Axel Pähler

Computational toxicology in drug development

Computational tools for predicting toxicity have been envisaged for their potential to considerably impact the attrition rate of compounds in drug discovery and development. In silico techniques like knowledge-based expert systems (quantitative) structure activity relationship tools and modeling approaches may therefore help to significantly reduce drug development costs by succeeding in predicting adverse drug reactions in preclinical studies. It has been shown that commercial as well as proprietary systems can be successfully applied in the pharmaceutical industry. As the prediction has been exhaustively optimized for early safety-relevant endpoints like genotoxicity, future activities will now be directed to prevent the occurrence of undesired toxicity in patients by making these tools more relevant to human disease.

Increased brain levels of 4-hydroxy-2-nonenal glutathione conjugates in severe Alzheimer's disease.

In the last decade an important role for the progression of neuronal cell death in Alzheimers disease (AD) has been ascribed to oxidative stress. trans-4-Hydroxy-2-nonenal, a product of lipid peroxidation, forms conjugates with a variety of nucleophilic groups such as thiols or amino moieties. Here we report for the first time the quantitation of glutathione conjugates of trans-4-hydroxy-2-nonenal (HNEGSH) in the human postmortem brain using the specific and very sensitive method of electrospray ionization triple quadrupole mass spectrometry (ESI-MS-MS). Levels of HNEGSH conjugates calculated as the sum of three chromatographically separated diastereomers were determined in hippocampus, entorhinal cortex, substantia innominata, frontal and temporal cortex, as well as cerebellum from patients with AD and controls matched for age, gender, postmortem delay and storage time. Neither age, nor postmortem delay, nor storage time did correlate with levels of HNEGSH conjugates which ranged between 1 and 500 pmol/g fresh weight in the brain areas examined. The brain specimen from patients with clinically and neuropathologically probable AD diagnosed according to criteria of the consortium to establish a registry for AD (CERAD) show increased levels of HNEGSH in the temporal and frontal cortex, as well as in the substantia innominata. Classification of disease severity according to Braak and Braak, which takes into consideration the amount of neurofibrillary tangles and neuritic plaques, revealed highest levels of HNEGSH in the substantia innominata and the hippocampus, two brain regions known to be preferentially affected in AD. These results substantiate the link between conjugates of glutathione with a product of lipid peroxidation and Alzheimers disease and justify further studies to evaluate the role of HNE metabolites as potential biomarkers for disease progression in AD.

Metabotropic Glutamate Receptor 5 Negative Allosteric Modulators: Discovery of 2-Chloro-4-[1-(4-fluorophenyl)-2,5-dimethyl-1H-imidazol-4-ylethynyl]pyridine (Basimglurant, RO4917523), a Promising Novel Medicine for Psychiatric Diseases

Negative allosteric modulators (NAMs) of metabotropic glutamate receptor 5 (mGlu5) have potential for the treatment of psychiatric diseases including depression, fragile X syndrome (FXS), anxiety, obsessive-compulsive disorders, and levodopa induced dyskinesia in Parkinsons disease. Herein we report the optimization of a weakly active screening hit 1 to the potent and selective compounds chloro-4-[1-(4-fluorophenyl)-2,5-dimethyl-1H-imidazol-4-ylethynyl]pyridine (basimglurant, 2) and 2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)ethynyl)pyridine (CTEP, 3). Compound 2 is active in a broad range of anxiety tests reaching the same efficacy but at a 10- to 100-fold lower dose compared to diazepam and is characterized by favorable DMPK properties in rat and monkey as well as an excellent preclinical safety profile and is currently in phase II clinical studies for the treatment of depression and fragile X syndrome. Analogue 3 is the first reported mGlu5 NAM with a long half-life in rodents and is therefore an ideal tool compound for chronic studies in mice and rats.

Post-acquisition analysis of untargeted accurate mass quadrupole time-of-flight MSE data for multiple collision-induced neutral losses and fragment ions of glutathione conjugates

RATIONALE Analytical methods to assess glutathione (GSH) conjugate formation based on mass spectrometry usually take advantage of the specific fragmentation behavior of the glutathione moiety. However, most methods used for GSH adduct screening monitor only one specific neutral loss or one fragment ion, even though the peptide moiety of GSH adducts shows a number of other specific neutral fragments and fragment ions which can be used for identification. METHODS Nine reference drugs well known to form GSH adducts were incubated with human liver microsomes. Mass spectrometric analysis was performed with a quadrupole time-of-flight mass spectrometer in untargeted accurate mass MS(E) mode. The data analysis and evaluation was achieved in an automated approach with software to extract and identify GSH conjugates based on the presence of multiple collision-induced neutral losses and fragment ions specific for glutathione conjugates in the high-energy MS spectra. RESULTS In total 42 GSH adducts were identified. Eight (18%) adducts did not show the neutral loss of 129 but were identified based on the appearance of other GSH-specific neutral losses or fragment ions. In high-energy MS(E) spectra the GSH-specific fragment ions of m/z 308 and 179 as well as the neutral loss of 275 Da were complementary to the commonly used neutral loss of 129 Da. Further, one abundant (yet unpublished) GSH conjugate of troglitazone formed in human liver microsomes was found. CONCLUSIONS A software-aided approach was developed to reliably retrieve GSH adduct formation data out of untargeted complex full scan QTOFMS(E) data in a fast and efficient way. The present approach to detect and analyze multiple collision-induced neutral losses and fragment ions of glutathione conjugates in untargeted MS(E) data might be applicable to higher throughput to assess reactive metabolite formation in drug discovery.

