HLM chip - A microfluidic approach to study the mechanistic basis of cytochrome P450 inhibition using immobilized human liver microsomes.

Cytochrome P450 (CYP) system is a critical elimination route to most pharmaceuticals in human, but also prone to drug-drug interactions arising from the fact that concomitantly administered pharmaceuticals inhibit one another's CYP metabolism. The most severe form of CYP interactions is irreversible inhibition, which results in permanent inactivation of the critical CYP pathway and is only restored by de novo synthesis of new functional enzymes. In this study, we conceptualize a microfluidic approach to mechanistic CYP inhibition studies using human liver microsomes (HLMs) immobilized onto the walls of a polymer micropillar array. We evaluated the feasibility of these HLM chips for CYP inhibition studies by establishing the stability and the enzyme kinetics for a CYP2C9 model reaction under microfluidic flow and determining the half-maximal inhibitory concentrations (IC50) of three human CYP2C9 inhibitors (sulfaphenazole, tienilic acid, miconazole), including evaluation of their inhibition mechanisms and nonspecific microsomal binding on chip. Overall, the enzyme kinetics of CYP2C9 metabolism on the HLM chip (KM = 127 ± 55 µM) was shown to be similar to that of static HLM incubations (KM = 114 ± 14 µM) and the IC50 values toward CYP2C9 derived from the microfluidic assays (sulfaphenazole 0.38 ± 0.09 µM, tienilic acid 3.4 ± 0.6 µM, miconazole 0.54 ± 0.09 µM) correlated well with those determined using current standard IC50 shift assays. Most importantly, the HLM chip could distinguish between reversible (sulfaphenazole) and irreversible (tienilic acid) enzyme inhibitors in a single, automated experiment, indicating the great potential of the HLM chip to simplify current workflows used in mechanistic CYP inhibition studies. Furthermore, the results suggest that the HLM chip can also identify irreversible enzyme inhibitors, which are not necessarily resulting in a time-dependent inhibition (like suicide inhibitors), but whose inhibition mechanism is based on other kind of covalent or irreversible interaction with the CYP system. With our HLM chip approach, we could identify miconazole as such a compound that nonselectively inhibits the human CYP system with a prolonged, possibly irreversible impact in vitro, even if it is not a time-dependent inhibitor according to the IC50 shift assay.

Chloramine Disinfection of Levofloxacin and Sulfaphenazole: Unraveling Novel Disinfection Byproducts and Elucidating Formation Mechanisms for an Enhanced Understanding of Water Treatment.

The unrestricted utilization of antibiotics poses a critical challenge to global public health and safety. Levofloxacin (LEV) and sulfaphenazole (SPN), widely employed broad-spectrum antimicrobials, are frequently detected at the terminal stage of water treatment, raising concerns regarding their potential conversion into detrimental disinfection byproducts (DBPs). However, current knowledge is deficient in identifying the potential DBPs and elucidating the precise transformation pathways and influencing factors during the chloramine disinfection process of these two antibiotics. This study conducts a comprehensive analysis of reaction pathways, encompassing piperazine ring opening/oxidation, Cl-substitution, OH-substitution, desulfurization, and S-N bond cleavage, during chloramine disinfection. Twelve new DBPs were identified in this study, exhibiting stability and persistence even after 24 h of disinfection. Additionally, an examination of DBP generation under varying disinfectant concentrations and pH values revealed peak levels at a molar ratio of 25 for LEV and SPN to chloramine, with LEV contributing 11.5% and SPN 23.8% to the relative abundance of DBPs. Remarkably, this research underscores a substantial increase in DBP formation within the molar ratio range of 1:1 to 1:10 compared to 1:10 to 1:25. Furthermore, a pronounced elevation in DBP generation was observed in the pH range of 7 to 8. These findings present critical insights into the impact of the disinfection process on these antibiotics, emphasizing the innovation and significance of this research in assessing associated health risks.

Sulfaphenazole reduces thermal and pressure injury severity through rapid restoration of tissue perfusion.

