Biotransformation of metoprolol by the fungus Cunninghamella blakesleeana

Aim： To investigate the biotransformation of metoprolol, a β-cardioselective adrenoceptor antagonist, by filamentous fungus, and to compare the parallels between microbial transformation and mammalian metabolism. Methods： Five strains of Cunninghamella （ C elegans AS 3.156, C elegans AS 3.2028, C echinulata AS 3.2004, C blakesleeana AS 3.153 and AS 3.910） were screened for the ability to transform metoprolol. The metabolites of metoprolol produced by C blakesleeana AS 3.153 were separated and assayed by liquid chromatography-tandem mass spectrometry （LC/MSn）. The major metabolites were isolated by semipreparative HPLC and the structures were identified by a combination of LC/MSn and nuclear magnetic resonance analysis. Results： Metoprolol was transformed to 7 metabolites; 2 were identified as new metabolites and 5 were known metabolites in mammals. Conclusion： The microbial transformation of metoprolol was similar to the metabolism in mammals. The fungi belonging to Cunninghamella species could be used as complementary models for predicting in vivo metabolism and producing quantities of metabolite references for drugs like metoprolol.     

小克银汉霉菌对萘普生的微生物转化

目的：通过分离和鉴定萘普生由真菌转化生成的代谢产物,研究微生物转化和哺乳动物体内药物代谢之间的相似性．方法：选用3种小克银汉霉菌对萘普生进行微生物转化研究．采用液相色谱—质谱联用法检测代谢产物,并通过半制备高效液相色谱法分离出主要代谢物,经质谱和核磁共振光谱确证其结构．结果：萘普生被转化成去甲萘普生和去甲萘普生硫酸结合物,这两种代谢产物都是已知的哺乳动物代谢产物．其中去甲萘普生硫酸结合物是首次在微生物转化样品中发现．结论：小克银汉霉菌对萘普生的转化结果与哺乳动物的体内代谢有某些相似性,有可能成为一种新的体外模型进行药物代谢研究,用于预测和制备可能的药物代谢产物．     

Biotransformation of indomethacin by the fungus Cunninghamella blakesleeana

AIM: To investigate the biotransformation of indomethacin, the first of the newer nonsteroidal anti-inflammatory drugs, by filamentous fungus and to compare the similarities between microbial transformation and mammalian metabolism of indomethacin. METHODS: Five strains of Cunninghamella (C elegans AS 3.156, C elegans AS 3.2028, C blakesleeana AS 3.153, C blakesleeana AS 3.910 and C echinulata AS 3.2004) were screened for their ability to catalyze the biotransformation of indomethacin. Indomethacin was partially metabolized by five strains of Cunninghamella, and C blakesleeana AS 3.910 was selected for further investigation. Three metabolites produced by C blakesleeana AS 3.910 were isolated using semi-preparative HPLC, and their structures were identified by a combination analysis of LC/MS(n) and NMR spectra. These three metabolites were separated and quantitatively assayed by liquid chromatography-ion trap mass spectrometry. RESULTS: After 120 h of incubation with C blakesleeana AS 3.910, approximately 87.4% of indomethacin was metabolized to three metabolites: O-desmethylindomethacin (DMI, M1, 67.2%), N-deschlorobenzoylindomethacin (DBI, M2, 13.3%) and O-desmethyl-N-deschlorobenzoylindomethacin (DMBI, M3, 6.9%). Three phase I metabolites of indomethacin produced by C blakesleeana AS 3.910 were identical to those obtained in humans. CONCLUSION: C blakesleeana could be a useful tool for generating the mammalian phase I metabolites of indomethacin.     

