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Autoimmune deficiency and destruction in either β-cell mass or function can cause insufficient insulin levels and, as a result, hyperglycemia and diabetes. Thus, promoting β-cell proliferation could be one approach toward diabetes intervention. In this report we describe the discovery of a potent and selective DYRK1A inhibitor GNF2133, which was identified through optimization of a 6-azaindole screening hit. In vitro, GNF2133 is able to proliferate both rodent and human β-cells. In vivo, GNF2133 demonstrated significant dose-dependent glucose disposal capacity and insulin secretion in response to glucose-potentiated arginine-induced insulin secretion (GPAIS) challenge in rat insulin promoter and diphtheria toxin A (RIP-DTA) mice. The work described here provides new avenues to disease altering therapeutic interventions in the treatment of type 1 diabetes (T1D).

Significance We have completed a screen of ∼106 small molecules to identify compounds that induce cell death in multipotent glioblastoma multiforme (GBM) cancer stem cells (CSCs). This resulted in the identification of a hit class (RIPGBM) that was found to induce apoptosis in GBM CSCs in a cell type-selective manner. Metabolite profiling experiments led to the identification of a proapoptotic derivative of RIPGBM (cRIPGBM), which was found to be selectively formed in GBM CSCs. Mechanistic studies revealed that cRIPGBM induces apoptosis by binding to receptor-interacting protein kinase 2 (RIPK2) in a mode that results in the formation of a proapoptotic RIPK2/caspase 1 complex. In a physiologically relevant orthotopic intracranial GBM CSC tumor xenograft mouse model, RIPGBM was found to significantly inhibit in vivo tumor formation., Glioblastoma multiforme (GBM; grade IV astrocytoma) is the most prevalent and aggressive form of primary brain cancer. A subpopulation of multipotent cells termed GBM cancer stem cells (CSCs) play a critical role in tumor initiation, tumor maintenance, metastasis, drug resistance, and recurrence following surgery. Here we report the identification of a small molecule, termed RIPGBM, from a cell-based chemical screen that selectively induces apoptosis in multiple primary patient-derived GBM CSC cultures. The cell type-dependent selectivity of this compound appears to arise at least in part from redox-dependent formation of a proapoptotic derivative, termed cRIPGBM, in GBM CSCs. cRIPGBM induces caspase 1-dependent apoptosis by binding to receptor-interacting protein kinase 2 (RIPK2) and acting as a molecular switch, which reduces the formation of a prosurvival RIPK2/TAK1 complex and increases the formation of a proapoptotic RIPK2/caspase 1 complex. In an orthotopic intracranial GBM CSC tumor xenograft mouse model, RIPGBM was found to significantly suppress tumor formation in vivo. Our chemical genetics-based approach has identified a drug candidate and a potential drug target that provide an approach to the development of treatments for this devastating disease.

INTRODUCTION Glioblastoma multiforme (GBM) is the most aggressive form of primary brain cancer. A subpopulation of multipotent cells termed GBM cancer stem cells (CSCs) play a critical role in tumor initiation and maintenance, drug resistance, and recurrence following surgery. New therapeutic strategies for the treatment of GBM have recently focused on targeting CSCs. Here we have used an unbiased large-scale screening approach to identify drug-like small molecules that induce apoptosis in GBM CSCs in a cell type-selective manner. METHODS A luciferase-based survival assay of patient-derived GBM CSC lines was established to perform a large-scale screen of ∼one million drug-like small molecules with the goal of identifying novel compounds that are selectively toxic to chemoresistant GBM CSCs. Compounds found to kill GBM CSC lines as compared to control cell types were further characterized. A caspase activation assay was used to evaluate the mechanism of induced cell death. A xenograft animal model using patient-derived GBM CSCs was employed to test the leading candidate for suppression of in vivo tumor formation. RESULTS We identified a small molecule, termed RIPGBM, from the cell-based chemical screen that induces apoptosis in primary patient-derived GBM CSC cultures. The cell type-dependent selectivity of RIPGBM appears to arise at least in part from redox-dependent formation of a proapoptotic derivative, termed cRIPGBM, in GBM CSCs. cRIPGBM induces caspase 1-dependent apoptosis by binding to receptor-interacting protein kinase 2 (RIPK2) and acting as a molecular switch, which reduces the formation of a prosurvival RIPK2/TAK1 complex and increases the formation of a proapoptotic RIPK2/caspase 1 complex. In an intracranial GBM xenograft mouse model, RIPGBM was found to significantly suppress tumor formation. CONCLUSIONS Our chemical genetics-based approach has identified a small molecule drug candidate and a potential drug target that selectively targets cancer stem cells and provides an approach for the treatment of GBMs.

