Comparison of Toxicities among Different Bumped Kinase Inhibitor Analogs for Treatment of Cryptosporidiosis.

Recent advances on the development of bumped kinase inhibitors for treatment of cryptosporidiosis have focused on the 5-aminopyrazole-4-carboxamide scaffold, due to analogs that have less hERG inhibition, superior efficacy, and strong in vitro safety profiles. Three compounds, BKI-1770, -1841, and -1708, showed strong efficacy in C. parvum infected mice. Both BKI-1770 and BKI-1841 had efficacy in the C. parvum newborn calf model, reducing diarrhea and oocyst excretion. However, both compounds caused hyperflexion of the limbs seen as dropped pasterns. Toxicity experiments in rats and calves dosed with BKI-1770 showed enlargement of the epiphyseal growth plate at doses only slightly higher than the efficacious dose. Mice were used as a screen to check for bone toxicity, by changes to the tibia epiphyseal growth plate, or neurological causes, by use of a locomotor activity box. These results showed neurological effects from both BKI-1770 and BKI-1841 and bone toxicity in mice from BKI-1770, indicating one or both effects may be contributing to toxicity. However, BKI-1708 remains a viable treatment candidate for further evaluation as it showed no signs of bone toxicity or neurological effects in mice.

Symmetric Dimethylarginine Is a Sensitive Biomarker of Glomerular Injury in Rats.

Glomerular filtration rate is the gold-standard method for assessment of renal function but is rarely performed in routine toxicity studies. Standard serum biomarkers of renal function are insensitive and become elevated only with significant loss of organ function. Symmetric dimethylarginine (SDMA) is a ubiquitous analyte that is freely filtered by the glomerulus and can be detected in serum. It has shown utility for the detection of renal injury in dogs and cats in clinical veterinary practice, but the potential utility of SDMA to detect renal injury in preclinical species or toxicity studies has not been thoroughly investigated. We utilized a well-characterized glomerular toxicant, puromycin aminonucleoside, to induce podocyte injury and subsequent proteinuria in young male Sprague-Dawley rats. At the end of 1 or 2 weeks, blood, urine, and kidney tissue were collected for analysis. One week following a single 50 mg/kg dose, urea nitrogen, creatinine, and albumin mean values were within historical control ranges, while SDMA was increased. Glomerular changes in these animals included periodic acid-Schiff positive globules within podocytes, podocyte hypertrophy by light microscopy, and podocyte degeneration with effacement of foot processes by electron microscopy (EM). Taken together, our data indicate that SDMA may be a useful biomarker for early detection of glomerular toxicities in rats.

Automated blood sampling in canine telemetry model: Enhanced assessment of immune liabilities.

INTRODUCTION: This manuscript presents a successful integration of multi-timepoint biomarker blood sampling (e.g., cytokines) in a conscious dog cardiovascular study using automated blood sampling via vascular access ports in telemetry instrumented dogs. In addition to determining plasma exposure of the test compound, the assessment of biomarkers of interest allows for more comprehensive preclinical evaluation on a traditional conscious dog cardiovascular (CV) telemetry study especially for immunology and immune-oncology molecules. This model system provides a rapid and efficient means to quickly gain understanding of potential effects on key cardiovascular parameters in large species that are commonly used for preclinical safety evaluations while collecting multiple blood samples for drug and cytokine analysis. METHODS: Male beagle dogs were chronically implanted with telemetry devices (PhysioTel™ model D70-PCTP) and vascular access ports (SPMID-GRIDAC-5NC). BASi Culex-L automated blood sampling (ABS) (Bioanalytical Systems, Inc) system was used to collect blood samples at multiple time points for cytokine analysis. Four beagles received low-dose lipopolysaccharide solution (LPS) (0.1 and 0.5 µg/mL). The following cytokines were measured by Milliplex® map Canine Cytokine Magnetic Bead Panel: Interleukin (IL) 2, IL-6, IL-7, IL-8, IL-10, IL-15, IL-18, TNF-α, MCP-1, KC-like, GM-CSF, IFN gamma, and IP10. RESULTS: Low dose LPS administration induced a pronounced dose-dependent, transient release of key inflammatory cytokines (IL-2, IL-6, IL-10, TNF-α, MCP-1, and KC-like). Cytokine responses were similar to other canine and human endotoxin models. LPS administration led to an increase in body temperature, heart rate, and mean arterial pressure, as well as a decrease in QTcV interval. CONCLUSION: Successful incorporation of cytokine analysis in telemetry instrumented dogs with vascular access ports allows for translational PK/PD modeling of both efficacy and safety of compounds in the immunology as well as the immune-oncology therapeutic areas designed to modulate the immune system. Remote collection of blood samples simultaneously with CV endpoints is a significant enhancement for assessment of biomarkers that are sensitive to animal handling and excitement associated with room disturbances which are obligatory with manual blood collection. Furthermore, implementing this approach has also refined our animal welfare procedure by reducing the handling during a study and thereby reducing stress (positive refinement 3R impact).