短刺小克银汉霉AS 3.153菌株对丁咯地尔的代谢转化

建立能够模拟丁咯地尔在哺乳动物体内代谢的微生物模型,研究丁咯地尔在微生物体内的转化途径,并应用该模型制备代谢产物。菌株筛选实验中考察了4种小克银汉霉对丁咯地尔的转化能力,确定最佳菌株为短刺小克银汉霉AS 3.153;然后针对该菌株进行转化条件(培养基初始pH、底物浓度和转化时间)优化,建立微生物模型。应用LC-MSn法对转化样品进行分析,共检测到3种代谢产物,其中2种为首次发现的丁咯地尔代谢产物,进一步采用半制备液相色谱法制备获得对位-O-去甲基丁咯地尔和12-C-氧化丁咯地尔。哺乳动物对比实验表明,短刺小克银汉霉AS 3.153对丁咯地尔的代谢情况与人和比格犬类似,而与大鼠差异较大。     

Carbon–carbon bond cleavage and formation reactions in drug metabolism and the role of metabolic enzymes

Elimination of xenobiotics from the human body is often facilitated by a transformation to highly water soluble and more ionizable molecules. In general, oxidation–reduction, hydrolysis, and conjugation reactions are common biotransformation reactions that are catalyzed by various metabolic enzymes including cytochrome P450s (CYPs), non-CYPs, and conjugative enzymes. Although carbon–carbon (C–C) bond formation and cleavage reactions are known to exist in plant secondary metabolism, these reactions are relatively rare in mammalian metabolism and are considered exceptions. However, various reactions such as demethylation, dealkylation, dearylation, reduction of alkyl chain, ring expansion, ring contraction, oxidative elimination of a nitrile through C–C bond cleavage, and dimerization, and glucuronidation through C–C bond formation have been reported for drug molecules. Carbon–carbon bond cleavage reactions for drug molecules are primarily catalyzed by CYP enzymes, dimerization is mediated by peroxidases, and C-glucuronidation is catalyzed by UGT1A9. This review provides an overview of C–C bond cleavage and formation reactions in drug metabolism and the metabolic enzymes associated with these reactions.     

活性黄酮类成分生物转化的研究进展

黄酮类化合物具有抗感染、抗肿瘤、抗炎镇痛、抗氧化等多种生物活性,是开发活性先导物或新药的宝库.利用植物细胞、微生物和酶对黄酮类化合物进行生物转化是提供新型活性黄酮、解决黄酮溶解度差、生物利用率低等问题的有效手段.综述近年来利用植物细胞、微生物、酶转化黄酮类活性成分及其生物转化过程中影响因子的研究文献,并对其研究进展作了分析.     

Microbial models of mammalian metabolism of xenobiotics: An updated review

The utilization of microbes as models for mammalian metabolism of xenobiotics has been well established since the concept was first introduced by Smith and Rosazza in the early seventies. The core assumption of this concept rests on the fact that fungi are eukaryotic organisms that possess metabolizing enzyme systems similar to those present in mammalian systems. Hence, the outcome of xenobiotic metabolism in both systems is expected to be similar, if not identical, and, thus, fungi can be used to predict the outcome of mammalian metabolism of various xenobiotics, including drugs. Utilizing microbial models offers a number of advantages over the use of animals in metabolism studies, mainly reduction in use of animals, ease of setup and manipulation, higher yield and diversity of metabolite production, and lower cost of production. In a continuation to our contribution to this field, this review will outline the results of studies that were conducted over the last seven years to emphasize the similarities between the microbial and mammalian metabolic pathways of xenobiotics through the endorsement of the concept of microbial models of mammalian metabolism .     

Production of drugs by microbial biosynthesis and biotransformation. Possibilities, limits and future developments (1st communication)

