Meeting report of the 3rd European Biotransformation Workshop.

Challenges, strategies and new technologies in the field of biotransformation were presented and discussed at the 3rd European Biotransformation Workshop which was held in collaboration with the DMDG on 5-6 October 2022 in Amsterdam. In this meeting report we summarise the presentations and discussions from this workshop. The topics covered are listed below:Accelerator mass spectrometry (AMS) for the support of microtracer studiesBiotransformation of the novel myeloperoxidase inhibitor AZD4831 in preclinical species and humansAMS in biotransformation studies: unusual case studiesDiscussion on new FDA draft guidance and AMSMultimodal molecular imaging and ion mobility applications in drug discovery and developmentMetabolites in Safety Testing considerations for large molecules.

Unusual Biotransformation Reactions of Drugs and Drug Candidates.

Detailed assessment of the fate of drugs in nonclinical test species and humans is essential to ensure the safety and efficacy of medicines in patients. In this context, biotransformation of drugs and drug candidates has been an area of keen interest over many decades in the pharmaceutical industry as well as academia. Although many of the enzymes and biotransformation pathways involved in the metabolism of xenobiotics and more specifically drugs have been well characterized, each drug molecule is unique and constitutes specific challenges for the biotransformation scientist. In this mini-review written for the special issue on the occasion of the 50th Anniversary celebration of Drug Metabolism and Disposition and to celebrate contributions of F. Peter Guengerich, one of the pioneers of the drug metabolism field, recently reported "unusual" biotransformation reactions are presented. Scientific and technological advances in the "toolbox" of the biotransformation scientists are summarized. As the pharmaceutical industry continues to explore therapeutic modalities different from the traditional small molecule drugs, the new challenges confronting the biotransformation scientist as well as future opportunities are discussed. SIGNIFICANCE STATEMENT: For the biotransformation scientists, it is essential to share and be aware of unexpected biotransformation reactions so that they can increase their confidence in predicting metabolites of drugs in humans to ensure the safety and efficacy of these metabolites before the medicines reach large numbers of patients. The purpose of this review is to highlight recent observations of "unusual" metabolites so that the scientists working in the area of drug metabolism can strengthen their readiness in expecting the unexpected.

Multimodal molecular imaging in drug discovery and development.

In addition to individual imaging techniques, the combination and integration of several imaging techniques, so-called multimodal imaging, can provide large amounts of anatomical, functional, and molecular information accelerating drug discovery and development processes. Imaging technologies aid in understanding the disease mechanism, finding new pharmacological targets, and assessment of new potential drug candidates and treatment response. Here, we describe how different imaging techniques can be used in different phases of drug discovery and development and highlight their strengths, related innovations, and future potential with a focus on the implementation of artificial intelligence (AI) and radiomics for imaging technologies.

Setup of human liver-chips integrating 3D models, microwells and a standardized microfluidic platform as proof-of-concept study to support drug evaluation.

Human 3D liver microtissues/spheroids are powerful in vitro models to study drug-induced liver injury (DILI) but the small number of cells per spheroid limits the models' usefulness to study drug metabolism. In this work, we scale up the number of spheroids on both a plate and a standardized organ-chip platform by factor 100 using a basic method which requires only limited technical expertise. We successfully generated up to 100 spheroids using polymer-coated microwells in a 96-well plate (= liver-plate) or organ-chip (= liver-chip). Liver-chips display a comparable cellular CYP3A4 activity, viability, and biomarker expression as liver spheroids for at least one week, while liver-plate cultures display an overall reduced hepatic functionality. To prove its applicability to drug discovery and development, the liver-chip was used to test selected reference compounds. The test system could discriminate toxicity of the DILI-positive compound tolcapone from its less hepatotoxic structural analogue entacapone, using biochemical and morphological readouts. Following incubation with diclofenac, the liver-chips had an increased metabolite formation compared to standard spheroid cultures. In summary, we generated a human liver-chip model using a standardized organ-chip platform which combines up to 100 spheroids and can be used for the evaluation of both drug safety and metabolism.

Human hepatocytes and cytochrome P450-selective inhibitors predict variability in human drug exposure more accurately than human recombinant P450s.

