In vitro and in vivo metabolic activation and hepatotoxicity of chlorzoxazone mediated by CYP3A.

Chlorzoxazone (CZX), a benzoxazolone derivative, has been approved for the treatment of musculoskeletal disorders to relieve localized muscle spasm. However, its idiosyncratic toxicity reported in patients brought attention, particularly for hepatotoxicity. The present study for the first time aimed at the relationship between CZX-induced hepatotoxicity and identification of oxirane intermediate resulting from metabolic activation of CZX. Two N-acetylcysteine (NAC) conjugates (namely M1 and M2) and two glutathione (GSH) conjugates (namely M3 and M4) were detected in rat & human microsomal incubations with CZX (200 µM) fortified with NAC or GSH, respectively. The formation of M1-M4 was NADPH-dependent and these metabolites were also observed in urine or bile of SD rats given CZX intragastrically at 10 mg/kg or 25 mg/kg. NAC was found to attach at C-6' of the benzo group of M1 by sufficient NMR data. CYPs3A4 and 3A5 dominated the metabolic activation of CZX. The two GSH conjugates were also observed in cultured rat primary hepatocytes after exposure to CZX. Inhibition of CYP3A attenuated the susceptibility of hepatocytes to the cytotoxicity of CZX (10-400 µM). The in vitro and in vivo studies provided solid evidence for the formation of oxirane intermediate of CZX. This would facilitate the understanding of the underlying mechanisms of toxic action of CZX.

Quantification of ternary mixture of paracetamol, chlorzoxazone and ibuprofen present in tablet dosage form using ratio subtraction spectrophotometric approaches.

A sensitive and selective spectrophotometric approach comprising of successive ratio subtraction was developed for quantification and resolution of spectrum of mixture containing three components without prior separation. Three components, namely paracetamol, chlorzoxazone and ibuprofen were present in tablet dosage form. The linearity studies were carried out by recording zero order spectra and measuring absorbances at 285.0, 282.0 and 220.0 nm for paracetamol, chlorzoxazone and ibuprofen respectively. The drugs exhibited linear response in the concentration range of 6.0-18.0, 3.0-15.0 and 4.0-20.0 µg / mL for paracetamol, chlorzoxazone and ibuprofen respectively. The spectrum of paracetamol was most extended which was subsequently followed by moderately extended spectrum of chlorzoxazone and unextended spectrum of ibuprofen. The ratio spectra were manipulated successfully for quantification of paracetamol, chlorzoxazone, and ibuprofen. The developed method was validated as per ICH guidelines for the parameters of specificity, linearity, precision and accuracy. The percent recoveries for all the three drugs were in the range of 98.0-102.0 % with mean recovery of paracetamol, chlorzoxazone and ibuprofen were 99.72, 99.02 and 100.34 % respectively. Additionally the validity of the method is assured by analyzing marketed formulation.

Tree shrew cytochrome P450 2E1 is a functional enzyme that metabolises chlorzoxazone and p-nitrophenol.

Cytochromes P450 (CYPs or P450s) are important enzymes for drug metabolism. Tree shrews are non-primate animal species used in various fields of biomedical research, including infection (especially hepatitis viruses), depression, and myopia. A recent tree shrew genome analysis indicated that the sequences and the numbers of P450 genes are similar to those of humans; however, P450s have not been adequately identified and analysed in this species.In this study, a novel CYP2E1 was isolated from tree shrew liver and was characterised in comparison with human, dog, and pig CYP2E1. Tree shrew CYP2E1 and human CYP2E1 showed high amino acid sequence identity (83%) and were closely related in a phylogenetic tree.Gene and genome structures of CYP2E1 were generally similar in humans, dogs, pigs, and tree shrews. Tissue expression patterns showed that tree shrew CYP2E1 mRNA was predominantly expressed in liver, just as for dog and pig CYP2E1 mRNAs. In tree shrews, recombinant CYP2E1 protein and liver microsomes metabolised chlorzoxazone and p-nitrophenol, probe substrates of human CYP2E1, just as they do in dogs and pigs.These results suggest that tree shrew CYP2E1 encodes a functional drug-metabolising enzyme that plays a role in the liver, similar to human CYP2E1.