Before the advent of alchemy the therapeutic aids for man and animals consisted exclusively of using natural products in many different forms. Chemical syntheses have been used for little more than 100 years as a means of obtaining drugs. The discovery of penicillin and the first industrial production of this compound in 1941/42 opened the door to a third way for the preparation of drugs by exploitation of the manifold biosynthetic capabilities of microorganisms to produce antibiotics or more recently other pharmacologically active substances. The selective use of individual enzymatic transformation stages with microorganisms in chemical production pathways in particular by biotransformations of steroids in 1950 expanded the field of biotechnological production of pharmaceuticals. The increasing knowledge in the regulation of the biosynthesis of primary and secondary metabolites, the growing experience in the use of microorganisms as biocatalysts and source of valuable enzymes and the development of new economical technical procedures raised the number and volume of drugs prepared by microbial biosynthesis and biotransformation. The modern method of the genetic engineering supported by the chemical DNA-synthesis enabled the preparation of important proteohormones and physiologically active peptides in microorganisms. Finally, the development of monoclonal antibodies, although at present still formed in mammalian cells, will lead to new ways of therapy in future. A review is given on the present state of biotechnological productions of antibiotics, vitamins, steroids, alkaloids, amino acids and pharmaceutical enzymes combined with new developments in the preparation of blood factors, enzyme inhibitors, hormones and physiologically active peptides and the possible future use of monoclonal antibodies.     

Genetic polymorphisms of human N-acetyltransferase, cytochrome P450, glutathione-S-transferase, and epoxide hydrolase enzymes: relevance to xenobiotic metabolism and toxicity

In this review, an overview is presented of the current knowledge of genetic polymorphisms of four of the most important enzyme families involved in the metabolism of xenobiotics, that is, the N-acetyltransferase (NAT), cytochrome P450 (P450), glutathione-S-transferase (GST), and microsomal epoxide hydrolase (mEH) enzymes. The emphasis is on two main topics, the molecular genetics of the polymorphisms and the consequences for xenobiotic metabolism and toxicity. Studies are described in which wild-type and mutant alleles of biotransformation enzymes have been expressed in heterologous systems to study the molecular genetics and the metabolism and pharmacological or toxicological effects of xenobiotics. Furthermore, studies are described that have investigated the effects of genetic polymorphisms of biotransformation enzymes on the metabolism of drugs in humans and on the metabolism of genotoxic compounds in vivo as well. The effects of the polymorphisms are highly dependent on the enzyme systems involved and the compounds being metabolized. Several polymorphisms are described that also clearly influence the metabolism and effects of drugs and toxic compounds, in vivo in humans. Future perspectives in studies on genetic polymorphisms of biotransformation enzymes are also discussed. It is concluded that genetic polymorphisms of biotransformation enzymes are in a number of cases a major factor involved in the interindividual variability in xenobiotic metabolism and toxicity. This may lead to interindividual variability in efficacy of drugs and disease susceptibility.     

Biotransformation of pantoprazole by the fungus Cunninghamella blakesleeana

To investigate the biotransformation of pantoprazole, a proton-pump inhibitor, by filamentous fungus and further to compare the similarities between microbial transformation and mammalian metabolism of pantoprazole, four strains of Cunninghamella (C. blakesleeana AS 3.153, C. echinulata AS 3.2004, C. elegans AS 3.156, and AS 3.2028) were screened for the ability to catalyze the biotransformation of pantoprazole. Pantoprazole was partially metabolized by four strains of Cunninghamella, and C. blakesleeana AS 3.153 was selected for further investigation. Three metabolites produced by C. blakesleeana AS 3.153 were isolated using semi-preparative HPLC, and their structures were identified by a combination analysis of LC/MS(n) and NMR spectra. Two further metabolites were confirmed with the aid of synthetic reference compounds. The structure of a glucoside was tentatively assigned by its chromatographic behavior and mass spectroscopic data. These six metabolites were separated and quantitatively assayed by liquid chromatography-ion trap mass spectrometry. After 96h of incubation with C. blakesleeana AS 3.153, approximately 92.5% of pantoprazole was metabolized to six metabolites: pantoprazole sulfone (M1, 1.7%), pantoprazole thioether (M2, 12.4%), 6-hydroxy-pantoprazole thioether (M3, 1.3%), 4'-O-demethyl-pantoprazole thioether (M4, 48.1%), pantoprazole thioether-1-N-beta-glucoside (M5, 20.6%), and a glucoside conjugate of pantoprazole thioether (M6, 8.4%). Among them, M5 and M6 are novel metabolites. Four phase I metabolites of pantoprazole produced by C. blakesleeana were essentially similar to those obtained in mammals. C. blakesleeana could be a useful tool for generating the mammalian phase I metabolites of pantoprazole.     

