Inhibition Of Cytochrome P450 Enzymes Research Articles

Inhibition of Cytochrome P450 Enzymes by Drugs-Molecular Basis and Practical Applications.

Drug-drug interactions are a major cause of hospitalization and deaths related to drug use. A large fraction of these is due to inhibition of enzymes involved in drug metabolism and transport, particularly cytochrome P450 (P450) enzymes. Understanding basic mechanisms of enzyme inhibition is important, particularly in terms of reversibility and the use of the appropriate parameters. In addition to drug-drug interactions, issues have involved interactions of drugs with foods and natural products related to P450 enzymes. Predicting drug-drug interactions is a major effort in drug development in the pharmaceutical industry and regulatory agencies. With appropriate in vitro experiments, it is possible to stratify clinical drug-drug interaction studies. A better understanding of drug interactions and training of physicians and pharmacists has developed. Finally, some P450s have been the targets of drugs in some cancers and other disease states.

CYPlebrity: Machine learning models for the prediction of inhibitors of cytochrome P450 enzymes

The vast majority of approved drugs are metabolized by the five major cytochrome P450 (CYP) isozymes, 1A2, 2C9, 2C19, 2D6 and 3A4. Inhibition of CYP isozymes can cause drug-drug interactions with severe pharmacological and toxicological consequences. Computational methods for the fast and reliable prediction of the inhibition of CYP isozymes by small molecules are therefore of high interest and relevance to pharmaceutical companies and a host of other industries, including the cosmetics and agrochemical industries. Today, a large number of machine learning models for predicting the inhibition of the major CYP isozymes by small molecules are available. With this work we aim to go beyond the coverage of existing models, by combining data from several major public and proprietary sources. More specifically, we used up to 18815 compounds with measured bioactivities to train random forest classification models for the individual CYP isozymes. A major advantage of the new data collection over existing ones is the better representation of the minority class, the CYP inhibitors. With the new data collection we achieved inhibitor-to-non-inhibitor ratios in the order of 1:1 (CYP1A2) to 1:3 (CYP2D6). We show that our models reach competitive performance on external data, with Matthews correlation coefficients (MCCs) ranging from 0.62 (CYP2C19) to 0.70 (CYP2D6), and areas under the receiver operating characteristic curve (AUCs) between 0.89 (CYP2C19) and 0.92 (CYPs 2D6 and 3A4). Importantly, the models show a high level of robustness, reflected in a good predictivity also for compounds that are structurally dissimilar to the compounds represented in the training data. The best models presented in this work are freely accessible for academic research via a web service.

Repercussion of piperine on pharmacokinetics parameters of eletriptan in albino rats: Involvement of CYP3A and P-GP inhibition

Eletriptan is at the type of triptan medication suggested for the management of episodes of severe headache with or without aura. Piperine (1-piperoylpiperidine) is a pure alkaloid as well as the basic tanginess material from the pipli and peppercorn. There are signs that piperine inhibits Cytochrome P-450 enzymes and P-glycoprotein, rather than arouses, drug metabolism generally, thus increasing the bioavailability and effect of several medications. The analysis was undertaken to assess the results of Piperine on the pharmacokinetics of Eletriptan. Samples of blood were taken at different time points for example 0 (predose), 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 6.0, 8.0, 12, 24 hours post-treatment. The plasma concentration of Eletriptan was estimated with the HPLC procedure. In our study Cmax, Tmax, AUC0-24 , AUC0-8, AUC%, AUMC0-24, AUMC0-8, t1/2, MRT0-24, MRT0-8 and Volume of distribution were increased by approximately 51.76%, 7.55%, 72.93%, 84.42%, 42.72%, 93.56%, 128.82%, 19.97%, 11.74%, 23.96% and 3% respectively, where as clearance decreased by 42.10% when eletriptan co-administered with piperine. In summary, the results obtained herein imply that Piperine is improving the bioavailability of Eletriptan by strengthening the exposure (AUC) of their Eletriptan when concomitantly administered by the oral route. The Piperine improved the oral bioavailability of eletriptan by inhibiting CYP3A and P-GP in rats. This observation indicates the possibility that the combo of piperine along with other CYP3A and P-GP double substrates can also enhance bioavailability.