A chlorzoxazone-folic acid combination improves cognitive affective decline in SCA2-58Q mice.

Spinocerebellar ataxia type 2 (SCA2) is a polyglutamine disorder caused by a pathological expansion of CAG repeats in ATXN2 gene. SCA2 is accompanied by cerebellar degeneration and progressive motor decline. Cerebellar Purkinje cells (PCs) seem to be primarily affected in this disorder. The majority of the ataxia research is focused on the motor decline observed in ataxic patients and animal models of the disease. However, recent evidence from patients and ataxic mice suggests that SCA2 can also share the symptoms of the cerebellar cognitive affective syndrome. We previously reported that SCA2-58Q PC-specific transgenic mice exhibit anxiolytic behavior, decline in spatial memory, and a depressive-like state. Here we studied the effect of the activation of the small conductance calcium-activated potassium channels (SK channels) by chlorzoxazone (CHZ) combined with the folic acid (FA) on the PC firing and also motor, cognitive and affective symptoms in SCA2-58Q mice. We realized that CHZ-FA combination improved motor and cognitive decline as well as ameliorated mood alterations in SCA2-58Q mice without affecting the firing rate of their cerebellar PCs. Our results support the idea of the combination therapy for both ataxia and non-motor symptoms in ataxic mice without affecting the firing frequency of PCs.

Hyperacusis in the Adult Fmr1-KO Mouse Model of Fragile X Syndrome: The Therapeutic Relevance of Cochlear Alterations and BKCa Channels.

Hyperacusis, i.e., an increased sensitivity to sounds, is described in several neurodevelopmental disorders (NDDs), including Fragile X Syndrome (FXS). The mechanisms underlying hyperacusis in FXS are still largely unknown and effective therapies are lacking. Big conductance calcium-activated potassium (BKCa) channels were proposed as a therapeutic target to treat several behavioral disturbances in FXS preclinical models, but their role in mediating their auditory alterations was not specifically addressed. Furthermore, studies on the acoustic phenotypes of FXS animal models mostly focused on central rather than peripheral auditory pathways. Here, we provided an extensive characterization of the peripheral auditory phenotype of the Fmr1-knockout (KO) mouse model of FXS at adulthood. We also assessed whether the acute administration of Chlorzoxazone, a BKCa agonist, could rescue the auditory abnormalities of adult mutant mice. Fmr1-KO mice both at 3 and 6 months showed a hyperacusis-like startle phenotype with paradoxically reduced auditory brainstem responses associated with a loss of ribbon synapses in the inner hair cells (IHCs) compared to their wild-type (WT) littermates. BKCa expression was markedly reduced in the IHCs of KOs compared to WT mice, but only at 6 months, when Chlorzoxazone rescued mutant auditory dysfunction. Our findings highlight the age-dependent and progressive contribution of peripheral mechanisms and BKCa channels to adult hyperacusis in FXS, suggesting a novel therapeutic target to treat auditory dysfunction in NDDs.

Green Constant-wavelength Concurrent Selective Fluorescence Method for Assay of Ibuprofen and Chlorzoxazone in Presence of Chlorzoxazone Degradation Product.