Bumped Kinase Inhibitors as therapy for apicomplexan parasitic diseases: lessons learned.

Bumped Kinase Inhibitors, targeting Calcium-dependent Protein Kinase 1 in apicomplexan parasites with a glycine gatekeeper, are promising new therapeutics for apicomplexan diseases. Here we will review advances, as well as challenges and lessons learned regarding efficacy, safety, and pharmacology that have shaped our selection of pre-clinical candidates.

Scientific and Regulatory Policy Committee Points to Consider*: Review of Scientific and Regulatory Policy Committee Points to Consider: Review of Current Practices for Ultrastructural Pathology Evaluations in Support of Nonclinical Toxicology Studies.

Anatomic pathology and clinical pathology end points are standard components of almost every nonclinical general toxicity study conducted during the risk assessment of novel pharmaceuticals and chemicals. On occasion, an ultrastructural pathology evaluation using transmission electron microscopy (TEM) may be included in nonclinical toxicity studies. Transmission electron microscopy is most commonly used when a light microscopic finding may require further characterization that could inform on the pathogenesis and/or mechanism of action. Regulatory guidance do not address the use of TEM in general study designs nor whether these assessments should be performed in laboratories conducted in compliance with Good Laboratory Practices. The Scientific and Regulatory Policy Committee of the Society of Toxicologic Pathology (STP) formed a Working Group to assess the current practices on the use of TEM in nonclinical toxicity studies. The Working Group constructed a survey sent to members of societies of toxicologic pathology in the United States, Europe, Britain, and Japan, and responses were collected through the STP for evaluation by the Working Group. The survey results and regulatory context are discussed, as are "points to consider" from the collective experience of the Working Group. This survey indicates that TEM remains an essential diagnostic option for complementing toxicologic pathology evaluations. *This Points to Consider article is a product of a Society of Toxicologic Pathology (STP) Working Group commissioned by the Scientific and Regulatory Policy Committee (SRPC) of the STP. It has been reviewed and approved by the SRPC and Executive Committee of the STP but it does not represent a formal Best Practice recommendation of the Society; rather, it is intended to provide key "points to consider" in designing nonclinical studies or interpreting data from toxicity and safety studies intended to support regulatory submissions. The points expressed in this document are those of the authors and do not reflect views or policies of the employing institutions. Readers of Toxicologic Pathology are encouraged to send their thoughts on these articles or ideas for new topics to the Editor.

From the Cover: Inhibitors of Nicotinamide Phosphoribosyltransferase Cause Retinal Damage in Larval Zebrafish.

Nicotinamide phosphoribosyltransferase (NAMPT) has been investigated as a target for oncology because it catalyzes a rate-limiting step in cellular energy metabolism to produce nicotinamide adenine dinucleotide. Small molecule inhibitors of NAMPT have been promising drug candidates but preclinical development has been hindered due to associated retinal toxicity. Here we demonstrate that larval zebrafish can predict retinal toxicity associated with this mechanism revealing an attractive alternative method for identifying such toxicities. Zebrafish permit higher throughput testing while using far lower quantities of test article compared with mammalian systems. NAMPT inhibitor-associated toxicity manifested in zebrafish as a loss of response to visual cues compared with auditory cues. Zebrafish retinal damage associated with NAMPT inhibitor treatment was confirmed through histopathology. Ranking 6 NAMPT inhibitors according to their impact on zebrafish vision revealed a positive correlation with their in vitro potencies on human tumor cells. This correlation indicates translatable pharmacodynamics between zebrafish and human NAMPT and is consistent with on-target activity as the cause of retinal toxicity associated with NAMPT inhibition. Together, these data illustrate the utility of zebrafish for identifying compounds that may cause ocular toxicity in mammals, and, likewise, for accelerating development of compounds with improved safety margins.