BACKGROUND AND PURPOSE: Drugs metabolically eliminated by several enzymes are less vulnerable to variable compound exposure in patients due to drug-drug interactions (DDI) or if a polymorphic enzyme is involved in their elimination. Therefore, it is vital in drug discovery to accurately and efficiently estimate and optimize the metabolic elimination profile. EXPERIMENTAL APPROACH: CYP3A and/or CYP2D6 substrates with well described variability in vivo in humans due to CYP3A DDI and CYP2D6 polymorphism were selected for assessment of fraction metabolized by each enzyme (fmCYP ) in two in vitro systems: (i) human recombinant P450s (hrP450s) and (ii) human hepatocytes combined with selective P450 inhibitors. Increases in compound exposure in poor versus extensive CYP2D6 metabolizers and by the strong CYP3A inhibitor ketoconazole were mathematically modelled and predicted changes in exposure were compared with in vivo data. KEY RESULTS: Predicted changes in exposure were within twofold of reported in vivo values using fmCYP estimated in human hepatocytes and there was a strong linear correlation between predicted and observed changes in exposure (r2  = 0.83 for CYP3A, r2  = 0.82 for CYP2D6). Predictions using fmCYP in hrP450s were not as accurate (r2  = 0.55 for CYP3A, r2  = 0.20 for CYP2D6). CONCLUSIONS AND IMPLICATIONS: The results suggest that variability in human drug exposure due to DDI and enzyme polymorphism can be accurately predicted using fmCYP from human hepatocytes and CYP-selective inhibitors. This approach can be efficiently applied in drug discovery to aid optimization of candidate drugs with a favourable metabolic elimination profile and limited variability in patients.

An assessment of Movement Disorder Society Task Force diagnostic criteria for mild cognitive impairment in Parkinson's disease.

BACKGROUND AND PURPOSE: Cognitive impairment is one of the most disabling non-motor symptoms of Parkinson's disease. Mild cognitive impairment constitutes a major risk for the development of Parkinson's disease dementia in the course of the disease. A Movement Disorder Society Task Force proposed diagnostic criteria for mild cognitive impairment in Parkinson's disease (PD-MCI), comprising two operational levels: Level I and Level II. The objective of our study was to test the accuracy of Level I versus Level II diagnostic criteria. METHODS: Eighty-six consecutive patients with Parkinson's disease were screened and 68 patients without dementia or depression were included in the study. We used the Montreal Cognitive Assessment, Mini-Mental State Examination and Addenbrooke's Cognitive Evaluation-R screening tools for Level I and an extensive neuropsychological battery for Level II assessment. We first diagnosed PD-MCI on the basis of Level II assessment and then calculated sensitivity, specificity and area under the receiver-operator characteristics curve, comparing the performance of the three screening batteries. RESULTS: None of the three screening batteries proposed for Level I assessment provided satisfactory combined sensitivity and specificity for detecting PD-MCI, and their performance was similar. Using the Level II criteria, 29 patients (43%) were diagnosed as having PD-MCI. Lowest cut-off levels that provided at least 80% sensitivity were 24 for the Montreal Cognitive Assessment, 29 for the Mini-Mental State Examination and 87 for the Addenbrooke's Cognitive Evaluation-R. However, specificity levels were below 80% at these cut-off levels. CONCLUSIONS: We conclude that Level I assessment alone using screening batteries is not sufficiently sensitive/specific to detect PD-MCI.

Cloning and heterologous expression of chlorophyll a synthase in Rhodobacter sphaeroides.

Rhodobacter sphaeroides is a purple non-sulfur bacterium which photoheterotrophically produces hydrogen from organic acids under anaerobic conditions. A gene coding for putative chlorophyll a synthase (chlG) from cyanobacterium Prochlorococcus marinus was amplified by nested polymerase chain reaction and cloned into an inducible-expression plasmid which was subsequently transferred to R. sphaeroides for heterologous expression. Induced expression of chlG in R. sphaeroides led to changes in light absorption spectrum within 400-700 nm. The hydrogen production capacity of the mutant strain was evaluated on hydrogen production medium with 15 mM malate and 2 mM glutamate. Hydrogen yield and productivity were increased by 13.6 and 22.6%, respectively, compared to the wild type strain. The results demonstrated the feasibility of genetic engineering to combine chlorophyll and bacteriochlorophyll biosynthetic pathways which utilize common intermediates. Heterologous expression of key enzymes from biosynthetic pathways of various pigments is proposed here as a general strategy to improve absorption spectra and yield of photosynthesis and hydrogen gas production in bacteria.

