Synthesis Of Drug Metabolites Research Articles

Importance and review of drug metabolite synthesis

Importance and review of drug metabolite synthesis

Late Microaerobic Growth for Efficient Production of Human Cytochrome P450 3A4 in E. coli.

Detailed preclinical characterization of metabolites formed in vivo from candidate drug substances is mandatory prior to the initiation of clinical trials. Therefore, inexpensive and efficient methods for drug metabolite synthesis are of high importance for rapid advancement of the drug development process. A large fraction of small molecule drugs is modified by monooxygenase cytochrome P450 3A4 produced in the human liver and intestine. Therefore, this enzyme is frequently employed to catalyze metabolite synthesis in vitro, making 3A4 availability a critical requirement in early drug development. Unfortunately, the recombinant production of this enzyme in microbial hosts is notoriously difficult. Maintaining low oxygen transfer rates and the use of rich media for host cultivation are required for P450 3A4 production. However, detailed studies on the relationship between oxygen supply and P450 3A4 space-time yields are missing. We describe an improved biotechnological process for the heterologous expression of P450 3A4 together with its redox partner, cytochrome P450 reductase, in Escherichia coli. Enzyme production was most efficient under so-called "late microaerobic" growth conditions, in which the cells have just not yet made the switch to anaerobic metabolism, characterized by a limited oxygen supply leading to oxygen concentrations in the liquid phase that are far below the detection limit of standard oxygen electrodes. Furthermore, feeding the carbon source glycerol as well as controlling cellular acetate formation improved process productivity. The presented protocol resulted in the formation of functional recombinant 3A4 at concentrations up to 680nmol L-1.

Green electrosynthesis of drug metabolites.

In this concise review, the field of electrosynthesis (ES) as a green methodology for understanding drug metabolites linked to toxicology is exemplified. ES describes the synthesis of chemical compounds in an electrochemical cell. Compared to a conventional chemical reaction, ES operates under green conditions (the electron is the reagent) and has several industrial applications, including the synthesis of drug metabolites for toxicology testing. Understanding which circulating drug metabolites are formed in the body is a crucial stage in the development of new medicines and gives insight into any potential toxic pathologies resulting from the metabolites formed. Current methods to prepare drug metabolites directly from the drug molecule often involve time-consuming multistep syntheses. Throughout this review, the application of green ES to (i) identify drug metabolites, (ii) enable their efficient synthesis, and (iii) investigate the toxicity of the metabolites generated are highlighted.

One-Step Regio- and Stereoselective Electrochemical Synthesis of Orexin Receptor Antagonist Oxidative Metabolites.

Synthesis of drug metabolites, which often have complex structures, is an integral step in the evaluation of drug candidate metabolism, pharmacokinetic (PK) properties, and safety profiles. Frequently, such synthetic endeavors entail arduous, multiple-step de novo synthetic routes. Herein, we present the one-step Shono-type electrochemical synthesis of milligrams of chiral α-hydroxyl amide metabolites of two orexin receptor antagonists, MK-8133 and MK-6096, as revealed by a small-scale (pico- to nano-mole level) reaction screening using a lab-built online electrochemistry (EC)/mass spectrometry (MS) (EC/MS) platform. The electrochemical oxidation of MK-8133 and MK-6096 was conducted in aqueous media and found to produce the corresponding α-piperidinols with exclusive regio- and stereoselectivity, as confirmed by high-resolution nuclear magnetic resonance (NMR) characterization of products. Based on density functional theory (DFT) calculations, the exceptional regio- and stereoselectivity for this electrochemical oxidation are governed by more favorable energetics of the transition state, leading to the preferred secondary carbon radical α to the amide group and subsequent steric hindrance associated with the U-shaped conformation of the cation derived from the secondary α-carbon radical, respectively.

Biosynthesis using cytochrome P450 enzymes: Focus on synthesis of drug metabolites.