Lower back pain is a universal dilemma leaving a negative effect on both health and life quality. It was found that a fixed dose combination of chlorzoxazone and ibuprofen gave a higher efficiency than analgesic alone in treatment of acute lower back pain. Based on the significant benefit of that combination, a green, sensitive, rapid, direct, and cost-effective method is created for concurrent determination of ibuprofen and chlorzoxazone in presence of 2-amino para chlorophenol (a synthetic precursor and potential impurity of chlorzoxazone) adopting the synchronous spectrofluorimetric technique. Synchronous spectrofluorimetric technique is adopted to avoid the highly overlapped native spectra of both drugs. The synchronous spectrofluorometric method was applied at Δλ = 50 nm, ibuprofen was measured at 227 nm while chlorzoxazone was measured at 282 nm with no hindering from one to another. The various experimental variables affecting the performance of the suggested technique were explored and adjusted. The suggested technique showed good linearity from 0.02 to 0.6 and 0.1 to 5.0 µg/mL for ibuprofen and chlorzoxazone, respectively. The produced detection limits were 0.27 × 10-3 and 0.03, while the quantitation limits were 0.82 × 10-3 and 0.09 µg/mL for ibuprofen and chlorzoxazone, respectively. The suggested approach was successfully applied for the analysis of the studied drugs in the synthetic mixture, different pharmaceutical preparations, and spiked human plasma. The suggested technique was validated with respect to the International Council of Harmonization (ICH) recommendations. The suggested technique was found to be simpler and greener with lower cost compared to the earlier reported methods which required complicated techniques, longer time of analysis, and less safe solvents and reagents. Green profile assessment for the developed method compared with the reported spectrofluorometric method was performed using four assessment tools. These tools confirmed that the recommended technique attained the most possible green parameters, so it could be used as a greener option in routine quality control for analyzing the two drugs in genuine form and pharmaceutical preparations.

Influence of solvent choice and operating conditions on Chlorzoxazone crystal shape and size

Abstract Solubility of pharmaceutical drugs in organic solvents is one of the important parameters to understand the equilibrium concentration of solute-solvent, which helps optimize and design crystallization conditions to obtain the desired product crystals. In the present study, Chlorzoxazone (CHZ) is used as a model pharmaceutical compound to investigate the equilibrium solubility, the influence of solvent and the operating conditions on the shape, and the size distribution. The solubility of CHZ is determined in organic solvents like Isopropanol, Ethanol, and 2-Ethoxyethylacetate, Ethylacetate and Ethyllactate using shake flask method from -5ºC to 60ºC. The solubility of CHZ in these solvents shows an increasing trend as the temperature increases in the following order: ethyllactate + water (0.5+0.5) < ethylacetate < isopropanol < ethanol < 2-ethoxyethylacetate < ethyllactate + water (0.75+0.25). The solvents, isopropanol, ethanol, and ethyl lactate, produce needle-shaped crystals, while 2-ethoxyethylacetate and ethyl acetate tend to produce plate shaped crystals. CHZ crystals obtained from 2-ethoxyethylacetate tend to have plate shaped crystals with a lower aspect ratio and are selected for batch cooling crystallization experiments performed at different cooling rates, and agitation. It is found that the agitation at 300 rpm and the cooling rate 0.2ºC/min produce more uniform crystal size distribution

Silymarin Protects against Acute Liver Injury Induced by Acetaminophen by Downregulating the Expression and Activity of the CYP2E1 Enzyme.

Previous studies have shown that silymarin protects against various types of drug-induced liver injury, but whether the protective mechanism of silymarin against acetaminophen-induced liver injury is related to the CYP2E1 enzyme remains unclear. In this study, we investigated the effect of silymarin on the activity and expression of CYP2E1 in vitro and in vivo. The results of in vitro studies showed that silymarin not only inhibited the activity of CYP2E1 in human and rat liver microsomes but also reduced the expression of CYP2E1 in HepG2 cells. In vivo studies showed that silymarin pretreatment significantly reduced the conversion of chlorzoxazone to its metabolite 6-OH-CLX and significantly increased the t1/2, area under the curve (AUC) and mean residence time (MRT) of chlorzoxazone. In addition, silymarin pretreatment significantly inhibited the upregulation of Cyp2e1 expression, reduced the production of 3-cysteinylacetaminophen trifluoroacetic acid salt (APAP-CYS), and restored the liver glutathione level. The results of our study show that silymarin plays an important protective role in the early stage of acetaminophen-induced acute liver injury by reducing the activity and expression of CYP2E1, reducing the generation of toxic metabolites, and alleviating liver injury.

Comparative Risk of Opioid Overdose With Concomitant Use of Prescription Opioids and Skeletal Muscle Relaxants.

