Maturity-related Variability of the Thymus in Cynomolgus Monkeys (Macaca fascicularis).

Terminal body weights (TBWs), thymus weight parameters, and thymus morphology were retrospectively evaluated in 453 cynomolgus monkeys assigned to control groups on nonclinical toxicity studies. Morphology of bone, ovary, and testis/epididymis were used to determine maturity status of individual animals. There was no correlation between TBW and thymus weight (absolute and/or relative to TBW or brain weight). Thymus weight parameters and grades of decreased lymphocytes in the thymus were highly variable in immature animals compared to mature animals. There was also high (up to 11-fold) variability of thymus weight parameters within a given control group on the same study (generally 3 or 4 animals per sex). Several parameters evaluated had more pronounced age-related changes in males when compared to females. Our results demonstrate the inherent variability of thymus weight parameters and morphologic observations for cynomolgus monkeys on toxicology studies. Changes in thymus parameters in cynomolgus monkeys are unreliable indicators of immunomodulation or immunotoxicity in the absence of other relevant findings. Therefore, the thymus parameters commonly evaluated in preclinical safety assessments should not be the primary data set used to determine the presence of a direct test article-related effect on the immune system.

High hematocrit resulting from administration of erythropoiesis-stimulating agents is not fully predictive of mortality or toxicities in preclinical species.

We conducted a retrospective analysis of publicly available preclinical toxicology studies with erythropoiesis-stimulating agents (ESAs) to examine common adverse events in rats, Beagle dogs, and cynomolgus monkeys. Mortality and/or thrombotic events were reported sporadically in a subset of studies and attributed to the high hematocrit (HCT) achieved in the animals. However, similarly high HCT was achieved in both high-dose and low-dose groups, but there were no reported adverse events in the low-dose group suggesting HCT was not the sole contributing factor leading to toxicity. Our analysis indicated that increased dose, dose frequency, and dosing duration in addition to high HCT contributed to mortality and thrombosis. To further evaluate this relationship, the incidence of toxicities was compared in rats administered an experimental hyperglycosylated analog of recombinant human erythropoietin (AMG 114) at varying dosing schedules in 1-month toxicity studies. The incidence of mortality and thrombotic events increased in higher dose groups and when dosed more frequently, despite a similarly high HCT in all animals. The results from the investigative study and retrospective analysis demonstrate that ESA-related toxicities in preclinical species are associated with dose level, dose frequency, and dosing duration, and not solely dependent upon a high HCT.

Utility of quantitative whole-body autoradiography (QWBA) and imaging mass spectrometry (IMS) by matrix-assisted laser desorption/ionization (MALDI) in the assessment of ocular distribution of drugs.

INTRODUCTION: Assessment of drug candidate properties and potential liabilities can greatly benefit from issue driven studies that are designed to address specific toxicological effects such as ocular phototoxicity. If a compound absorbs light in the wavelength range of 290-700 nm (UV-A, UV-B, and visible light) and generates a positive response in a standard in vitro neutral red uptake phototoxicity assay in Balb/c 3T3 mouse fibroblasts, a single-dose in vivo study may be conducted to assess the potential for drug-induced phototoxicity in the eyes and skin of pigmented Long-Evans rats. Critical to ocular phototoxicity assessment is the hypothesis that the drug or drug-related material must be present in the affected substructures such as the uveal tract, retina, lens, or cornea. For compounds that induce a positive ocular response in the in vivo phototoxicity assay, data on distribution patterns to substructures of the eye can inform decisions regarding the nature of the ocular findings and possibly influence compound advancement. METHODS: Quantitative whole-body autoradiography (QWBA) and imaging mass spectrometry (IMS) by matrix-assisted laser desorption ionization (MALDI) on an ion trap mass spectrometer employing higher order mass spectrometric scanning functions were utilized for localization of dosed drug or metabolites in eye substructures. RESULTS: In investigative studies designed to simulate an in vivo phototoxicity study, rats were administered radio-labeled test article for QWBA analysis and un-labeled test article for IMS analysis. Autoradiograms from the QWBA study indicated that the radio-labeled analyte(s) preferentially distributed to the uveal tract and not the cornea. However, QWBA did not provide information on the nature of the detected analyte(s); i.e. intact parent drug versus potential metabolites or degradants. Multistage MS experiments performed directly on tissue sections demonstrated semi-quantitative localization in the uveal tract and unequivocal identification of the analyte as the dosed parent drug; no potential metabolites were detected. DISCUSSION: Image analysis by QWBA and IMS by MALDI proved complementary in the localization and identification of small molecule drug distribution within the eye.