A regenerative erythropoietic response does not increase the frequency of Pig-a mutant reticulocytes and erythrocytes in Sprague-Dawley rats.

The in vivo rodent Pig-a mutation assay is a sensitive test to identify exposure to mutagenic substances, and has been proposed as an assay for the identification of impurities for pharmaceuticals. Red blood cells (RBCs) and reticulocytes (RETs) are analyzed by flow cytometry after exposure to potentially mutagenic chemicals for cells deficient in the cell surface anchored protein CD59, representing mutation in the X-linked Pig-a gene. The full potential of the assay as well as its limitations are currently being explored. The current study investigated the effects of regenerative erythropoietic bone marrow responses on the frequency of Pig-a mutated reticulocytes (RETCD59- ) and erythrocytes (RBCCD59- ). We hypothesized that a robust regenerative erythropoietic response would not increase the basal frequency of RETCD59- or RBCCD59- cells. Two groups of six male Sprague-Dawley rats either had 2 mL of blood sampled each day via an indwelling catheter over a period of 5 days or were minimally sampled for hematology and used as controls. Blood was also then collected and evaluated 5, 18, and 49 days after the initial bleed period for the number of Pig-a mutant cells in either the RET or RBC population. Despite the expected decrease in hematocrit and the correlative increase in reticulocytes after bleeding, no increase in the number of Pig-a mutant cells was observed in male Sprague-Dawley rats that were bled for five consecutive days. These results indicate that changes in erythropoiesis and hematology parameters in rats appear to have no effect on the background levels of Pig-a mutated RETs and RBCs. Environ. Mol. Mutagen. 59:91-95, 2018. © 2017 Wiley Periodicals, Inc.

Measuring drug absorption improves interpretation of behavioral responses in a larval zebrafish locomotor assay for predicting seizure liability.

INTRODUCTION: Unanticipated effects on the central nervous system are a concern during new drug development. A larval zebrafish locomotor assay can reveal seizure liability of experimental molecules before testing in mammals. Relative absorption of compounds by larvae is lacking in prior reports of such assays; having those data may be valuable for interpreting seizure liability assay performance. METHODS: Twenty-eight reference drugs were tested at multiple dose levels in fish water and analyzed by a blinded investigator. Responses of larval zebrafish were quantified during a 30min dosing period. Predictive metrics were calculated by comparing fish activity to mammalian seizure liability for each drug. Drug level analysis was performed to calculate concentrations in dose solutions and larvae. Fifteen drug candidates with neuronal targets, some having preclinical convulsion findings in mammals, were tested similarly. RESULTS: The assay has good predictive value of established mammalian responses for reference drugs. Analysis of drug absorption by larval fish revealed a positive correlation between hyperactive behavior and pro-convulsive drug absorption. False negative results were associated with significantly lower compound absorption compared to true negative, or true positive results. The predictive value for preclinical toxicology findings was inferior to that suggested by reference drugs. DISCUSSION: Disproportionately low exposures in larvae giving false negative results demonstrate that drug exposure analysis can help interpret results. Due to the rigorous testing commonly performed in preclinical toxicology, predicting convulsions in those studies may be more difficult than predicting effects from marketed drugs.

Frontiers in preclinical safety biomarkers: microRNAs and messenger RNAs.