While modern synthetic chemistry provides the ability to synthesize an incredible variety of new structures, the natural world provides unmatched chemical diversity. This diversity can be employed in the drug discovery process either through isolation of an organic molecule from a biological source as a drug candidate, usually referred to as natural product chemistry, or by providing enzymes that are capable to performing chemistry not available through synthetic chemistry approaches. Natural or engineered enzymes can be used in candidate discovery to generate chemical diversity in conjunction with synthetic efforts. As a candidate progresses into develop there is often a need to characterize metabolites, thus a need for metabolite standard synthesis. Metabolite synthesis is best accomplished with a flexible application of both chemical and biosynthetic approaches. This overview of the use of biosynthesis to aid in the drug discovery and development process will cover multiple methodologies with a focus on the use of microbes as a flexible and cost-effective resource.

Identification of functional cytochrome P450 and ferredoxin from Streptomyces sp. EAS-AB2608 by transcriptional analysis and their heterologous expression

Bioconversion using microorganisms and their enzymes is an important tool in many industrial fields. The discovery of useful new microbial enzymes contributes to the development of industries utilizing bioprocesses. Streptomyces sp. EAS-AB2608, isolated from a soil sample collected in Japan, can convert the tetrahydrobenzotriazole CPD-1 (a selective positive allosteric modulator of metabotropic glutamate receptor 5) to its hydroxylated form at the C4-(R) position. The current study was performed to identify the genes encoding the enzymes involved in CPD-1 bioconversion and to verify their function. To identify gene products responsible for the conversion of CPD-1, we used RNA sequencing to analyze EAS-AB2608; from its 8333 coding sequences, we selected two genes, one encoding cytochrome P450 (easab2608_00800) and the other encoding ferredoxin (easab2608_00799), as encoding desirable gene products involved in the bioconversion of CPD-1. The validity of this selection was tested by using a heterologous expression approach. A bioconversion assay using genetically engineered Streptomyces avermitilis SUKA24 ∆saverm3882 ∆saverm7246 co-expressing the two selected genes (strain ES_SUKA_63) confirmed that these gene products had hydroxylation activity with respect to CPD-1, indicating that they are responsible for the conversion of CPD-1. Strain ES_SUKA_63 also showed oxidative activity toward other compounds and therefore might be useful not only for bioconversion of CPD-1 but also as a tool for synthesis of drug metabolites and in optimization studies of various pharmaceutical lead compounds. We expect that this approach will be useful for bridging the gap between the latest enzyme optimization technologies and conventional enzyme screening using microorganisms. KEY POINTS: • Genes easab2608_00800 (cyp) and easab2608_00799 (fdx) were selected by RNA-Seq. • Selection validity was evaluated by an engineered S. avermitilis expression system. • Strain ES_SUKA_63 showed oxidative activity toward CPD-1 and other compounds.

Magnetic Nanoparticles with Dual Surface Functions—Efficient Carriers for Metalloporphyrin-Catalyzed Drug Metabolite Synthesis in Batch and Continuous-Flow Reactors

The dual functionalization of magnetic nanoparticles with inert (methyl) and reactive (aminopropyl) groups enables efficient immobilization of synthetic metalloporphyrins (such as 5,10,15,20-tetrakis(2,3,4,5,6-pentafluorophenyl)iron(II) porphyrin and 5,10,15,20-tetrakis-(4-sulfonatophenyl)iron(II) porphyrin) via covalent or ionic interactions. The proportion of reactive function on the surface has significant effect on the biomimetic activity of metalloporphyrins. The optimized magnetic nanocatalyst containing porphyrin was successfully applied for biomimetic oxidation of antihypertensive drug Amlodipine in batch and continuous-flow reactors as well.

The Development of Biocatalysis as a Tool for Drug Discovery.

