Human Drug Metabolites Research Articles

Immobilization of unspecific peroxygenases (EC 1.11.2.1) in PVA/PEG gel and hollow fiber modules

The immobilization of enzymes has many advantages, such as higher stability, easier handling, and reuse of the catalyst. Here we report, for the first time, two effective methods for the immobilization of unspecific peroxygenase (UPO; EC 1.11.2.1). This biocatalyst type comprises heavily glycosylated heme-thiolate proteins that catalyze various biotechnologically relevant oxyfunctionalizations. Both the encapsulation in cryogel and the retention of the enzyme in hollow fiber modules were found to be efficient methods for their immobilization. After encapsulation, the enzyme still exhibited 60% of its initial activity. Interestingly, we did not find differences in the kinetic parameters of free and immobilized UPOs. In long-term experiments, the conversion of the pharmaceutical diclofenac with immobilized UPOs in different reactor types yielded between 62mg and 154mg of the major human drug metabolite 4′-hydroxydiclofenac. The maximal total turnover number was about 60-fold higher compared to the free enzyme. A test over 5 months showed that storage of encapsulated UPOs in non-polar solvents (e.g., cyclohexane) helps to preserve the enzyme stability and increases their relative activity (by about ∼150%, in the case of diclofenac hydroxylation). In addition to the hydrophilic substrate diclofenac, encapsulated UPOs also oxidized the hydrophobic model compound cyclohexane.

Conversions of tricyclic antidepressants and antipsychotics with selected P450s from Sorangium cellulosum So ce56.

Human cytochromes P450 (P450s) play a major role in the biotransformation of drugs. The generated metabolites are important for pharmaceutical, medical, and biotechnological applications and can be used for derivatization or toxicological studies. The availability of human drug metabolites is restricted and alternative ways of production are requested. For this, microbial P450s turned out to be a useful tool for the conversion of drugs and related derivatives. Here, we used 10 P450s from the myxobacterium Sorangium cellulosum So ce56, which have been cloned, expressed, and purified. The P450s were investigated concerning the conversion of the antidepressant drugs amitriptyline, clomipramine, imipramine, and promethazine; the antipsychotic drugs carbamazepine, chlorpromazine, and thioridazine, as well as their precursors, iminodibenzyl and phenothiazine. Amitriptyline, chlorpromazine, clomipramine, imipramine, and thioridazine are efficiently converted during the in vitro reaction and were chosen to upscale the production by an Escherichia coli-based whole-cell bioconversion system. Two different approaches, a whole-cell system using M9CA medium and a system using resting cells in buffer, were used for the production of sufficient amounts of metabolites for NMR analysis. Amitriptyline, clomipramine, and imipramine are converted to the corresponding 10-hydroxylated products, whereas the conversion of chlorpromazine and thioridazine leads to a sulfoxidation in position 5. It is shown for the first time that myxobacterial P450s are efficient to produce known human drug metabolites in a milligram scale, revealing their ability to synthesize pharmaceutically important compounds.

Drug metabolism in microorganisms.

Several wild type and recombinant microorganisms can transform drugs to the equivalent human metabolites. Fungi, such as Cunninghamella spp., and Streptomyces bacteria express cytochrome P450 (CYP) enzymes that enable analogous phase I (oxidative) reactions with a wide range of drugs. The gene encoding the bifunctional CYP102A1 in Bacillus megaterium can be expressed easily in E. coli, and extensive mutagenesis experiments have generated numerous variants that can produce human drug metabolites. Additionally, human CYP isoforms have been expressed in various hosts. The application of microbial CYPs to the production of human drug metabolites is reviewed, and additional applications in the field of drug development are considered.

Pragmatic Approaches to Determine the Exposures of Drug Metabolites in Preclinical and Clinical Subjects in the MIST Evaluation of the Clinical Development Phase

The recent stream of regulatory guidelines on the Safety Testing of Drug Metabolites by the FDA in 2008 and the ICH in 2009 and 2012 has cast light on the importance of qualifying metabolite exposure as part of the safety evaluation of new drugs and has provided a much needed framework for the drug safety researcher. Since then, numerous publications interpreting the practicalities of the guidelines have appeared in the literature focusing on strategic approaches and/or adaptation of modern analytical methodologies, e.g., NMR and AMS, for the identification and quantification of metabolites in the species used in preclinical safety assessments and in humans. Surprisingly, there are few literature accounts demonstrating how, in practice, a particular strategy or analytical method has been used to qualify drug metabolites during the safety evaluation of a drug during clinical development. At the same time as the initial FDA and ICH guideline releases, the neuroscience therapy area of AstraZeneca had a number of projects in clinical development, or approaching this phase, which gave the authors a scaffold upon which to build knowledge regarding the safety testing of drug metabolites. In this article, we present how the MIST strategy was developed to meet the guidelines. Pragmatic approaches have evolved from the experience learned in various projects in DMPK at AstraZeneca, Södertälje, Sweden. Our experience dictates that there is no single strategy for qualifying the safety of drug metabolites in humans; however, all activities should be tied to two unifying themes: first that the exposure to drug metabolites should be compared between species at repeated administration using the relative method or a similar one; and second that the internal regulatory documentation of the metabolite qualification should be agnostic to external criteria (guidelines), indication, dose given, and timing.