Pressure injuries, also known as pressure ulcers, are regions of localized damage to the skin and/or underlying tissue. Repeated rounds of ischemia-reperfusion (I/R) have a major causative role for tissue damage in pressure injury. Ischemia prevents oxygen/nutrient supply, and restoration of blood flow induces a burst of reactive oxygen species that damages blood vessels, surrounding tissues and can halt blood flow return. Minimizing the consequences of repeated I/R is expected to provide a protective effect against pressure injury. Sulfaphenazole (SP), an off patent sulfonamide antibiotic, is a potent CYP 2C6 and CYP 2C9 inhibitor, functioning to decrease post-ischemic vascular dysfunction and increase blood flow. The therapeutic effect of SP on pressure injury was therefore investigated in apolipoprotein E knockout mice, a model of aging susceptible to ischemic injury, which were subjected to repeated rounds of I/R-induced skin injury. SP reduced overall severity, improved wound closure and increased wound tensile strength compared to vehicle-treated controls. Saliently, SP restored tissue perfusion in and around the wound rapidly to pre-injury levels, decreased tissue hypoxia, and reduced both inflammation and fibrosis. SP also demonstrated bactericidal activity through enhanced M1 macrophage activity. The efficacy of SP in reducing thermal injury severity was also demonstrated. SP is therefore a potential therapeutic option for pressure injury and other ischemic skin injuries.

Differential contribution of renal cytochrome P450 enzymes to kidney endothelial dysfunction and vascular oxidative stress in obesity.

Arachidonic acid (AA)-derived cytochrome P450 (CYP) derivatives, epoxyeicosatrienoic acids (EETs) and 20-hidroxyeicosatetranoic acid (20-HETE), play a key role in kidney tubular and vascular functions and blood pressure. Altered metabolism of CYP epoxygenases and CYP hydroxylases has differentially been involved in the pathogenesis of metabolic disease-associated vascular complications, although the mechanisms responsible for the vascular injury are unclear. The present study aimed to assess whether obesity-induced changes in CYP enzymes may contribute to oxidative stress and endothelial dysfunction in kidney preglomerular arteries. Endothelial function and reactive oxygen species (ROS) production were assessed in interlobar arteries of obese Zucker rats (OZR) and their lean counterparts lean Zucker rats (LZR) and the effects of CYP2C and CYP4A inhibitors sulfaphenazole and HET0016, respectively, were examined on the endothelium-dependent relaxations and O2- and H2O2 levels of preglomerular arteries. Non-nitric oxide (NO) non-prostanoid endothelium-derived hyperpolarization (EDH)-type responses were preserved but resistant to the CYP epoxygenase blocker sulfaphenazole in OZR in contrast to those in LZR. Sulfaphenazole did not further inhibit reduced arterial H2O2 levels, and CYP2C11/CYP2C23 enzymes were downregulated in intrarenal arteries from OZR. Renal EDH-mediated relaxations were preserved in obese rats by the enhanced activity and expression of endothelial calcium-activated potassium channels (KCa). CYP4A blockade restored impaired NO-mediated dilatation and inhibited augmented O2- production in kidney arteries from OZR. The current data demonstrate that both decreased endothelial CYP2C11/ CYP2C23-derived vasodilator H2O2 and augmented CYP4A-derived 20-HETE contribute to endothelial dysfunction and vascular oxidative stress in obesity. CYP4A inhibitors ameliorate arterial oxidative stress and restore endothelial function which suggests its therapeutic potential for the vascular complications of obesity-associated kidney injury.

The optimization and characterization of functionalized sulfonamides derived from sulfaphenazole against Mycobacterium tuberculosis with reduced CYP 2C9 inhibition.