Genetic Polymorphisms of Human N-Acetyltransferase,Cytochrome P450, Glutathione-S-Transferase,and Epoxide Hydrolase Enzymes: Relevance to Xenobiotic Metabolism and Toxicity

In this review, an overview is presented of the current knowledge of genetic polymorphisms of four of the most important enzyme families involved in the metabolism of xenobiotics, that is, the N-acetyltransf erase (NAT), cytochrome P450 (P450), glutathione-S-transferase (GST), and microsomal epoxide hydrolase (mEH) enzymes. The emphasis is on two main topics, the molecular genetics of the polymorphisms and the consequences for xenobiotic metabolism and toxicity. Studies are described in which wild-type and mutant alleles of biotransformation enzymes have been expressed in heterologous systems to study the molecular genetics and the metabolism and pharmacological or toxicological effects of xenobiotics. Furthermore, studies are described that have investigated the effects of genetic polymorphisms of biotransformation enzymes on the metabolism of drugs in humans and on the metabolism of genotoxic compounds in vivo as well. The effects of the polymorphisms are highly dependent on the enzyme systems involved and the compounds being metabolized. Several polymorphisms are described that also clearly influence the metabolism and effects of drugs and toxic compounds, in vivo in humans. Future perspectives in studies on genetic polymorphisms of biotransformation enzymes are also discussed. It is concluded that genetic polymorphisms of biotransformation enzymes are in a number of cases a major factor involved in the interindividual variability in xenobiotic metabolism and toxicity. This may lead to interindividual variability in efficacy of drugs and disease susceptibility.     

Bioactivation to an aldehyde metabolite—Possible role in the onset of toxicity induced by the anti-HIV drug abacavir

Aldehydes are highly reactive molecules, which can be generated during numerous physiological processes, including the biotransformation of drugs. Several non-P450 enzymes participate in their metabolism albeit alcohol dehydrogenase and aldehyde dehydrogenase are the ones most frequently involved in this process. Endogenous and exogenous aldehydes have been strongly implicated in multiple human pathologies. Their ability to react with biomacromolecules (e.g. proteins) yielding covalent adducts is suggested to be the common primary mechanism underlying the toxicity of these reactive species.     

Human microsomal carbonyl reducing enzymes in the metabolism of xenobiotics: well-known and promising members of the SDR superfamily

The best known, most widely studied enzyme system in phase I biotransformation is cytochrome P450 (CYP), which participates in the metabolism of roughly 9 of 10 drugs in use today. The main biotransformation isoforms of CYP are associated with the membrane of the endoplasmatic reticulum (ER). Other enzymes that are also active in phase I biotransformation are carbonyl reducing enzymes. Much is known about the role of cytosolic forms of carbonyl reducing enzymes in the metabolism of xenobiotics, but their microsomal forms have been mostly poorly studied. The only well-known microsomal carbonyl reducing enzyme taking part in the biotransformation of xenobiotics is 11β-hydroxysteroid dehydrogenase 1, a member of the short-chain dehydrogenase/reductase superfamily. Physiological roles of microsomal carbonyl reducing enzymes are better known than their participation in the metabolism of xenobiotics. This review is a summary of the fragmentary information known about the roles of the microsomal forms. Besides 11β-hydroxysteroid dehydrogenase 1, it has been reported, so far, that retinol dehydrogenase 12 participates only in the detoxification of unsaturated aldehydes formed upon oxidative stress. Another promising group of microsomal biotransformation carbonyl reducing enzymes are some members of 17β-hydroxysteroid dehydrogenases. Generally, it is clear that this area is, overall, quite unexplored, but carbonyl reducing enzymes located in the ER have proven very interesting. The study of these enzymes could shed new light on the metabolism of several clinically used drugs or they could become an important target in connection with some diseases.     