Strain-related differences in urine composition of male rats of potential relevance to urolithiasis.

In carcinogenicity studies with PPAR gamma and alpha/gamma agonists, urinary bladder tumors have been reported in Harlan Sprague-Dawley (HSD) and Charles River Sprague-Dawley (SD) but not Wistar (WI) rats, with urolithiasis purported to be the inciting event. In two 3-month studies, the authors investigated strain-related differences in urine composition by sampling urine multiple times daily. Urine pH, electrolytes, creatinine, protein, citrate and oxalate levels, and serum citrate were assessed; urine sediment was analyzed by scanning electron microscopy and energy dispersive x-ray spectroscopy. HSD rats had significantly higher urine calcium than SD or WI rats, primarily as calcium phosphate-containing precipitate. When compared to SD rats, HSD rats had lower urine volume, higher urine protein, and a comparable (week 4) to lower (week 13) burden of MgNH(4)PO(4) aggregates. Relative to WI rats, HSD rats had higher urine protein and magnesium and lower serum and urine citrate. Overall, the susceptibility to urolithiasis in male rats was HSD > SD > WI; this was likely due to strain-related differences in the amount of urine protein (a nidus for crystal formation), lithogenic ions, citrate (an inhibitor of lithogenesis), and/or volume. Strain-related differences in urine composition need to be considered when interpreting the outcome of studies with compounds that alter urine composition.

Dietary ammonium chloride for the acidification of mouse urine.

A novel therapeutic compound was found to induce bladder tumors in male rats. Given the location of the tumors and the increased amounts of calcium- and magnesium-containing solids found in the urine of treated animals, we hypothesized that tumorigenesis was secondary to urine crystal formation rather than a direct effect of the drug on urothelium. To investigate the basis for the response, a method of acidifying rodent urine was needed. This study tested the efficacy of 1% dietary NH(4)Cl in reducing the urinary pH of male mice. After 1 wk, urinary pH (mean +/- SD) at 1 h after light onset was 7.51 +/- 0.32 among controls compared with 6.21 +/- 0.31 for the NH(4)Cl-fed group. After 2 wk of supplementation, urinary pH was 7.78 +/- 0.41 for controls and 6.20 +/- 0.30 for the NH(4)Cl-fed group. To investigate whether the time of collection altered urinary pH, samples also were collected 8 h after the start of the light cycle on the day of the 2-wk collection. Urinary pH was 7.12 +/- 0.28 for the control group and 5.80 +/- 0.23 for the NH(4)Cl-fed mice. The pH differences between control and NH(4)Cl-fed groups and the differences in pH within groups at 1 and 8 h were statistically significant. Dietary NH(4)Cl is an effective urinary acidifier for mice. When evaluating the pH of mouse urine, care should be taken to compare samples collected at the same time after the start of the light cycle.

In vitro cyclooxygenase-2 protein expression and enzymatic activity in neoplastic cells.