Software aided approaches to structure-based metabolite identification in drug discovery and development.

Technological advances in mass spectrometry (MS) such as accurate mass high resolution instrumentation have fundamentally changed the approach to systematic metabolite identification over the past decade. Despite technological break-through on the instrumental side, metabolite identification still requires tedious manual data inspection and interpretation of huge analytical datasets. The process of metabolite identification has become largely facilitated and partly automated by cheminformatics approaches such as knowledge base metabolite prediction using, for example, Meteor, MetaDrug, MetaSite and StarDrop that are typically applied pre-acquisition. Likewise, emerging new technologies in postacquisition data analysis like mass defect filtering (MDF) have moved the technology driven analytical methodology to metabolite identification toward generic, structure-based workflows. The biggest challenge for automation however remains the structural assignment of drug metabolites. Software-guided approaches for the unsupervised metabolite identification still cannot compete with expert user manual data interpretation yet. Recently MassMetaSite has been introduced for the automated ranked output of metabolite structures based on the combination of metabolite prediction and interrogation of analytical mass spectrometric data. This approach and others are promising milestones toward an unsupervised process to metabolite identification and structural characterization moving away from a sample focused per-compound approach to a structure-driven generic workflow.

In vivo and in vitro characterization of ethyl methanesulfonate pharmacokinetics in animals and in human

Pharmacokinetics of ethyl methanesulfonate (EMS) were characterized in mice, rats and in cynomolgus monkeys with unlabelled and (14)C-radiolabelled EMS by either quantification of unchanged EMS or the N-ethyl-valine haemoglobin (Hb) adduct. EMS was well absorbed and exhibited close to 100% oral bioavailability. EMS showed some species differences in systemic clearance (intermediate in mice, low in rats and monkeys) representing only 1-4% of the cardiac output. The volume of distribution (0.5-0.8L/kg) was constant across species and corresponded to extracellular water. As a result of the species differences in clearance, the half-life ranged from 10 min in mouse (at low dose) to 5h in monkey. The systemic exposure of free EMS and the levels of its Hb adduct increased nearly dose proportionately from 1 to 5 and 0.5 to 80 mg/kg, respectively. The persistence of the N-ethyl-valine Hb adduct was much longer than EMS itself, consistent with the long life span of haemoglobin. No species differences were evident for the binding of EMS to Hb in whole blood ex vivo as determined by the second order rate constants. Following administration of Viracept tablets of the contaminated production batches (to monkeys leading to EMS doses of 0.08-27 microg/kg, concentrations of ethyl-valine Hb adducts) were near or below the detection limit of the assay (0.043 nmol/g Hb).

Neoantigen formation and clastogenic action of HCFC-123 and perchloroethylene in human MCL-5 cells

In this study, the metabolic activation of 2,2-dichloro-1,1,1-trifluoroethane (hydrochlorofluorocarbons-123, HCFC-123), halothane or 1,1-dichloro-1-fluoroethane (HCFC-141b) was compared to that of perchloroethylene, using lymphoblastoma derived cell lines expressing human CYP1A1, CYP1A2, CYP2E1, CYP2A6 and CYP3A4 (MCL-5 cells). A dose dependent increase in micronucleus formation was detected over a nominal concentration range of 0.05-2 mM for HCFC-123 and halothane, but this was not seen with HCFC-141b. No dose response for HCFC-123 was seen in a control cHo1 cell line not expressing this cytochrome P450s. Cell lines expressing individual human cytochrome P-450 (CYP) forms were also used to define the enzymes responsible for the clastogenic events and to investigate the formation of immunoreactive protein by microsomal fractions. It was shown that CYP2E1 or CYP2B6 catalysed the clastogenic response, but CYP2D6, CYP3A4, CYP1A2 or CYP1A1 all appeared to be inactive. The formation of neoantigenic trifluoroacetylated protein adducts by microsomal mixtures incubated with HCFC-123 and NADPH was catalysed primarily by CYP2E1 and to a lesser extent by CYP2C19, whereas, only trace levels of immunoreactive protein were seen with microsomes expressing CYP2B6 or CYP2C8. With perchloroethylene as a substrate, the extent of activation was low in comparison with HCFC-123, as judged by the absence of micronuclei formation in the MCL-5 cell line and the weak immunoreactivity of proteins following Western blotting. CYP1A2, CYP2B6 and CYP2C8 appeared to be responsible for perchloroethylene immunoreactivity and in contrast to the findings with the HCFCs, no activation of perchloroethylene by CYP2E1 could be detected. These results show that even though both saturated and unsaturated halocarbons can result in neoantigen formation, there is a marked difference in the specificity of the CYP enzymes involved in their metabolic activation.

