Gastric Neuroendocrine Tumors With Parietal Cell Atrophy in a Long-term Carcinogenicity Study in Rats.

Malignant neuroendocrine tumors were diagnosed in the stomach of two out of sixty female Sprague-Dawley rats treated for 89 weeks with a high dose of a novel, small molecule, cannabinoid-1 antagonist. The tumors were associated with parietal cell atrophy accompanied by foveolar hyperplasia of the glandular stomach mucosa. Parietal cell atrophy/foveolar hyperplasia was considered test article related at the high dose, given the higher incidence and severity relative to untreated controls, although the precise mechanism of the parietal cell atrophy was undetermined. Spontaneous gastric neuroendocrine tumors are very rare in rats, and the current cases were considered secondary to parietal cell atrophy causing reduced gastric acid secretion and subsequent overstimulation of gastrin release through a feedback loop.

Publication Categories in Toxicologic Pathology.

Toxicologic Pathology is the official journal of the Society of Toxicologic Pathology (STP), the British Society of Toxicological Pathology, and the European STP (ESTP). Toxicologic Pathology publishes articles related to topics in various aspects of toxicologic pathology such as anatomic pathology, clinical pathology, experimental pathology, and biomarker research. Publications include society-endorsed Best Practice/Position and Points to Consider publications and ESTP Expert Workshop articles that are relevant to toxicologic pathology and scientific regulatory processes, Opinion articles under the banner of the STP Toxicologic Pathology Forum, Original Articles, Review Articles (unsolicited/contributed, mini, and invited), Brief Communications, Letters to the Editor, Meeting Reports, and Book Reviews. This article provides details on the various publication categories in Toxicologic Pathology and will serve as a reference for authors and readers.

Successful rescue treatment of right posterior inferior cerebellar artery (PICA) vascular stasis with tirofiban following endovascular coil implantation.

This report describes a patient who developed intraprocedural vascular stasis immediately following elective endovascular coil emboliation. Urgent antiplatelet treatment with the GpIIb/IIIa agent tirofiban was used. It was infused intra-arterially during the procedure, followed by a fixed rate intravenous continuous infusion, and successfully restored normal circulation. There were no reports of further bleeding or haemodynamic compromise during the hospital stay. The patient's condition returned to baseline and he was discharged the following day with no neurological deficits.

An in vitro alveolar epithelial cell model recapitulates LRRK2 inhibitor-induced increases in lamellar body size observed in preclinical models.

Alveolar type II (ATII) epithelial cells contain lamellar bodies (LBs) which synthesize and store lung surfactants. In animals, the inhibition or knockout of leucine-rich repeat kinase 2 (LRRK2) causes abnormal enlargement of LBs in ATII cells. This effect of LRRK2 inhibition in lung is largely accepted as being mediated directly through blocking of the kinase function; however, downstream consequences in the lung remain unknown. In this work we established an in vitro alveolar epithelial cell (AEC) model that recapitulates the in vivo phenotype of ATII cells and developed an assay to quantify changes in LB size in response to LRRK2 inhibitors. Culture of primary human AECs at the air-liquid interface on matrigel and collagen-coated transwell inserts in the presence of growth factors promoted the LB formation and apical microvilli and induced expression of LRRK2 and ATII cell markers. Treatment with a selective LRRK2 inhibitor resulted in pharmacological reduction of phospho-LRRK2 and a significant increase in LB size; effects previously reported in lungs of non-human primates treated with LRRK2 inhibitor. In summary, our human in vitro AEC model recapitulates the abnormal lung findings observed in LRRK2-perturbed animals and holds the potential for expanding current understanding of LRRK2 function in the lung.

Characterization of cachexia in the human fibrosarcoma HT-1080 mouse tumour model.