NOD2 (nucleotide-binding oligomerization domain-containing protein 2) is an internal pattern recognition receptor that recognizes bacterial peptidoglycan and stimulates host immune responses. Dysfunction of NOD2 pathway has been associated with a number of autoinflammatory disorders. To date, direct inhibitors of NOD2 have not been described due to technical challenges of targeting the oligomeric protein complex. Receptor interacting protein kinase 2 (RIPK2) is an intracellular serine/threonine/tyrosine kinase, a key signaling partner, and an obligate kinase for NOD2. As such, RIPK2 represents an attractive target to probe the pathological roles of NOD2 pathway. To search for selective RIPK2 inhibitors, we employed virtual library screening (VLS) and structure based design that eventually led to a potent and selective RIPK2 inhibitor 8 with excellent oral bioavailability, which was used to evaluate the effects of inhibition of RIPK2 in various in vitro assays and ex vivo and in vivo pharmacodynamic models.

Over the past decade, first and second generation EGFR inhibitors have significantly improved outcomes for lung cancer patients with activating mutations in EGFR. However, both resistance through a secondary T790M mutation at the gatekeeper residue and dose-limiting toxicities from wild-type (WT) EGFR inhibition ultimately limit the full potential of these therapies to control mutant EGFR-driven tumors and new therapies are urgently needed. Herein, we describe our approach toward the discovery of 47 (EGF816, nazartinib), a novel, covalent mutant-selective EGFR inhibitor with equipotent activity on both oncogenic and T790M-resistant EGFR mutations. Through molecular docking studies we converted a mutant-selective high-throughput screening hit (7) into a number of targeted covalent EGFR inhibitors with equipotent activity across mutants EGFR and good WT-EGFR selectivity. We used an abbreviated in vivo efficacy study for prioritizing compounds with good tolerability and efficacy that ultimately led to the se...

The Plainfield Road corridor is a major commuter route in Blue Ash, Ohio. This corridor is plagued by extreme peak hour congestion and intersection crash rates that are the largest in the city. The City intends to construct a series of roundabouts to address both congestion and safety issues. This presentation summarizes the evaluation process used to identify roundabouts as the preferred alternative to address both issues.

Neuroblastoma (NB) is one of the most common and deadly pediatric solid tumors. NB is characterized by clinical heterogeneity, from spontaneous regression to relentless progression despite intensive multimodality therapy. There is compelling evidence that members of the tropomyosin receptor kinase (Trk) family play important roles in these disparate clinical behaviors. Indeed, TrkB and its ligand, brain-derived neurotrophic factor (BDNF), are expressed in 50-60 % of high-risk NBs. The BDNF/TrkB autocrine pathway enhances survival, invasion, metastasis, angiogenesis and drug resistance.We tested a novel pan-Trk inhibitor, GNF-4256 (Genomics Institute of the Novartis Research Foundation), in vitro and in vivo in a nu/nu athymic xenograft mouse model to determine its efficacy in inhibiting the growth of TrkB-expressing human NB cells (SY5Y-TrkB). Additionally, we assessed the ability of GNF-4256 to enhance NB cell growth inhibition in vitro and in vivo, when combined with conventional chemotherapeutic agents, irinotecan and temozolomide (Irino-TMZ).GNF-4256 inhibits TrkB phosphorylation and the in vitro growth of TrkB-expressing NBs in a dose-dependent manner, with an IC₅₀ around 7 and 50 nM, respectively. Furthermore, GNF-4256 inhibits the growth of NB xenografts as a single agent (p0.0001 for mice treated at 40 or 100 mg/kg BID, compared to controls), and it significantly enhances the antitumor efficacy of irinotecan plus temozolomide (Irino-TMZ, p0.0071 compared to Irino-TMZ alone).Our data suggest that GNF-4256 is a potent and specific Trk inhibitor capable of significantly slowing SY5Y-TrkB growth, both in vitro and in vivo. More importantly, the addition of GNF-4256 significantly enhanced the antitumor efficacy of Irino-TMZ, as measured by in vitro and in vivo growth inhibition and increased event-free survival in a mouse xenograft model, without additional toxicity. These data strongly suggest that inhibition of TrkB with GNF-4256 can enhance the efficacy of current chemotherapeutic treatment for recurrent/refractory high-risk NBs with minimal or no additional toxicity.