BACKGROUND: Cyclooxygenase-2 (COX-2) and its principle enzymatic metabolite, prostaglandin E2 (PGE2), are implicated in cancer progression. Based upon immunohistochemical (IHC) evidence that several tumor types in animals overexpress COX-2 protein, COX-2 inhibitors are used as anticancer agents in dogs and cats. HYPOTHESIS: IHC is inaccurate for assessing tumor-associated COX-2 protein and enzymatic activity. METHODS: Five mammalian cell lines were assessed for COX-2 protein expression by IHC and Western blot analysis (WB), and functional COX-2 activity was based upon PGE2 production. RESULTS: Detection of COX-2 protein by IHC and WB were in agreement in 4 of 5 cell lines. In 1 cell line that lacked COX-2 gene transcription because of promoter hypermethylation (HCT-116), IHC produced false-positive staining for COX-2 protein expression. Functional COX-2 enzymatic activity was dissociated from relative IHC-based COX-2 protein expression in 2 cell lines (RPMI 2650 and SCCF1). The RPMI 2650 cell line demonstrated strong COX-2 protein expression but minimal PGE2 production. CONCLUSIONS AND CLINICAL IMPORTANCE: Western blot is more accurate than IHC for the detection of COX-2 protein in the cell lines studied. Furthermore, the semiquantitative identification of COX-2 protein by IHC or WB does not necessarily correlate with enzymatic activity. Based upon the potential inaccuracy of IHC and dissociation of COX-2 protein expression from enzymatic activity, the practice of instituting treatment of tumors with COX-2 inhibitors based solely on IHC results should be reconsidered.

Rodent carcinogenicity profile of the antidiabetic dual PPAR alpha and gamma agonist muraglitazar.

The carcinogenic potential of muraglitazar, a dual human peroxisome proliferator-activated receptor alpha/gamma agonist, was evaluated in 2-year studies in mice (1, 5, 20, and 40 mg/kg) and rats (1, 5, 30, and 50 mg/kg). Benign gallbladder adenomas occurred at low incidences in male mice at 20 and 40 mg/kg (area under the curve [AUC] exposures > or = 62 times human exposure at 5 mg/day) and were considered drug related due to an increased incidence of gallbladder mucosal hyperplasia at these doses. There was a dose-related increased incidence of transitional cell papilloma and carcinoma of the urinary bladder in male rats at 5, 30, and 50 mg/kg (AUC exposures > or = 8 times human exposure at 5 mg/day). At 30 and 50 mg/kg, the urinary bladder tumors were accompanied by evidence of increased urine solids. Subsequent investigative studies established that the urinary bladder carcinogenic effect was mediated by urolithiasis rather than a direct pharmacologic effect on urothelium. Incidences of subcutaneous liposarcoma in male rats and subcutaneous lipoma in female rats were increased at 50 mg/kg (AUC exposures > or = 48 times human exposure at 5 mg/day) and attributed, in part, to persistent pharmacologic stimulation of preadipocytes. Toxicologically relevant nonneoplastic changes in target tissues included thinning of cortical bone in mice and hyperplastic and metaplastic adipocyte changes in mice and rats. Considering that muraglitazar is nongenotoxic, the observed tumorigenic effects in mice and rats have no established clinical relevance since they occurred at either clinically nonrelevant exposures (gallbladder and adipose tumors) or by a species-specific mechanism (urinary bladder tumors).

Parathyroid hormone-related protein and ezrin are up-regulated in human lung cancer bone metastases.

Lung cancer often metastasizes to bone in patients with advanced disease. Identification of the factors involved in the interactions between lung cancer cells and bone will improve the prevention and treatment of bone metastases. We identified changes in metastasis-related gene expression of human HARA lung squamous carcinoma cells co-cultured with neonatal mouse calvariae using a pathway-specific microarray analysis. Nine genes were up-regulated and two genes down-regulated in HARA cells co-cultured with mouse calvariae. Five of the nine up-regulated genes, including caveolin 1, CD44, EphB2, ezrin, and Parathyroid hormone-related protein (PTHrP), and one down-regulated gene, SLPI, were further confirmed by Reverse transcription-polymerase chain reaction (RT-PCR). A mouse model was subsequently used to study the role of PTHrP and ezrin in bone metastasis in vivo. PTHrP (all three isoforms) and ezrin were up-regulated in HARA cells at sites of bone metastasis as detected by RT-PCR and immunohistochemistry. The PTHrP 141 mRNA isoform was increased by the greatest extent (13.9-fold) in bone metastases compared to PTHrP 139 and PTHrP 173 mRNA. We then generated a HARA cell line in which PTHrP expression was inducibly silenced by RNA interference. Silencing of PTHrP expression caused significant reduction of submembranous F-actin and decreased HARA cell invasion. Ezrin up-regulation was confirmed by Western blots on HARA cells co-cultured with adult mouse long bones. Further, Transforming growth factor beta (TGF-beta) was identified as one of the factors in the bone microenvironment that was responsible for the up-regulation of ezrin. The identification of PTHrP and ezrin as important regulators of lung cancer bone metastasis offers new mechanistic insights into the metastasis of lung cancer and provides potential targets for the prevention and treatment of lung cancer metastasis.