Generation of Major Human Excretory and Circulating Drug Metabolites Using a Hepatocyte Relay Method

The prediction of human drug metabolites using in vitro experiments containing human-derived reagents is an important approach in modern drug research; however, this can be challenging for drugs that are slowly metabolized. In this report, we describe the use of a recently developed human hepatocyte relay method for the purpose of predicting human drug metabolite profiles. Five compounds for which in vivo human metabolism data were available were selected for the investigation of this method, and the results were compared with data gathered in hepatocyte suspensions as well as previous data from a micropatterned hepatocyte coculture method. The hepatocyte relay method demonstrated an improved performance (generation of 75% of human in vivo metabolites) for those drugs for which previous methods showed a relatively low rate of success (50% of human in vivo metabolites). Metabolites included those arising from both oxidative and conjugative reactions and metabolites that required sequential reactions. Two 4-hour relays were shown to adequately generate metabolites, and no further benefit was derived from more relays. Overall, it can be concluded that the hepatocyte relay assay method can be successfully used in the generation of relevant human metabolites, even for challenging drugs.

Chimeric cytochromes P450 engineered by domain swapping and random mutagenesis for producing human metabolites of drugs

Human drug metabolites produced by cytochrome P450 enzymes are critical for safety testing and may themselves act as drugs or leads in the drug discovery and development process. Here, highly active chimeric fusion proteins (chimeras) were obtained by reductase domain swapping of mutants at key catalytic residues of the heme domain with that of a natural variant (CYP102A1.2) of P450 BM3 (CYP102A1.1) from Bacillus megaterium. Random mutagenesis at the heme domain of the chimera was also used to generate chimeric mutants that were more active and diverse than the chimeras themselves. To determine whether the chimeras and several mutants of the highly active chimera displayed enhanced catalytic activity and, more importantly, whether they acquired activities of biotechnological importance, we measured the oxidation activities of the chimeras and chimeric mutants toward human P450 substrates, mainly drugs. Some of the chimeric mutants showed high activity toward typical human P450 substrates including drugs. Statin leads, especially chiral products, with inhibitory effects toward HMG-CoA reductase could be obtained from metabolites of statin drugs generated using these chimeric mutants. This study reveals the critical role of the reductase domain for the activity of P450 BM3 and shows that chimeras generated by domain swapping can be used to develop industrial enzymes for the synthesis of human metabolites from drugs and drug leads.

Biotransformations of Prenylated Hop Flavonoids for Drug Discovery and Production

In this review we aim to present current knowledge on biotransformation of flavonoids from hop cones with respect to type of product, catalyst and conversion. Subsequently, a comparative analysis of biological activity of prenylated hop flavonoids and their biotransformation products has been performed in order to indicate these research efforts that have good potential for application in pharmaceutical industry. There is increasing evidence that the products of biotransformation of hop prenylflavonoids, which have been little studied until recently, can be used as drugs or drug ingredients and also as standards of human drug metabolites. They can also serve as an inspiration for the design and chemical synthesis of new derivatives with higher or different biological activity. Nevertheless, much additional work, among others on determining the mechanism of action in in vivo systems, is needed to open up the way to biomedical application of these compounds.

Identification of Drug Metabolites in Human Plasma or Serum Integrating Metabolite Prediction, LC–HRMS and Untargeted Data Processing

Comprehensive identification of human drug metabolites in first-in-man studies is crucial to avoid delays in later stages of drug development. We developed an efficient workflow for systematic identification of human metabolites in plasma or serum that combines metabolite prediction, high-resolution accurate mass LC-MS and MS vendor independent data processing. Retrospective evaluation of predictions for 14 (14)C-ADME studies published in the period 2007-January 2012 indicates that on average 90% of the major metabolites in human plasma can be identified by searching for accurate masses of predicted metabolites. Furthermore, the workflow can identify unexpected metabolites in the same processing run, by differential analysis of samples of drug-dosed subjects and (placebo-dosed, pre-dose or otherwise blank) control samples. To demonstrate the utility of the workflow we applied it to identify tamoxifen metabolites in serum of a breast cancer patient treated with tamoxifen. Previously published metabolites were confirmed in this study and additional metabolites were identified, two of which are discussed to illustrate the advantages of the workflow.