Coexistent CYP24A1 and PHEX Gene Mutations With Hypervitaminosis D Plus Hypercalcemia Treated With Fluconazole

Background: CYP24A1 and PHEX gene mutations are rare and can cause hypercalcemia, hypervitaminosis D and elevated FGF23 levels. Fluconazole, an antifungal medication, has shown therapeutic benefit in achieving normocalcemia plus normalisation of vitamin D levels in this case report. Clinical Case: A 42 year old man was referred to the endocrine clinic with a history of severe nephrocalcinosis and recurrent nephrolithiasis requiring surgical intervention and gradual decline in kidney function over 20 years. Biochemical investigations revealed hypercalcaemia with adjusted calcium levels of 2.83 mmol/L (R 2.2–2.6 nmol/L) and suppressed PTH 1.1 pmol/L(R 1.6–6.9 pmol/L). Twenty-four hour urine calcium/creatinine clearance ratio was above 0.0578 mmol/mmol indicating hypercalciuria. Vitamin D metabolites 25 OH Vitamin D was elevated at 201 nmol/L, (R 50–120 nmol/L) along with intermittently elevated 1,25 OH Vitamin D 147 pmol/L(R 55–139 pml/L). 24,25 Vitamin D was low at 2.0 nmol/L producing a 25:24,25 dihydroxyvitamin D ratio of 80 (n<25). This biochemical data was highly suggestive of a loss of function mutation in the CYP24A1 gene that codes for the enzyme 24-hydroxylase, which is responsible for conversion of 1,25 vitamin D to 24,25 vitamin D. A pathogenic variant (heterozygous c.756G>A) was confirmed on genetic testing.Plasma FGF23 (immutopics) was raised (with a peak of 596 RU/mL, n<100 RU/mL) but a full body octreotide scan did not reveal malignancy or other paraneoplastic syndromes such as oncogenic osteomalacia. A pathogenic variant in his PHEX gene (homozygous c.1874A>T) was also identified that has been associated with increased levels of FGF23 plus hypophosphataemia. Fluconazole at 50 mg once daily was initiated. Azoles inhibit cytochrome P450 enzymes and have been used in sarcoidosis to block vitamin D-synthesizing enzymes such as 25-hydroxylases and 1-α-hydroxylase that are P450 dependent. Few cases of CYP24A1 gene defects have been treated with fluconazole, which has a favourable side effect profile and yields good results. Adjusted calcium reduced to 2.62 nmol/L, 25 OH Vitamin D normalised to 111 nmol/L and 24:24,25 dihydroxyvitamin D ratio is now 17. Patient’s liver functions and full blood count has been monitored regularly during the course of treatment and the drug was well tolerated. Conclusion: Genetic causes of hypercalcemia can be left undiagnosed for long periods and there is a lack of proven or definitive therapeutic agents for correction of elevated calcium. Here fluconazole has been shown to reduce the hypercalcaemic burden and effectively lowered the Vitamin D levels in this case of a CYP24A1 mutation. This study augments fluconazole use in these cases but further studies are needed to elucidate the long term safe usage.

Cytochrome P450 Enzyme Inhibition and Herb-Drug Interaction Potential of Medicinal Plant Extracts Used for Management of Diabetes in Nigeria.

The use of herbal medicines is common in Africa, and patients often use a combination of herbs and drugs. Concurrent herbal and pharmaceuticals treatments can cause adverse effects through herb-drug interactions (HDI). This study evaluated the potential risk of HDI for five medicinal plants, Vernonia amygdalina, Ocimum gratissimum, Moringa oleifera, Azadirachta indica, and Picralima nitida, using in vitro assays. Patients with diabetes and some other disease conditions commonly use these medicinal plants in Nigeria, and little is known regarding their potential for drug interaction, despite their enormous use. Crude extracts of the medicinal plants were evaluated for reversible and time-dependent inhibition (TDI) activity of six cytochrome P450 (CYP) enzymes using pooled human liver microsomes and cocktail probe-based assays. Enzyme activity was determined by quantifying marker metabolites' formation using liquid chromatography-mass spectrometry/mass spectrometry. The drug interaction potential was predicted for each herbal extract using the in vitro half-maximal inhibitory concentration (IC50) values and the percentage yield. O. gratissimum methanol extracts reversibly inhibited CYP 1A2, 2C8, 2C9 and 2C19 enzymes (IC50: 6.21 µg/ml, 2.96 µg/ml, 3.33 µg/ml and 1.37 µg/ml, respectively). Additionally, V. amygdalina methanol extract inhibited CYP2C8 activity (IC50: 5.71 µg/ml); P. nitida methanol and aqueous extracts inhibited CYP2D6 activity (IC50: 1.99 µg/ml and 2.36 µg/ml, respectively) while A. indica methanol extract inhibited CYP 3A4/5, 2C8 and 2C9 activity (IC50: 7.31 µg/ml, 9.97 µg/ml and 9.20 µg/ml, respectively). The extracts showed a potential for TDI of the enzymes when incubated at 200 µg/ml; V. amygdalina and A. indica methanol extracts exhibited TDI potential for all the major CYPs. The medicinal plants inhibited CYP activity in vitro, with the potential to cause in vivo HDI. Clinical risk assessment and proactive monitoring are recommended for patients who use these medicinal plants concurrently with drugs that are cleared through CYP metabolism.