Database Extraction of Metabolite Information of Drug Candidates: Analysis of 27 AstraZeneca Compounds with Human Absorption, Distribution, Metabolism, and Excretion Data.

As part of the drug discovery and development process, it is important to understand the human metabolism of a candidate drug prior to clinical studies. Preclinical in vitro and in vivo experiments across species are conducted to build knowledge concerning human circulating metabolites in preparation for clinical studies; therefore, the quality of these experiments is critical. Within AstraZeneca, all metabolite identification (Met-ID) information is stored in a global database using ACDLabs software. In this study, the Met-ID information derived from in vitro and in vivo studies for 27 AstraZeneca drug candidates that underwent human absorption, distribution, metabolism, and excretion studies was extracted from the database. The retrospective analysis showed that 81% of human circulating metabolites were previously observed in preclinical in vitro and/or in vivo experiments. A detailed analysis was carried out to understand which human circulating metabolites were not captured in the preclinical experiments. Metabolites observed in human hepatocytes and rat plasma but not seen in circulation in humans (extraneous metabolites) were also investigated. The majority of human specific circulating metabolites derive from multistep biotransformation reactions that may not be observed in in vitro studies within the limited time frame in which cryopreserved hepatocytes are active. Factors leading to the formation of extraneous metabolites in preclinical studies seemed to be related to species differences with respect to transporter activity, secondary metabolism, and enzyme kinetics. This retrospective analysis assesses the predictive value of Met-ID experiments and improves our ability to discriminate between metabolites expected to circulate in humans and irrelevant metabolites seen in preclinical studies.

Reactive Metabolites: Current and Emerging Risk and Hazard Assessments.

Although idiosyncratic adverse drug reactions are rare, they are still a major concern to patient safety. Reactive metabolites are widely accepted as playing a pivotal role in the pathogenesis of idiosyncratic adverse drug reactions. While there are today well established strategies for the risk assessment of stable metabolites within the pharmaceutical industry, there is still no consensus on reactive metabolite risk assessment strategies. This is due to the complexity of the mechanisms of these toxicities as well as the difficulty in identifying and quantifying short-lived reactive intermediates such as reactive metabolites. In this review, reactive metabolite risk and hazard assessment approaches are discussed, and their pros and cons highlighted. We also discuss the nature of idiosyncratic adverse drug reactions, using acetaminophen and nefazodone to exemplify the complexity of the underlying mechanisms of reactive metabolite mediated hepatotoxicity. One of the key gaps moving forward is our understanding of and ability to predict the contribution of immune activation in idiosyncratic adverse drug reactions. Sections are included on the clinical phenotypes of immune mediated idiosyncratic adverse drug reactions and on the present understanding of immune activation by reactive metabolites. The advances being made in microphysiological systems have a great potential to transform our ability to risk assess reactive metabolites, and an overview of the key components of these systems is presented. Finally, the potential impact of systems pharmacology approaches in reactive metabolite risk assessments is highlighted.

Significantly Different Covalent Binding of Oxidative Metabolites, Acyl Glucuronides, and S-Acyl CoA Conjugates Formed from Xenobiotic Carboxylic Acids in Human Liver Microsomes.