In this study, a series of sulfonamide compounds was designed and synthesized through the systematic optimization of the antibacterial agent sulfaphenazole for the treatment of Mycobacterium tuberculosis (M. tuberculosis). Preliminary results indicate that the 4-aminobenzenesulfonamide moiety plays a key role in maintaining antimycobacterial activity. Compounds 10c, 10d, 10f and 10i through the optimization on phenyl ring at the R2 site on the pyrazole displayed promising antimycobacterial activity paired with low cytotoxicity. In particular, compound 10d displayed good activity (MIC = 5.69 µg/mL) with low inhibition of CYP 2C9 (IC50 > 10 µM), consequently low potential risk of drug-drug interaction. These promising results provide new insight into the combination regimen using sulfonamide as one component for the treatment of M. tuberculosis.

Small Molecule Rescue of ATXN3 Toxicity in C. elegans via TFEB/HLH-30.

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is a polyglutamine expansion disease arising from a trinucleotide CAG repeat expansion in exon 10 of the gene ATXN3. There are no effective pharmacological treatments for MJD, thus the identification of new pathogenic mechanisms, and the development of novel therapeutics is urgently needed. In this study, we performed a comprehensive, blind drug screen of 3942 compounds (many FDA approved) and identified small molecules that rescued the motor-deficient phenotype in transgenic ATXN3 Caenorhabditis elegans strain. Out of this screen, five lead compounds restoring motility, protecting against neurodegeneration, and increasing the lifespan in ATXN3-CAG89 mutant worms were identified. These compounds were alfacalcidol, chenodiol, cyclophosphamide, fenbufen, and sulfaphenazole. We then investigated how these molecules might exert their neuroprotective properties. We found that three of these compounds, chenodiol, fenbufen, and sulfaphenazole, act as modulators for TFEB/HLH-30, a key transcriptional regulator of the autophagy process, and require this gene for their neuroprotective activities. These genetic-chemical approaches, using genetic C. elegans models for MJD and the screening, are promising tools to understand the mechanisms and pathways causing neurodegeneration, leading to MJD. Positively acting compounds may be promising candidates for investigation in mammalian models of MJD and preclinical applications in the treatment of this disease.

Characterization of kinetics of human cytochrome P450s involved in bioactivation of flucloxacillin: inhibition of CYP3A-catalysed hydroxylation by sulfaphenazole.

BACKGROUND AND PURPOSE: The aim of this study was to characterize the human cytochrome P450s (CYPs) involved in oxidative bioactivation of flucloxacillin to 5-hydroxymethyl flucloxacillin, a metabolite with high cytotoxicity towards biliary epithelial cells. EXPERIMENTAL APPROACH: The CYPs involved in hydroxylation of flucloxacillin were characterized using recombinant human CYPs, pooled liver microsomes in the presence of CYP-specific inhibitors and by correlation analysis using a panel of liver microsomes from 16 donors. KEY RESULTS: Recombinant CYPs showing the highest specific activity were CYP3A4, CYP3A7 and to lower extent CYP2C9 and CTP2C8. Michaelis-Menten enzyme kinetics were determined for pooled human liver microsomes, recombinant CYP3A4, CYP3A7 and CYP2C9. Surprisingly, sulfaphenazole appeared to be a potent inhibitor of 5'-hydroxylation of flucloxacillin by both recombinant CYP3A4 and CYP3A7. CONCLUSIONS AND IMPLICATIONS: The combined results show that the 5'-hydroxylation of flucloxacillin is primarily catalysed by CYP3A4, CYP3A7 and CYP2C9. The large variability of the hepatic expression of these enzymes could affect the formation of 5'-hydroxymethyl flucloxacillin, which may determine the differences in susceptibility to flucloxacillin-induced liver injury. Additionally, the strong inhibition in CYP3A-catalysed flucloxacillin metabolism by sulfaphenazole suggests that unanticipated drug-drug interactions could occur with coadministered drugs.

Cytochrome P450 2C9-natural antiarthritic interactions: Evaluation of inhibition magnitude and prediction from in vitro data.