Preparation of labeled human drug metabolites and drug‐drug interaction‐probes with fungal peroxygenases

Enzymatic conversion of a drug can be an efficient alternative for the preparation of a complex metabolite compared with a multi-step chemical synthesis approach. Limitations exist for chemical methods for direct oxygen incorporation into organic molecules often suffering from low yields and unspecific oxidation and also for alternative whole-cell biotransformation processes, which require specific fermentation know-how. Stable oxygen-transferring biocatalysts such as unspecific peroxygenases (UPOs) could be an alternative for the synthesis of human drug metabolites and related stable isotope-labeled analogues. This work shows that UPOs can be used in combination with hydrogen/deuterium exchange for an efficient one-step process for the preparation of 4'-OH-diclofenac-d6. The scope of the reaction was investigated by screening of different peroxygenase subtypes for the transformation of selected deuterium-labeled substrates such as phenacetin-d3 or lidocaine-d3. Experiments with diclofenac-d7 revealed that the deuterium-labeling does not affect the kinetic parameters. By using the latter substrate and H2 (18) O2 as cosubstrate, it was possible to prepare a doubly isotope-labeled metabolite (4'-(18) OH-diclofenac-d6). UPOs offer certain practical advantages compared with P450 enzyme systems in terms of stability and ease of handling. Given these advantages, future work will expand the existing 'monooxygenation toolbox' of different fungal peroxygenases that mimic P450 in vitro reactions.

Filamentous fungal biofilm for production of human drug metabolites

In drug development, access to drug metabolites is essential for assessment of toxicity and pharmacokinetic studies. Metabolites are usually acquired via chemical synthesis, although biological production is potentially more efficient with fewer waste management issues. A significant problem with the biological approach is the effective half-life of the biocatalyst, which can be resolved by immobilisation. The fungus Cunninghamella elegans is well established as a model of mammalian metabolism, although it has not yet been used to produce metabolites on a large scale. Here, we describe immobilisation of C. elegans as a biofilm, which can transform drugs to important human metabolites. The biofilm was cultivated on hydrophilic microtiter plates and in shake flasks containing a steel spring in contact with the glass. Fluorescence and confocal scanning laser microscopy revealed that the biofilm was composed of a dense network of hyphae, and biochemical analysis demonstrated that the matrix was predominantly polysaccharide. The medium composition was crucial for both biofilm formation and biotransformation of flurbiprofen. In shake flasks, the biofilm transformed 86% of the flurbiprofen added to hydroxylated metabolites within 24 h, which was slightly more than planktonic cultures (76%). The biofilm had a longer effective lifetime than the planktonic cells, which underwent lysis after 2×72 h cycles, and diluting the Sabouraud dextrose broth enabled the thickness of the biofilm to be controlled while retaining transformation efficiency. Thus, C. elegans biofilm has the potential to be applied as a robust biocatalyst for the production of human drug metabolites required for drug development.

Optimization of the bacterial cytochrome P450 BM3 system for the production of human drug metabolites.

Drug metabolism in human liver is a process involving many different enzymes. Among them, a number of cytochromes P450 isoforms catalyze the oxidation of most of the drugs commercially available. Each P450 isoform acts on more than one drug, and one drug may be oxidized by more than one enzyme. As a result, multiple products may be obtained from the same drug, and as the metabolites can be biologically active and may cause adverse drug reactions (ADRs), the metabolic profile of a new drug has to be known before this can be commercialized. Therefore, the metabolites of a certain drug must be identified, synthesized and tested for toxicity. Their synthesis must be in sufficient quantities to be used for metabolic tests. This review focuses on the progresses done in the field of the optimization of a bacterial self-sufficient and efficient cytochrome P450, P450 BM3 from Bacillus megaterium, used for the production of metabolites of human enzymes. The progress made in the improvement of its catalytic performance towards drugs, the substitution of the costly NADPH cofactor and its immobilization and scale-up of the process for industrial application are reported.