BACKGROUND AND OBJECTIVES: The concomitant use of prescription opioids and skeletal muscle relaxants has been associated with opioid overdose, but little data exist on the head-to-head safety of these drug combinations. The objective of this study was to compare the risk of opioid overdose among patients on long-term opioid therapy who concurrently initiate skeletal muscle relaxants. METHODS: We conducted an active comparator cohort study spanning 2000 to 2019 using healthcare utilization data from 4 US commercial and public insurance databases. Individuals were required to have at least 180 days of continuous enrollment and at least 90 days of continuous prescription opioid use immediately before and on the date of skeletal muscle relaxant initiation. Exposures were the concomitant use of prescription opioids and skeletal muscle relaxants, and the main outcome was the hazard ratio (HR) and bootstrapped 95% CI of opioid overdose resulting in an emergency department visit or hospitalization. The primary analysis quantified opioid overdose risk across 7 prescription opioid-skeletal muscle relaxant therapies and a negative control outcome (sepsis) to assess potential confounding by unmeasured illicit opioid use. Secondary analyses evaluated two-group and five-group comparisons in patients with similar baseline characteristics; individuals without previous recorded substance abuse; and subgroups stratified by baseline opioid dosage, benzodiazepine codispensing, and oxycodone or hydrocodone use. RESULTS: Weighted HR of opioid overdose relative to cyclobenzaprine was 2.52 (95% CI 1.29-4.90) for baclofen; 1.64 (95% CI 0.81-3.34) for carisoprodol; 1.14 (95% CI 0.53-2.46) for chlorzoxazone/orphenadrine; 0.46 (95% CI 0.17-1.24) for metaxalone; 1.00 (95% CI 0.45-2.20) for methocarbamol; and 1.07 (95% CI 0.49-2.33) for tizanidine in the 30-day intention-to-treat analysis. Findings were similar in the as-treated analysis, 2-group and 5-group comparisons, and patients without previous recorded substance abuse. None of the therapies relative to cyclobenzaprine were associated with sepsis, and no subgroups indicated an increased risk of opioid overdose. DISCUSSION: Concomitant use of prescription opioids and baclofen relative to cyclobenzaprine is associated with opioid overdose. Clinical interventions may focus on prescribing alternatives in the same drug class or providing access to opioid antagonists if treatment with both medications is necessary for pain management.

Gut microbiota and host Cyp450s co-contribute to pharmacokinetic variability in mice with non-alcoholic steatohepatitis: Effects vary from drug to drug.

INTRODUCTION: Pharmacokinetic variability in disease state is common in clinical practice, but its underlying mechanism remains unclear. Recently, gut microbiota has been considered to be pharmacokinetically equivalent to the host liver. Although some studies have explored the roles of gut microbiota and host Cyp450s in drug pharmacokinetics, few have explored their effects on pharmacokinetic variability, especially in disease states. OBJECTIVES: In this study, we aim to investigate the effects of gut microbiota and host Cyp450s on pharmacokinetic variability in mice with non-alcoholic steatohepatitis (NASH), and to elucidate the contribution of gut microbiota and host Cyp450s to pharmacokinetic variability in this setting. METHODS: The pharmacokinetic variability of mice with NASH was explored under intragastric and intravenous administrations of a cocktail mixture of omeprazole, phenacetin, midazolam, tolbutamide, chlorzoxazone, and metoprolol, after which the results were compared with those obtained from the control group. Thereafter, the pharmacokinetic variabilities of all drugs and their relations to the changes in gut microbiota and host Cyp450s were compared and analyzed. RESULTS: The exposures of all drugs, except metoprolol, significantly increased in the NASH group under intragastric administration. However, no significant increase in the exposure of all drugs, except tolbutamide, was observed in the NASH group under intravenous administration. The pharmacokinetic variabilities of phenacetin, midazolam, omeprazole, and chlorzoxazone were mainly associated with decreased elimination activity in the gut microbiota. By contrast, the pharmacokinetic variability of tolbutamide was mainly related to the change in the host Cyp2c65. Notably, gut microbiota and host Cyp450s exerted minimal effects on the pharmacokinetic variability of metoprolol. CONCLUSION: Gut microbiota and host Cyp450s co-contribute to the pharmacokinetic variability in mice with NASH, and the degree of contribution varies from drug to drug. The present findings provide new insights into the explanation of pharmacokinetic variability in disease states.