BACKGROUND: Cancer cachexia is a complex metabolic disease with unmet medical need. Although many rodent models are available, none are identical to the human disease. Therefore, the development of new preclinical models that simulate some of the physiological, biochemical, and clinical characteristics of the human disease is valuable. The HT-1080 human fibrosarcoma tumour cell line was reported to induce cachexia in mice. Therefore, the purpose of this work was to determine how well the HT-1080 tumour model could recapitulate human cachexia and to examine its technical performance. Furthermore, the efficacy of ghrelin receptor activation via anamorelin treatment was evaluated, because it is one of few clinically validated mechanisms. METHODS: Female severe combined immunodeficient mice were implanted subcutaneously or heterotopically (renal capsule) with HT-1080 tumour cells. The cachectic phenotype was evaluated during tumour development, including body weight, body composition, food intake, muscle function (force and fatigue), grip strength, and physical activity measurements. Heterotopic and subcutaneous tumour histology was also compared. Energy balance was evaluated at standard and thermoneutral housing temperatures in the subcutaneous model. The effect of anamorelin (ghrelin analogue) treatment was also examined. RESULTS: The HT-1080 tumour model had excellent technical performance and was reproducible across multiple experimental conditions. Heterotopic and subcutaneous tumour cell implantation resulted in similar cachexia phenotypes independent of housing temperature. Tumour weight and histology was comparable between both routes of administration with minimal inflammation. Subcutaneous HT-1080 tumour-bearing mice presented with weight loss (decreased fat mass and skeletal muscle mass/fibre cross-sectional area), reduced food intake, impaired muscle function (reduced force and grip strength), and decreased spontaneous activity and voluntary wheel running. Key circulating inflammatory biomarkers were produced by the tumour, including growth differentiation factor 15, Activin A, interleukin 6, and TNF alpha. Anamorelin prevented but did not reverse anorexia and weight loss in the subcutaneous model. CONCLUSIONS: The subcutaneous HT-1080 tumour model displays many of the perturbations of energy balance and physical performance described in human cachexia, consistent with the production of key inflammatory factors. Anamorelin was most effective when administered early in disease progression. The HT-1080 tumour model is valuable for studying potential therapeutic targets for the treatment of cachexia.

LRRK2 inhibitors induce reversible changes in nonhuman primate lungs without measurable pulmonary deficits.

The kinase-activating mutation G2019S in leucine-rich repeat kinase 2 (LRRK2) is one of the most common genetic causes of Parkinson's disease (PD) and has spurred development of LRRK2 inhibitors. Preclinical studies have raised concerns about the safety of LRRK2 inhibitors due to histopathological changes in the lungs of nonhuman primates treated with two of these compounds. Here, we investigated whether these lung effects represented on-target pharmacology and whether they were reversible after drug withdrawal in macaques. We also examined whether treatment was associated with pulmonary function deficits. We conducted a 2-week repeat-dose toxicology study in macaques comparing three different LRRK2 inhibitors: GNE-7915 (30 mg/kg, twice daily as a positive control), MLi-2 (15 and 50 mg/kg, once daily), and PFE-360 (3 and 6 mg/kg, once daily). Subsets of animals dosed with GNE-7915 or MLi-2 were evaluated 2 weeks after drug withdrawal for lung function. All compounds induced mild cytoplasmic vacuolation of type II lung pneumocytes without signs of lung degeneration, implicating on-target pharmacology. At low doses of PFE-360 or MLi-2, there was ~50 or 100% LRRK2 inhibition in brain tissue, respectively, but histopathological lung changes were either absent or minimal. The lung effect was reversible after dosing ceased. Lung function tests demonstrated that the histological changes in lung tissue induced by MLi-2 and GNE-7915 did not result in pulmonary deficits. Our results suggest that the observed lung effects in nonhuman primates in response to LRRK2 inhibitors should not preclude clinical testing of these compounds for PD.

Spontaneous Ectopic Choroid Plexus with Sclerosis in Adult Beagle Dogs.

Microscopic examination of the brain of adult Beagle dogs from four different general toxicity studies revealed the presence of ectopic choroid plexus tissue in six individual dogs (4 females and 2 males) with ages ranging from 12 to 18 months. In each dog, this finding was characterized by a well-circumscribed mass localized to a region above and along the corpus callosum without any apparent compression of adjacent brain tissue. Each mass was composed of columnar ependymal cells forming tubular structures surrounded by variable amounts of fibrovascular connective tissue and had the appearance of small rests of ependymal cells that had been penetrated by the leptomeninges during neural development. There were no associated clinical signs or macroscopic correlates. Based on morphologic appearance, a diagnosis of spontaneous ectopic choroid plexus with secondary sclerosis was made. To the authors' knowledge, ectopic choroid plexus has not been reported in Beagle dogs and is rare in humans and horses.

Design and Synthesis of Clinical Candidate PF-06751979: A Potent, Brain Penetrant, ß-Site Amyloid Precursor Protein Cleaving Enzyme 1 (BACE1) Inhibitor Lacking Hypopigmentation.

A major challenge in the development of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors for the treatment of Alzheimer's disease is the alignment of potency, drug-like properties, and selectivity over related aspartyl proteases such as Cathepsin D (CatD) and BACE2. The potential liabilities of inhibiting BACE2 chronically have only recently begun to emerge as BACE2 impacts the processing of the premelanosome protein (PMEL17) and disrupts melanosome morphology resulting in a depigmentation phenotype. Herein, we describe the identification of clinical candidate PF-06751979 (64), which displays excellent brain penetration, potent in vivo efficacy, and broad selectivity over related aspartyl proteases including BACE2. Chronic dosing of 64 for up to 9 months in dog did not reveal any observation of hair coat color (pigmentation) changes and suggests a key differentiator over current BACE1 inhibitors that are nonselective against BACE2 in later stage clinical development.