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.

Animal models of bone metastasis.

Animal models will continue to be indispensable to investigate the pathogenesis of bone metastasis in vivo, conduct preclinical chemotherapeutic, chemoprevention and genetic therapy studies, test gene delivery mechanisms, and identify metastasis suppressor and inducer genes. It is likely that the bone marrow microenvironment, such as the endothelial cells, stromal cells, hematopoietic cells, bone cells, and the intercellular matrix play important roles in the localization and clonal growth of cancer cells in bone. Given the complexity of bone metastasis, many genes are expected to be involved in the pathogenesis and few are likely indispensable. The use of genomic and proteomic approaches to study these animal models will identify key targets for therapeutic intervention. As we further refine these models and use imaging for real-time evaluation of cells, and eventually target genes, these models will more closely mirror human disease and will hopefully become more predictive of the human response to therapy.

Animal models of bone metastasis.

BACKGROUND: Animal models are important tools to investigate the pathogenesis and develop treatment strategies for bone metastases in humans. However, there are few spontaneous models of bone metastasis despite the fact that rodents (rats and mice) and other animals (dogs and cats) often spontaneously develop cancer. Therefore, most experimental models of bone metastasis in rodents require injection or implantation of neoplastic cells into orthotopic locations, bones, or the left ventricle of the heart. METHODS: The current study reviews the natural incidence and clinical manifestation of bone metastases of mammary and prostate carcinoma in animals, as well as the experimental models developed in mice using animal and human-derived neoplasms. RESULTS: Rats, mice, dogs, and cats often develop spontaneous mammary carcinoma, but bone metastases are rare. Intact and neutered dogs develop prostate carcinoma that is usually androgen independent and may be associated with regional bone invasion or distant bone metastasis. Normal dog prostate tissue induces new bone formation in vivo and can serve as a model of osteoblastic metastasis without concurrent bone destruction. Experimental models of osteolytic, osteoblastic, and mixed osteolytic/osteoblastic bone metastases include syngeneic rodent neoplasms or human xenografts implanted at orthotopic sites (e.g., breast or prostate glands) in immunodeficient mice, injection of cancer cells into the left ventricle of the heart, or direct injection into bones. New transgenic mouse models of cancer have a low incidence of spontaneous bone metastasis, but cell lines derived from these tumors can be selected in vivo for increased incidence of bone metastasis. It is essential to validate and correctly interpret the lesions in models of bone metastasis to accurately correlate the data from animal models to human disease. Animal models have provided support for the "seed and soil" hypothesis of bone metastasis. However, the roles of vascular patterns in the metaphyses of long bones and rapid bone turnover in young animals in the pathogenesis of metastasis in experimental models are uncertain. Improvements in the imaging of experimental animals in vivo using fluorescent markers or light emitted from luciferase have led to increased sensitivity of detection and more accurate quantification of bone metastases. For example, imaging of human prostate carcinoma PC-3M cells transfected with luciferase, following injection into the left ventricle, has demonstrated that there is rapid localization of tumor cells to bones and other organs, such as the kidneys and lungs. CONCLUSIONS: Animal models of metastasis have supported drug development and have been useful for identification of metastasis suppressor and promoter genes as novel targets for the development of novel therapies. Further refinement of these models will involve spatiotemporal analysis of the metastatic process by imaging and use of image data to stage disease and guide tissue sampling for gene expression profiling via gene array technology. In the future, integrated analyses of these models will be needed to understand the complexities of this important disease process.