The measurement of plasma microRNAs (miRNAs) and messenger RNAs (mRNAs) is the most recent effort to identify novel biomarkers in preclinical safety. These genomic markers often display tissue-specific expression, may be released from the tissues into the plasma during toxic events, change early and with high magnitude in tissues and in the blood during specific organ toxicities, and can be measured using multiplex formats. Their validation as biomarkers has been challenged by the technical difficulties. In particular, the concentration of miRNAs in the plasma depends on contamination by miRNAs originating from blood cells and platelets, and the relative fraction of miRNAs in complexes with Argonaute 2, high-density lipoproteins, and in exosomes and microvesicles. In spite of these hurdles, considerable progress has recently been made in assessing the potential value of miRNAs in the clinic, especially in cancer patients and cardiovascular diseases. The future of miRNAs and mRNAs as biomarkers of disease and organ toxicity depends on our ability to characterize their kinetics and to establish robust collection and measurement methods. This review covers the basic biology of miRNAs and the published literature on the use of miRNAs and mRNAs as biomarkers of specific target organ toxicity.

Computer-assisted imaging algorithms facilitate histomorphometric quantification of kidney damage in rodent renal failure models.

INTRODUCTION: Surgical 5/6 nephrectomy and adenine-induced kidney failure in rats are frequently used models of progressive renal failure. In both models, rats develop significant morphological changes in the kidneys and quantification of these changes can be used to measure the efficacy of prophylactic or therapeutic approaches. In this study, the Aperio Genie Pattern Recognition technology, along with the Positive Pixel Count, Nuclear and Rare Event algorithms were used to quantify histological changes in both rat renal failure models. METHODS: Analysis was performed on digitized slides of whole kidney sagittal sections stained with either hematoxylin and eosin or immunohistochemistry with an anti-nestin antibody to identify glomeruli, regenerating tubular epithelium, and tubulointerstitial myofibroblasts. An anti-polymorphonuclear neutrophil (PMN) antibody was also used to investigate neutrophil tissue infiltration. RESULTS: Image analysis allowed for rapid and accurate quantification of relevant histopathologic changes such as increased cellularity and expansion of glomeruli, renal tubular dilatation, and degeneration, tissue inflammation, and mineral aggregation. The algorithms provided reliable and consistent results in both control and experimental groups and presented a quantifiable degree of damage associated with each model. CONCLUSION: These algorithms represent useful tools for the uniform and reproducible characterization of common histomorphologic features of renal injury in rats.

Ultrastructural analysis in preclinical safety evaluation.

The first electron microscopic images of biological specimens were made in the 1940s, and the next 30 years comprised an era of descriptive ultrastructure during which transmission electron microscopy (TEM) was integral to an explosion in cellular and molecular biology. However, when questions could no longer be answered by ultrastructural information alone, the use of TEM in biological research declined. Innovative molecular techniques and newer imaging technologies such as confocal fluorescence microscopy filled the gap, providing faster answers with less rigorous training as a prerequisite to data collection. The use of TEM in toxicologic pathology has paralleled the rise and fall of its popularity in other disciplines. However, TEM remains an essential resource that provides direct and unequivocal data to explain and address safety concerns in preclinical toxicity studies. There is still an important place for TEM in preclinical safety evaluation and mechanistic studies, particularly when visualization of subcellular structures provides a link to other endpoints. This review reinforces the value of TEM in preclinical safety testing and model development and encourages best practices for ultrastructural evaluation.

Copy number abnormalities in sporadic canine colorectal cancers.

Human colorectal cancer (CRC) is one of the better-understood systems for studying the genetics of cancer initiation and progression. To develop a cross-species comparison strategy for identifying CRC causative gene or genomic alterations, we performed array comparative genomic hybridization (aCGH) to investigate copy number abnormalities (CNAs), one of the most prominent lesion types reported for human CRCs, in 10 spontaneously occurring canine CRCs. The results revealed for the first time a strong degree of genetic homology between sporadic canine and human CRCs. First, we saw that between 5% and 22% of the canine genome was amplified/deleted in these tumors, and that, reminiscent of human CRCs, the total altered sequences directly correlated to the tumor's progression stage, origin, and likely microsatellite instability status. Second, when mapping the identified CNAs onto syntenic regions of the human genome, we noted that the canine orthologs of genes participating in known human CRC pathways were recurrently disrupted, indicating that these pathways might be altered in the canine CRCs as well. Last, we observed a significant overlapping of CNAs between human and canine tumors, and tumors from the two species were clustered according to the tumor subtypes but not the species. Significantly, compared with the shared CNAs, we found that species-specific (especially human-specific) CNAs localize to evolutionarily unstable regions that harbor more segmental duplications and interspecies genomic rearrangement breakpoints. These findings indicate that CNAs recurrent between human and dog CRCs may have a higher probability of being cancer-causative, compared with CNAs found in one species only.