Use of Rat Primary Mesenteric Cells for the Prediction of PDE4 Inhibitor Drug-Induced Vascular Injury.

Drug-induced vascular injury (DIVI) in preclinical studies can delay, if not terminate, a drug development program. Clinical detection of DIVI can be very difficult as there are no definitive biomarkers known to reliably detect this disorder in all instances. The preclinical identification of DIVI requires detailed microscopic examination of a wide range of tissues although one of the most commonly affected areas in rats is the mesenteric vasculature. The reason for this predisposition of mesenteric arteries in rats as well as the exact mechanism and cell types involved in the initial development of these lesions have not been fully elucidated. We hypothesized that by using a mixed culture of cells from rat mesenteric tissue, we would be able to identify an RNA expression signature that could predict the invivo development of DIVI. Five compounds designed to inhibit Phosphodiesterase 4 activity (PDE4i) were chosen as positive controls. PDE4i's are well known to induce DIVI in the mesenteric vasculature of rats and there is microscopic evidence that this is associated, at least in part, with a proinflammatory mechanism. We surveyed, by qRT-PCR, the expression of 96 genes known to be involved in inflammation and using a Random-Forest model, identified 12 genes predictive of invivo DIVI outcomes in rats. Using these genes, we were able to cross-validate the ability of the Random-Forest modeling to predict the concentration at which PDE4i caused DIVI invivo.

Discovery and Characterization of (R)-6-Neopentyl-2-(pyridin-2-ylmethoxy)-6,7-dihydropyrimido[2,1-c][1,4]oxazin-4(9H)-one (PF-06462894), an Alkyne-Lacking Metabotropic Glutamate Receptor 5 Negative Allosteric Modulator Profiled in both Rat and Nonhuman Primates.

We previously observed a cutaneous type IV immune response in nonhuman primates (NHP) with the mGlu5 negative allosteric modulator (NAM) 7. To determine if this adverse event was chemotype- or mechanism-based, we evaluated a distinct series of mGlu5 NAMs. Increasing the sp3 character of high-throughput screening hit 40 afforded a novel morpholinopyrimidone mGlu5 NAM series. Its prototype, (R)-6-neopentyl-2-(pyridin-2-ylmethoxy)-6,7-dihydropyrimido[2,1-c][1,4]oxazin-4(9H)-one (PF-06462894, 8), possessed favorable properties and a predicted low clinical dose (2 mg twice daily). Compound 8 did not show any evidence of immune activation in a mouse drug allergy model. Additionally, plasma samples from toxicology studies confirmed that 8 did not form any reactive metabolites. However, 8 caused the identical microscopic skin lesions in NHPs found with 7, albeit with lower severity. Holistically, this work supports the hypothesis that this unique toxicity may be mechanism-based although additional work is required to confirm this and determine clinical relevance.

Mechanisms of Skin Toxicity Associated with Metabotropic Glutamate Receptor 5 Negative Allosteric Modulators.

Cutaneous reactions represent one of the most common adverse drug effects observed in clinical trials leading to substantial compound attrition. Three negative allosteric modulators (NAMs) of metabotropic glutamate receptors (mGluRs), which represent an important target for neurological diseases, developed by Pfizer, were recently failed in preclinical development due to delayed type IV skin hypersensitivity observed in non-human primates (NHPs). Here we employed large-scale phenotypic profiling in standardized panels of human primary cell/co-culture systems to characterize the skin toxicity mechanism(s) of mGluR5 NAMs from two different series. Investigation of a database of chemicals tested in these systems and transcriptional profiling suggested that the mechanism of toxicity may involve modulation of nuclear receptor targets RAR/RXR, and/or VDR with AhR antagonism. The studies reported here demonstrate how phenotypic profiling of preclinical drug candidates using human primary cells can provide insights into the mechanisms of toxicity and inform early drug discovery and development campaigns.

Identification of MHC Haplotypes Associated with Drug-induced Hypersensitivity Reactions in Cynomolgus Monkeys.