Chlorophyll sensitized (BiO)2CO3/ CdWO4/rGO nano-hybrid assembly for solar assisted photo-degradation of chlorzoxazone.

In the present laboratory scale experiment, we report the fabrication of chlorophyll sensitized (BiO)2CO3/CdWO4/rGO (BCR) photo-catalyst. The green approach has been adopted for boosting the optical activity by chlorophyll as a sensitizer. The functionality, nature and surface compositions of synthesized photo-catalyst have been identified by FTIR, XRD and XPS instrumentation. The internal and surface morphology has been studied using FE-SEM and HR-TEM. The optical activity has been investigated by UV-vis and photoluminescence spectroscopy. The catalytic activity of chlorophyll sensitized BCR have been tested for the photo degradation of Chlorzoxazone (CZX) under simulated visible light for 90 min. The detailed comparison has been studied for the different loading amount of chlorophyll and RGO onto BCR photo-catalyst. The potential of BCR for the photo-degradation of CZX was investigated under various operational parameters such as catalysts dosage, pollutant concentration, effect of pH and ions etc. Approximately, 96.2% of CZX has been degraded over 90 min with the optimum catalyst amount 250 mgL-1 at pH 7. The ●OH radical has been identified as major reactive species using radical scavenging experiment. The mineralization of CZX has been evaluated in terms of HR-MS and TOC-COD analysis.

Cholesterol's inhibition effect on entering of chlorzoxazone into phosphatidylethanolamine bilayer: Relevance to cytochrome P450 drug metabolism at endoplasmic reticulum membranes.

Many drugs are metabolized by cytochrome P450 (CYP) in the endoplasmic reticulum (ER) membrane. Recent studies have shown that CYP-substrate drugs reach the CYP active site after entering the lipid hydrophobic part of the ER membrane. To clarify the role of cholesterol (Chol) in the CYP-related drug metabolic process, we investigated the lipid bilayer entry of CYP-substrate drugs using a model membrane system as follows. The model membrane system comprised palmitoyl-oleoyl-phosphatidylethanolamine (POPE) and Chol. Phosphatidylethanolamine is the second major phospholipid component of ER membranes. Chlorzoxazone (CZX) was used as the CYP-substrate drug. Calorimetric measurements showed that the addition of CZX to POPE bilayers decreased the gel-liquid crystal phase transition temperature; X-ray diffraction indicated that CZX distributes into the liquid crystal phase bilayers but not practically the gel phase POPE bilayers. In the presence of Chol, dialysis and X-ray structural analyses showed that Chol inhibited CZX entry into the bilayer with an increase in Chol concentration. The Chol concentration in the ER membrane (5-10 mol%) is much lower than that in the plasma membrane (approximately 30 mol%). This fact may allow CYP-substrate drugs to enter the hydrophobic portion of the ER membrane more easily than other organelle membranes, yielding efficient drug metabolism.

Comparison of mouse and human cytochrome P450 mediated-drug metabolising activities in hepatic and extrahepatic microsomes.

Despite the importance of mice as a preclinical species in drug testing, their hepatic and extrahepatic drug-metabolising characteristics are poorly understood. Here, we compared the P450-dependent drug oxidation activity in tissue microsomes and distribution patterns of P450 protein/mRNA between humans and mice.The activities of midazolam 1'-/4-hydroxylation in the liver and intestine and chlorzoxazone 6-hydroxylation in the liver were similar in humans and mice. The activities of coumarin 7-hydroxylation, flurbiprofen 4'-hydroxylation, and S-mephenytoin 4'-hydroxylation in the liver were higher in humans than in mice. The activities of 7-ethoxyresorufin O-deethylation in the liver, 7-pentoxyresorufin O-depentylation in the lung/liver/intestine, bufuralol 1'-hydroxylation in the liver/intestine, propafenone 4'-hydroxylation in liver/intestine, and diazepam N-demethylation in the liver/intestine were higher in mice than in humans.CYP1A2/2E1 mRNAs were mainly expressed in the livers of humans and mice. Cyp2b9/2b10 mRNAs were abundant in the mouse lung/liver/intestine, but CYP2B6 was mainly expressed in the human liver. CYP2C/2D/3A mRNAs were expressed in the liver and intestine, with the respective proteins detected in tissue microsomes of both humans and mice.These information on P450-dependent drug-metabolising characteristics in hepatic and extrahepatic tissues is useful to understand the similarities and differences between humans and mice in drug metabolism.