Many dietary supplements are promoted to patients with osteoarthritis (OA) including the three naturally derived compounds, glucosamine, chondroitin and diacerein. Despite their wide spread use, research on interaction of these antiarthritic compounds with human hepatic cytochrome P450 (CYP) enzymes is limited. This study aimed to examine the modulatory effects of these compounds on CYP2C9, a major CYP isoform, using in vitro biochemical assay and in silico models. Utilizing valsartan hydroxylase assay as probe, all forms of glucosamine and chondroitin exhibited IC50 values beyond 1000 µM, indicating very weak potential in inhibiting CYP2C9. In silico docking postulated no interaction with CYP2C9 for chondroitin and weak bonding for glucosamine. On the other hand, diacerein exhibited mixed-type inhibition with IC50 value of 32.23 µM and Ki value of 30.80 µM, indicating moderately weak inhibition. Diacerein's main metabolite, rhein, demonstrated the same mode of inhibition as diacerein but stronger potency, with IC50 of 6.08 µM and Ki of 1.16 µM. The docking of both compounds acquired lower CDOCKER interaction energy values, with interactions dominated by hydrogen and hydrophobic bondings. The ranking with respect to inhibition potency for the investigated compounds was generally the same in both in vitro enzyme assay and in silico modeling with order of potency being diacerein/rhein > various glucosamine/chondroitin forms. In vitro-in vivo extrapolation of inhibition kinetics (using 1 + [I]/Ki ratio) demonstrated negligible potential of diacerein to cause interaction in vivo, whereas rhein was predicted to cause in vivo interaction, suggesting potential interaction risk with the CYP2C9 drug substrates.

Physiologically Based Pharmacokinetic Modelling of Cytochrome P450 2C9-Related Tolbutamide Drug Interactions with Sulfaphenazole and Tasisulam.

BACKGROUND AND OBJECTIVES: Cytochrome P450 2C9 (CYP2C9) is involved in the biotransformation of many commonly used drugs, and significant drug interactions have been reported for CYP2C9 substrates. Previously published physiologically based pharmacokinetic (PBPK) models of tolbutamide are based on an assumption that its metabolic clearance is exclusively through CYP2C9; however, many studies indicate that CYP2C9 metabolism is only responsible for 80-90% of the total clearance. Therefore, these models are not useful for predicting the magnitude of CYP2C9 drug-drug interactions (DDIs). This paper describes the development and verification of SimCYP®-based PBPK models that accurately describe the human pharmacokinetics of tolbutamide when dosed alone or in combination with the CYP2C9 inhibitors sulfaphenazole and tasisulam. METHODS: A PBPK model was optimized in SimCYP® for tolbutamide as a CYP2C9 substrate, based on published in vitro and clinical data. This model was verified to replicate the magnitude of DDI reported with sulfaphenazole and was further applied to simulate the DDI with tasisulam, a small molecule investigated for the treatment of cancer. A clinical study (CT registration # NCT01185548) was conducted in patients with cancer to assess the pharmacokinetic interaction of tasisulum with tolbutamide. A PBPK model was built for tasisulam, and the clinical study design was replicated using the optimized tolbutamide model. RESULTS: The optimized tolbutamide model accurately predicted the magnitude of tolbutamide AUC increase (5.3-6.2-fold) reported for sulfaphenazole. Furthermore, the PBPK simulations in a healthy volunteer population adequately predicted the increase in plasma exposure of tolbutamide in patients with cancer (predicted AUC ratio = 4.7-5.4; measured mean AUC ratio = 5.7). CONCLUSIONS: This optimized tolbutamide PBPK model was verified with two strong CYP2C9 inhibitors and can be applied to the prediction of CYP2C9 interactions for novel inhibitors. Furthermore, this work highlights the utility of mechanistic models in navigating the challenges in conducting clinical pharmacology studies in cancer patients.

CYP epoxygenase-derived H2O2 is involved in the endothelium-derived hyperpolarization (EDH) and relaxation of intrarenal arteries.

