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

Study the effect of 3,4-Methylenedioxy methamphetamine on cytochrome P450 2E1 activity

Abstract Evaluating the effects of ecstasy on CYP2E1 activity is of great concern, mainly due to growing trends in abuse and co-administration of MDMA with ethanol and the dominant role of this isoenzyme on ethanol metabolism. This study aimed to evaluate the effects of MDMA on CYP2E1 activity. A total of 24 male rats were selected and divided into three groups. The first and second groups consisted of 12 rats and were employed to optimize the perfusion method, and the third group was employed for studying the alteration of CYP2E1 activity after liver exposure to MDMA (300 and 600 ng/ml). The amount of chlorzoxazone and 6-hydroxy chlorzoxazone in a sample obtained from liver perfusion before and after exposure to a buffer containing MDMA was determined by HPLC-FL. The enzymatic activity of rat CYP2E1 decreased after liver perfusion with a buffer containing 600 ng/ml of MDMA. However, no significant changes were observed in chlorzoxazone and 6-hydroxy chlorzoxazone concentration in perfusate before and after liver perfusion with a buffer containing 300 ng/ml of MDMA. Our findings suggest that the activity of CYP2E1 in rats might decrease only after administration of MDMA at a lethal dose. However, further animal and human studies are needed to confirm our assumption.

Validated analytical study of the effect of Lycopene on the pharmacokinetics of Paracetamol and Chlorzoxazone in rats

Lycopene was reported to influence some cytochrome P450 enzymes activity. The present study investigates the effect of lycopene on the pharmacokinetics of paracetamol and chlorzoxazone. Lycopene (20 mg/kg) was intra-peritoneally administered to two groups of rats for eight consecutive days and two other groups were given vehicle. On the eighth day, chlorzoxazone and paracetamol were separately intravenously administered to a lycopene group and a control group. Blood samples were collected at different time intervals, treated and analyzed using HPLC. The HPLC method used for paracetamol analysis was based on isocratic elution using a mobile phase consisting of water: methanol, (77:23 v/v) at a flow rate 1 mL min−1, Kromasil C18 column, and UV detection at 254 nm using caffeine as internal standard. About chlorzoxazone, separation was carried out using water: acetonitrile (60: 40, v/v) as the mobile phase at a flow rate 1 mL min−1, Inertsil ODS-3 C18 column, UV detection at 283 nm and esomeprazole as internal standard. Statistical analysis of the pharmacokinetic data using student t test showed a significant increase in AUC 0-t , AUC 0-Inf and t1/2 of paracetamol (P<0.05) and of chlorzoxazone (P<0.05) in the groups pretreated with lycopene (20 mg/kg), significant increase in the volume of distribution of paracetamol (P < 0.05), but no significant difference in that of chlorzoxazone. In other words, paracetamol and chlorzoxazone showed significant decrease (P < 0.05), respectively. These results demonstrate that treatment of rats with Lycopene (20mg/kg, ip) has a significant effect on the metabolic clearance and the pharmacokinetics of both drugs

Determination of Plasma Concentration of Caffeine,Dapsone and Chlorzoxazone by Solid Phase Extrac-tion-HPLC and Pharmacokinetic Study / 中国药房

OBJECTIVE:To determine plasma concentration of caffeine,dapsone and chlorzoxazone in rats,and to calculate pharmacokinetic parameters. METHODS:6 rats were given the mixture of caffeine,dapsone and chlorzoxazone intragastrically, 1.5,2 and 3 mg/kg,respectively. 0.2-0.3 ml blood were collected before medication and 0.5,1,2,3,4,6,8,12,24 h after medication.The plasma sample was treated with solid phase extraction. The plasma concentration of caffeine,dapsone and chlorzoxa-zone were determined by HPLC using N-(2-Hydroxyethyl) phthalimide as internal standard. The pharmacokinetic parameters were calculated using DAS 2.0 software. RESULTS:The linear ranges of caffeine,dapsone and chlorzoxazone were all 0.2-30 μg/ml (r were 0.996 4,0.996 1,0.998 8,respectively). The limit of quantitation were 0.2 μg/ml. The recoveries of low-concentration, medium-concentration and high concentration were(84.8±3.6)%-(111.4±10.2)%(RSD were 4.3%-9.8%,n=3),(107.0±13.3)%-(113.5±8.1)%(RSD were 7.1%-14.0%,n=3),(104.2±10.8)%-(111.1±12.2)%(RSD were 8.0%-11.0%,n=3). Pharmacoki-netic parameters were as follows as tmax(1.70±0.99),(1.50±1.00),(1.92±0.80)h;t1/2(0.73±0.22),(2.77±1.35),(2.78±2.34) h;cmax (2.60 ± 0.50),(5.78 ± 1.19),(9.76 ± 1.37) mg/L;AUC0-t (8.43 ± 0.79),(20.68 ± 1.91),(26.71 ± 2.45) mg·h/L(n=6). CONCLUSIONS:The method is simple,sensitive and accurate,and can be used for the plasma concentration determination and pharmacokinetic study of caffeine,dapson and chlorzoxazone.

