In Vitro Prediction and in Vivo Verification of Enantioselective Human Tofisopam Metabolite Profiles

Abstract

ABSTRACT:In vitro studies were conducted to elucidate the metabolic profiles of and the enzymes responsible for the metabolism of (R) The use of pure enantiomers as pharmaceuticals has greatly increased the awareness of the effect of chirality on the interactions of small molecules with proteins. In addition to the development of new drugs, several older racemic drugs such as nefopam and thioridazine have been reintroduced as pure enantiomers (Brian, 2001). In cases where one of the enantiomers shows greater activity, the use of pure enantiomers can have key advantages such as improved pharmacokinetics or decreasing nonspecific toxicity problems. There are instances in which toxicity has been linked to one member of a pair of stereoisomers, not necessarily the active isomer (FDA, 1992).-Racemic tofisopam (1-(3,4-dimethoxyphenyl)-5-ethyl-7,8-dimethoxy-4-methyl-5H-2,3-benzodiazepine) is approved in 18 countries including Japan, South Korea, Russia, and Hungary and has been used for many years to treat disorders of the autonomic nervous system (Pakkanen et al., 1980;Hovi-Viander et al., 1985;Andrasi et al., 1987;Nedogoda and Parshev, 2000). Recently the individual enantiomers have been evaluated for their pharmacological activity. Animal models on both (R)-and (S)-tofisopam suggest that they decrease visceral hypersensitivity, have no effect on normal gastrointestinal function, and normalize gastrointestinal motility in stressed conditions. (R)-Tofisopam has an improved activity for irritable bowl syndrome and is currently in phase IIb clinical trials.Metabolism of racemic tofisopam in microsomal incubations from multiple species was reported previously (Elekes et al., 1981;Tomori et al., 1982Tomori et al., , 1984 and the primary metabolites were identified. These metabolites corresponded to demethylation of one or more of the four tofisopam methoxy groups by cytochrome P450. All previous studies detailing tofisopam metabolism were conducted on racemic tofisopam instead of individual enantiomers and did not evaluate the specific enzymes responsible for tofisopam metabolism.In the present study, we present data on the stereoselective metabolism of the individual enantiomers of tofisopam. The concentrations of tofisopam, 50 and 500 ng/ml (Ϸ0.13 and 1.3 M), were chosen to represent clinical concentrations. In vitro studies with recombinant P450 enzymes and with human liver microsomes, (R)-and (S)-tofisopam, showed different metabolite profiles for the two enantiomers. The primary enzymes responsible for generation of the individual metabolites were identified. These in vitro results were confirmed by analyses of samples from human clinical trials. Materials and MethodsChemicals Used. Pure enantiomers of tofisopam, racemic metabolites, and d 6 -tofisopam were synthesized by Cerilliant (Round Rock, TX). Other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) in the highest purity available. Human liver microsomes (lot 821-1) pooled from 15 individuals (male and female) were obtained from CellzDirect (Aust...

New Discoveries in Enantiomeric Separation of Racemic Tofisopam

“…All four advantaged structures have a sedative effect-so they are currently in the racemic form commercially available; however, their efficacy [10,11] and their metabolism [12] are significantly different.…”

Tofisopam: Prospects for Non-Hormonal Therapy of Neuro-Vegetative and Psycho-Emotional Disorders Associated With Menopausal Syndrome

“…Основные метаболиты: R-Тофизопам образуется при участии фермента катализировать CYP2C9 и S-Тофизопам -при участии CYP3А4. Выведение из организма происходит в первую очередь путем глюкоронидации [16].…”

Analytical QbD Approach for Development and Validation of RP-High Performance Liquid Chromatography Method for Determination of Tofisopam in Pure Form and Tablets

“…It is a white, pale yellowish-white crystalline powder, practically insoluble in water, sparingly soluble in ethanol, freely soluble in methanol, and acetonitrile with melting range 155-159°C. [2] There are analytical methods for the estimation of tofisopam with spectrophotometric and spectrofluorimetic [3] chromatographic, [4][5][6] enantiomeric separation, [7,8] gas-liquid chromatography (GLC), [9] and super-critical chromatography [10] method. All these methods require a large sample volume (1-5 mL) and time-consuming sample clean up procedure.…”