Xenobiotic carboxylic acids may be metabolized to oxidative metabolites, acyl glucuronides, and/or S-acyl-CoA thioesters (CoA conjugates) in vitro, e.g., in hepatocytes, and in vivo. These metabolites can potentially be reactive species and bind covalently to tissue proteins and are generally considered to mediate adverse drug reactions in humans. Acyl glucuronide metabolites have been the focus of reactive metabolite research for decades, whereas drug-CoA conjugates, which have been shown to be up to 40-70 times more reactive, have been given much less attention. In an attempt to dissect the contribution of different pathways to covalent binding, we utilized human liver microsomes supplemented with NADPH, uridine 5'-diphosphoglucuronic acid (UDPGA), or CoA to evaluate the reactivity of each metabolite separately. Seven carboxylic acid drugs were included in this study. While ibuprofen and tolmetin are still on the market, ibufenac, fenclozic acid, tienilic acid, suprofen, and zomepirac were stopped before their launch or withdrawn. The reactivities of the CoA conjugates of ibuprofen, ibufenac, fenclozic acid, and tolmetin were higher compared to those of their corresponding oxidative metabolites and acyl glucuronides, as measured by the level of covalent binding to human liver microsomal proteins. The highest covalent binding was observed for ibuprofenyl-CoA and ibufenacyl-CoA, to levels of 1000 and 8600 pmol drug eq/mg protein, respectively. In contrast and in agreement with the proposed P450-mediated toxicity for these drug molecules, the reactivities of oxidative metabolites of suprofen and tienilic acid were higher compared to the reactivities of their conjugated metabolites, with NADPH-dependent covalent binding of 250 pmol drug eq/mg protein for both drugs. The seven drugs all formed UDPGA-dependent acyl glucuronides, but none of these resulted in covalent binding. This study shows that, unlike studies with hepatocytes or in vivo, human liver microsomes provide an opportunity to investigate the reactivity of individual metabolites.

Use of electrochemical oxidation and model peptides to study nucleophilic biological targets of reactive metabolites: the case of rimonabant.

Electrochemical oxidation of drug molecules is a useful tool to generate several different types of metabolites. In the present study we developed a model system involving electrochemical oxidation followed by characterization of the oxidation products and their propensity to modify peptides. The CB1 antagonist rimonabant was chosen as the model drug. Rimonabant has previously been shown to give high covalent binding to proteins in human liver microsomes and hepatocytes and the iminium ion and/or the corresponding aminoaldehyde formed via P450 mediated α-carbon oxidation of rimonabant was proposed to be a likely contributor. This proposal was based on the observation that levels of covalent binding were significantly reduced when iminium species were trapped as cyanide adducts but also following addition of methoxylamine expected to trap aldehydes. Incubation of electrochemically oxidized rimonabant with peptides resulted in peptide adducts to the N-terminal amine with a mass increment of 64 Da. The adducts were shown to contain an addition of C5H4 originating from the aminopiperidine moiety of rimonabant. Formation of the peptide adducts required further oxidation of the iminium ion to short-lived intermediates, such as dihydropyridinium species. In addition, the metabolites and peptide adducts generated in human liver microsomes were compared with those generated by electrochemistry. Interestingly, the same peptide modification was found when rimonabant was coincubated with one of the model peptides in microsomes. This clearly indicated that reactive metabolite(s) of rimonabant identical to electrochemically generated species are also present in the microsomal incubations. In summary, electrochemical oxidation combined with peptide trapping of reactive metabolites identified a previously unobserved bioactivation pathway of rimonabant that was not captured by traditional trapping agents and that may contribute to the in vitro covalent binding.

Bioactivation of the cannabinoid receptor antagonist rimonabant to a cytotoxic iminium ion metabolite.

The cannabinoid type 1 receptor (CB1r) antagonist rimonabant was approved in 2006 for the treatment of obesity but was withdrawn in 2008 due to serious drug-related psychiatric disorders. In vitro metabolism studies with rimonabant have revealed high levels of reactive metabolite formation, which resulted in irreversible time-dependent P450 3A4 inhibition and in covalent binding to microsomal proteins. In the present study, an in vitro approach has been used to explore whether metabolic bioactivation of rimonabant might result in cell toxicity. A panel of SV40-T-antigen-immortalized human liver derived (THLE) cells that had been transfected with vectors encoding various human cytochrome P450 enzymes (THLE-1A2, 2C9, 2C19, 2D6, and 3A4) or with an empty vector (THLE-Null) were exposed to rimonabant. Cell toxicity and covalent binding to cellular proteins were evaluated, as was metabolite formation. Rimonabant exhibited markedly potentiated dose and time dependent cytotoxicity to THLE-3A4 cells, compared to that of all other THLE cell lines. This was accompanied by high levels of covalent binding of [(14)C]-rimonabant to THLE-3A4 cell proteins (1433 pmol drug equivalents/mg protein) and the formation of several metabolites that were not generated by THLE-Null cells. These included N-aminopiperidine (NAP) and an iminium ion species. However, no toxicity was observed when THLE cells were incubated with NAP. Glutathione depletion did not alter the observed potent cell cytotoxicity of rimonabant to THLE-3A4 cells. Preincubation of THLE-3A4 cells with the cytochrome P450 3A4 inhibitor ritonavir blocked the selective toxicity of rimonabant to these cells. In addition, ritonavir pretreatment blocked the metabolism of the compound in the cells and thereby significantly decreased the covalent binding of [(14)C]-rimonabant to THLE-3A4 cell proteins. We conclude that the potent toxicity of rimonabant in THLE-3A4 cells occurs by a mechanistic sequence, which is initiated by cytochrome P450 3A4 mediated formation of a highly cytotoxic reactive iminium ion metabolite that binds covalently to cellular proteins.