Enzymes are versatile biocatalysts capable of performing selective reactions. The advantages of enzymes in comparison to classical chemistry including chemical catalysts are the generally milder process conditions and avoidance of harmful reactants. Their high selectivity and specificity are especially beneficial for the enzymatic synthesis of new products with potential applications in drug research. Therefore, in the past decades, the utilization of isolated enzymes or whole-cell biocatalysts has spread through a growing number of biotechnological industries. The applications comprise the production of chiral building blocks for the pharmaceutical and fine chemical industry, the enzymatic synthesis of drug metabolites for testing of toxicity, function, biological activity, degradation and the production of biocatalytically modified natural products, which all play a role in drug discovery. Especially Oreste Ghisalba's contributions, which paved the way for the industrial use of enzymes, will be considered in this review.

Production of drug metabolites by human FMO3 in Escherichia coli

BackgroundIn the course of drug discovery and development process, sufficient reference standards of drug metabolites are required, especially for preclinical/clinical or new therapeutic drugs. Whole-cell synthesis of drug metabolites is of great interest due to its low cost, low environmental impact and specificity of the enzymatic reaction compared to chemical synthesis. Here, Escherichia coli (E. coli) JM109 cells over-expressing the recombinant human FMO3 (flavin-containing monooxygenase isoform 3) were used for the conversions of clomiphene, dasatinib, GSK5182 and tozasertib to their corresponding N-oxide metabolites.ResultsThe effects of NADPH regeneration, organic solvents as well as C-terminal truncations of human FMO3 were investigated. Under the optimized conditions, in excess of 200 mg/L of N-oxide metabolite of each of the four drugs could be produced by whole-cell catalysis within 24 h. Of these, more than 90% yield conversions were obtained for the N-oxidation of clomiphene and dasatinib. In addition, FMO3 shows high regio-selectivity in metabolizing GSK5182 where only the (Z) isomer is monooxygenated.ConclusionsThe study shows the successful use of human FMO3-based whole-cell as a biocatalyst for the efficient synthesis of drug metabolites including regio-selective reactions involving GSK5182, a new candidate against type 2 diabetes mellitus.

Mimicking of Phase I Metabolism Reactions of Molindone by HLM and Photocatalytic Methods with the Use of UHPLC-MS/MS.

Establishing the metabolism pathway of the drug undergoing the hepatic biotransformation pathway is one of the most important aspects in the preclinical discovery process since the presence of toxic or reactive metabolites may result in drug withdrawal from the market. In this study, we present the structural elucidation of six, not described yet, metabolites of an antipsychotic molecule: molindone. The elucidation of metabolites was supported with a novel photocatalytical approach with the use of WO3 and WS2 assisted photochemical reactions. An UHPLC-ESI-Q-TOF combined system was used for the registration of all obtained metabolite profiles as well as to record the high resolution fragmentation spectra of the observed transformation products. As a reference in the in vitro metabolism simulation method, the incubation with human liver microsomes was used. Chemometric comparison of the obtained profiles pointed out the use of the WO3 approach as being more convenient in the field of drug metabolism studies. Moreover, the photocatalysis was used in the direction of the main drug metabolite synthesis in order to further isolation and characterization.

Liver-on-a-Chip‒Magnetic Nanoparticle Bound Synthetic Metalloporphyrin-Catalyzed Biomimetic Oxidation of a Drug in a Magnechip Reactor.

Biomimetic oxidation of drugs catalyzed by metalloporphyrins can be a novel and promising way for the effective and sustainable synthesis of drug metabolites. The immobilization of 5,10,15,20-tetrakis(2,3,4,5,6-pentafluorophenyl)iron(II) porphyrin (FeTPFP) and 5,10,15,20-tetrakis-(4-sulfonatophenyl)iron(II) porphyrin (FeTSPP) via stable covalent or rapid ionic binding on aminopropyl-functionalized magnetic nanoparticles (MNPs-NH2) were developed. These immobilized catalysts could be efficiently applied for the synthesis of new pharmaceutically active derivatives and liver related phase I oxidative major metabolite of an antiarrhythmic drug, amiodarone integrated in a continuous-flow magnetic chip reactor (Magnechip).