Reversible and Irreversible Inhibition of Cytochrome P450 Enzymes by Methylophiopogonanone A.

Methylophiopogonanone A (MOA), an abundant homoisoflavonoid bearing a methylenedioxyphenyl moiety, is one of the major constituents in the Chinese herb Ophiopogon japonicas This work aims to assess the inhibitory potentials of MOA against cytochrome P450 enzymes and to decipher the molecular mechanisms for P450 inhibition by MOA. The results showed that MOA concentration-dependently inhibited CYP1A, 2C8, 2C9, 2C19, and 3A in human liver microsomes (HLMs) in a reversible way, with IC50 values varying from 1.06 to 3.43 μM. By contrast, MOA time-, concentration-, and NADPH-dependently inhibited CYP2D6 and CYP2E1, along with KI and kinact values of 207 µM and 0.07 minute-1 for CYP2D6, as well as 20.9 µM and 0.03 minutes-1 for CYP2E1. Further investigations demonstrated that a quinone metabolite of MOA could be trapped by glutathione in an HLM incubation system, and CYP2D6, 1A2, and 2E1 were the major contributors to catalyze the metabolic activation of MOA to the corresponding O-quinone intermediate. Additionally, the potential risks of herb-drug interactions triggered by MOA or MOA-related products were also predicted. Collectively, our findings verify that MOA is a reversible inhibitor of CYP1A, 2C8, 2C9, 2C19, and 3A but acts as an inactivator of CYP2D6 and CYP2E1. SIGNIFICANCE STATEMENT: Methylophiopogonanone A (MOA), an abundant homoisoflavonoid isolated from the Chinese herb Ophiopogon japonicas, is a reversible inhibitor of CYP1A, 2C8, 2C9, 2C19, and 3A but acts as an inactivator of CYP2D6 and CYP2E1. Further investigations demonstrated that a quinone metabolite of MOA could be trapped by glutathione in a human liver microsome incubation system, and CYP2D6, 1A2, and 2E1 were the major contributors to catalyze the metabolic activation of MOA to the corresponding O-quinone intermediate.

Plant essential oil constituents enhance deltamethrin toxicity in a resistant population of bed bugs (Cimex lectularius L.) by inhibiting cytochrome P450 enzymes

Plant essential oils (EOs) are secondary metabolites derived from aromatic plants that are composed of complex mixtures of chemical constituents. EOs have been proposed as one of the alternative methods for bed bug (Cimex lectularius L.) control. In insecticide resistant mosquitoes and tobacco cutworm, EOs synergize pyrethroid toxicity by inhibiting detoxification enzymes. However, whether EOs and their constituents enhance pyrethroid toxicity in C. lectularius has remained unknown. Therefore, this study was designed to (i) determine the effects of binary mixtures of deltamethrin (a pyrethroid insecticide) with EOs or EO constituents or EcoRaider® (an EO-based product) on mortality of insecticide resistant and susceptible bed bugs, and (ii) evaluate the effects of EO constituent pre-treatment on detoxification enzyme activities of resistant and susceptible populations. Topical bioassays with binary mixtures of deltamethrin and individual EOs (e.g., thyme, oregano, clove, geranium or coriander oils) or their major constituents (e.g., thymol, carvacrol, eugenol, geraniol or linalool) or EcoRaider® at doses that kill approximately 25% of bed bugs caused significant increases in mortality of resistant bed bugs. However, in the susceptible population, only coriander oil, EcoRaider®, thymol, and carvacrol significantly increased the toxicity of deltamethrin. Detoxification enzyme assays with protein extracts from bed bugs pre-treated with EO constituents suggested selective inhibition of cytochrome P450 activity in the resistant population, but no impacts were observed on esterase and glutathione transferase activities in either population. Inhibition of P450 activity by EO constituents thus appears to be one of the mechanisms of deltamethrin toxicity enhancement in resistant bed bugs.