The synthesis, preclinical profile, and in vivo efficacy in rat xenograft models of the novel and selective anaplastic lymphoma kinase inhibitor 15b (LDK378) are described. In this initial report, preliminary structure–activity relationships (SARs) are described as well as the rational design strategy employed to overcome the development deficiencies of the first generation ALK inhibitor 4 (TAE684). Compound 15b is currently in phase 1 and phase 2 clinical trials with substantial antitumor activity being observed in ALK-positive cancer patients.

Over the past decade, first and second generation EGFR inhibitors have significantly improved outcomes for lung cancer patients with activating mutations in EGFR. However, both resistance through a secondary T790M mutation at the gatekeeper residue and dose-limiting toxicities from wild-type (WT) EGFR inhibition ultimately limit the full potential of these therapies to control mutant EGFR-driven tumors and new therapies are urgently needed. Herein, we describe our approach toward the discovery of 47 (EGF816, nazartinib), a novel, covalent mutant-selective EGFR inhibitor with equipotent activity on both oncogenic and T790M-resistant EGFR mutations. Through molecular docking studies we converted a mutant-selective high-throughput screening hit (7) into a number of targeted covalent EGFR inhibitors with equipotent activity across mutants EGFR and good WT-EGFR selectivity. We used an abbreviated in vivo efficacy study for prioritizing compounds with good tolerability and efficacy that ultimately led to the selection of 47 as the clinical candidate.

With the extensive use of different strains of mice and rats in in vivo efficacy models, lack of relevant metabolic clearance data among strains has been a concern. Metabolic clearance is an important parameter impacting drug discovery, and it is often used as a compound selection filter. Metabolically stable compounds are often preferred, and will have a better chance to achieve the desired exposure in vivo. The present study examined strain differences in mouse and rat, using 96 compounds which spanned a wide range of intrinsic clearances. The in vitro clearances were determined using liver microsomes from commonly used strains of mouse (BALB/c, C57BL/6J, and CD-1) and of rat (Sprague-Dawley, Fischer, and Wistar Han). There were few discrepancies in the interpretation of the in vitro intrinsic clearance results within species for the 96 compounds tested in mouse and rat liver microsomes. This data gives us confidence that the phase I hepatic clearance can be determined using only one strain of mouse or rat liver microsomes.

Described in this article are strategies implemented to increase the throughput of in vivo rodent pharmacokinetic (PK) studies using the snapshot PK study design and automated methods for compound submission, sample processing, data analysis and reporting. Applying snapshot PK studies to categorize the oral exposure of >1300 discovery compounds as low, moderate or high resulted in an attrition rate of 86%. The follow up full PK studies on the remaining compounds found that 98% of the compounds were predicted in the correct (69%) or adjacent (29%) oral exposure category by the snapshot PK studies. These results demonstrate that the snapshot PK screen in rodents can serve as an effective and efficient in vivo tool in the compound selection process in drug discovery.

The aim of the present study was to determine whether Cremophor EL is a suitable surfactant that can be routinely applied to pharmacokinetic (PK) studies in early drug discovery without influencing the intrinsic PK characteristics of the new chemical entities (NCEs). Cremophor EL, a polyoxyl 35 castor oil, has been used as a solubilization aid for water-insoluble compounds in pre-clinical drug discovery. The effect of Cremophor EL on the PK properties of NCEs was examined in seven structurally diverse discovery compounds after intravenous administration. Significant effects of Cremophor EL on plasma volume of distribution (Vss) and plasma clearance (CL) were observed in compounds with moderate to high Vss or CL. The plasma Vss decreased more than 2-fold and the Vss binning category decreased by one unit (e.g. from moderate to low Vss) in 6 of 7 test compounds. Two to five-fold reduction of CL was observed with these 6 compounds. Effect on the terminal half-life (T1/2) was minimal. Using one of these 7 NCEs, concentration dependent effect of Cremophor EL in the vehicle was also determined. Higher percentage of Cremophor EL in vehicle resulted in progressively increased alterations on the plasma CL and Vss. Taken together, these findings indicated that Cremophor EL altered the intrinsic PK properties of these discovery compounds in a concentration dependent manner.