Drug metabolite synthesis by immobilized human FMO3 and whole cell catalysts

BackgroundSufficient reference standards of drug metabolites are required in the drug discovery and development process. However, such drug standards are often expensive or not commercially available. Chemical synthesis of drug metabolite is often difficulty due to the highly regio- and stereo-chemically demanding. The present work aims to construct stable and efficient biocatalysts for the generation of drug metabolites in vitro.ResultIn this work, using benzydamine as a model drug, two easy-to-perform approaches (whole cell catalysis and enzyme immobilization) were investigated for the synthesis of FMO3-generated drug metabolites. The whole cell catalysis was carried out by using cell suspensions of E. coli JM109 harboring FMO3 and E. coli BL21 harboring GDH (glucose dehydrogenase), giving 1.2 g/L benzydamine N-oxide within 9 h under the optimized conditions. While for another approach, two HisTrap HP columns respectively carrying His6-GDH and His6-FMO3 were connected in series used for the biocatalysis. In this case, 0.47 g/L benzydamine N-oxide was generated within 2.5 h under the optimized conditions. In addition, FMO3 immobilization at the C-terminal (membrane anchor region) significantly improved its enzymatic thermostability by more than 10 times. Moreover, the high efficiency of these two biocatalytic approaches was also confirmed by the N-oxidation of tamoxifen.ConclusionsThe results presented in this work provides new possibilities for the drug-metabolizing enzymes-mediated biocatalysis.

Phase I and phase II metabolism simulation of antitumor-active 2-hydroxyacridinone with electrochemistry coupled on-line with mass spectrometry

Here, we report the metabolic profile and the results of associated metabolic studies of 2-hydroxy-acridinone (2-OH-AC), the reference compound for antitumor-active imidazo- and triazoloacridinones.Electrochemistry coupled with mass spectrometry was applied to simulate the general oxidative metabolism of 2-OH-AC for the first time. The reactivity of 2-OH-AC products to biomolecules was also examined. The usefulness of the electrochemistry for studying the reactive drug metabolite trapping (conjugation reactions) was evaluated by the comparison with conventional electrochemical (controlled-potential electrolysis) and enzymatic (microsomal incubation) approaches.2-OH-AC oxidation products were generated in an electrochemical thin-layer cell. Their tentative structures were assigned based on tandem mass spectrometry in combination with accurate mass measurements. Moreover, the electrochemical conversion of 2-OH-AC in the presence of reduced glutathione and/or N-acetylcysteine unveiled the formation of reactive metabolite-nucleophilic trapping agent conjugates (m/z 517 and m/z 373, respectively) through the thiol group. This glutathione S-conjugate was also identified after electrolysis experiment as well as was detected in liver microsomes.Summing up, the present work illustrates that the electrochemical simulation of metabolic reactions successfully supports the results of classical electrochemical and enzymatic studies. Therefore, it can be a useful tool for synthesis of drug metabolites, including reactive metabolites.

Microbial biotransformation – an important tool for the study of drug metabolism

Metabolite identification is an integral part of both preclinical and clinical drug discovery and development. Synthesis of drug metabolites is often required to support definitive identification, preclinical safety studies and clinical trials.Here we describe the use of microbial biotransformation as a tool to produce drug metabolites, complementing traditional chemical synthesis and other biosynthetic methods such as hepatocytes, liver microsomes and recombinant human drug metabolizing enzymes.A workflow is discussed whereby microbial strains are initially screened for their ability to form the putative metabolites of interest, followed by a scale-up to afford quantities sufficient to perform definitive identification and further studies.Examples of the microbial synthesis of several difficult-to-synthesize hydroxylated metabolites and three difficult-to-synthesize glucuronidated metabolites are described, and the use of microbial biotransformation in drug discovery and development is discussed.

Human Enzymes for Organic Synthesis.

