for the catabolism of emerging organic pollutants.

Cannabinoid Hyperemesis Syndrome Survey and Genomic Investigation.

Background:Cannabinoid hyperemesis syndrome (CHS) is a diagnosis of exclusion with intractable nausea, cyclic vomiting, abdominal pain, and hot bathing behavior associated with ongoing tetrahydrocannabinol (THC) exposure. Increasing cannabis use may elevate CHS prevalence, exacerbating a public health issue with attendant costs and morbidity.Objective, Design, and Data Source:This study, the largest contemporaneous database, investigated genetic mutations underlying CHS. Patients with CHS diagnosis and ongoing symptoms were compared with current cannabis users lacking symptoms.Target Population:A screening questionnaire was posted online. Of 585 respondents, 205 qualified as the CHS pool and 54 as controls; a reduced pool of 28 patients and 12 controls ultimately completed genomic testing.Results:Patients and controls were high-frequency users of cannabis flower or concentrates (93%), using multiple grams/day of THC-predominant material. Among patients, 15.6% carried diagnoses of cannabis dependency or addiction, and 56.6% experienced withdrawal symptoms. About 87.7% of patients improved after cannabis cessation, most suffering recurrence rapidly after resumption. Findings in patients included mutations in genes COMT {odds ratio, 12 (95% confidence limit [CL], 1.3–88.1) p=0.012}, transient receptor potential vanilloid receptor 1 (TRPV1) (odds ratio, 5.8 [95% CL, 1.2–28.4] p=0.015), CYP2C9 (odds ratio, 7.8 [95% CL, 1.1–70.1] p=0.043), gene coding dopamine-2 receptor (DRD2) (odds ratio, 6.2 [95% CL, 1.1–34.7] p=0.031), and ATP-binding cassette transporter gene (ABCA1) (odds ratio, 8.4 [95% CL, 1.5–48.1] p=0.012).Limitations:Some participants were reluctant to undergo genetic testing; only 28 of 99 CHS patients who agreed to testing ultimately returned a kit.Conclusion:This is the largest patient cohort of CHS examined to date, and first to note associated mutations in genes affecting neurotransmitters, the endocannabinoid system, and the cytochrome P450 complex associated with cannabinoid metabolism. Although the sample size was smaller than desired, these preliminary findings may contribute to the growing body of knowledge, stimulate additional investigation, help elucidate the pathophysiology of CHS, and, ultimately, direct future treatment.

Dimethyl Sulfoxide: Morphological, Histological, and Molecular View on Developing Chicken Liver.

Dimethyl sulfoxide (DMSO) is widely used as a solvent for small hydrophobic drug molecules. However, the safe volume allowing to avoid its embryotoxic effect has been poorly studied. In this study, we documented the effects of dimethyl sulfoxide (DMSO) in the developing chicken embryo at morphological, histological, and molecular levels. We focused on the developing chicken liver as the main organ involved in the process of detoxification. In our study, 100% DMSO was administered subgerminally onto the eggshell membrane (membrana papyracea) at various volumes (5, 10, 15, 20, 25, 30, 35, and 50 µL) on 4th embryonic day (ED). We focused on histopathological alterations of the liver structure, and noticed the overall impact of DMSO on developing chicken embryos (embryotoxicity, malformation). At the molecular level, we studied cytochrome P450 complex (CYP) isoform’s activities in relation to changes of CYP1A5, CYP3A37, and CYP3A80 gene expression. Total embryotoxicity after application of different doses of DMSO on ED 4, and the embryo lethality increased with increasing DMSO amounts. Overall mortality after DMSO administration ranged below 33%. Mortality was increased with higher amounts of DMSO, mainly from 20 µL. The highest mortality was observed for the highest dose of DMSO over 35 µL. The results also showed a decrease in body weight with increased application volumes of DMSO. At the histological level, we observed mainly the presence of lipid droplets and dilated bile canaliculi and sinusoids in samples over the administration of 25 µL of DMSO. While these findings were not statistically significant, DMSO treatment caused a significant different up-regulation of mRNA expression in all studied genes. For CYP1A5, CYP3A37, and CYP3A80 DMSO volumes needed were 15 µL, 10 µL, and 20 µL, respectively. A significant down-regulation of all studied CYP isoform was detected after application of a DMSO dose of 5 µL. Regarding the morphological results, we can assume that the highest safe dose of DMSO without affecting chicken embryo development and its liver is up to 10 µL. This conclusion is corroborated with the presence of number of malformations and body weight reduction, which correlates with histological findings. Moreover, the gene expression results showed that even the lowest administered DMSO volume could affect hepatocytes at the molecular level causing down-regulation of cytochrome P450 complex (CYP1A5, CYP3A37, CYP3A80).