Repercussion of piperine on pharmacokinetics parameters of eletriptan in albino rats: Involvement of CYP3A and P-GP inhibition

Eletriptan is at the type of triptan medication suggested for the management of episodes of severe headache with or without aura. Piperine (1-piperoylpiperidine) is a pure alkaloid as well as the basic tanginess material from the pipli and peppercorn. There are signs that piperine inhibits Cytochrome P-450 enzymes and P-glycoprotein, rather than arouses, drug metabolism generally, thus increasing the bioavailability and effect of several medications. The analysis was undertaken to assess the results of Piperine on the pharmacokinetics of Eletriptan. Samples of blood were taken at different time points for example 0 (predose), 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 6.0, 8.0, 12, 24 hours post-treatment. The plasma concentration of Eletriptan was estimated with the HPLC procedure. In our study Cmax, Tmax, AUC0-24 , AUC0-8, AUC%, AUMC0-24, AUMC0-8, t1/2, MRT0-24, MRT0-8 and Volume of distribution were increased by approximately 51.76%, 7.55%, 72.93%, 84.42%, 42.72%, 93.56%, 128.82%, 19.97%, 11.74%, 23.96% and 3% respectively, where as clearance decreased by 42.10% when eletriptan co-administered with piperine. In summary, the results obtained herein imply that Piperine is improving the bioavailability of Eletriptan by strengthening the exposure (AUC) of their Eletriptan when concomitantly administered by the oral route. The Piperine improved the oral bioavailability of eletriptan by inhibiting CYP3A and P-GP in rats. This observation indicates the possibility that the combo of piperine along with other CYP3A and P-GP double substrates can also enhance bioavailability.

Fosravuconazole to treat severe onychomycosis in the elderly.

Fosravuconazole is a novel oral antifungal drug developed in Japan and used to treat tinea unguium since 2018. Its excellent oral absorbability and systemic bioavailability has enabled short-duration therapy of 3months. Furthermore, no concomitant drugs are contraindicated due to the presence of the mild inhibitor of cytochrome P450 enzyme which is responsible for polypharmacy adverse effects. Therefore, it can be safely administrated to elderly patients. Elderly patients (≥65years old) with severe onychomycosis (≥50% nail involvement) were treated with oral fosravuconazole 100mg once daily for 12weeks. The rate of involvement improved from 86.6% to 28.1% (P<0.01). The efficacy (i.e. percentage of those rated as "improved" and better) and cure rate was 83.8% (31/37) and 29.7% (11/37), respectively. Furthermore, when focusing on the thin nail group (<3mm), the efficacy and cure rate was 88.2% (15/17) and 58.8% (10/17), respectively. Although the serum γ-glutamyltransferase levels increased in 21.6% (8/37), all patients recovered without any specific treatments.

Development of a method to determine cytochrome P450 1A2, 2C9, 2D6 and 3A4 activity sheep hepatic microsomes

IntroductionEx vivo studies of human fetal hepatic drug metabolism are uncommon as it requires access to functional liver tissue and therefore raises practical and ethical concerns. Large animal models provide an alternative opportunity to study changes in cytochrome P450 (CYP) activity in the mother and fetus during pregnancy. We aimed to develop methods to determine the activity of CYP1A2, CYP2C9, CYP2D6 and CYP3A4 in sheep hepatic microsomes. MethodsWe identified optimal conditions to determine the activity of CYP1A2 (using the probe drug phenacetin), CYP2C9 (diclofenac), CYP2D6 (dextromethorphan) and CYP3A4 (midazolam) by varying techniques for microsome extraction, probe drug concentration, incubation time and microsome concentration. The specificity of each probe drug was assessed by determining the rate of metabolism when specific CYP enzyme inhibitors were included in the reaction. ResultsThe optimum incubation time and probe drug concentration was six hours with 5 μM phenacetin (CYP1A2), four hours with 10 μM diclofenac (CYP2C9), 30 min with 1 μM of midazolam (CYP3A4) and 10 min with 1 μM dextromethorphan (CYP2D6). For both CYP2D6 and CYP3A4 reactions required 20 μg of microsomal protein, whereas for CYP1A2 and CYP2C9, reactions required 40 μg of microsomal protein. Metabolism of phenacetin, dextromethorphan and midazolam was reduced by specific enzyme inhibitors, but the specific CYP2C9 inhibitor sulfaphenazole did not substantially inhibit diclofenac metabolism. DiscussionThis study identifies the optimal conditions for determining CYP activity in maternal sheep hepatic microsomes. In doing so, we have developed a standardised protocol for assessment of microsomal activity of CYP3A4, CYP1A2 and CYP2D6, but we were unable to optimise conditions for assessment of CYP2C9. This approach can be applied to investigate the impact of pregnancy complications on maternal and fetal hepatic drug metabolism.

Azole pharmaceuticals induce germinal vesicle breakdown (GVBD) in preovulatory oocytes of zebrafish (Danio rerio): an in vitro study.