Study of Cytochrome P450 2E1 and its allele Variants in Liver Injury of Nondiabetic, Nonalcoholic Steatohepatitis Obese Women

CYP2E1 enzyme is related to nonalcoholic steatohepatitis (NASH) due to its ability for reactive oxygen species production, which can be influenced by polymorphisms in the gene. The aim of this study was to investigate hepatic levels, activity, and polymorphisms of the CYP2E1 gene to correlate it with clinical and histological features in 48 female obese NASH patients. Subjects were divided into three groups: (i) normal; (ii) steatosis; and (iii) steatohepatitis. CYP2E1 protein level was assayed in microsomes from liver biopsies, and in vivo chlorzoxazone hydroxylation was determined by HPLC. Genomic DNA was isolated for genotype analysis through PCR. The results showed that liver CYP2E1 content was significantly higher in the steatohepatitis (45 percent; p=0.024) and steatosis (22 percent; p=0.032) group compared with normal group. Chlorzoxazone hydroxylase activity showed significant enhancement in the steatohepatitis group (15 percent, p=0.027) compared with the normal group. c2 rare allele of RsallPstl polymorphisms but no C allele of Dral polymorphism was positively associated with CHZ hydroxylation, which in turn is correlated with liver CYP2E1 content (r=0.59; p=0.026). In conclusion, c2 allele is positively associated with liver injury in NASH. This allele may determine a higher transcriptional activity of the gene, with consequent enhancement in pro-oxidant activity of CYP2E1 thus affording liver toxicity.

Effects of chlorzoxazone on HepG2 tumor cells / 中国药理学通报

Aim To investigate effects of chlorzoxazone on survival and apoptosis of HepG2 cells.Methods The necrosis of HepG2 cells was evaluated by measurement of LDH release.The effects of chlorzoxazone on survival of HepG2 cells were assayed by MTT dyereduction.The effects of chlorzoxazone on cell apoptosis was analyzed by TUNEL method.The ultrastructure of HepG2 cells was observed by transmission electron microscope.Results Chlorzoxazone at concentrations of 100～500 ?mol?L-1 inhibited survival ratios of HepG2 cells in a dose-dependent manner significantly.Typical apoptotic changes were observed in HepG2 cells under the fluorescence microscope and transmission electron microscope.Apoptosis of HepG2 cells was induced after treatment of chlorzoxazone at concentrations from 100 ?mol?L-1 to 500 ?mol?L-1 for 48h,which showed obvious concentration-effect relationship.The apoptotic ratios of HepG2 cells were also increased when chlorzoxazone(100,200,300 and 500 ?mol?L-1) was treated for 24,48 and 72 h,which showed obvious time-effect relationship.Conclusion Chlorzoxazone inhibited HepG2 cells survival and induced cell apoptosis.

Determination of the activity of human hepatic cytochromes P450 CYP2C8/9, CYP2E1 / 中国药理学通报

AIM: To establish the methods for assaying the activities of tolbutamide hydroxylase (CYP2C8/9) and chlorzoxazone 6-hydroxylase (CYP2E1) from adult human livers microsomes. METHODS: The microsomes was isolated from human adults liver and the contents of microsomal protein were determined. Using the tolbutamide and chlorzoxazone as substrates, the amount of hydroxytoblbutamide and hydroxychlorzoxazone formed during the incubation period was quantified by extroppolating from the standard curve and the specific activity of CYP2C8/9 and CYP2E1 were calculated. RESULTS: There was no siginificant influence on the activities of CYP2C8/9 and CYP2E1 in different times. CONCLUSIONS: The methods utilized to estimate the activity of CYP2C8/9 and CYP2E1 were simple, stable, and repeatable. These methods can be used in new drug screening, safety evaluation and reseasch on pathology and toxicology of liver.