Elucidating the Bio Degradation Pathway and Catabolic Genes of B Enzophenone-3, a Xenobiotic, in &lt;i&gt;Rhodococcus&lt;/i&gt; Sp. S2-17

A new bacterium, Rhodococcus sp. S2-17, which could completely degrade the xenobiotic compound benzophenone-3 (BP-3), was isolated from contaminated sediment through an enrichment procedure, and its BP-3 catabolic pathway and genes were identified through metabolic intermediate and transcriptomic analyses and biochemical and genetic studies. Metabolic intermediate analysis suggested that strain S2-17 may degrade BP-3 using a catabolic pathway progressing via the intermediates BP-1, 2,4,5-trihydroxy-benzophenone, 3-hydroxy-4-benzoyl-2,4-hexadienedioic acid, 4-benzoyl-3-oxoadipic acid, 3-oxoadipic acid, and benzoic acid. A putative BP-3 catabolic gene cluster including cytochrome P450, flavin-dependent oxidoreductase, hydroxyquinol 1,2-dioxygenase, maleylacetate reductase, and α/β hydrolase genes was identified through genomic and transcriptomic analyses. Genes encoding the cytochrome P450 complex that demethylates BP-3 to BP-1 were functionally verified through protein expression, and the functions of the other genes were also verified through knockout mutant construction and intermediate analysis. This study suggested that strain S2-17 might have acquired the ability to catabolize BP-3 by recruiting the cytochrome P450 complex and α/β hydrolase, which hydrolyzes 4-benzoyl-3-oxoadipic acid to benzoic acid and 3-oxoadipic acid, genes, providing insights into the acquisition and reconstitution of the catabolic pathways of xenobiotic compounds.

The interactions of a number of steroid-metabolizing cytochromes P450 with abiraterone D4A metabolite: spectral analysis and molecular docking

The interactions of pharmacologically active 3-keto-Δ4-metabolite of anticancer drug abiraterone (D4A) with steroid-metabolizing cytochromes P450 (CYP51A1, CYP11A1, CYP19A1) was studied by absorption spectroscopy and molecular docking. Both abiraterone and D4A induce type I spectral changes of CYP51A1, one of the enzymes of cholesterol biosynthesis. We have revealed that D4A did not induce spectral changes of CYP11A1, the key enzyme of pregnenolone biosynthesis, unlike abiraterone (type II ligand of CYP11A1). On the contrary, D4A interacts with the active site of CYP19A1, the key enzyme of estrogen biosynthesis, inducing type II spectral changes, while abiraterone does not. Spectral analysis allowed us to calculate spectral dissociation constant (KS) for each complex of cytochrome P450 with respective ligands. The data were supported by molecular docking. The obtained results broaden understanding of interactions of D4A with some of the key steroid-metabolizing cytochromes P450 and allow one to predict possible disproportions of steroid metabolism.