Azoles, the antifungal pharmaceuticals are emerging as a new class of water contaminants with a potential to influence the endocrine physiology of surrounding aquatic fauna. In this study, we made an attempt to assess the relative efficacy of widely used azoles belonging to two subclasses, i.e., (i) triazoles (letrozole, fluconazole, itraconazole) and (ii) imidazoles (ketaconazole, ornidazole, clotrimazole), on the onset of germinal vesicle breakdown (GVBD) (an initial step in the final maturation of oocytes) in fully grown preovulatory oocytes of zebrafish (Danio rerio) using an in vitro model. Oocytes (> 650 μm) isolated manually from gravid ovaries were exposed to (i) 0.01 and/or 0.1, 1.0, 5.0, 10, 15, and 20 ng/ml and (ii) 1.0, 2.0, 3.0, 4.0, and 5.0 μg/ml of drugs. Zebrafish Ringer's solution (vehicle) and 0.01%ethyl alcohol (solvent) were used as negative controls. 17α, 20 β-Dihydroxy-4-pregnen-3-one (17α-DHP) and diethylstibestrol (DES), potent inducers of GVBD in fish, were used as positive controls. GVBD was scored hourly from 0-6 h. In negative controls, there were no indications of GVBD even at the 6th hour, while in 17α-DHP- and DES-exposed oocytes, GVBD was initiated from the 1st hour, reaching 80% and 76% respectively at the 6th hour. Among azoles, letrozole induced GVBD in 73-85%, fluconazole (30-33%), itraconazole (23-33%), ketaconazole (46-53%), ornidazole (36-40%), and clotrimazole (30-33%) of oocytes. These results suggest that azole pharmaceuticals induce GVBD in fish oocytes that may be attributed to their variable degree of cytochrome P450 enzyme inhibitor activity.

Undervirilized male infant with in utero exposure to maternal use of high dose antifungal therapy

BackgroundAntifungals act on fungal sterols structurally similar to human cholesterol. Ketoconazole reversibly suppresses steroidogenesis by inhibiting cytochrome P450 enzymes and interferes with dihydrotestosterone (DHT) activity by binding to the androgen receptor. Hypospadias was reported in infants exposed to nystatin in utero.Case presentationA male infant exposed to antepartum nystatin presented with severe under-undervirilization and transient adrenal corticosteroid abnormalities. He was born in USA at 31 weeks gestation to a mother treated with vaginal Polygynax capsules (nystatin-100,000 international units, neomycin sulphate-35,000 international units and polymyxin B-35,000 international units) for vaginal discharge in the Ivory Coast. She used approximately 60 capsules between the first trimester until delivery. The infant was born with micropenis, chordee, perineo-scrotal hypospadias and bifid scrotum with bilaterally palpable gonads. The karyotype was 46,XY. No Mullerian structures were seen on ultrasound. Serum 17-hydroxyprogesterone (17 OHP) on newborn screening was high (304 ng/ml, normal < 35). Cortisol response to cosyntropin on the 3rd day of life (DOL) was 10 mcg/ml; the subnormal cortisol response may have resulted from prematurity and the predelivery treatment with betamethasone. The elevation of several adrenal corticosteroids was not consistent with any specific enzymatic defect. Hydrocortisone and fludrocortisone were initiated at another hospital for suspected mild glucocorticoid and mineralocorticoid deficiencies. Genetic screening for adrenal and gonadal developmental defects performed when transferred to our care were normal. All medications were gradually discontinued over 5–8 months. Adrenal and testicular responses to cosyntropin and human chorionic gonadotropin (hCG) were normal at 8 months.ConclusionsWe report severe undervirilization in a 46,XY infant born to a mother treated with prolonged and high dose nystatin during pregnancy. This presentation suggests that prolonged antepartum use of high dose nystatin could lead to severe but transient defects in androgen synthesis and/or action possibly by acting as an endocrine disruptor. Further studies are warranted to confirm this finding. Thus, endocrine disruptors should be considered in male newborns with atypical genitalia not explained by common pathologies.