Using Chimeric Mice with Humanized Livers to Predict Human Drug Metabolism and a Drug-Drug Interaction

Interspecies differences in drug metabolism have made it difficult to use preclinical animal testing data to predict the drug metabolites or potential drug-drug interactions (DDIs) that will occur in humans. Although chimeric mice with humanized livers can produce known human metabolites for test substrates, we do not know whether chimeric mice can be used to prospectively predict human drug metabolism or a possible DDI. Therefore, we investigated whether they could provide a more predictive assessment for clemizole, a drug in clinical development for the treatment of hepatitis C virus (HCV) infection. Our results demonstrate, for the first time, that analyses performed in chimeric mice can correctly identify the predominant human drug metabolite before human testing. The differences in the rodent and human pathways for clemizole metabolism were of importance, because the predominant human metabolite was found to have synergistic anti-HCV activity. Moreover, studies in chimeric mice also correctly predicted that a DDI would occur in humans when clemizole was coadministered with a CYP3A4 inhibitor. These results demonstrate that using chimeric mice can improve the quality of preclinical drug assessment.

Production of human cytochrome P450 2D6 drug metabolites with recombinant microbes – a comparative study

The processes of drug development require efficient strategies to produce the respective drug metabolites, which are often difficult to obtain. Biotransformations employing recombinant microorganisms as whole-cell biocatalysts have become an attractive alternative to the chemical syntheses of such metabolites. For the first time, the potential of four different microbial systems expressing the human cytochrome P450 2D6 (CYP2D6), which is one of the most important drug-metabolizing enzymes, were compared and evaluated for such applications. The microbial host Pichia pastoris was the most efficient at expressing CYP2D6. Without additional over-expression of chaperons, the achieved yield of CYP2D6 was the highest of microbial hosts reported so far. Therefore, the system described in this study outperformed the previously reported expression of the N-terminally modified enzyme. It was also shown that the activities of the whole-cell conversions of bufuralol in recombinant P. pastoris were significantly higher than the Escherichia coli catalyst, which expressed the same unmodified gene.

Perspectives on non-clinical safety evaluation of drug metabolites through the JSOT workshop

The prompt and appropriate safety assessment of drug metabolite(s) was mentioned in regulatory guidances such as an International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidance, entitled "Guidance on Non-clinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals" (ICH M3(R2)) implemented in January 1 of 2011 in Japan, and has become a significant issue in the drug development. Upon release of ICH M3(R2) Step 4, a survey was conducted between March and April 2010 on the safety assessment of drug metabolites in 63 member companies of the Japan Pharmaceutical Manufacturers Association (JPMA). The Pharmacokinetics Team in the Non-Clinical Evaluation Expert Committee in JPMA conducted a questionnaire survey and compiled the results to comprehend how safety of drug metabolites are currently assessed at research-based pharmaceutical companies in Japan. The assessment of "Metabolites in Safety Testing" (MIST) can be divided into three stages based on the research purpose as follows: MIST 1 is a stage of estimating human drug metabolites and predicting their potential risks, MIST 2 is a stage of deciding the necessity for non-clinical safety studies, and MIST 3 is a stage of conducting non-clinical safety studies. In this paper, we propose typical approaches on safety assessment of metabolites that meet the purpose of each stage, considering the current level of scientific technology. Our proposals are based on the results from our survey and a symposium about the safety assessment of drug metabolites at the 37th annual meeting of the Japanese Society of Toxicology held in June 2010.

Expression of recombinant human flavin monooxygenase and moclobemide-N-oxide synthesis on multi-mg scale

A panel of human flavin monooxygenases were heterologously expressed in E. coli to obtain ready-to-use biocatalysts for the in vitro preparation of human drug metabolites. Moclobemide-N-oxide (65 mg) was the first high-priced metabolite prepared with recombinant hFMO3 on the multi-milligram scale.

Preparation of human drug metabolites using fungal peroxygenases

The synthesis of hydroxylated and O- or N-dealkylated human drug metabolites (HDMs) via selective monooxygenation remains a challenging task for synthetic organic chemists. Here we report that aromatic peroxygenases (APOs; EC 1.11.2.1) secreted by the agaric fungi Agrocybe aegerita and Coprinellus radians catalyzed the H 2O 2-dependent selective monooxygenation of diverse drugs, including acetanilide, dextrorphan, ibuprofen, naproxen, phenacetin, sildenafil and tolbutamide. Reactions included the hydroxylation of aromatic rings and aliphatic side chains, as well as O- and N-dealkylations and exhibited different regioselectivities depending on the particular APO used. At best, desired HDMs were obtained in yields greater than 80% and with isomeric purities up to 99%. Oxidations of tolbutamide, acetanilide and carbamazepine in the presence of H 2 18O 2 resulted in almost complete incorporation of 18O into the corresponding products, thus establishing that these reactions are peroxygenations. The deethylation of phenacetin- d 1 showed an observed intramolecular deuterium isotope effect [( k H/ k D) obs] of 3.1 ± 0.2, which is consistent with the existence of a cytochrome P450-like intermediate in the reaction cycle of APOs. Our results indicate that fungal peroxygenases may be useful biocatalytic tools to prepare pharmacologically relevant drug metabolites.