The synthesis and structure-activity relationship of a series of arylaminoethyl amide cathepsin S inhibitors are reported. Optimization of P3 and P2 groups to improve overall physicochemical properties resulted in significant improvements in oral bioavailability over early lead compounds. An X-ray structure of compound 37 bound to the active site of cathepsin S is also reported.

Purpose. The aim of this study was to reduce or prevent nonspecific binding (NSB) of compounds to ultrafiltration (UF) protein binding (PB) testing units. Methods. UF units (regenerated cellulose, MWCO 10K) were used for PB and NSB measurements with or without pretreatment with 5% tween 80 (TW 80) or 5% benzalkonium chloride (BAK) on the filter membrane. Dosing solutions (10 μM) in human serum and pH 7.4 phosphate-buffered saline were centrifuged at 3,000 g and room temperature after 1-h incubation in UF testing units. In parallel, a 96-well equilibrium dialyzer was used for PB and NSB measurements in equilibrium dialysis (ED) at 37°C for 4 h. Samples of UF and ED were analyzed by LC/MS or LSC. Results. Severe NSB was observed for etoposide, hydrocortisone, propranolol, and vinblastine in UF. In contrast, TW 80 or BAK pretreatment on the filter membrane decreased the NSB from 87-95% to 13-64% without causing a significant change in membrane integrity. When NSB was below 50% as a result of pretreating agents, PB data of marker compounds were comparable to those of ED. Conclusions. The pretreated membrane with TW 80 or BAK showed significantly less NSB for compounds that had a tendency toward high membrane binding. A modified UF method with pretreatment improved the performance of UF and was able to produce comparable PB results to ED.

Herbal supplements are often regarded as "natural", and are, therefore, considered by many to be safer than pharmaceuticals; however, the adverse effects of these supplements are under-reported in many cases. Many herbal supplements, such as pyrrolizidine alkaloids, kava, chaparral and germander, are known to induce liver injury, which, in general, is one of the main toxicity liabilities observed in the clinic and accounts for about half of total liver failures. One example is the hepatotoxicity of pennyroyal oil, which is ingested as an abortifacient, among other uses. For three decades, the late Professor Sidney Nelson contributed to our understanding of the mechanism of toxicity of pennyroyal and broadened our understanding of chemical toxicology. Here we present the studies and review the findings on acute hepatotoxicity of pennyroyal oil. These studies involved the isolation and characterization of pennyroyal components, determination of the appropriate animal models, identification of the structural requirement for toxicity and determination of the target enzymes and the enzymes involved in the process of bioactivation. Studies with stable isotope labeled pennyroyal metabolites, pulegone and menthofuran, furthered our understanding of the role of cytochrome P450 in the oxidation of these compounds. This review presents the investigational tools used in the study of pennyroyal oil, allowing the reader to not only appreciate these methods but also utilize them to tackle and better understand metabolism-based toxicity in their own projects.

Activation of the G-protein coupled receptor (GPCR) Takeda G-protein receptor 5 (TGR5), also known as G-protein bile acid receptor 1 (GPBAR1), has been shown to play a key role in pathways associated with diabetes, metabolic syndrome, and autoimmune disease. Nipecotamide 5 was identified as an attractive starting point after a high-throughput screen (HTS) for receptor agonists. A comprehensive hit-to-lead effort culminated in the discovery of 45h as a potent, selective, and bioavailable TGR5 agonist to test in preclinical metabolic disease models. In genetically obese mice (ob/ob), 45h was as effective as a dipeptidyl peptidase-4 (DPP-4) inhibitor at reducing peak glucose levels in an acute oral glucose tolerance test (OGTT), but this effect was lost upon chronic dosing.

The synthesis, preclinical profile, and in vivo efficacy in rat xenograft models of the novel and selective anaplastic lymphoma kinase inhibitor 15b (LDK378) are described. In this initial report, preliminary structure-activity relationships (SARs) are described as well as the rational design strategy employed to overcome the development deficiencies of the first generation ALK inhibitor 4 (TAE684). Compound 15b is currently in phase 1 and phase 2 clinical trials with substantial antitumor activity being observed in ALK-positive cancer patients.