Modeling of cytochrome P-450 enzyme inhibitors activities using 2D/3D QSAR

In this research, quantitative structure–activity relationship (QSAR) studies were carried out on the inhibitory activities of a set of nicotine derivatives against the cytochrome-p450 2A6 (CYP2A6) enzyme. Two-dimensional quantitative structure–activity relationship (2D-QSAR) models were developed using multiple linear regression (MLR) and linear square-support vector machine (LS-SVM) methods. The result of statistical parameters of the MLR method show that the correlation coefficient (R2) and standard error (SE) for the training set respectively are R2 = 0.702, SE = 0.49 and for the test set R2 = 0.689, SE = 0.52 and the results of statistical parameters of the LS-SVM method for the training set R2 = 0.993, SE = 0.10 and for the test set R2 = 0.977, SE = 0.20. The obtained results reveal the superiority of LS-SVM over MLR model. Then three-dimensional quantitative structure–activity relationship (3D-QSAR) model was developed using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) on the same dataset of nicotine derivatives. The acquired statistical parameters of the CoMFA model for the training set are R2 = 0.884, SE = 0.316 and for the test set R2 = 0.847, SE = 0.33, F = 41.27, Q2 = 0.581, while the statistical values of CoMSIA model for the training set are R2 = 0.889, SE = 0.31 and for the test set R2 = 0.670, SE = 0.54, F = 39.049, Q2 = 0.554. The results of this study revealed that the CoMFA model was more predictive and could be helpful in designing novel potent nicotine derivatives with enhanced inhibitory activity.

Discovery of a Conformationally Constrained Oxazolidinone with Improved Safety and Efficacy Profiles for the Treatment of Multidrug-Resistant Tuberculosis.

Tuberculosis (TB) remains a serious public health challenge, and the research and development of new anti-TB drugs is an essential component of the global strategy to eradicate TB. In this work, we discovered a conformationally constrained oxazolidinone 19c with improved anti-TB activity and safety profile through a focused lead optimization effort. Compound 19c displayed superior in vivo efficacy in a mouse TB infection model compared to linezolid and sutezolid. The druggability of compound 19c was demonstrated in a panel of assays including microsomal stability, cytotoxicity, cytochrome P450 enzyme inhibition, and pharmacokinetics in animals. Compound 19c demonstrated an excellent safety profile in a battery of safety assays, including mitochondrial protein synthesis, hERG K+, hCav1.2, and Nav1.5 channels, monoamine oxidase, and genotoxicity. In a 4 week repeated dose toxicology study in rats, 19c appeared to have less bone marrow suppression than linezolid, which has been a major liability of the oxazolidinone class.

Exploring the effect of khat (Catha edulis) chewing on the pharmacokinetics of the antiplatelet drug clopidogrel in rats using the newly developed LC-MS/MS technique

AbstractClopidogrel (CLOP) is widely used worldwide for cardiovascular complications. CLOP is highly metabolized in the liver to its active metabolite by cytochrome P450 enzymes. Studies have shown that khat, an addictive substance, is a powerful inhibitor of cytochrome P450 enzymes and can influence the metabolism of drugs that are concomitantly used. Therefore, this study was designed to evaluate the effects of khat on the pharmacokinetics of CLOP in rats. In this study, rats were administered either CLOP alone or CLOP combined with khat and their plasma were obtained at different time intervals and analyzed using the newly developed and validated liquid chromatography with tandem mass spectrometry (LC-MS/MS) method using foretinib (FTB) as the internal standard. The corresponding peak area of the analyte versus FTB was used for calculating the peak ratio. The validated LC-MS/MS method resulted in the separation of the well-defined quantifiable peaks of CLOP, FTB, and CLOP metabolite within 7 min. Results showed a significant influence of khat on the peak ratio of CLOP metabolite, which was found to be significantly decreased (P &lt; 0.05) in comparison to CLOP alone, suggesting significant decrease in the conversion of CLOP to its active metabolite due to the inhibition of CYP450 enzymes by khat. Therefore, there might be a need for dose adjustment for regular khat chewers using CLOP.

Global Trends in Genetic Markers of Pediculus humanus capitis Resistance Mechanisms

Head lice infestations remain an important public health problem, albeit poorly defined in most endemic countries. Reports of increasing resistance to first-line treatment have renewed scientific research into this neglected ectoparasitosis. Mapping and understanding resistance mechanisms are essential for the development of more effective treatments, as well as for prolonging the life of existing pediculicides. This review aims to synthetize recent data on the type, frequency, and distribution of genetic mutations associated with head lice resistance to chemical treatments. Head lice resistance is reported in all continents. The most studied mechanism of resistance is through Knockdown resistance (kdr) mutations, with biomarkers M815I, T917I, and L920F, most commonly reported. Other reported mechanisms such as cytochrome P450 enzyme inhibition, altered acetylcholinesterase binding, and GluCl mutations are still being investigated. Allele mutations linked to pyrethroid resistance differ between regions and countries, which highlights the need for monitoring resistance through region-specific genetic markers. In geographic areas where resistance is well-established, louse populations are predominantly homozygous. In regions where heterozygosity still prevails, selective pressure through the use of chemical pediculicides will cause resistance to increase. Continuous surveillance of resistance is needed to monitor the frequency of resistance alleles, which is expected to increase as lice populations move into fixation. Further research should be conducted on new treatments with physical mechanisms of action.