Squamous cell carcinoma.

Squamous cell carcinoma (SCC) is a relatively common, malignant neoplasm of dogs and cats that can arise in a variety of locations. The gross appearance of SCC can be variable and nonspecific, so definitive diagnosis requires microscopic examination of the tissue (cytology or histology). Several treatment modalities exist, but surgical excision, if possible, is regarded as the best treatment option. Early diagnosis and treatment of SCC are key because small, early-stage tumors are the most amenable to treatment and carry the best prognosis.

Prostate cancer in dogs: comparative and clinical aspects.

The canine prostate gland shares many morphological and functional similarities with the human prostate and dogs are the only other large mammals that commonly develop spontaneous prostate cancer. However, the incidence of prostate cancer is much lower in dogs and the precise cell of origin is not known. Dogs with prostate cancer usually present with advanced disease that does not respond to androgen deprivation therapy. Similar to humans, affected dogs often develop osteoblastic bone metastases in the pelvis and/or lumbar spine with associated pain and neurological deficits. Other clinical signs include weight loss, lethargy, and abnormal urination and/or defecation. Surgery, chemotherapy, and radiation have been used to treat dogs with prostate cancer, but success has been limited by the location and aggressive nature of the disease. It is evident that better methods of early detection and more effective therapies are needed for prostate cancer in dogs and advanced prostate carcinoma in men. Dogs with naturally-occurring prostate cancer are relevant models for the disease in humans and pre-clinical studies of new diagnostics and therapies in dogs may benefit both humans and dogs with prostate cancer.

Serum bile acids concentrations in healthy and clinically ill neonatal foals.

BACKGROUND: Reference ranges for serum bile acids (SBA) concentration are well established in healthy adult horses. Increased values are indicative of hepatic disease. HYPOTHESES: SBA concentrations are significantly greater in the neonatal period compared with mature horses, and illness in the neonatal period will further increase SBA. ANIMALS: Ten healthy mature horses, 12 healthy foals, and 31 clinically ill foals. METHODS: Prospective cross-sectional study. Blood samples were obtained once from the mature horses, from healthy foals immediately after birth, at 2 days, and at 1, 2, 3, 4, and 6 weeks of age; and from ill foals less than 1 month of age at the time of admission to the Veterinary Teaching Hospital. SBA concentrations were determined enzymatically and by radioimmunoassay. Total and direct bilirubin and triglyceride concentrations were measured, as well as sorbitol dehydrogenase (SDH) and gamma-glutamyltransferase (GGT) activities. RESULTS: There was a significant negative correlation between age and SBA concentration. Compared with mature horses, SBA concentrations were significantly greater in healthy foals at each collection time over the first 6 weeks of life. Radioimmunoassay values were lower than enzymatic SBA values, with increasing bias as the mean difference between values increased. When comparing age-matched values between healthy and ill foals, there were no significant differences in SBA. None of the ill foals had a primary diagnosis of hepatic disease. There was no significant correlation between the SBA concentration and the bilirubin or triglyceride concentrations or the GGT activity. There was a significant direct correlation between increased SBA and serum SDH activity in healthy foals only. CONCLUSION AND CLINICAL IMPORTANCE: SBA concentrations in foals are significantly higher in the early neonatal period, underscoring the importance of using age-matched references when evaluating clinical pathology values during the neonatal period.

A subcutaneous mass on the neck of a horse.

A 25-year-old Arabian gelding was presented for investigation of a subcutaneous neck mass. Fine-needle aspirates and impression smears revealed mast cells with widely varying degrees of cytoplasmic granulation and scattered eosinophils. Histopathology revealed a poorly circumscribed mass composed of sheets and bundles of mast cells with a large population of eosinophils. The mast cells were separated into numerous lobules by a heavy collagenous stroma, and multifocal collagen necrosis was present. Strong reactivity of the tumor cells for both Giemsa and toluidine blue stains confirmed the diagnosis of a mast cell tumor, and a Luna stain accentuated the eosinophilic infiltrates. Cutaneous mast cell tumors are found in many domestic animals but are uncommonly encountered in horses. Equine cutaneous mast cell tumors are usually benign, and there are no reports of visceral metastasis. Surgical excision is thought to be curative.