Abstract TP253: Clopidogrel Pharmacogenetics in the POINT Trial

Introduction: Across 269 international sites, the POINT trial randomly assigned 4881 patients with minor ischemic stroke or high-risk transient ischemic attack to receive either clopidogrel at a loading dose of 600 mg on day 1 followed by 75 mg per day, plus aspirin at a dose of 50-325 mg per day or the same range of doses of aspirin alone. Dual antiplatelet therapy significantly reduced the incidence of major ischemic events, but also significantly increased the incidence of major hemorrhage. Clopidogrel is a prodrug that requires metabolism by the cytochrome P450 complex to act on the P2Y12 platelet receptor. CYP2C19 (cytochrome P450 family 2 subfamily C polypeptide 19) variants are associated with variable rates of metabolism of clopidogrel and thus pharmacogenomic effects may be clinically relevant. Methods: Genomic DNA was extracted from peripheral blood monocytes using the standard protocols. Genotyping for CYP2C19 *2, *3 and *17 alleles (rs4244285, rs4986893 and rs12248560) defines metabolizer/activator status and was performed using custom TaqMan Allelic Discrimination Assays on a QuantStudio™ 7 Flex Real-Time PCR System. Results: As of June 28, 2018, a total of 445 trial participants had DNA available for analysis. CYP2C19 allele genotyping demonstrated approximately one quarter of subjects (n=127; 28.6%) would be defined as poor or Intermediate metabolizers of clopidogrel. Conclusions: Based on targeted cytochrome P 450 genotyping, about one quarter of POINT patients have reduced clopidogrel activation. Additional genotyping is ongoing. We plan to present results assessing whether the treatment effect of clopidogrel is limited to those with CYP2C19 genotypes associated with normal drug metabolism.

A Minimal Functional Complex of Cytochrome P450 and FBD of Cytochrome P450 Reductase in Nanodiscs.

Structural interactions that enable electron transfer to cytochrome-P450 (CYP450) from its redox partner CYP450-reductase (CPR) are a vital prerequisite for its catalytic mechanism. The first structural model for the membrane-bound functional complex to reveal interactions between the full-length CYP450 and a minimal domain of CPR is now reported. The results suggest that anchorage of the proteins in a lipid bilayer is a minimal requirement for CYP450 catalytic function. Akin to cytochrome-b5 (cyt-b5 ), Arg 125 on the C-helix of CYP450s is found to be important for effective electron transfer, thus supporting the competitive behavior of redox partners for CYP450s. A general approach is presented to study protein-protein interactions combining the use of nanodiscs with NMR spectroscopy and SAXS. Linking structural details to the mechanism will help unravel the xenobiotic metabolism of diverse microsomal CYP450s in their native environment and facilitate the design of new drug entities.

A Minimal Functional Complex of Cytochrome P450 and FBD of Cytochrome P450 Reductase in Nanodiscs

AbstractStructural interactions that enable electron transfer to cytochrome‐P450 (CYP450) from its redox partner CYP450‐reductase (CPR) are a vital prerequisite for its catalytic mechanism. The first structural model for the membrane‐bound functional complex to reveal interactions between the full‐length CYP450 and a minimal domain of CPR is now reported. The results suggest that anchorage of the proteins in a lipid bilayer is a minimal requirement for CYP450 catalytic function. Akin to cytochrome‐b5 (cyt‐b5), Arg 125 on the C‐helix of CYP450s is found to be important for effective electron transfer, thus supporting the competitive behavior of redox partners for CYP450s. A general approach is presented to study protein–protein interactions combining the use of nanodiscs with NMR spectroscopy and SAXS. Linking structural details to the mechanism will help unravel the xenobiotic metabolism of diverse microsomal CYP450s in their native environment and facilitate the design of new drug entities.