Chlorzoxazone Alleviates Experimental Autoimmune Encephalomyelitis via Inhibiting IL-6 Secretion of Dendritic Cells.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating autoimmune disease with chronic inflammatory demyelination of the CNS. Experimental autoimmune encephalomyelitis (EAE) is an important animal model to study MS, with many pathological phenomena similar to MS. Th17 cells are important regulators of EAE and MS pathogenesis. Most cytokines needed for Th cell development are secreted by APCs, such as dendritic cells (DCs). Consequently, MS could be improved by inhibiting cytokine secretion from DCs. In this study, we reported that chlorzoxazone could ameliorate EAE pathogenesis via inhibiting IL-6 production by DCs. The EAE signs in the chlorzoxazone-treated group of mice were relieved, which was mainly manifested as lower clinical scores, a decrease in the number of immune cells, and a reduction of demyelination in the CNS. Moreover, the proportion of Th17 cells in the spleen and CNS decreased significantly. In vitro experiments showed that chlorzoxazone treatment significantly reduced DC-derived IL-6 production. In the DC-T cell coculture experiment, significantly decreased Th17 differentiation was observed after chlorzoxazone treatment. In addition, mass spectrometric analysis was performed to elucidate the mechanism by which chlorzoxazone affected EAE and DC function. We showed that the effect of chlorzoxazone on inhibiting the secretion of IL-6 by DCs may be mediated via the AMP-activated protein kinase pathway. Overall, our study elucidated the key role of chlorzoxazone in regulating EAE pathogenesis and suggested that it might be used as a new drug for MS patients.

[Effect of Laportea bulbifera extract on CYP450 enzyme activities in rats by Cocktail probe drug method].

The Cocktail probe drug method was used to evaluate the effect of Laportea bulbifera extract on the different subtypes of CYP450 enzyme activities in rats, and to provide references for its clinical rational drug use. The rats were randomly divided into a high-dose L. bulbifera group(0.45 g·kg~(-1)·d~(-1)) and a low-dose L. bulbifera group(0.09 g·kg~(-1)·d~(-1)). After continuous gavage for 7 and 14 days, the Cocktail probe mixing solution, including caffeine, midazolam, tolbutamide, omeprazole, metoprolol, and chlorzoxazone, was injected into the tail vein, and the blood sample was obtained from the tail vein at different time points. Ultra-high performance liquid chromatography-mass spectrometry(UPLC-MS) was established to determine the concentration of six probe drugs in rat plasma. DAS 2.0 was used to calculate its pharmacokinetic parameters, and the effect of L. bulbifera extract on CYP1 A2, CYP2 C9, CYP2 C19, CYP2 D6, CYP2 E1, and CYP3 A4 in rats was investigated. As compared with the blank control group, under the omeprazole index, the AUC_(0-t) and AUC_(0-∞) of the 7-day administration groups and the 14-day high-dose group were significantly decreased, and the CLz and Vz were significantly increased. Under the midazolam index, the AUC_(0-t) and AUC_(0-∞) of the 7-day low-dose group and the 14-day administration group were significantly decreased, and the CLz was significantly increased. In addition, the AUC_(0-∞) of the 7-day high-dose group was also significantly decreased. Under the index of metoprolol, the AUC_(0-t) and AUC_(0-∞) of each experimental group were decreased significantly, and the CLz and Vz of the 7-day low-dose group and the 14-day low-dose group were increased significantly. Under the caffeine index, the AUC_(0-t) and AUC_(0-∞) of the 7-day administration groups were increased significantly, the CLz was decreased significantly, and the t_(1/2 z) of the 14-day high-dose group was increased significantly. Under the chlorzoxazone index, the AUC_(0-t) and AUC_(0-∞) of the 7-day low-dose group were increased significantly, and the CLz was decreased significantly. Under the tolbutamide index, there was no statistical difference in the pharmacokinetic parameters. In conclusion, L. bulbifera extract induces the activities of CYP2 C19, CYP3 A4, and CYP2 D6, inhibits the activities of CYP1 A2 and CYP2 E1, and does not affect the activity of CYP2 C9.