Phosphatidic acids (PAs) are a molecularly diverse group of phospholipids. Certain species of PA are highly active intracellular signaling molecules associated with cellular activation and mitogenesis. Not surprisingly, the PA species active in signaling are not produced by normal cells under homeostatic conditions. Recently the molecular species of PA associated with specific cellular activating events have been identified and compounds that inhibit the formation of specific PA species have been synthesised. We have characterised four classes of PA not derived from phosphatidylinositol hydrolysis that are involved in intracellularsignaling. PA1α is produced following exposure of cells to lipopolysaccharide, IL-1, hypoxia-reoxygenation, IL-8, or platelet activating factor. Lisofylline, a functional inhibitor of PA1a formation, is in Phase II-III trials as a therapeutic to reduce toxicities of anti-cancer therapies and for cytokine-mediated acute systemic inflammatory conditions. PA1α, PA1β and PA2 are pro...

Most malaria drug development focuses on parasite stages detected in red blood cells, even though, to achieve eradication, next-generation drugs active against both erythrocytic and exo-erythrocytic forms would be preferable. We applied a multifactorial approach to a set of >4000 commercially available compounds with previously demonstrated blood-stage activity (median inhibitory concentration < 1 micromolar) and identified chemical scaffolds with potent activity against both forms. From this screen, we identified an imidazolopiperazine scaffold series that was highly enriched among compounds active against Plasmodium liver stages. The orally bioavailable lead imidazolopiperazine confers complete causal prophylactic protection (15 milligrams/kilogram) in rodent models of malaria and shows potent in vivo blood-stage therapeutic activity. The open-source chemical tools resulting from our effort provide starting points for future drug discovery programs, as well as opportunities for researchers to investigate the biology of exo-erythrocytic forms.

1. (R)-(+)-Pulegone is a monoterpene that is oxidized by cytochromes P-450 to reactive metabolites that initiate events in the pathogenesis of hepatotoxicity in mice, rats and humans. 2. Selective labelling of (R)-(+)-pulegone with deuterium revealed that menthofuran was a proximate hepatotoxic metabolite formed by oxidation of the allylic methyl groups of pulegone. Incubations of pulegone with mouse liver microsomes in an atmosphere of 18O2 resulted in the formation of menthofuran that contained only oxygen-18 in the furan moiety. These results are consistent with oxidation of pulegone to an allylic alcohol that reacts intramolecularly with the ketone moiety to form a hemiketal that subsequently dehydrates to generate menthofuran. 3. Studies on the metabolism of menthofuran revealed that it is oxidized by cytochromes P-450 to an electrophilic gamma-ketoenal that reacts with nucleophilic groups on proteins to form covalent adducts. In addition, diastereomeric mintlactones are formed. Investigations with H2(18)O and 18O2 are indicative of a furan epoxide intermediate, or a precursor, in the formation of the gamma-ketoenal and mintlactones.

A novel series of benzo[a]carbazole-based small molecule agonists of the thrombopoietin (Tpo) receptor is reported. Starting from a 3.4 microM high throughput screen hit, members of this series have been identified which are full agonists with functional potency50 nM and oral bioavailability in mice.

Peptidic, non-covalent inhibitors of lysosomal cysteine protease cathepsin S (1 and 2) were investigated due to low oral bioavailability, leading to an improved series of peptidomimetic inhibitors. Utilizing phenyl succinamides as the P2 residue increased the oral exposure of this lead series of compounds, while retaining selective inhibition of the cathepsin S isoform. Concurrent investigation of the P1 and P2 subsites resulted in the discovery of several potent and selective inhibitors of cathepsin S with good pharmacokinetic properties due to the elimination of saturated aliphatic P2 residues.

A systematic study of anilines led to the discovery of a metabolically robust fluoroindoline replacement for the alkoxy aniline toxicophore in 1. Investigations of the P1 pocket resulted in the discovery of a wide tolerance of functionality leading to the discovery of 11 as a potent and selective inhibitor of cathepsin S.

Screening of our compound collection using Staphylococcus aureus Ni-Peptide deformylase (PDF) afforded a very potent PDF inhibitor with an IC(50) in the low nanomolar range but with poor antibacterial activity (MIC). Three-dimensional structural information obtained from Pseudomonas aeruginosa Ni-PDF complexed with the inhibitor suggested the synthesis of a variety of analogues that would maintain high binding affinity while attempting to improve antibacterial activity. Many of the compounds synthesized proved to be excellent PDF-Ni inhibitors and some showed increased antibacterial activity in selected strains.