Biodegradation of fluoroquinolone antibiotics and the climbazole fungicide by Trichoderma species.

Filamentous fungi Trichoderma have been able to efficiently degrade fluoroquinolone antibiotics namely ciprofloxacin (CIP) and ofloxacin (OFL) as well as the fungicide climbazole (CLB) that are persistent in conventional activated sludge processes. All targeted compounds were biotransformed by whole cells of Trichoderma spp., exactly T. harzanium and T. asperellum, and biosorption played a limited role in their elimination. However, contrasting results were obtained with the two strains. T. asperellum was more efficient against CIP, with a 81% degradation rate in 13days of incubation, while T. harzianum was more efficient against CLB, with a 91% degradation rate. While in the case of OFL, both strains showed same efficiency with degradation rate around 40%. Adding a cytochrome P450 enzyme inhibitor hardly resulted in the modification of degradation kinetics supporting the implication of extracellular enzymes in chemical biotransformation. Transformation products were identified by liquid chromatography-high resolution-mass spectrometry and transformation pathways were proposed. Biotransformation of selected compounds included hydroxylation, oxidation/reduction and N- and O-dealkylation reactions, similarly to those reported with white rot fungi. CIP underwent transformations at the piperazinyl ring through oxidation and conjugation reactions, while OFL mainly underwent hydroxylation processes and CLB carbonyl reduction into alcohol. Consequently, Trichoderma spp. likely possess a machinery of unspecific enzymes, which makes their application in removal of pharmaceutical and personal care products attractive.

Assessment of CYP2A6 and CYP2C19 Genetic Variation on Cotinine‐N‐Oxide Formation and their Potential Effect on the Nicotine Metabolic Ratio (NMR)

SignificanceThe nicotine metabolic ratio (NMR) of 3‐hydroxycotinine (3HC)/cotinine in smokers is a determinant of CYP2A6 activity and is widely used in clinical practice to help select smoking cessation pharmacotherapy. The major mode of metabolism of nicotine is via the formation of cotinine and subsequently to 3HC by the enzyme CYP2A6. Cotinine is also metabolized to cotinine‐N‐glucuronide and to cotinine‐N‐oxide (COX), accounting for ~7 and 5% of urinary nicotine metabolites, respectively, in smokers. The goal of the present study was to investigate the enzymes that catalyze COX formation and determine whether genetic variation in these enzymes may affect this pathway.MethodsWe used specific inhibitors of the major hepatic cytochrome P450 (CYP) enzymes including CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 in cotinine‐N‐oxidation reactions using pooled human liver microsomes (HLM). COX formation was monitored by UPLC‐MS/MS, and enzyme kinetic analysis was performed using microsomal fractions from CYP‐overexpressing HEK293 cell lines. Additionally, genotype‐phenotype analysis was performed for a panel of 113 HLM specimens.ResultsInhibition of COX formation was only observed when using inhibitors of CYPs 2A6, 2B6, 2C19, 2E1, and 3A4. CYPs 2A6 and 2C19 exhibited similar oxidation activity against cotinine with Vmax/KM values of 4.4, and 4.2 nL•min−1•mg−1, respectively; the Vmax/KM value for the 2B6 isoform was of 2.2 nL•min−1•mg−1. The activity observed for both CYPs 2E1 and 3A4 were low, with KM’s greater than 10 mM. We measured the levels of COX formation in 113 HLM specimens and genotyped variants in CYPs 2A6 (*2, *9, and *14), 2B6 (*2, *5, *9, rs4803419 and rs8109525) and 2C19 (*2 and *17) using qPCR. An association between decreased levels of COX formation and CYP variants in the 2C19 (up to 35% reduction) and 2A6 (up to 38% reduction) isoforms was observed. No statistically significant correlation was observed with variants in the 2B6 isoform.ConclusionsThese results demonstrate that genetic variants in CYPs 2A6 and 2C19 are associated with decreased COX formation, potentially affecting the levels of free cotinine and consequently the calculated NMR in smokers and clinical practices surrounding smoking cessation therapies.Support or Funding InformationThis work is supported by grants from NIH, National Institutes of Environmental Health Sciences (grant R01‐ES025460; to P. Lazarus), the Fulbright‐Garcia Robles Program and CONACyT I2T2 (to Y. Perez‐Paramo), Health Sciences and Services Authority of Spokane, Washington (grant WSU002292 to College of Pharmacy and Pharmaceutical Sciences, Washington State University)

Optimization and biological evaluation of imidazopyridine derivatives as a novel scaffold for γ-secretase modulators with oral efficacy against cognitive deficits in Alzheimer’s disease model mice

