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.

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.

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.

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.

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.

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.

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.

New bone formation in nude mouse calvaria induced by canine prostate tissue.

Osteoblastic metastases are common in patients with advanced prostate cancer. The pathophysiology of the new bone formation at metastatic sites is not currently known, but it is hypothesized that growth factors secreted by the prostate may be involved. Unfortunately, most rodent models of prostate cancer with metastasis to bone are osteolytic and not osteoblastic. Significant osteolysis by tumor cells at metastatic sites also may lead to fractures or bone instability. Misinterpretation of new periosteal bone due to bone instability as tumor-cell osteo-induction is another disadvantage of the osteolytic models. To circumvent these problems, we have developed a model system of new bone formation in the calvaria of nude mice stimulated by normal canine prostate tissue. Collagenase-digested normal prostate tissue was implanted adjacent to the calvaria of nude mice. Calvaria were examined at 2 weeks post-implantation for changes in the bone microenvironment by histology, calcein uptake at sites of bone mineralization, and tartrate-resistant acid phosphatase staining for osteoclasts. The prostate tissue remained viable and induced abundant new woven bone formation on the adjacent periosteal surface. In some cases new bone formation also was induced on the distant or concave calvarial periosteum. The new bone stained intensely with calcein, which demonstrated mineralization of the bone matrix. The new bone formation on prostate-implanted calvaria significantly increased (1.7-fold) the thickness of the calvaria compared with control calvaria. New bone formation was not induced in calvaria of mice implanted with normal canine kidney, urinary bladder, spleen, or skeletal muscle tissue, or mice with surgically-induced disruption of the periosteum. Osteoclast numbers in the medullary spaces and periosteum of calvaria were mildly increased (61%) in mice with implanted prostate tissue. In conclusion, this animal model will be useful for investigating the roles of prostate-derived growth factors on new bone formation in vivo.

Prostatic carcinoma causing urethral obstruction and obstipation in a cat.

A 9-year-old intact male cat was presented for vomiting and straining to defecate. A large abdominal mass was palpated. The urinary bladder was full and non-expressible. Exploratory laparotomy revealed that the mass was compressing the colon and encircling the urethra caudal to the bladder. The mass was removed, the urethra transected, and the urinary bladder marsupialized to the ventral abdominal wall to allow urine drainage. Histopathologic examination of the mass revealed a prostatic carcinoma. The cat died approximately 6 weeks after removal of the mass. This is the first reported case of a prostatic carcinoma causing urethral obstruction and obstipation in a cat.

Hypercalcemia and high serum parathyroid hormone-related protein concentration in a horse with multiple myeloma.

A 13-year-old gelding was examined because of weight loss, hyperglobulinemia, and hypercalcemia. Possible causes of hypercalcemia that were considered included renal failure, primary hyperparathyroidism, vitamin D toxicosis, and malignancy. There was no history of vitamin D ingestion, and serum creatinine and parathyroid hormone concentrations were normal, making renal failure and primary hyperparathyroidism unlikely. The hypercalcemia was suspected to be a result of malignancy, but thorough testing did not reveal any neoplastic disease. Eight months later, serum parathyroid hormone-related protein (PTHrP) concentration was high, supporting the suggestion that hypercalcemia was a result of malignancy. In addition, radial immunodiffusion confirmed a selective 300-fold increase in serum IgA concentration. The horse was euthanatized, and postmortem examination revealed neoplastic infiltrates in the kidneys, lymph nodes, liver, and bone marrow. Neoplastic cells had morphologic characteristics of plasma cells, and immunohistochemical staining confirmed that neoplastic cells were expressing PTHrP and IgA. The final diagnosis was multiple myeloma with expression of IgA paraprotein.

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.

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.

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.

Canine prostate induces new bone formation in mouse calvaria: A model of osteoinduction by prostate tissue.

BACKGROUND: Osteoblastic metastases are common in patients with advanced prostate cancer. The pathophysiology of the new bone formation at metastatic sites is not currently known, but it is hypothesized that growth factors secreted by the prostate may be involved. Unfortunately, most rodent models of prostate cancer with metastasis to bone are osteolytic and not osteoblastic. Significant osteolysis by tumor cells at metastatic sites may also lead to fractures or bone instability. Misinterpretation of new periosteal bone due to bone instability as tumor-cell osteoinduction is another disadvantage of the osteolytic models. To circumvent these problems, we have developed a model system of new bone formation in the calvaria of nude mice stimulated by normal canine prostate tissue. METHODS: Collagenase-digested normal prostate tissue was implanted adjacent to the calvaria of nude mice. Calvaria were examined at 2 weeks post-implantation for changes in the bone microenvironment by histology, calcein uptake at sites of bone mineralization, and tartrate-resistant acid phosphatase staining for osteoclasts. RESULTS: The prostate tissue remained viable and induced abundant new woven bone formation on the adjacent periosteal surface. In some cases new bone formation also was induced on the distant or concave calvarial periosteum. The new bone stained intensely with calcein, which demonstrated mineralization of the bone matrix. The new bone formation on prostate-implanted calvaria significantly increased (1.7-fold) the thickness of the calvaria compared with control calvaria. New bone formation was not induced in calvaria of mice implanted with normal canine kidney, urinary bladder, spleen, or skeletal muscle tissue, or mice with surgically-induced disruption of the periosteum. Osteoclast numbers in the medullary spaces and periosteum of calvaria were mildly increased (61%) in mice with implanted prostate tissue. CONCLUSIONS: This animal model will be useful for investigating the roles of prostate-derived growth factors on new bone formation in vivo.

Canine prostate stimulates osteoblast function using the endothelin receptors.

BACKGROUND: Bone metastases are common in humans and dogs with late-stage prostate cancer. A unique feature of prostate cancer metastases is new bone formation at metastatic sites ("osteoblastic metastases"). Many carcinomas that metastasize to bone cause bone destruction, not new bone formation. The mechanisms by which prostate cancer induces bone formation at sites of bone metastasis are not well understood. We hypothesized that stimulation of osteoblasts by prostate tissue at metastatic sites was due to the paracrine actions of growth factors produced by prostate epithelial cells. METHODS: We have previously shown that normal canine prostate tissue induced new bone formation when implanted adjacent to the calvarium of nude mice. To complement this in vivo model, we developed an in vitro system of prostate-stimulated osteoblast function to investigate mechanisms of prostate-induced new bone formation. RESULTS: We found that treatment of cultured rat calvaria for 24 hr with proteins from normal dog prostate stimulated alkaline phosphatase activity in a dose-dependent manner 4-6 fold compared to controls. Stimulation began approximately 8 hr after treatment, and was diminished after 72 hr. Calvaria treated with homogenates of normal dog salivary gland, kidney, bladder, and muscle did not increase ALP activity. Pretreatment of the calvaria for 1 hr with endothelin antagonists, but not anti-parathyroid hormone-related protein (PTHrP) antibody or indomethacin, abrogated the stimulation of ALP. CONCLUSIONS: Our results indicated that osteoblast activation by canine prostate occurs via an endothelin-dependent mechanism, and that PTHrP or prostaglandin synthase-mediated pathways are likely not involved. This is a reliable, reproducible assay for determining the roles of molecules important in the activation of osteoblasts by the prostate.