微生物转化在药学中的应用

目的对近十年来微生物转化在药学研究和工业生产中的应用及发展进行综述。方法在查阅国内外文献的基础上,简介了微生物转化的几种主要化学反应和微生物转化在手性药物合成、药物代谢及天然药物中的应用。结果与讨论通过综述微生物转化在药学研究和生产中的应用,说明了微生物转化的重要性及广阔的发展前景。     

Toxicity of orally inhaled drug formulations at the alveolar barrier: parameters for initial biological screening

Oral delivery is the most common mode of systemic drug application. Inhalation is mainly used for local therapy of lung diseases but may also be a promising route for systemic delivery of drugs that have poor oral bioavailability. The thin alveolar barrier enables fast and efficient uptake of many molecules and could deliver small molecules and proteins, which are susceptible to degradation and show poor absorption by oral application. The low rate of biotransformation and proteolytic degradation increases bioavailability of drugs but accumulation of not absorbed material may impair normal lung function. This limitation is more relevant for compounds that should be systematically active because higher doses have to be applied to the lung. The review describes processes that determine absorption of orally inhaled formulations, namely dissolution in the lung lining fluid and uptake and degradation by alveolar epithelial cells and macrophages. Dissolution testing in simulated lung fluid, screening for cytotoxicity and pro-inflammatory action in respiratory cells and study of macrophage morphology, and phagocytosis can help to identify adverse effects of pulmonary formulations.     

细胞色素P450活性与癌症治疗

本文通过总结影响细胞色素P450活性的因素和抗肿瘤药的生物转化途径,以期探索细胞色素P450同工酶介导的代谢在抗癌药物体内生物转化中的作用,进而探索该酶活性与癌症患者的疗效之间的相关性。发现化疗药物的个体间药代动力学差异会引起治疗的疗效和安全性的不同结果。年龄、性别、单个基因多态性等单一因素并不能阐明个体间对药物反应的差异性,化疗方案中多个药物的相互作用在临床上非常重要。为了更好地评估细胞色素P450酶类对机体代谢的作用,药物的吸收、排泄、活化,以及代谢整个药代动力学途径涉及的酶的多态性都应进行研究。     

Influencing mucosal homeostasis and immune responsiveness: the impact of nutrition and pharmaceuticals

Both nutrition and orally ingested drugs pass the gastrointestinal mucosa and may affect the balance between the mucosal immune system and microbial community herein, i.e. affecting composition of the microbial community as well as the status of local immune system that controls microbial composition and maintains mucosal integrity. Numerous ways are known by which the microbial community stimulates mammalian host's immune system and vice versa. The communication between microbiota and immune system is principally mediated by interaction of bacterial components with pattern recognition receptors expressed by intestinal epithelium and various local antigen-presenting cells, resulting in activation or modulation of both innate and adaptive immune responses. Current review describes some of the factors influencing development and maintenance of a proper mucosal/immune balance, with special attention to Toll like receptor signaling and regulatory T cell development. It further describes examples (antibiotic use, HIV and asthma will be discussed) showing that disruption of the balance can be linked to immune function failure. The therapeutic potential of nutritional pharmacology herein is the main focus of discussion.     