Acute effects of moderate and strenuous running on trace element distribution in the brain, liver, and spleen of trained rats.

OBJECTIVE: Trace elements such as manganese (Mn), cobalt (Co) and chromium (Cr) play key roles in metabolic reactions and are important in many physiological enzymatic processes. In this study, we aimed to investigate the acute effects of moderate and strenuous running (treadmill) exercise on the levels of Mn, Co and Cr in the brain, liver, and spleen of trained rats. STUDY DESIGN: Animal experiment. MATERIAL AND METHODS: Twenty-one Wistar-Albino adult male rats were used in the study. Rats were grouped as control group (no mandated exercise; n=8), moderate exercise group (30 min exercise duration; n=7), and strenuous exercise group (60 min exercise duration; n=6). The levels of Mn, Co, and Cr in the frontal lobe, temporal lobe, brain stem, liver, and spleen were determined by atomic absorption spectrophotometer. RESULTS: Cr levels in liver of rats increased in parallel to the time course of running supporting the exercise training effect on the action of insulin. Compared to the control group, the level of Co significantly decreased in the brain stem of rats in the moderate exercise group (p=0.009) and in the frontal lobe of rats in the strenuous exercise group (p=0.004). In the strenuous exercise group, an examination of the brain stem revealed that the level of Mn significantly decreased (p=0.001), and levels of Co and Cr were apparently depleted to the extent that these elements were no longer detectable. CONCLUSION: A notable finding is that during or after single bout strenuous exercise, levels of Co decreased in the spleen and particularly decreased in the brain stem of regularly trained rats. From this study, it can be inferred that sportsmen should aware trace element disturbances among the body parts or depletion of some trace elements after single bout of chronic strenuous running exercise.

In vitro approach to assess the potential for risk of idiosyncratic adverse reactions caused by candidate drugs.

Idiosyncratic adverse drug reactions (IADRs) in humans can result in a broad range of clinically significant toxicities leading to attrition during drug development as well as postlicensing withdrawal or labeling. IADRs arise from both drug and patient related mechanisms and risk factors. Drug related risk factors, resulting from parent compound or metabolites, may involve multiple contributory mechanisms including organelle toxicity, effects related to compound disposition, and/or immune activation. In the current study, we evaluate an in vitro approach, which explored both cellular effects and covalent binding (CVB) to assess IADR risks for drug candidates using 36 drugs which caused different patterns and severities of IADRs in humans. The cellular effects were tested in an in vitro Panel of five assays which quantified (1) toxicity to THLE cells (SV40 T-antigen-immortalized human liver epithelial cells), which do not express P450s, (2) toxicity to a THLE cell line which selectively expresses P450 3A4, (3) cytotoxicity in HepG2 cells in glucose and galactose media, which is indicative of mitochondrial injury, (4) inhibition of the human bile salt export pump, BSEP, and (5) inhibition of the rat multidrug resistance associated protein 2, Mrp2. In addition, the CVB Burden was estimated by determining the CVB of radiolabeled compound to human hepatocytes and factoring in both the maximum prescribed daily dose and the fraction of metabolism leading to CVB. Combining the aggregated results from the in vitro Panel assays with the CVB Burden data discriminated, with high specificity (78%) and sensitivity (100%), between 27 drugs, which had severe or marked IADR concern, and 9 drugs, which had low IADR concern, we propose that this integrated approach has the potential to enable selection of drug candidates with reduced propensity to cause IADRs in humans.