Solar-Powered Whole-Cell P450 Catalytic Platform for C-Hydroxylation Reactions.

Invited for this month's cover is the joint research group of Prof. Chan Beum Park at the Korea Advanced Institute of Science and Technology (KAIST) and Prof. Chul-Ho Yun at the Chonnam National University (CNU). The image shows how the use of a natural photosensitizer, flavin mononucleotide, and visible light can lead to a cost-effective, green, and sustainable process for P450-catalyzed reactions in a whole-cell system. The Communication itself is available at 10.1002/cssc.202100944.

Amalgamation of in-silico, in-vitro and in-vivo approach to establish glabridin as a potential CYP2E1 inhibitor.

CYP2E1 is directly or indirectly involved in the metabolism of ethanol and endogenous fatty acids but it plays a major role in the bio-activation of toxic substances that produce reactive metabolites leading to hepatotoxicity. Therefore, identification of CYP2E1 inhibitor from bioflavonoids class having useful pharmacological properties has dual benefit regarding avoidance of severe food-drug/nutraceutical-drug interaction and scope to develop a phytotherapeutics through an intended pharmacokinetic interaction.In the present study, we aimed to identify CYP2E1 inhibitor from experimental bioflavonoids which are unexplored for CYP2E1 inhibition till date using in-silico, in-vitro and in-vivo approaches.Results of in-vitro CYP2E1 inhibitory studies using CYP2E1-mediated chlorzoxazone 6-hydroxylation in human liver microsomes showed that glabridin have the highest potential than fisetin, epicatechin, nobiletin, and chrysin to inhibit CYP2E1 enzyme. Mechanistic investigations indicate that glabridin is a competitive CYP2E1 inhibitor. Molecular docking study results demonstrate that glabridin strongly interacted with the active site of human CYP2E1 enzyme. Pharmacokinetics of a CYP2E1 substrate in mice model indicates a significant alteration of chlorzoxazone and 6-hydroxychlorzoxazone plasma levels in the presence of glabridin. Further studies are needed to confirm the results at clinical level.Overall, glabridin is found to be a potential CYP2E1 inhibitor.

Green spectrofluorimetric methods for tramadol assay with ibuprofen or chlorzoxazone: comparison of greenness profiles.

At this time, green analytical chemistry is gaining more interest and concern. The present work details three green spectrofluorimetric methods for tramadol (TRM) determination using ibuprofen (IBU) (mixture 1) and chlorzoxazone (CLZ) (mixture 2). In first method, two excitation wavelengths (λex ), 220 and 280 nm, were used to record the emission spectra for IBU and TRM, respectively (mixture 1) followed by a first derivative treatment. For mixture 2, one λex (280 nm) was optimum for both drugs followed by a first derivative technique for TRM and a second derivative for CLZ determinations. The second method measured the first derivative values for synchronous spectra using constant-wavelength mode at 280 nm for TRM and 260 nm for IBU, and at 270 nm for TRM and 292 nm for CLZ. The third method used constant-energy mode to record synchronous spectra. First derivative values were computed at 282 nm for TRM and 260 nm for IBU in mixture 1 and at 272 nm for TRM and 292 nm for CLZ in mixture 2. ICH validation guidelines were assessed in full and assay of the two TRM binary mixtures in their drug products was successful. Green profile evaluation for the developed methods compared with the reported chromatographic methods was performed using the 'analytical eco-scale' and the 'green analytical procedure index'. These two assessment tools corroborated that the proposed methods achieved the most green parameters, therefore recommending their use as a green option for analyzing the studied drugs in bulk and dosage forms for routine quality control.