Conventional and novel approaches in generating and characterization of reactive intermediates from drugs/drug candidates

Despite several thousands of drugs are in use currently, research on new drug molecules is continuing. Because, there are diseases still without medication, successor/better drugs make the predecessor ones obsolete, and advancement in both life sciences and analytical technologies provide identification of previously unknown mechanisms of diseases, and discovery of novel drug targets. The two main criteria which a drug candidate should meet are high affinity for the target, and no or acceptable/tolerable toxicity in humans. Among these two, toxicity is the limiting one; developing a drug candidate with unacceptable toxicity has to be discontinued, even if it has an extremely high pharmacological activity. Drug would be withdrawn, if serious toxicity arises after marketing. Since drug development is a long (approximately 10 years), expensive, and infertile (one lead in 10.000 molecules) process, it is extremely important to detect the potential toxicity of drug candidate as early as possible. Today, it is believed that a great majority of toxic effects are caused by reactive intermediates generated by biotransformation of the parent drug. However, there are experimental difficulties in identifying such metabolite(s) in vivo. Their formation is affected by multi-factorial events; they can further be metabolized to structurally different products, and/or they may bind to a huge variety of biological sites or macromolecules. Hence, some reactive intermediates and their corresponding stable derivatives are generated in trace amounts, which make their determination more difficult. The ability of cytochrome P450s (CYP450) and other biotransformation enzymes to function in vitro offers a great flexibility to researchers, biotransformation of any compound can be simulated in a test tube, and metabolites/reactive intermediates are generated in an environment which has relatively much less background and less interfering multi-factorial events compared to in vivo. To simulate biotransformation, microsomal fraction is used most frequently from human and non-human sources. Purified or recombinant enzymes are used in determining the individual isoenzymes responsible for certain metabolites. Because of the chemical reactivity of intermediates, relevant, usually nucleophilic trapping agent(s) such as glutathione (GSH), N-acetylcysteine (NAC) and cyanide (CN-) are used to stabilize the metabolite. Trapped metabolites are subjected to spectrometric and/or nuclear magnetic resonance spectroscopic analyses for structural identification. Vertiginous advances especially in mass spectrometric technologies offer researchers new challenges in this area. This review is aimed at briefly summarizing the state of the art and compiling the highlighted studies in characterization of the reactive metabolites from drug molecules.     

临床药师要关注药物的代谢转化

药物进入体内后，经有关酶的催化，进行化学变化，称代谢转化。药物经转化后水溶性增高，有利于排泄体外。多数情况代谢产物的活性或毒性降低，但也有不少实例经代谢转化后代谢产物药理活性或毒性增高。药物的酶促代谢不只通过单一途径，产生的也不仅单一产物，每个代谢产物的药理活性或毒性不同，而每个病人的酶活性有所不同，对通过这些途径的速率相异，可表现为药物效应或毒性的个体差异。临床药师观察到病人所表现的不同反应往往可用代谢转化的观点解释，因而临床药师要关注药物的代谢转化，以便协助医师更适当、更安全有效地用药。     

Regioselective biotransformation of CNS drugs and its clinical impact on adverse drug reactions

INTRODUCTION: Adverse drug reactions (ADRs) continue to be one of the major causes of failure in drug development while limiting the clinical utilities of many drugs. Contribution of the metabolites formed in vivo to ADRs could be more significant than we might have expected. AREAS COVERED: This review focuses on the relationship between regioselectivity in biotransformation and the ADRs of drugs acting on the central nervous system (CNS). "Regioselectivity" is defined as an exclusively or significantly preferential metabolic reaction at one (or several) site(s) on the substrate molecule. Several CNS drugs and toxicants, of which the metabolites play pivotal roles in ADRs, are summarized in details with the highlight on the roles of metabolism in both toxification and detoxification. The article also discusses in silico predictions of regioselectivity and the formation of toxic metabolites which are becoming increasingly important. EXPERT OPINION: Researchers working on CNS drugs face particular challenges in predicting drug metabolism and potential toxicities of their metabolites. A number of factors contribute to the difficulty of accurate prediction of metabolite disposition in the human brain. Better knowledge of regioselectivity in biotransformation and elucidation of the relationships between biotransformations and ADRs would definitely help designing new compounds with lower bioactivation potentials and rejuvenating the older drugs whose clinical applications are restricted by their ADRs. Administrating drugs by alternative routes such as the intranasal, transdermal, sublingual, and buccal routes could also be a strategy to reduce unwanted metabolite formations.