Reactive oxygen species (ROS) like hydrogen peroxide (H2O2) are involved in the in endothelium-derived hyperpolarization (EDH)-type relaxant responses of coronary and mesenteric arterioles. The role of ROS in kidney vascular function has mainly been investigated in the context of harmful ROS generation associated to kidney disease. The present study was sought to investigate whether H2O2 is involved in the endothelium-dependent relaxations of intrarenal arteries as well the possible endothelial sources of ROS generation involved in these responses. Under conditions of cyclooxygenase (COX) and nitric oxide (NO) synthase inhibition, acetylcholine (ACh) induced relaxations and stimulated H2O2 release that were reduced by catalase and by the glutathione peroxidase (GPx) mimetic ebselen in rat renal interlobar arteries, suggesting the involvement of H2O2 in the endothelium-dependent responses. ACh relaxations were also blunted by the CYP2C inhibitor sulfaphenazole and by the NADPH oxidase inhibitor apocynin. Acetylcholine stimulated both superoxide (O2•-) and H2O2 production that were reduced by sulfaphenazole and apocynin. Expression of the antioxidant enzyme CuZnSOD and of the H2O2 reducing enzymes catalase and GPx-1 was found in both intrarenal arteries and renal cortex. On the other hand, exogenous H2O2 relaxed renal arteries by decreasing vascular smooth muscle (VSM) intracellular calcium concentration [Ca2+]i and markedly enhanced endothelial KCa currents in freshly isolated renal endothelial cells. CYP2C11 and CYP2C23 epoxygenases were highly expressed in interlobar renal arteries and renal cortex, respectively, and were co-localized with eNOS in renal endothelial cells. These results demonstrate that H2O2 is involved in the EDH-type relaxant responses of renal arteries and that CYP 2C epoxygenases are physiologically relevant endothelial sources of vasodilator H2O2 in the kidney.

Development of a Specific Substrate-Inhibitor Panel (Liver-on-a-Chip) for Evaluation of Cytochrome P450 Activity.

We developed a cytochrome P450 substrate-inhibitor panel for preclinical in vitro evaluation of drugs in a 3D histotypical microfluidic cell model of human liver (liver-on-a-chip technology). The concentrations of substrates and inhibitors were optimized to ensure reliable detection of the principal metabolites by HPLC-mass-spectroscopy. The selected specific substrate-inhibitor pairs, namely bupropion/2-phenyl-2-(1-piperidinyl)propane) for evaluation of CYP2B6B activity, tolbutamide/sulfaphenazole for CYP2C9, omeprazole/(+)-N-benzylnirvanol for CYP2C19, and testosterone/ketoconazole for CYP3A4, enable reliable evaluation of the drug metabolism pathway. In contrast to animal models characterized by species-specific expression profile and activity of cytochrome P450 isoforms, our in vitro model reflects the metabolism of human hepatocytes in vivo.

Improvement of the chemical inhibition phenotyping assay by cross-reactivity correction.

BACKGROUND: The fraction of an absorbed drug metabolized by the different hepatic cytochrome P450 (CYP) enzymes, relative to total hepatic CYP metabolism (fmCYP), can be estimated by measuring the inhibitory effects of presumably selective CYP inhibitors on the intrinsic metabolic clearance of a drug using human liver microsomes. However, the chemical inhibition data are often affected by cross-reactivities of the chemical inhibitors used in this assay. METHODS: To overcome this drawback, the cross-reactivities exhibited by six chemical inhibitors (furafylline, montelukast, sulfaphenazole, ticlopidine, quinidine and ketoconazole) were quantified using specific CYP enzyme marker reactions. The determined cross-reactivities were used to correct the in vitro fmCYPs of nine marketed drugs. The corrected values were compared with reference data obtained by physiologically based pharmacokinetics simulation using the software SimCYP. RESULTS: Uncorrected in vitro fmCYPs of the nine drugs showed poor linear correlation with their reference data (R2=0.443). Correction by factoring in inhibitor cross-reactivities significantly improved the correlation (R2=0.736). CONCLUSIONS: Correcting in vitro chemical inhibition results for cross-reactivities appear to offer a straightforward and easily adoptable approach to provide improved fmCYP data for a drug.