Efficient Screening of Cytochrome P450 BM3 Mutants for Their Metabolic Activity and Diversity toward a Wide Set of Drug-Like Molecules in Chemical Space

In the present study, the diversity of a library of drug-metabolizing bacterial cytochrome P450 (P450) BM3 mutants was evaluated by a liquid chromatography-mass spectrometry (LC-MS)-based screening method. A strategy was designed to identify a minimal set of BM3 mutants that displays differences in regio- and stereoselectivities and is suitable to metabolize a large fraction of drug chemistry space. We first screened the activities of six structurally diverse BM3 mutants toward a library of 43 marketed drugs (encompassing a wide range of human P450 phenotypes, cLogP values, charges, and molecular weights) using a rapid LC-MS method with an automated method development and data-processing system. Significant differences in metabolic activity were found for the mutants tested and based on this drug library screen; nine structurally diverse probe drugs were selected that were subsequently used to study the metabolism of a library of 14 BM3 mutants in more detail. Using this alternative screening strategy, we were able to select a minimal set of BM3 mutants with high metabolic activities and diversity with respect to substrate specificity and regiospecificity that could produce both human relevant and BM3 unique drug metabolites. This panel of four mutants (M02, MT35, MT38, and MT43) was capable of producing P450-mediated metabolites for 41 of the 43 drugs tested while metabolizing 77% of the drugs by more than 20%. We observed this as the first step in our approach to use of bacterial P450 enzymes as general reagents for lead diversification in the drug development process and the biosynthesis of drug(-like) metabolites.

Engineered Bacterial Mimics of Human Drug Metabolizing Enzyme CYP2C9

AbstractSimple and universal methods for the preparation of human drug metabolites are required to produce quantities sufficient for their characterization and toxicity testing. Synthetic chemistry lacks general catalysts for selective oxidation of unactivated CH bonds, a transformation that plays a key role in metabolism; bioconversions using P450 enzymes have emerged as a powerful alternative. Variants of P450BM3 from Bacillus megaterium act on diverse substrates, including drugs. Acidic substrates, such as the compounds metabolized by CYP2C9, which is one of three main hepatic human P450s, are not accepted by P450BM3 variants engineered to date. Herein, we report bacterial mimics of CYP2C9, which are active on two widely administered drugs, naproxen and ibuprofen, that are CYP2C9 substrates in vivo. These P450BM3 variants can also act on desmethylnaproxen, the human metabolite of naproxen, and convert it to the 1,4‐naphthoquinone derivative. We analyzed the crystal structure of the heme domain of an early intermediate in the directed‐evolution experiment. The active site mutation, L75R, which initially conferred activity on charged substrates, dramatically increased structural flexibility in the B′‐helix. This increased flexibility, which was accompanied by a dramatic decrease in enzyme stability, may contribute to the variant’s ability to accept a broader range of substrates.

Analytical Strategies for Assessment of Human Metabolites in Preclinical Safety Testing

Strategies for the detection, identification, and quantification of in vivo drug metabolites from non-radiolabeled studies are reviewed with an emphasis on the utilization of accurate-mass-based data mining tools. New approaches to the determination of coverage of human drug metabolites in preclinical species without using radiolabeled drugs or synthetic standards are also discussed.

Chiral Sulfoxides as Metabolites of 2-Thioimidazole-Based p38α Mitogen-Activated Protein Kinase Inhibitors: Enantioselective Synthesis and Biological Evaluation

A number of pharmaceutically important drugs contain asymmetric sulfinyl moieties, so the biological evaluation of chiral sulfoxides as human drug metabolites is important for the development of safe and effective pharmaceuticals. Asymmetric oxidation is one of the most attractive ways to prepare chiral sulfoxides. In combination with different chiral ligands, the iron- and titanium-catalyzed asymmetric oxidations of tri- and tetrasubstituted 2-thioimidazoles afford the corresponding sulfoxides with enantiomeric excesses up to 99% as novel p38α mitogen-activated protein kinase (p38α MAPK) inhibitors. The enantiomerically pure sulfoxides were evaluated on their inhibitory potency against p38α MAPK compared to the respective sulfides and sulfoxide racemates and showed differences in their affinities for the enzyme with IC(50) in the low nanomolar range. In addition, the ability to inhibit the release of tumor necrosis factor-α (TNF-α) from human whole blood (HWB) was examined. Some pyridinylimidazole derivatives showed excellent HWB activity with IC(50) as low as 52 nM.