Human enzymes have been widely studied in various disciplines. The number of reactions taking place in the human body is vast, and so is the number of potential catalysts for synthesis. Herein, we focus on the application of human enzymes that catalyze chemical reactions in course of the metabolism of drugs and xenobiotics. Some of these reactions have been explored on the preparative scale. The major field of application of human enzymes is currently drug development, where they are applied for the synthesis of drug metabolites.

A novel cytochrome P450 mono-oxygenase from Streptomyces platensis resembles activities of human drug metabolizing P450s.

Cytochrome P450 mono-oxygenases (P450) are versatile enzymes which play essential roles in C-source assimilation, secondary metabolism, and in degradations of endo- and exogenous xenobiotics. In humans, several P450 isoforms constitute the largest part of phase I metabolizing enzymes and catalyze oxidation reactions which convert lipophilic xenobiotics, including drugs, to more water soluble species. Recombinant human P450s and microorganisms are applied in the pharmaceutical industry for the synthesis of drug metabolites for pharmacokinetics and toxicity studies. Compared to the membrane-bound eukaryotic P450s, prokaryotic ones exhibit some advantageous features, such as high stability and generally easier heterologous expression. Here, we describe a novel P450 from Streptomyces platensis DSM 40041 classified as CYP107L that efficiently converts several commercial drugs of various size and properties. This P450 was identified by screening of actinobacterial strains for amodiaquine and ritonavir metabolizing activities, followed by genome sequencing and expression of the annotated S. platensis P450s in Escherichia coli. Performance of CYP107L in biotransformations of amodiaquine, ritonavir, amitriptyline, and thioridazine resembles activities of the main human metabolizing P450s, namely CYPs 3A4, 2C8, 2C19, and 2D6. For application in the pharmaceutical industry, an E. coli whole-cell biocatalyst expressing CYP107L was developed and evaluated for preparative amodiaquine metabolite production.

Biomimetic Synthesis of Drug Metabolites in Batch and Continuous-Flow Reactors.

A medium-throughput screening (MTS) of biomimetic drug metabolite synthesis is developed by using an iron porphyrin catalyst. The microplate method, in combination with HPLC-MS analysis, was shown to be a useful tool for process development and parameter optimization in the production of targeted metabolites and/or oxidation products of forty-three different drug substances. In the case of the biomimetic oxidation of amiodarone, the high quantity and purity of the isolated products enabled detailed HRMS and NMR spectroscopic studies. In addition to identification of known metabolites, several new oxidation products of the drug that was studied were characterized. Fast degradation and poor recovery of the catalyst under batch conditions was overcome by immobilization of 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin iron(III) chloride (FeTSPP) on the surface of 3-aminopropyl-functionalized silica by electrostatic interaction. The supported catalyst was successfully applied in a packed-bed reactor under continuous-flow reaction conditions for the large-scale synthesis of amiodarone metabolites.

ChemInform Abstract: Accessing Drug Metabolites via Transition‐Metal Catalyzed C—H Oxidation: The Liver as Synthetic Inspiration

AbstractReview: [87 refs.

Accessing Drug Metabolites via Transition-Metal Catalyzed C-H Oxidation: The Liver as Synthetic Inspiration.

Can classical and modern chemical C-H oxidation reactions complement biotransformation in the synthesis of drug metabolites? We have surveyed the literature in an effort to try to answer this important question of major practical significance in the pharmaceutical industry. Drug metabolites are required throughout all phases of the drug discovery and development process; however, their synthesis is still an unsolved problem. This Review, not intended to be comprehensive or historical, highlights relevant applications of chemical C-H oxidation reactions, electrochemistry and microfluidic technologies to drug templates in order to access drug metabolites, and also highlights promising reactions to this end. Where possible or appropriate, the contrast with biotransformation is drawn. In doing so, we have tried to identify gaps where they exist in the hope to spur further activity in this very important research area.

Bacterial surface display of human cytochrome P450 1A2 and cytochrome P450 reductase for drug metabolite synthesis

Bacterial surface display of human cytochrome P450 1A2 and cytochrome P450 reductase for drug metabolite synthesis