The aim of this study was to reduce or prevent nonspecific binding (NSB) of compounds to ultrafiltration (UF) protein binding (PB) testing units.UF units (regenerated cellulose, MWCO 10K) were used for PB and NSB measurements with or without pretreatment with 5% tween 80 (TW 80) or 5% benzalkonium chloride (BAK) on the filter membrane. Dosing solutions (10 microM) in human serum and pH 7.4 phosphate-buffered saline were centrifuged at 3,000 g and room temperature after 1-h incubation in UF testing units. In parallel, a 96-well equilibrium dialyzer was used for PB and NSB measurements in equilibrium dialysis (ED) at 37 degrees C for 4 h. Samples of UF and ED were analyzed by LC/MS or LSC.Severe NSB was observed for etoposide, hydrocortisone, propranolol, and vinblastine in UF. In contrast, TW 80 or BAK pre-treatment on the filter membrane decreased the NSB from 87-95% to 13-64% without causing a significant change in membrane integrity. When NSB was below 50% as a result of pretreating agents, PB data of marker compounds were comparable to those of ED.The pretreated membrane with TW 80 or BAK showed significantly less NSB for compounds that had a tendency toward high membrane binding. A modified UF method with pretreatment improved the performance of UF and was able to produce comparable PB results to ED.

The disposition of 8-methyl-8-azabicyclo[3,2,1]octan-3-yl 3,5-dichlorobenzoate (MDL 72,222; 1), a potent 5-hydroxytryptamine antagonist, and its N-demethylated and N-oxide metabolites was studied in dogs and monkeys. After single, intravenous doses of 1 at 5 mg/kg, the mean terminal half-lives of 1 in plasma were 2.6 h in the dog and 3.8 h in the monkey. The mean half-life of the N-demethylated metabolite in dogs (approximately 20 h) was very similar to that in monkeys. However, the mean half-life of the N-oxide metabolite in dogs (10.8 h) was different from that in monkeys (3.9 h). The steady-state volume of distribution of 1 was 16 L/kg in dogs and 8.9 L/kg in monkeys. Examination of the mean residence times revealed that 1, in both species, and the N-oxide metabolite, in dogs, distributed to the peripheral tissue, whereas the distribution of the N-demethylated metabolite in both species and the N-oxide metabolite in monkeys was limited mainly to the systemic circulation. Compound 1 was metabolized extensively in both species. In dogs, 0.7, 2.5 and 40.6% of the administered dose were excreted in 0-120-h urine samples as 1 and its N-demethylated and N-oxide metabolites, respectively. In monkeys, however, the corresponding percentages were 0.8, 0.7, and 1.8%. Most of the administered dose in monkeys was excreted in urine as 3,5-dichlorobenzoic acid and its glycine conjugate.

(R)-(+)-Pulegone is a major monoterpene constituent of pennyroyal oil, a mint oil obtained from the leaves of plants Mentha pulegiwn or Hedeoma pulegoides (1). Pennyroyal oil is widely used as a fragrance and flavoring agent (2), and has been used, based on folklore, as a herbal medicine to control menses and terminate pregnancy (3). The ingestion of large quantities of the oil has been associated with toxic effects (4–8), and hepatotoxicity observed in humans has been modeled in mice (9, 10) and rats (11, 12).

The hepatocarcinogen acetamide, in single doses of 100 and 400 mg/kg b.wt., was shown to act as an initiator in a dose-dependent fashion in rat liver using the Solt-Farber method. Acetamide and its putative metabolite N-hydroxy-acetamide did not cause liver necrosis in single dose experiments. Acetamide showed no evidence for genotoxicity in tests for mutations in Salmonella typhimurium, for DNA damage in rat hepatoma cells or for DNA repair in isolated rat hepatocytes. In contrast, N-hydroxy-acetamide displayed genotoxic activity in all 3 test systems. Neither acetamide nor N-hydroxy-acetamide induced transformation of primary Syrian hamster embryo cells or gave evidence of inhibition of metabolic cooperation in V79 cells. Radiolabeled acetamide and N-hydroxy-acetamide were not bound covalently to proteins in the presence of various metabolic activation systems (microsomes plus NADPH or xanthine/xanthine oxidase, cytosol or cytosol plus acetyl CoA or proline plus ATP). N-Hydroxy-acetamide was cytotoxic to monolayers of isolated hepatocytes at concentrations above 2.5 mM. This cytotoxicity was increased after diethyl maleate treatment, but N-hydroxy-acetamide did not deplete cellular glutathione. A HPLC system was developed for the separation and quantification of acetamide, N-hydroxy-acetamide and acetic acid. No significant excretion of N-hydroxy-acetamide or acetic acid in the urine could be demonstrated after treatment of rats with 100 or 1,000 mg/kg b.wt. of acetamide. The underlying mechanism for the observed initiating effect of acetamide is obscure.