Gamma-secretase modulators (GSMs) selectively lower amyloid-β42 (Aβ42) and are therefore potential disease-modifying drugs for Alzheimer’s disease (AD). Here, we report the discovery of imidazopyridine derivatives as GSMs with oral activity on not only Aβ42 levels but also cognitive function. Structural optimization of the biphenyl group and pyridine-2-amide moiety of compound 1a greatly improved GSM activity and rat microsomal stability, respectively. 5-{8-[(3,4′-Difluoro[1,1′-biphenyl]-4-yl)methoxy]-2-methylimidazo[1,2-a]pyridin-3-yl}-N-methylpyridine-2-carboxamide (1o) showed high in vitro potency and brain exposure, induced a robust reduction in brain Aβ42 levels, and exhibited undetectable inhibition of cytochrome p450 enzymes. Moreover, compound 1o showed excellent efficacy against cognitive deficits in AD model mice. These findings suggest that compound 1o is a promising candidate for AD therapeutics.

Влияние газотрансмиттера сероводорода на микрореологические свойства эритроцитов здоровых лиц и больных сахарным диабетом второго типа

Цель исследования: изучение влияния донора газотрансмиттера (ГТ) сероводорода (NaНS) и активатора его внутриклеточного синтеза (Lцистеина) на микрореологические характеристики эритроцитов в норме и при сахарном диабете 2-го типа (СД2). Материалы и методы. Эритроциты 16 здоровых лиц и 16 больных СД2 инкубировали с донором сероводорода гидросульфидом натрия (NaHS) в концентрации 10, 50 и 100 мкмоль и стимулятором его эндогенного синтеза Lцистеином в концентрации 10, 50 и 100 мкмоль. Блокировали АТФзависимые калиевые каналы глибенкламидом (50 мкмоль). Ингибировали растворимую гуанилатциклазу с помощью 0,5 мкмоль ODQ (1H-1,2,4-oxadiazolo4,3-a quinoxalin-l-one). Регистрировали деформируемость (ИУЭ) и агрегацию эритроцитов. Результаты. Под влиянием NaHS наблюдали дозозависимые положительные изменения микрореологии эритроцитов. Микрореологические ответы эритроцитов на NaHS были более выраженными у здоровых лиц, чем у больных СД2. Сходную картину наблюдали и при воздействии на эритроциты Lцистеином. Прединкубация с глибенкламидом не устраняла положительного влияния донора ГТ на микрореологию эритроцитов, тогда как ингибирование растворимой гуанилатциклазы с помощью ODQ полностью устраняло положительные изменения ИУЭ под действием ГТ и существенно уменьшало его влияние на агрегацию клеток. Заключение. Таким образом, сероводород как газотрансмиттер улучшает микрореологию эритроцитов. При одинаковой концентрации донора сероводорода (NaHS) и стимулятора его синтеза (Lцистеина) более выраженные сдвиги микрореологических параметров эритроцитов наблюдаются у здоровых лиц по сравнению с больными СД2. Aim: to study the effect of donor gasotransmitter (GT) hydrogen sulphide (NaHS) and the activator of its intracellular synthesis (Lcysteine) on the red blood cells (RBCs) microrheological characteristics in normal and diabetes mellitus type 2 (DM2). Materials and methods. RBCs of 16 healthy individuals and 16 patients with DM2 were incubated with hydrogen sulfide donor sodium hydrosulfide (NaHS) in concentrations of 10, 50 and 100 mol and a stimulator of its endogenous synthesis Lcysteine (10, 50 and 100 mol). ATPdependent potassium channels were blocked by glibenclamide (50 mol). Soluble guanylate cyclase was inhibited using 0.5 mol ODQ (1H-1,2,4-oxadiazolo4,3-a quinoxalin-l-one). Deformability (RBCD) and RBCs aggregation were recorded. Results. Under the influence of NaHS, dose-dependent positive changes in RBCs microrheology were observed. Cellular microrheological responses to GT donor were more pronounced in healthy individuals than in patients with DM2. A similar pattern was observed when RBCs were exposed to Lcysteine. Preincubation with glibenclamide did not eliminate the positive effect of GT donor on RBCs microrheology. Whereas inhibition of soluble guanylate cyclase with the help of ODQ completely eliminated positive changes in RBCD under the action of GT and significantly reduced its effect on aggregation. Conclusion. Thus, hydrogen sulfide as a gasotransmitter improves RBCs microrheology. With the same concentration of hydrogen sulphide donor (NaHS) and its synthesis stimulator (Lcysteine), more pronounced shifts in the microrheological parameters of erythrocytes are observed in healthy individuals as compared with DM2 patients.