Thiol dioxygenase turnover yields benzothiazole products from 2-mercaptoaniline and O2-dependent oxidation of primary alcohols

Thiol dioxygenases are non-heme mononuclear iron enzymes that catalyze the O2-dependent oxidation of free thiols (-SH) to produce the corresponding sulfinic acid (-SO2-). Previous chemical rescue studies identified a putative FeIII-O2− intermediate that precedes substrate oxidation in Mus musculus cysteine dioxygenase (Mm CDO). Given that a similar reactive intermediate has been identified in the extradiol dioxygenase 2, 3-HCPD, it is conceivable that these enzymes share other mechanistic features with regard to substrate oxidation. To explore this possibility, enzymatic reactions with Mm CDO (as well as the bacterial 3-mercaptopropionic acid dioxygenase, Av MDO) were performed using a substrate analogue (2-mercaptoaniline, 2ma). This aromatic thiol closely approximates the catecholic substrate of homoprotocatechuate of 2, 3-HPCD while maintaining the 2-carbon thiol-amine separation preferred by Mm CDO. Remarkably, both enzymes exhibit 2ma-gated O2-consumption; however, none of the expected products for thiol dioxygenase or intra/extradiol dioxygenase reactions were observed. Instead, benzothiazoles are produced by the condensation of 2ma with aldehydes formed by an off-pathway oxidation of primary alcohols added to aqueous reactions to solubilize the substrate. The observed oxidation of 1º-alcohols in 2ma-reactions is consistent with the formation of a high-valent intermediate similar to what has been reported for cytochrome P450 and mononuclear iron model complexes.

The impact of tobacco smoking in patients taking long-action injection drugs – A retrospective comparative study between haloperidol and risperidone

IntroductionSmoking rate seems to be higher among patients with schizophrenia, comparing to other psychiatric entities, mainly in those who are on typical antipsychotics. Tobacco is known to have enzyme inducer properties, due to cytochrome P450 complex activity: CYP1A1, CYP1A2, CYP2E1 and CYP2D6. CYP2D6 and CYP1A2 play an important role in antipsychotics metabolism, mainly in the first generation ones, like haloperidol, despite its importance in risperidone metabolism.AimTo analyze the importance of tobacco smoking in patients taking long-action injections.ObjectivesTo investigate how sexual dysfunction varies with tobacco smoking, in patients taking long-action injections.MethodsIndividuals from both sexes, from 18 to 55 years old, taking antipsychotic long-action injections, answered the Arizona Sexual Experience Scale (ASEX).ResultsIn the studied population (n = 44), there were 20 individuals on haloperidol and 24 individuals on risperidone. In a total of 18 (40.9%) positive results for sexual dysfunction, 6 were on haloperidol (30%), 12 (50%) were on risperidone. Seventeen individuals of the 20 who were on haloperidol were smokers, but only 4 were considered to have sexual dysfunction, 35.3%; 12 of the 24 individuals who were on risperidone were smokers, but only 5 were considered to have sexual dysfunction, 41.7%.ConclusionsPatients treated with haloperidol smoke more, comparing to risperidone. Sexual dysfunction is more frequent in patients taking risperidone than in patients taking haloperidol. This data supports that CYP2D6-CYP1A2 induction by tobacco, mainly interacts with haloperidol, which may be helpful for patients to try less side effects.Disclosure of interestThe authors have not supplied their declaration of competing interest.

Impact of the CYP4F2 gene polymorphisms on the warfarin maintenance dose: A systematic review and meta-analysis.