Drug-induced hypersensitivity reactions can significantly impact drug development and use. Studies to understand risk factors for drug-induced hypersensitivity reactions have identified genetic association with specific human leukocyte antigen (HLA) alleles. Interestingly, drug-induced hypersensitivity reactions can occur in nonhuman primates; however, association between drug-induced hypersensitivity reactions and major histocompatibility complex (MHC) alleles has not been described. In this study, tissue samples were collected from 62 cynomolgus monkeys from preclinical studies in which 9 animals had evidence of drug-induced hypersensitivity reactions. Microsatellite analysis was used to determine MHC haplotypes for each animal. A total of 7 haplotypes and recombinant MHC haplotypes were observed, with distribution frequency comparable to known MHC I allele frequency in cynomolgus monkeys. Genetic association analysis identified alleles from the M3 haplotype of the MHC I B region (B*011:01, B*075:01, B*079:01, B*070:02, B*098:05, and B*165:01) to be significantly associated (χ2 test for trend, p < 0.05) with occurrence of drug-induced hypersensitivity reactions. Sequence similarity from alignment of alleles in the M3 haplotype B region and HLA alleles associated with drug-induced hypersensitivity reactions in humans was 86% to 93%. These data demonstrate that MHC alleles in cynomolgus monkeys are associated with drug-induced hypersensitivity reactions, similar to HLA alleles in humans.

Chemoproteomic profiling reveals that cathepsin D off-target activity drives ocular toxicity of ß-secretase inhibitors.

Inhibition of ß-secretase BACE1 is considered one of the most promising approaches for treating Alzheimer's disease. Several structurally distinct BACE1 inhibitors have been withdrawn from development after inducing ocular toxicity in animal models, but the target mediating this toxicity has not been identified. Here we use a clickable photoaffinity probe to identify cathepsin D (CatD) as a principal off-target of BACE1 inhibitors in human cells. We find that several BACE1 inhibitors blocked CatD activity in cells with much greater potency than that displayed in cell-free assays with purified protein. Through a series of exploratory toxicology studies, we show that quantifying CatD target engagement in cells with the probe is predictive of ocular toxicity in vivo. Taken together, our findings designate off-target inhibition of CatD as a principal driver of ocular toxicity for BACE1 inhibitors and more generally underscore the power of chemical proteomics for discerning mechanisms of drug action.

Using Histopathologic Evidence to Differentiate Reproductive Senescence from Xenobiotic Effects in Middle-aged Female Sprague-Dawley Rats.

The female reproductive cycle is orchestrated by cyclical and coordinated hormonal changes under the direction of the hypothalamic-pituitary-gonadal (HPG) axis. Any disruption of the HPG axis may lead to functional and structural alterations in the female reproductive system. Test article-related disturbances in the estrous cycle can be recognized in nonclinical toxicity studies by staging the cycle based on microscopic evaluation of female reproductive organs. In chronic rat toxicity studies, an additional complication is the development of reproductive senescence, which is associated with natural alterations in the reproductive cycle leading to changes in the female reproductive system that can potentially be confused with test article effects. The current article describes the features of persistent estrus, one stage of reproductive senescence, in middle-aged Sprague-Dawley rats and discusses elements to help differentiate senescence from induced effects.

Drug-induced Skin Lesions in Cynomolgus Macaques Treated with Metabotropic Glutamate Receptor 5 (mGluR5) Negative Allosteric Modulators.

Three orally administered metabotropic glutamate receptor 5 (mGluR5) negative allosteric modulators caused skin lesions consistent with delayed type-IV hypersensitivity in cynomolgus macaques in 2- and 12-week toxicity studies. Several monkeys developed macroscopic skin lesions in multiple locations after 8 to 9 days of dosing; the most prominent effects involved the genital region of males and generalized erythema occurred in both sexes. Microscopic lesions occurred in both clinically affected and unaffected areas and were characterized by lymphocytic interface inflammation, subepidermal bullae, and individual keratinocyte vacuolation/necrosis. In the 12-week study, clinical effects in 2 animals resolved with continued dosing, whereas in others the inflammatory process progressed with 1 female exhibiting systemic lymphocytic inflammation in multiple tissues. The inflammatory infiltrate consisted of CD3 and CD4 positive T lymphocytes with minimal CD68 positive macrophages and only rare CD8 positive T lymphocytes. A subset of animals given a dosing holiday was subsequently rechallenged with similar lesions developing but with a more rapid clinical onset. These skin lesions were consistent with type-IV delayed hypersensitivity with some features comparable to bullous drug eruptions in humans. A relationship between these findings and the intended mode of action for these compounds could not be ruled out, given the occurrence across different chemotypes.

Age-related testicular toxicity of mGluR5 negative allosteric modulators appears to be unrelated to testis drug transporter maturity.

