Emphasis Type="Italic"Cunninghamella/Emphasis

Authors: Elżbieta Pękala Paulina Kubowicz Dorota Łażewska

Publish Date: 2012/09/16

Volume: 168, Issue: 6, Pages: 1584-1593

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Abstract

The aim of the study was to analyze the ability of the microorganism Cunninghamella to carry out the biotransformation of 134tertbutylphenoxypropylpiperidine DL76 and to compare the obtained results with in silico models Biotransformation was carried out by three strains of filamentous fungus Cunninghamella echinulata Cunninghamella blakesleeana and Cunninghamella elegans Most probable direction of DL76 metabolic transition was the oxidation of the methyl group in the tertbutyl moiety leading to the formation of the metabolite with I° alcohol properties This kind of reaction was conducted by all three strains tested However only in the case of C blakesleeana that biotransformation product had a structure of carboxylic acid CYP2C19 was identified by Metasite software to be the isoform of major importance in the oxidation process in the tertbutyl moiety of DL76 In silico data coincide with the results of experiments conducted in vitro It was confirmed that Cunninghamella fungi are a very good model to study the metabolism of xenobiotics The computational methods and microbial models of metabolism can be used as useful tools in early ADMETox assays in the process of developing new drug candidatesEach drug before it is introduced to the market must undergo a complicated procedure At the beginning it must be checked if a given structure is active if yes it is not the end of the procedure The metabolism of such a substance and its toxicity is also very important 1 Before the drug reaches to clinical trials and is administered to humans there is another stage—preclinical screening 2 This stage gives us information about ADMET—absorption distribution metabolism excretion and toxicology 3Drug metabolism studies can rely on the use of animal systems like mouse 4 rat 5 or guinea pig 6 in vivo Unfortunately these models suffer from a number of limitations like ethical aspects time that must be spend on breeding animals and last but not least cost of experimental models Because of these disadvantages use of in vitro studies becomes an important tool for testing drugs and for production of valuable drug metabolites In vitro screening assays include human liver models like perfused liver 7 cell lines 8 hepatocytes 9 and liver slices 10 S9 fractions are very interesting because they contain both phase I and phase II activities and are useful in the study of xenobiotic metabolism and drug interaction 11 Human liver microsomes are another popular in vitro model 12 They are a rich source of drugmetabolizing enzymes 13 However in vitro studies are not only on human liver models To elucidate the metabolism of xenobiotics microbial cultures like bacteria 14 fungi 15 or yeast 16 can be used The cost of in vitro models is lower than in vivo 17 culture and extraction of cytochrome P450 are simple 18 and in such models the so called “nonsuffering organisms” are usedAnother way to study the metabolism of a drug is the so called in silico model It allows to predict the biological properties and parameters of ADMET by computational simulation 19 However neither in vitro models—cell or tissue culture microsomal preparations—nor in silico studies will replace animal system but the number of animals suffering from such tests may or even must be limited One of such widely used in vitro microbiological model is Cunninghamella 20Cunninghamella has specific properties which make this fungus very useful in studies of drug metabolism This property is the ability to metabolize a wide variety of drugs over a hundred of them in manners that are similar to those in mammalian enzyme systems 20 It was proven that Cunninghamella has enzymes that are synonymous to those involved in xenobiotic detoxification in mammals 21 Moreover there are many evidences 21 22 that Cunninghamella can predict the fate of the drug in the mammalian organism better than other microorganismsThe histamine H3 receptor H3R has been identified in the central nervous system CNS and peripheral nervous system as a presynaptic receptor controlling the release of histamine and numerous other neurotransmitters 23 In the past histamine H3R antagonists were imidazolecontaining compounds Imperfection of these structures appeared in unwanted hepatic cytochrome P450 inhibition and potential drug–drug interactions Therefore a new class of nonimidazole histamine H3R antagonists was designed and synthesized 24 Potential therapeutic use of histamine receptor ligands involves treatment of CNS diseases 25 In our department for many years we were looking for new ligands of the H3R in a group of nonimidazole derivatives One of the newly synthesized compounds 134tertbutylphenoxypropylpiperidine—DL76 was proved to be highly potent and orally available histamine H3 receptor antagonist hH3R K i = 22 ± 3 nM—affinity for the recombinant human H3R stably expressed in CHO ED50 28 ± 04 mg/kg 26The media required for the growth of Cunninghamella strains were purchased from BioShop Canada potato dextrose agar and from SigmaAldrich St Louis MO USA CSL Corn Steep Liquor DL76 was synthesized in the Department of Chemical Technology and Biotechnology of Drugs Faculty of Pharmacy Medical College of Jagiellonian University Cracow Poland HPLCgrade acetonitrile dichloromethane water and formic acid were obtained from Merck Darmstadt Germany All other chemicals were of analytical reagent grade and were obtained from SigmaAldrich

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