Warfarin is an oral anticoagulant with significant interpatient variability in dosage. A large number of studies have confirmed that the individual warfarin dose is mainly affected by the cytochrome P450 complex subunit 2C9 and vitamin K epoxide reductase complex subunit 1. However, the association between cytochrome P450 4F2 (CYP4F2) gene polymorphisms and warfarin dosage in the Asian population remains controversial. To investigate the impact of the CYP4F2 polymorphism rs2108622 (p.V433M) on warfarin dose requirement, a systematic review and meta-analysis were conducted. According to the strict inclusion and exclusion criteria set, a comprehensive literature search was performed, and the studies published before August 5, 2015 were searched for in PubMed, EMBASE and the China National Knowledge Infrastructure databases. The references were checked by two independent reviewers. The association between the warfarin maintenance dose and CYP4F2 polymorphism was analyzed. Twenty-two studies were included in the meta-analysis. Compared with the CYP4F2 genotype CC, carriers of the CT and TT genotypes required a 9 [95% confidence interval (CI): 6.0-13.0] and 20% (95% CI, 13.0-27.0) higher warfarin dose, respectively. In the combined analysis, T carriers (CT+TT) required an 11% (95% CI, 8.0-14.0) higher warfarin dose compared to the CC genotype. In addition, there was a 10% (95% CI, 5.0-15.0) higher warfarin dose in TT carriers compared to the CT genotype (all P<0.05). The results of the meta-analysis suggest that the effects of the CYP4F2 polymorphism on individual warfarin dose have a statistically significant difference, and the effect degree is variable in the subgroups. Further studies are expected to explore whether the pharmacogenetics model including the CYP4F2 polymorphism can strengthen the prediction of warfarin dose.

Influence of sesamin on CYP2C-mediated diclofenac metabolism: invitro and invivo analysis.

Our previous studies revealed that sesamin caused a mechanism-based inhibition (MBI) of CYP2C9 in human liver microsomes. Additionally, we observed a similar MBI of CYP2C by sesamin in the rat liver microsomes. Sesamin-induced difference spectra of rat or human liver microsomes in the presence of NADPH showed a peak at 459 nm, suggesting the formation of a metabolic–intermediate (MI) complex of cytochrome P450 and the methylenedioxyphenyl group of sesamin. However, the peak disappeared in both liver microsomes within 30 min after the termination of the metabolism. These results suggest that the MI complex of cytochrome P450 and sesamin is unstable, and the effects of sesamin on human CYP2C9- or rat CYP2C-mediated drug metabolism may be small. To confirm this, in vivo studies using rats were performed. The pharmacokinetics of diclofenac, which is mainly metabolized by CYP2C11 in male rats, were investigated after a 3-days administration of sesamin (0, 10, and 100 mg/kg bw). No significant differences were observed among the three groups in the pharmacokinetic parameters, Cmax, Tmax, and AUC. Furthermore, administration of sesamin to rats for 7 days had no significant effects on diclofenac hydroxylation activity in rat liver microsomes. These results demonstrate that no significant interaction occurs between diclofenac and sesamin in rats. Moreover, the results of these in vitro and in vivo studies suggest that no significant interaction may occur between sesamin and diclofenac when sesamin is administered to humans as a supplement, since the standard sesamin dose in humans is much lower than that administered to rats in this study.

Flexible docking-based molecular dynamics/steered molecular dynamics calculations of protein-protein contacts in a complex of cytochrome P450 1A2 with cytochrome b5.

Formation of transient complexes of cytochrome P450 (P450) with another protein of the endoplasmic reticulum membrane, cytochrome b5 (cyt b5), dictates the catalytic activities of several P450s. Therefore, we examined formation and binding modes of the complex of human P450 1A2 with cyt b5. Docking of soluble domains of these proteins was performed using an information-driven flexible docking approach implemented in HADDOCK. Stabilities of the five unique binding modes of the P450 1A2-cyt b5 complex yielded by HADDOCK were evaluated using explicit 10 ns molecular dynamics (MD) simulations in aqueous solution. Further, steered MD was used to compare the stability of the individual P450 1A2-cyt b5 binding modes. The best binding mode was characterized by a T-shaped mutual orientation of the porphyrin rings and a 10.7 Å distance between the two redox centers, thus satisfying the condition for a fast electron transfer. Mutagenesis studies and chemical cross-linking, which, in the absence of crystal structures, were previously used to deduce specific P450-cyt b5 interactions, indicated that the negatively charged convex surface of cyt b5 binds to the positively charged concave surface of P450. Our simulations further elaborate structural details of this interface, including nine ion pairs between R95, R100, R138, R362, K442, K455, and K465 side chains of P450 1A2 and E42, E43, E49, D65, D71, and heme propionates of cyt b5. The universal heme-centric system of internal coordinates was proposed to facilitate consistent classification of the orientation of the two porphyrins in any protein complex.