New bone formation and osteolysis by a metastatic, highly invasive canine prostate carcinoma xenograft.

BACKGROUND: Osteoblastic metastases are commonly induced by prostate cancer. A canine prostate carcinoma xenograft (Ace-1) was developed and used to evaluate neoplastic prostate cell growth, metastasis, and effects on bone formation in nude mice. METHODS: Characteristics of the Ace-1 cells were evaluated with histopathology, radiography, and bioluminescent imaging (BLI). Immunohistochemistry and quantitative RT-PCR were used to evaluate the expression of factors important in the development of osteoblastic metastases. RESULTS: The Ace-1 cells were invasive and induced bone formation and destruction. Radiographs demonstrated a mixed osteoblastic/osteolytic reaction. Lung and lymph node metastases occurred in 30% of mice. The tumor cells expressed parathyroid hormone-related protein (PTHrP-141 isoform), cathepsin K, keratins 8/18, and vimentin, but not keratins 5/14, and were androgen receptor negative. Intracardiac (IC) injections resulted in metastases in vertebrae and long bones. CONCLUSIONS: The Ace-1 xenograft is a useful model for investigating the pathogenesis of prostate cancer invasion and mixed osteoblastic/osteolytic bone metastases.

Risk factors associated with renal insufficiency in horses with primary gastrointestinal disease: 26 cases (2000-2003).

OBJECTIVE: To identify factors associated with renal insufficiency in colic- or colitis-affected horses with high serum creatinine (SCr) concentrations evaluated at a referral hospital. DESIGN: Retrospective case series. ANIMALS: 167 colic- or colitis-affected horses (88 represented a random sample [hospital population], and 79 had high SCr concentration at initial evaluation [study population]). PROCEDURE: Medical records were reviewed. Data collected included signalment; physical examination, clinicopathologic, and diagnostic findings; and outcome. The study population was categorized on the basis of whether SCr concentration did (AR group; n = 53) or did not (PA group; 26) normalize within 72 hours of fluid therapy. Characteristics of the study and hospital populations were compared. RESULTS: Males and Quarter Horses were significantly overrepresented in the study population. Compared with the hospital population, study-population horses were significantly more likely to have colitis, gastric reflux, and diarrhea at initial evaluation. Initial mean SCr concentration in the PA group was significantly higher than the AR group; identification of gastric reflux, abnormal rectal examination findings, and hypochloremia were significantly associated with persistent azotemia after 72 hours of fluid therapy. Compared with the AR group, PA group horses were 3 times as likely to die or be euthanized. CONCLUSIONS AND CLINICAL RELEVANCE: In colic- or colitis-affected horses, factors associated with renal insufficiency included gastric reflux, abnormal rectal examination findings, or hypochloremia initially; prognosis for horses in which azotemia resolves within 72 hours of treatment appears to be better than for horses with persistent azotemia.

Tail-base mass from a "horse of a different color".

A 14-year-old bay Thoroughbred gelding was presented for evaluation of a mass at the base of the tail. The mass had been present for 1 year, and recently had begun to increase in size. Additional masses were found around the eye and shoulder. A fine-needle aspirate of the tail-base mass revealed highly anaplastic round to polyhedral cells containing dark green to black cytoplasmic granules interpreted to be melanin. Histologically, the mass was composed of pleomorphic, poorly pigmented, round to polyhedral cells interpreted to be neoplastic melanocytes. With immunohistochemistry, the cells were positive for vimentin and S-100, but negative for pancytokeratin and Melan-A. The cytologic and histopathologic diagnoses were amelanotic melanoma. The horse was treated with cimetidine, but the tumor continued to progress. In this report, we describe the cytopathologic features of an aggressive amelanotic melanoma in a non-grey horse and emphasize the unique correlation between cytologic and histologic findings.