In vivo analysis of the spontaneous firing of cerebellar Purkinje cells in awake transgenic mice that model spinocerebellar ataxia type 2.

Cerebellar Purkinje cells (PCs) fire spontaneously in a tonic mode, although the precision of this pacemaking activity is disturbed in many abnormal conditions involving cerebellar atrophy, such as many spinocerebellar ataxias (SCAs). In our previous studies we used the single-unit extracellular recording method to analyze spontaneous PC firing in vivo in the anesthetized SCA2-58Q transgenic mice. We realized that PCs from aging SCA2-58Q mice fire much less regularly compared to PCs from their wild type (WT) littermates and this abnormal activity can be reversed with an intraperitoneal (i. p.) injection of SK channel-positive modulator chlorzoxazone (CHZ). Here we used the same single-unit extracellular recording method to analyze the spontaneous firing in vivo in awake SCA2-58Q transgenic mice. For this purpose, we used the Mobile HomeCage (Neurotar, Finland) floating platform to immobilize the experimental animal's head during the recording sessions. We discovered that generally PCs from awake animals fired much more frequently and much less regularly than previously observed PCs from anesthetized animals. In vivo recordings from awake SCA2/WT mice revealed that complex spikes, which are generated by PCs in reply to the excitation coming by climbing fibers, as well as simple spikes, were much less frequent in SCA2 mice compared to their WT littermates. To test the effect of the SK channel positive modulation on the PCs firing activity in awake SCA2 mice and also the effect on their motor coordination, we started the CHZ trial in these mice. We discovered that the long-term i. p. injections of CHZ did not affect the spike generation in SCA2-58Q mice, however, they did recover the precision of this spontaneous pacemaking activity. Furthermore, we also showed that treatment with CHZ alleviated the age-dependent motor impairment in SCA2-58Q mice. We propose that the lack of precision in PC spike generation might be a key cause for the progression of ataxic symptoms in different SCAs and that the activation of calcium-activated potassium channels, including SK channels, can be used as a potential way to treat SCAs on the physiological level of the disease.

A Chlorzoxazone-Baclofen Combination Improves Cerebellar Impairment in Spinocerebellar Ataxia Type 1.

BACKGROUND: A combination of central muscle relaxants, chlorzoxazone and baclofen (chlorzoxazone-baclofen), has been proposed for treatment of cerebellar symptoms in human spinocerebellar ataxia. However, central muscle relaxants can worsen balance. The optimal dose for target engagement without toxicity remains unknown. Using the genetically precise Atxn1154Q/2Q model of spinocerebellar ataxia type 1, we aimed to determine the role of cerebellar dysfunction in motor impairment. We also aimed to identify appropriate concentrations of chlorzoxazone-baclofen needed for target engagement without toxicity to plan for human clinical trials. METHODS: We use patch clamp electrophysiology in acute cerebellar slices and immunostaining to identify the specific ion channels targeted by chlorzoxazone-baclofen. Behavioral assays for coordination and grip strength are used to determine specificity of chlorzoxazone-baclofen for improving cerebellar dysfunction without off-target effects in Atxn1154Q/2Q mice. RESULTS: We identify irregular Purkinje neuron firing in association with reduced expression of ion channels Kcnma1 and Cacna1g in Atxn1154Q/2Q mice. Using in vitro electrophysiology in brain slices, we identified concentrations of chlorzoxazone-baclofen that improve Purkinje neuron spike regularity without reducing firing frequency. At a disease stage in Atxn1154Q/2Q mice when motor impairment is due to cerebellar dysfunction, orally administered chlorzoxazone-baclofen improves motor performance without affecting muscle strength. CONCLUSION: We identify a tight relationship between baclofen-chlorzoxazone concentrations needed to engage target and levels above which cerebellar function will be compromised. We propose to use this information for a novel clinical trial design, using sequential dose escalation within each subject, to identify dose levels that are likely to improve ataxia symptoms while minimizing toxicity. © 2020 International Parkinson and Movement Disorder Society.