Simultaneous Determination of Chlorzoxazone and Paracetamol in Compound Chlorzoxazone Tablets by Kalman Filter Spectrophotometry / 中国药房

OBJECTIVE: To determine the contents of chlorzoxazone and paracetamol in compound chlorzoxazone tablets by Kalman Filter spectrophotometry. METHODS: The absorbabilities of the two components were detected in an interval of 1mn over a wavelength range of 248～296 nm. The data obtained were computed by Kalman Filter program. RESULTS: Good linear relations were achieved when the concentration ranges of chlorzoxazone and paracetamol were 2～20 ?g?mL-1 (r=0.999 9) and 3～30 ?g?mL-1 (r=0.999 7) respectively; the recovery rates were 99.1%(RSD=1.01%) and 100.3% (RSD=0.92%), respectively. CONCLUSION: This method is simple, rapid and accurate, and it can be used for the simultaneonsly determination of 2 components in compound chlorzoxazone tablets without separation.

Studies on Pharmacokinetic Interaction Among CYP1A2,CYP2E1 and CYP3A4 Probes in Rats / 中药新药与临床药理

0.05).Conclusion When given concomitantly as a Cocktail,theophylline,chlorzoxazone and dapsone have no pharmacokinetic interaction,which could be used together to evaluate CYP1A2,CYP2E1 and CYP3A4 activity.

Effects of Huangkui Capsule on the P450 activities in rats / 中国临床药理学与治疗学

0.05), meanwhile,after being treated with higher-dose Huangkui Capsule,there were no significant differences for theophylline pharmacokinetics in rats,but AUC_(0-24 h) of chlorzoxazone after treatment was 1.75 times larger than that before treatment(P

Cytochrome P450 2E1 activity in a Korean population

Cytochrome P450 2E1 (CYP2E1) is involved in the toxicity and carcinogenicity of a number of solvents and xenobiotics. Like the various types of oxidation pharmacogenetics, the activity of the enzyme shows a discernible interindividual and interethnic variation. However, no pharmacogenetic information on CYP2E1 polymorphism has been available from a Korean population. The aim of this study was to explore the pharmacogenetics of CYP2E1 polymorphism in a native Koreans after an oral 400 mg dose of chlorzoxazone administered to 128 subjects. Urine samples were collected during the subsequent 8-hour period and urinary concentrations of chlorzoxazone and 6-hydroxychlorzoxazone were determined by a high performance liquid chromatography with an ultraviolet detector. The limit of detection in the samples was found to be 0.5 mug/ml. The mean value of the 6-hydroxychlorzoxazone excreted in 8 hr urine expressed as the percentage was 48.2 13.8%. The frequency distribution of percentage of the administered dose excreted as the 6-hydroxy metabolite was unimodally distributed in the subjects studied. However, the values showed wide (7-fold) interindividual difference, ranged from 11.6% to 79.8% of the dose of chlorzoxazone. Thus, it was considered that the pharmacogenetic characteristics of CYP2E1 in a Korean population did not-represent multimodal distribution in the 6-hydroxychlorzoxazone excreted in 8-hr urine expressed as the percentage. And the activity of the CYP2E1 in a Korean population seemed to be less compared with that of the Caucasian subjects.

Determination of the activity of human hepatic cytochromes P450 CYP2C8/9,CYP2E1 / 中国药理学通报

AIM To establish the methods for assaying the activities of tolbutamide hydroxylase (CYP2C8/9) and chlorzoxazone 6-hydroxylase(CYP2E1) from adult human livers microsomes. METHODS The microsomes was isolated from human adults liver and the contents of microsomal protein were determined. Using the tolbutamide and chlorzoxazone as substrates, the amount of hydroxytoblbutamide and hydroxychlorzoxazone formed during the incubation period was quantified by extroppolating from the standard curve and the specific activity of CYP2C8/9 and CYP2E1 were calculated. RESULTS There was no siginificant influence on the activities of CYP2C8/9 and CYP2E1 in different times. CONCLUSIONS The methods utilized to estimate the activity of CYP2C8/9 and CYP2E1 were simple, stable, and repeatable. These methods can be used in new drug screening, safety evaluation and reseasch on pathology and toxicology of liver.