Use of radiolabeled compounds in drug metabolism and pharmacokinetic studies.

As part of the drug discovery and development process, it is important to understand the fate of the drug candidate in humans and the relevance of the animal species used for preclinical toxicity and pharmacodynamic studies. Therefore, various in vitro and in vivo studies are conducted during the different stages of the drug development process to elucidate the absorption, distribution, metabolism, and excretion properties of the drug candidate. Although state-of-the-art LC/MS techniques are commonly employed for these studies, radiolabeled molecules are still frequently required for the quantification of metabolites and to assess the retention and excretion of all drug related material without relying on structural information and MS ionization properties. In this perspective, we describe the activities of Isotope Chemistry at AstraZeneca and give a brief overview of different commonly used approaches for the preparation of (14)C- and (3)H-labeled drug candidates. Also various drug metabolism and pharmacokinetic studies utilizing radiolabeled drug candidates are presented with in-house examples where relevant. Finally, we outline strategic changes to our use of radiolabeled compounds in drug metabolism and pharmacokinetic studies, with an emphasis on delaying of in vivo studies employing radiolabeled drug molecules.

Genetic bases and phenotypes of autosomal recessive Parkinson disease in a Turkish population.

BACKGROUND AND PURPOSE: To evaluate the phenotype and the frequencies of mutations in PRKN, DJ1 and PINK1 genes in patients with Parkinson disease (PD) in Turkey. METHODS: Eighty-six patients from 77 PD families participated in the study. Seventy-four families were originating from Turkey, two families from Greece and one family from Bulgaria. All patients underwent detailed neurological examination. PRKN, PINK1 and DJ1 genes were sequenced, and dosage analysis was performed by multiplex ligation-dependent probe amplification. RESULTS: Sixteen patients with PD were found to carry homozygous (n = 14) or compound heterozygous (n = 2) PRKN mutations. We identified exon rearrangements, three point mutations and one new point mutation in exon 2 (p.K27del). In two families, two new PINK1 point mutations (L31X and P416L) were identified. No pathogenic mutations were found in DJ1 gene. Clinical phenotypes of PRKN patients were comparable to previously described features, but only in four of 13 families, the pedigree structure was clearly consistent with an autosomal recessive (AR) mode of inheritance in comparison with nine families where also different pattern of transmission could have been possible. CONCLUSIONS: Our data suggest that the PRKN gene mutation is the most frequent form of ARPD in Turkey. The proportion of mutations with regard to the age of onset in our population is in the range of those previously described, but our pedigrees are characterized by high rate of consanguinity, which might explain the high proportion of families with homozygous mutations and of patients in more than one generation. Pathogenic DJ1 mutations do not seem to play a major role in Turkey.

Bioactivation pathways of the cannabinoid receptor 1 antagonist rimonabant.

In the present work, the characterization of the biotransformation and bioactivation pathways of the cannabinoid receptor 1 antagonist rimonabant (Acomplia) is described. Rimonabant was approved in Europe in 2006 for the treatment of obesity but was withdrawn in 2008 because of a significant drug-related risk of serious psychiatric disorders. The aim of the present work is to characterize the biotransformation and potential bioactivation pathways of rimonabant in vitro in human and rat liver microsomes. The observation of a major iminium ion metabolite led us to perform reactive metabolite trapping, covalent binding to proteins, and time-dependent inhibition of cytochrome P450 3A4 studies. The major biotransformation pathways were oxidative dehydrogenation of the piperidinyl ring to an iminium ion, hydroxylation of the 3 position of the piperidinyl ring, and cleavage of the amide linkage. In coincubations with potassium cyanide, three cyanide adducts were detected. A high level of covalent binding of rimonabant in human liver microsomes was observed (920 pmol equivalents/mg protein). In coincubations with potassium cyanide and methoxylamine, the covalent binding was reduced by approximately 40 and 30%, respectively, whereas GSH had no significant effect on covalent binding levels. Rimonabant was also found to inhibit cytochrome P450 3A4 irreversibly in a time-dependent manner. In view of these findings, it is noteworthy that, to date, no toxicity findings related to the formation of reactive metabolites from rimonabant have been reported.