Sembragiline: a novel, selective monoamine oxidase type B inhibitor for the treatment of Alzheimer’s disease

Monoamine oxidase B (MAO-B) has been implicated in the pathogenesis of Alzheimer’s disease (AD) and other neurodegenerative disorders. Increased MAO-B expression in astroglia has been observed adjacent to amyloid plaques in AD patient brains. This phenomenon is hypothesized to lead to increased production of hydrogen peroxide and reactive oxygen species (ROS), thereby contributing to AD pathology. Therefore, reduction of ROS-induced oxidative stress via inhibition of MAO-B activity may delay the progression of the disease. In the present study we report the pharmacological properties of sembragiline, a novel selective MAO-B inhibitor specifically developed for the treatment of AD, and on its effect on ROS-mediated neuronal injury and astrogliosis in MAO-B transgenic animals. Sembragiline showed potent and long-lasting MAO-B-selective inhibition and did not inhibit MAO-A at doses where full inhibition of MAO-B was observed. Such selectivity should translate into a favorable clinical safety profile. Indeed, sembragiline neither induced the serotonin syndrome when administered together with the serotonin precursor l-5-hydroxytryptophan in combination with antidepressants such as fluoxetine, nor potentiated the pressor effect of tyramine. Additionally, in experiments using a transgenic animal model conditionally overexpressing MAO-B in astroglia, sembragiline protected against neuronal loss and reduced both ROS formation and reactive astrogliosis. Taken together, these findings warrant further investigation of the potential therapeutic benefit of MAO-B inhibitors in patients with AD and other neurologic disorders.

Metabolism and mass balance of SGLT2 inhibitor tofogliflozin following oral administration to humans

Abstract 1. Tofogliflozin is a novel and selective SGLT2 inhibitor increasing glucosuria by inhibition of glucose re-absorption in the kidney for the treatment of type 2 diabetes mellitus. 2. In this study, the metabolism and the mass balance of tofogliflozin was evaluated following administration of a single oral dose of 20 mg [14C]-tofogliflozin to six healthy subjects. 3. Tofogliflozin underwent mainly oxidative metabolism in the ethylphenyl moiety, but also minor glucuronide conjugates of metabolites and the parent drug were formed. 4. In plasma, the parent drug and its major phenyl acetic acid metabolite M1 accounted for 42% and 52% of the total drug-related material, respectively. The hydroxyl metabolites and their successor ketone metabolite showed an exposure well below 5%, along with an acyl glucuronide of M1. 5. Tofogliflozin was completely absorbed with subsequent predominate metabolic clearance and a small contribution of direct urinary elimination. Approximately, 76% of the dose was excreted in urine and 20% in faeces within 72 h. The high absorption of tofogliflozin was exemplified by the small trace of parent drug in faeces. The phenyl acetic acid metabolite M1 was the major component excreted in urine and faeces accounting for more than half of the dose. Tofogliflozin demonstrated a high metabolic turnover.

Quantitation of Nϵ-(dichloroacetyl)-l-lysine in proteins after perchloroethene exposure by gas chromatography–mass spectrometry using chemical ionization and negative ion detection following immunoaffinity chromatography

Abstract An antibody specific to Nϵ-(dichloroacetyl)- l -lysine (DCA-Lys) was immobilized to immunoaffinity columns for the use in selective enrichment of dichloroacetylated proteins. These result from the reaction with dichlorothioketene the β-lyase cleavage product of the perchloroethene metabolite S-(trichlorovinyl)- l -cysteine. Dichloroacetylated proteins from rat kidney mitochondria, rat plasma and human blood plasma were isolated after exposure to 40 ppm tetrachloroethene (PER) for 6 h. After acid hydrolysis of the protein fraction, DCA-Lys was derivatized with 1,3-dichloro-1,1,3,3-tetrafluoroacetone using Nϵ-(trifluoroacetyl)- l -lysine as internal standard. Recovery of dichloroacetylated reference proteins from immunoaffinity columns was about 73%. Samples were analyzed by GC–MS with chemical ionization and negative ion (NCI) detection showing DCA-Lys in proteins with 2.26 (±0.02) pmol/mg protein in male rat kidney mitochondria and 1.92 (±0.05) pmol/mg total mitochondrial protein in female rats. In rat plasma 0.47 (±0.006) pmol DCA-Lys/mg protein in male and 0.34 (±0.02) in female animals were found. DCA-Lys could not be detected in blood plasma of human volunteers exposed to PER with a detection limit of 20 fmol for the DCA-Lys derivative 2,2-bis(chlorodifluoromethyl)-4-(1-dichloroacetamido)-butyl-1,3-oxazolidine-5-one. Immunoaffinity chromatography with specific antibodies provides a powerful tool for the enrichment of minor quantities of dichloroacetyled proteins in biological samples for GC–NCI-MS analysis of the modified amino acid lysine having broad utility in the biomonitoring of PER exposure.