Stereoselective analysis of thioridazine-2-sulfoxide and thioridazine-5-sulfoxide: An investigation of rac-thioridazine biotransformation by some endophytic fungi

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Abstract

The purpose of this study was to develop a method for the stereoselective analysis of thioridazine-2-sulfoxide (THD-2-SO) and thioridazine-5-sulfoxide (THD-5-SO) in culture medium and to study the biotransformation of rac-thioridazine (THD) by some endophytic fungi. The simultaneous resolution of THD-2-SO and THD-5-SO diastereoisomers was performed on a CHIRALPAK® AS column using a mobile phase of hexane:ethanol:methanol (92:6:2, v/v/v) + 0.5% diethylamine; UV detection was carried out at 262 nm. Diethyl ether was used as extractor solvent. The validated method was used to evaluate the biotransformation of THD by 12 endophytic fungi isolated from Tithonia diversifolia, Viguiera arenaria and Viguiera robusta. Among the 12 fungi evaluated, 4 of them deserve prominence for presenting an evidenced stereoselective biotransformation potential: Phomopsis sp. (TD2) presented greater mono-2-sulfoxidation to the form (S)-(SE) (12.1%); Glomerella cingulata (VA1) presented greater mono-5-sulfoxidation to the forms (S)-(SE) + (R)-(FE) (10.5%); Diaporthe phaseolorum (VR4) presented greater mono-2-sulfoxidation to the forms (S)-(SE) and (R)-(FE) (84.4% and 82.5%, respectively) and Aspergillus fumigatus (VR12) presented greater mono-2-sulfoxidation to the forms (S)-(SE) and (R)-(SE) (31.5% and 34.4%, respectively).

Introduction

Microorganisms have been used to produce chemicals, pharmaceuticals and perfumes for decades [1], [2] and also for pollutants degradation and recovery of the environment contaminated by chemicals [3]. The use of microorganisms can simplify or in some instances even enable the production process of complex chemicals and drug intermediates. In addition, they can add stereospecificity to the process, eliminating the need for complicated separation and purification steps. However, the use of microorganisms by the industry to obtain interesting compounds is still modest, considering the great availability of useful microorganisms and the large scope of reactions that can be accomplished by them.

Another interesting use of microorganisms is for studying biotransformation of drugs and other chemicals such as pesticides. The microbial biotransformation system is very similar to the mammalian phase I metabolic reactions. Therefore, these in vitro models can be an attractive alternative for tests of new drugs, making possible the production of metabolites in high amounts, facilitating the structural elucidation and toxicological tests [4].

Smith and Rosazza, in the early 1970's, established the use of microbial models for mammalian metabolism [5], [6]. It has been demonstrated that some fungi can generate a similar metabolic profile to mammalian and that the metabolism is also stereoselective [7]. Since them some studies have been reported describing the use of fungi for the biotransformation of drugs [8], [9], [10].

A special kind of fungi are the endophytic fungi, microorganisms that colonize and grow asymptomatically within internal plant tissues without causing harm to their host [11]. To present date, there are few studies using endophytic fungi in stereoselective biotransformation. Agusta et al. [12] reported the stereoselective oxidation at C-4 of flavans by the endophytic fungus Diaporthe sp. isolated from a tea plant Camelia sinensis.

Thioridazine (THD), a phenothiazine neuroleptic drug used for the treatment of schizophrenia and other psychiatric disorders, is commercially available as a racemic mixture of the enantiomers (−)-(S) and (+)-(R). It is extensively metabolized by S-oxidation to thioridazine-5-sulfoxide (THD-5-SO) and thioridazine-2-sulfoxide (THD-2-SO) which is further oxidized to thioridazine-2-sulfone (THD-2-SO2) [13], [14], [15]. THD-2-SO and THD-2-SO2 are metabolites considered pharmacologically active while THD-5-SO contributes to the cardio toxicity of the drug [16], [17]. A summary of rac-thioridazine's metabolic profile in humans is demonstrated in Fig. 1. THD-2-SO and THD-5-SO exist as two diastereoisomeric pairs, referred to fast-eluting (FE) THD-2-SO and slow-eluting (SE) THD-2-SO, and fast-eluting THD-5-SO and slow-eluting THD-5-SO, respectively, based on their chromatography properties [18], [19]. The (R) and (S) configurations of THD and their metabolites are related to the chiral carbon at position 2 in the piperidyl ring [19].

Considering that THD metabolites are also therapeutically active, but with undesirable side effects that can be attributed to specific stereoisomers, the study of its metabolism by endophytic fungi can be useful to obtain THD metabolites in the enantiomerically pure form and further accomplishment of clinical and/or toxicological studies.

Therefore, the aim of the present work was to develop a method for the stereoselective analysis of THD-2-SO and THD-5-SO in culture medium and to study the biotransformation of rac-thioridazine (THD) by some endophytic fungi. Although the stereoselective analyses of THD and/or its metabolites have already been described in the literature, none of the methods reported allows the simultaneous resolution of THD-2-SO and THD-5-SO stereoisomers [18], [20], [21], [22].

Section snippets

Standard solutions and chemicals

rac-thioridazine, thioridazine-2-sulfoxide and thioridazine-5-sulfoxide were kindly supplied by Novartis Pharma AG (Basel, Switzerland). Stock standard solutions were prepared in methanol at concentrations of 2.00, 4.00, 10.0, 20.0, 40.0, 80.0, 200 and 400 μg mL−1 and were stored at −20 °C in the absence of light. It is important to mention that the drugs used in the present work consist of the FE pair of enantiomers for THD-2-SO (racemic mixture of (R)-THD-2-SO (FE) and (S)-THD-2-SO (FE)) and of

Method validation

The chiral resolution of THD-2-SO and THD-5-SO has already been studied by our group. De Gaitani et al. [21] reported a method for the stereoselective analysis of THD-5-SO in human plasma by capillary electrophoresis using hydroxypropyl-β-cyclodextrin and sulfated-β-cyclodextrin as chiral selectors. Further, the degradation and epimerization THD-2-SO in human plasma, buffer and methanolic solutions were studied using an enantioselective HPLC method with a Chiralpak AD [22]. Unfortunately, these

Conclusions

A suitable high-performance liquid chromatography method was developed and validated for the stereoselective determination of thioridazine-2-sulfoxide and thioridazine-5-sulfoxide simultaneously in a fermentative medium.

This method was used to study the biotransformation of thioridazine by endophytic fungi, showing that different metabolites could be obtained by using different types of fungi and that metabolite production was stereoselective. The results reported here could be further used to

Acknowledgements

The authors are grateful to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), sub-program BIOTA/FAPESP (Rede BIOprospecTA), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for financial support and for granting research fellowships.

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