Chemical Compensation of Mitochondrial Phospholipid Depletion in Yeast and Animal Models of Parkinson's Disease.

We have been investigating the role that phosphatidylethanolamine (PE) and phosphatidylcholine (PC) content plays in modulating the solubility of the Parkinson's disease protein alpha-synuclein (α-syn) using Saccharomyces cerevisiae and Caenorhabditis elegans. One enzyme that synthesizes PE is the conserved enzyme phosphatidylserine decarboxylase (Psd1/yeast; PSD-1/worms), which is lodged in the inner mitochondrial membrane. We previously found that decreasing the level of PE due to knockdown of Psd1/psd-1 affects the homeostasis of α-syn in vivo. In S. cerevisiae, the co-occurrence of low PE and α-syn in psd1Δ cells triggers mitochondrial defects, stress in the endoplasmic reticulum, misprocessing of glycosylphosphatidylinositol-anchored proteins, and a 3-fold increase in the level of α-syn. The goal of this study was to identify drugs that rescue this phenotype. We screened the Prestwick library of 1121 Food and Drug Administration-approved drugs using psd1Δ + α-syn cells and identified cyclosporin A, meclofenoxate hydrochloride, and sulfaphenazole as putative protective compounds. The protective activity of these drugs was corroborated using C. elegans in which α-syn is expressed specifically in the dopaminergic neurons, with psd-1 depleted by RNAi. Worm populations were examined for dopaminergic neuron survival following psd-1 knockdown. Exposure to cyclosporine, meclofenoxate, and sulfaphenazole significantly enhanced survival at day 7 in α-syn-expressing worm populations whereby 50-55% of the populations displayed normal neurons, compared to only 10-15% of untreated animals. We also found that all three drugs rescued worms expressing α-syn in dopaminergic neurons that were deficient in the phospholipid cardiolipin following cardiolipin synthase (crls-1) depletion by RNAi. We discuss how these drugs might block α-syn pathology in dopaminergic neurons.

Impaired endothelial calcium signaling is responsible for the defective dilation of mesenteric resistance arteries from db/db mice to acetylcholine.

We aimed at assessing the role of endothelial cell calcium for the endothelial dysfunction of mesenteric resistance arteries of db/db mice (a model of type 2 diabetes) and determine whether treatment with sulfaphenazole, improves endothelial calcium signaling and function. Pressure myography was used to study acetylcholine (ACh) -induced vasodilation. Intracellular calcium ([Ca(2+)]i) transients was measured by confocal laser scanning microscopy and smooth muscle membrane potential with sharp microelectrodes. The impaired dilation to ACh observed in mesenteric resistance arteries from db/db mice was improved by treatment of the mice with sulfaphenazole for 8 weeks. The impaired dilation to ACh was associated with decreased endothelial [Ca(2+)]i and smooth muscle hyperpolarization. Sulfaphenazole applied in vitro improved endothelial mediated dilation of arteries from db/db mice both in the absence and the presence of inhibitors of nitric oxide and cyclooxygenase. Sulfaphenazole also increased the percentage of endothelial cells with ACh induced increases of [Ca(2+)]i. The study shows that impaired endothelial [Ca(2+)]i control can explain the reduced endothelial function in arteries from diabetic mice and that sulfaphenazole treatment improves endothelial [Ca(2+)]i responses to ACh and consequently endothelium-dependent vasodilation. These observations provide mechanistic insight into endothelial dysfunction in diabetes.

Nanoscale electron transport measurements of immobilized cytochrome P450 proteins.