Testicular degeneration was observed in exploratory toxicity studies in Wistar rats treated with several mGluR5 negative allosteric modulators. To determine if these testis effects were influenced by animal age, these compounds were administered to male Wistar rats of different ages (8, 10, and 12 weeks old) for 2 weeks followed by evaluation of male reproductive organ weights, testis histopathology, and inhibin B levels. Overall, seminiferous tubule degeneration was observed in 2/15, 5/15, and 0/15 compound treated rats from the 8, 10, and 12 week old cohorts and inhibin B was decreased in 8 and 10 week old animals, but not in 12 week old rats, suggesting that there is an age-related component to this testis toxicity. The gene expression profiles of drug transporters in the testis of rats aged PND 38 through PND 91 were very similar, indicating that immaturity of these transporters is an unlikely factor contributing to the age-related toxicity.

Two-year carcinogenicity study in rats with a nonnucleoside reverse transcriptase inhibitor.

Administration of lersivirine, a nonnucleotide reverse transcriptase inhibitor, daily by oral gavage to Sprague-Dawley rats for up to 2 yr was associated with decreased survival, decreased body weights, and an increase in neoplasms and related proliferative lesions in the liver, thyroid, kidney, and urinary bladder. Thyroid follicular adenoma and carcinoma, the associated thyroid follicular hypertrophy/hyperplasia, hepatocellular adenoma/adenocarcinoma, altered cell foci, and hepatocellular hypertrophy were consistent with lersivirine-related induction of hepatic microsomal enzymes. Renal tubular adenoma and renal tubular hyperplasia were attributed to the lersivirine-related exacerbation of chronic progressive nephropathy (CPN), while urinary bladder hyperplasia and transitional cell carcinoma in the renal pelvis and urinary bladder were attributed to urinary calculi. Renal tubular neoplasms associated with increased incidence and severity of CPN, neoplasms of transitional epithelium attributed to crystalluria, and thyroid follicular and hepatocellular neoplasms related to hepatic enzyme induction have low relevance for human risk assessment.

Discovery and preclinical profiling of 3-[4-(morpholin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]benzonitrile (PF-06447475), a highly potent, selective, brain penetrant, and in vivo active LRRK2 kinase inhibitor.

Leucine rich repeat kinase 2 (LRRK2) has been genetically linked to Parkinson's disease (PD) by genome-wide association studies (GWAS). The most common LRRK2 mutation, G2019S, which is relatively rare in the total population, gives rise to increased kinase activity. As such, LRRK2 kinase inhibitors are potentially useful in the treatment of PD. We herein disclose the discovery and optimization of a novel series of potent LRRK2 inhibitors, focusing on improving kinome selectivity using a surrogate crystallography approach. This resulted in the identification of 14 (PF-06447475), a highly potent, brain penetrant and selective LRRK2 inhibitor which has been further profiled in in vivo safety and pharmacodynamic studies.

Kinase domain inhibition of leucine rich repeat kinase 2 (LRRK2) using a [1,2,4]triazolo[4,3-b]pyridazine scaffold.

Leucine rich repeat kinase 2 (LRRK2) has been genetically linked to Parkinson's disease (PD). The most common mutant, G2019S, increases kinase activity, thus LRRK2 kinase inhibitors are potentially useful in the treatment of PD. We herein disclose the structure, potential ligand-protein binding interactions, and pharmacological profiling of potent and highly selective kinase inhibitors based on a triazolopyridazine chemical scaffold.

Primary cell cultures for understanding rat epididymal inflammation.

Treatment-induced epididymal inflammation and granuloma formation is only an occasional problem in preclinical drug development, but it can effectively terminate the development of that candidate molecule. Screening for backup molecules without that toxicity must be performed in animals (generally rats) that requires at least 2 to 3 weeks of in vivo exposure, a great deal of specially synthesized candidate compound, and histologic examination of the target tissues. We instead hypothesized that these treatments induced proinflammatory gene expression, and so used mixed-cell cultures from the rat epididymal tubule to monitor the induction of proinflammatory cytokines. Cells were exposed for 24 hr and then cytotoxicity was evaluated with the MTS assay and mRNA levels of Interleukin-6 (IL-6) and growth-related oncogene (GRO) were measured. We found that compounds that were more toxic in vivo stimulated a greater induction of IL-6 and GRO mRNA levels in vitro. By relating effective concentrations in vitro with the predicted C(eff), we could rank compounds by their propensity to induce inflammation in rats in vivo. This method allowed the identification of several compounds with very low inflammatory induction in vitro. When tested in rats, the compounds produced small degrees of inflammation at an acceptable margin (approximately 20×), and have progressed into further development.