Pennyroyal oil, an aromatic mint-like oil used as a flavoring and fragrance agent and as a herbal medicine, caused acute hepatic and lung damage at doses of 400 mg/kg, ip, and higher in male Swiss-Webster mice. Cellular necrosis was localized to the centrilobular regions of the liver and bronchiolar epithelial cells of the lung. Capillary gas chromatographic analysis of samples of pennyroyal oil that were obtained from health food stores showed the presence of several monoterpene constituents. R -(+)-Pulegone was the major terpene and constituted greater than 80% of the constituent terpenes in the oils that were examined. Pulegone and two other constituent terpenes, isopulegone and menthofuran, were found to be both hepatotoxic and lung toxic. Based on results of histologic scoring of necrosis, plasma GPT elevations, and hepatic glutathione depletion, R -(+)-pulegone is the terpene primarily responsible for the tissue necrosis. Furthermore, results of toxicity tests with several congeners of R -(+)-pulegone, including the enantiomeric S -(−)-pulegone, strongly implicated the α-isopropylidene ketone group as the structural unit required for eliciting hepatotoxicity, although the configurational orientation of the methyl group can modulate the hepatotoxic response.

Paracetamol and its major ultimate reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI) were studied for their genotoxic potential. Neither paracetamol nor NAPQI were found to cause mutations in Salmonella typhimurium, whereas NAPQI was severely cytotoxic to the bacteria. Radiolabelled paracetamol was found to bind covalently to DNA added to mouse-liver microsomal incubations at a rate of 2.6 pmoles/mg DNA/min. Paracetamol also bound covalently to hepatic DNA at a level of 15 pmoles/mg DNA after a hepatotoxic dose of paracetamol to mice. NAPQI caused extensive DNA single-strand breaks as evidenced by alkaline elution of DNA from treated Reuber hepatoma cells. This effect occurred at concentrations which later resulted in cytotoxicity. Paracetamol was shown to induce increased DNA-repair synthesis in isolated mouse-liver cells in monolayer culture, at concentrations where also cytotoxicity was evident. Increased DNA-repair synthesis occurred at lower paracetamol concentrations in cells isolated from mice pretreated with phenobarbital. Taken together, these data show that paracetamol can cause DNA interaction leading to damage at levels which are cytotoxic.

The potent mutagen 2-bromoacrolein is formed from the carcinogenic flame retardant tris(2,3-dibromopropyl)phosphate (Tris-BP) on incubation with hepatic microsomes. Substitution of deuterium for hydrogen at the terminal carbon atoms (C-3) of Tris-BP significantly decreased both the mutagenic response and the formation rate of 2-bromoacrolein. Mass spectral analysis of the 2-bromoacrolein that was formed from the selectively deuterated analogs of Tris-BP revealed that the primary mechanism for the formation of 2-bromoacrolein involves an initial oxidative dehalogenation at C-3 followed by a β-elimination reaction.

A novel metabolite, 2-(2-thienyl)propionic acid, is formed in vivo from 2-(2-thienyl)allylamine hydrochloride. Mass spectral analysis suggested 2-(2-thienyl)propionic acid formation involves loss of the amine moiety followed by reduction of the olefinic group.

The enantiomers of 2,3-dibromo-1-propanol were obtained by diazotization of the diastereomeric d-tartrate salts of 2,3-dibromopropylamine. The products of the reaction contained approximately 13% of the secondary alcohol 1,3-dibromo-2-propanol which was separated by either column chromatography on silica gel or preparative GLC to obtain the primary alcohols (+)-2,3-dibromo-1-propanol ((..cap alpha..)/sup 26/ /SUB D/ + 13.8/sup 0/) AND (-)-2,3-DIBROMO-1-PROPANOL ((..cap alpha..)/sup 24/ /SUB D/ -12.8/sup 0/). NMR and EI mass spectra of the primary and secondary dibromopropanols clearly distinguished the structural isomers from one another. The optical isomers will be used to examine possible stereoselective differences in mutagenic potency, since 2,3-dibromo-1-propanol is a mutagenic metabolite of the potent mutagen and carcinogen tris (2,3-dibromopropyl) phosphate.