Gold nanopillars, functionalized with an organic self-assembled monolayer, can be used to measure the electrical conductance properties of immobilized proteins without aggregation. Measurements of the conductance of nanopillars with cytochrome P450 2C9 (CYP2C9) proteins using conducting probe atomic force microscopy demonstrate that a correlation exists between the energy barrier height between hopping sites and CYP2C9 metabolic activity. Measurements performed as a function of tip force indicate that, when subjected to a large force, the protein is more stable in the presence of a substrate. This agrees with the hypothesis that substrate entry into the active site helps to stabilize the enzyme. The relative distance between hopping sites also increases with increasing force, possibly because protein functional groups responsible for electron transport (ETp) depend on the structure of the protein. The inhibitor sulfaphenazole, in addition to the previously studied aniline, increased the barrier height for electron transfer and thereby makes CYP2C9 reduction more difficult and inhibits metabolism. This suggests that P450 Type II binders may decrease the ease of ETp processes in the enzyme, in addition to occupying the active site.

Resveratrol alters human endothelial cells redox state and causes mitochondrial-dependent cell death.

Studies analyzing the impact of natural antioxidants (NA) on Endothelial Cells (ECs) have dramatically increased during the last years, since a deregulated ECs redox state is at the base of the onset and progression of several cardiovascular diseases. However, whether NA can provide cardiovascular benefits is still a controversial area of debate. Resveratrol (RES), a natural polyphenol found in grapes, is believed to provide cardiovascular benefits by virtue of its antioxidant effect on the endothelium. Here, we report that tissue-attainable doses of resveratrol increased the intracellular oxidative state, thus affecting mitochondrial membrane depolarization and inducing EC death. Cyclosporine A, a mitochondrial permeability transition pore inhibitor, prevented oxidative-mediated cell death, thus implicating mitochondria in resveratrol-induced EC impairment. The specific cytochrome P450 (CYP) 2C9 inhibitor, sulfaphenazole, counteracted both oxidative stress and mitochondrial membrane depolarization, providing EC protection against resveratrol-elicited pro-oxidant effects. Our findings strongly suggest that CYP2C9 mediates resveratrol-induced oxidative stress leading to mitochondria impairment and EC death.

Cytochrome P450 2C epoxygenases mediate photochemical stress-induced death of photoreceptors.

Degenerative loss of photoreceptors occurs in inherited and age-related retinal degenerative diseases. A chemical screen facilitates development of new testing routes for neuroprotection and mechanistic investigation. Herein, we conducted a mouse-derived photoreceptor (661W cell)-based high throughput screen of the Food and Drug Administration-approved Prestwick drug library to identify putative cytoprotective compounds against light-induced, synthetic visual chromophore-precipitated cell death. Different classes of hit compounds were identified, some of which target known genes or pathways pathologically associated with retinitis pigmentosa. Sulfaphenazole (SFZ), a selective inhibitor of human cytochrome P450 (CYP) 2C9 isozyme, was identified as a novel and leading cytoprotective compound. Expression of CYP2C proteins was induced by light. Gene-targeted knockdown of CYP2C55, the homologous gene of CYP2C9, demonstrated viability rescue to light-induced cell death, whereas stable expression of functional CYP2C9-GFP fusion protein further exacerbated light-induced cell death. Mechanistically, SFZ inhibited light-induced necrosis and mitochondrial stress-initiated apoptosis. Light elicited calcium influx, which was mitigated by SFZ. Light provoked the release of arachidonic acid from membrane phospholipids and production of non-epoxyeicosatrienoic acid metabolites. Administration of SFZ further stimulated the production of non-epoxyeicosatrienoic acid metabolites, suggesting a metabolic shift of arachidonic acid under inhibition of the CYP2C pathway. Together, our findings indicate that CYP2C genes play a direct causative role in photochemical stress-induced death of photoreceptors and suggest that the CYP monooxygenase system is a risk factor for retinal photodamage, especially in individuals with Stargardt disease and age-related macular degeneration that deposit condensation products of retinoids.

Sulfaphenazole and α-naphthoflavone attenuate the metabolism of the synthetic cannabinoids JWH-018 and AM2201 found in K2/spice.