Factors affecting hydrogen-tunneling contribution in hydroxylation reactions promoted by oxoiron(IV) porphyrin π-cation radical complexes.

Hydrogen atom transfer with a tunneling effect (H-tunneling) has been proposed to be involved in aliphatic hydroxylation reactions catalyzed by cytochrome P450 and synthetic heme complexes as a result of the observation of large hydrogen/deuterium kinetic isotope effects (KIEs). In the present work, we investigate the factors controlling the H-tunneling contribution to the H-transfer process in hydroxylation reaction by examining the kinetics of hydroxylation reactions at the benzylic positions of xanthene and 1,2,3,4-tetrahydronaphthalene by oxoiron(IV) 5,10,15,20-tetramesitylporphyrin π-cation radical complexes ((TMP(+•))Fe(IV)O(L)) under single-turnover conditions. The Arrhenius plots for these hydroxylation reactions of H-isotopomers have upwardly concave profiles. The Arrhenius plots of D-isotopomers, clear isosbestic points, and product analysis rule out the participation of thermally dependent other reaction processes in the concave profiles. These results provide evidence for the involvement of H-tunneling in the rate-limiting H-transfer process. These profiles are simulated using an equation derived from Bell's tunneling model. The temperature dependence of the KIE values (k(H)/k(D)) determined for these reactions indicates that the KIE value increases as the reaction temperature becomes lower, the bond dissociation energy (BDE) of the C-H bond of a substrate becomes higher, and the reactivity of (TMP(+•))Fe(IV)O(L) decreases. In addition, we found correlation of the slope of the ln(k(H)/k(D)) - 1/T plot and the bond strengths of the Fe═O bond of (TMP(+•))Fe(IV)O(L) estimated from resonance Raman spectroscopy. These observations indicate that these factors modulate the extent of the H-tunneling contribution by modulating the ratio of the height and thickness of the reaction barrier.

Stiripentol for the treatment of Dravet syndrome

Stiripentol (marketed by Biocodex as Diacomit ® ) is an anticonvulsant drug, structurally unrelated to any other compound, which has recently been approved as adjunctive therapy with clobazam and valproate for Dravet syndrome in Europe, Canada, and Japan. This rare form of early childhood epilepsy is associated with subsequent cognitive impairment, significant risk of death, and high pharmacoresistance. Based on an efficacy signal of stiripentol added to cloba - zam and valproate in an observational, prospectively conducted, exploratory study including 10% of children with Dravet syndrome, a randomized placebo-controlled trial was specifically dedicated to patients with Dravet syndrome inadequately controlled by clobazam and valproate. Results showed significantly higher responder rates (71% versus 5%; P,0.0001) and decrease in seizure frequency (-69% versus +7%; P,0.002) on stiripentol than on placebo. A second, independently performed, randomized controlled trial confirmed these results 2 years later, and both trials were plotted in a meta-analysis. Efficacy was supported by three subsequent obser - vational studies, with, respectively, 46 (France), 23 (Japan), and 82 (USA) children with Dravet syndrome treated with stiripentol for up to 5 years. Based on the experiences of more than 2,000 patients with Dravet syndrome who were exposed to stiripentol, drowsiness, loss of appetite, and weight loss are the most frequent adverse events and may be reduced by decreasing the dosage of co-medication. The inhibition of stiripentol by the cytochrome P450 complex (CYP2C19, and CYP3A4) leads to clinically significant interactions. Experimental data, both in vitro and in vivo, have definitively established that stiripentol is a GABAergic anticonvulsant and acts on different sites than benzodiazepines. The pharmacodynamic interactions also enhance the anticonvulsant effect of the stiripentol-clobazam combination in patients with Dravet syndrome, irrespective of the GABAergic effect. Stiripentol is currently the only drug specifically indicated in Dravet