"K2" or "Spice" is an emerging drug of abuse that is laced with psychoactive synthetic cannabinoids JWH-018 and AM2201. Previous studies have identified hydroxylated (OH) and carboxylated (COOH) species as primary human metabolites, and kinetic studies have implicated CYP2C9 and -1A2 as major hepatic P450s involved in JWH-018 and AM2201 oxidation. The present study extends these findings by testing the hypothesis that CYP2C9- and 1A2-selective chemical inhibitors, sulfaphenazole (SFZ) and α-naphthoflavone (ANF), block oxidation of JWH-018 and AM2201 in human liver microsomes (HLM). A concentration-dependent inhibition of JWH-018 and AM2201 oxidation was observed in the presence of increasing concentration of SFZ (0.5 - 50 µM) and ANF (0.1 - 5.0 µM). No metabolic inhibition was observed with omeprazole, quinidine, and ketoconazole. The results presented herein further demonstrate the importance of CYP2C9- and 1A2-mediated oxidation of JWH-018 and AM2201 and the likelihood of adverse toxicity in populations with polymorphic alleles of these enzymes.

Coumaric acid induces mitochondrial damage and oxidative-mediated cell death of human endothelial cells.

Evidence that higher natural antioxidants (NA) intake provides cardiovascular protection is contradictory. The endothelium plays a pivotal role in cardiovascular homeostasis, and for this reason, the molecular events resulting from the interaction of NA with endothelial cells (ECs) are actively investigated. Here, we show that moderately high doses of coumaric acid (CA) induced intracellular reactive oxygen species (ROS) production, mitochondrial membrane depolarization and ECs death. Treatment of ECs with cyclosporine A, a mitochondrial permeability transition pore inhibitor, prevented the oxidative-mediated cell damage indicating mitochondrial involvement in CA-induced ECs impairment. CA-induced intracellular ROS generation was counteracted by the specific cytochrome P450 (CYP) 2C9 inhibitor sulfaphenazole (SPZ). SPZ also prevented CA-induced mitochondrial membrane depolarization and ECs death, implicating CYP2C9 in mediating the cellular response upon CA treatment. Our results indicate that moderately high doses of CA can promote CYP2C9-mediated oxidative stress eliciting mitochondrial-dependent ECs death and may pave the way toward mechanistic insight into NA effects on cardiovascular cells.

[Metabolites and metabolic pathways of mesaconitine in rat liver microsomal investigated by using UPLC-MS/MS method in vitro].

Mesaconitine was incubated with rat liver microsomes in vitro. The metabolites of mesaconitine in rat liver microsomes were identified by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method with high resolution power. A typical reaction mixture of 100 mol L-1 Tris-HCI buffer (pH 7.4) containing 0.5 gL-1 microsomal protein and 50 micro molL-1 mesaconitine was prepared. The above reaction mixture was divided into six groups, and the volume of each group was 200 micro L. The incubation mixture was pre-incubated at 37 degrees C for 2 min and the reactions were initiated by adding NADPH generating system. After 90 min incubation at 37 degrees C, 200 micro L of acetonitrile was added to each group to stop the reaction. The metabolites of mesaconitine were investigated by UPLC-MS/MS method. Mesaconitine and 6 metabolites M1-M6 were found in the incubation system. The structures were characterized according to the data from MS/MS spectra and literatures. The metabolic reactions of mesaconitine in rat liver microsomes included the demethylation, deacetylation, dehydrogenation and hydroxylation. The major metabolic pathways of mesaconitine in rat liver microsomes were determined by UPLC-MS/MS on multiple reaction monitoring (MRM) mode combined with specific inhibitors of cytochrome P450 (CYP) isoforms, including alpha-naphthoflavone (CYP1A2), quinine (CYP2D), diethyldithiocarbamate (CYP2E1), ketoconazole (CYP3A) and sulfaphenazole (CYP2C), separately. Mesaconitine was mainly metabolized by CYP3A. CYP2C and CYP2D were also more important CYP isoforms for the metabolism reactions of mesaconitine, but CYP1A2 and CYP2E1 haven't any contribution to MA metabolism in rat liver microsomes.