Pathophysiology and human cancer risk assessment of pharmaceutical-induced thymoma in carcinogenicity studies.

Thymoma, a tumor of thymic lymphocytes or thymic epithelial cells (TECs), is a common spontaneous tumor in Wistar Han rats, especially in females with up to 18% incidence in controls. In addition to sex, there are rat strain differences in background incidence of thymomas such as Sprague Dawley versus Wistar Han rats. Human thymomas are very rare and without clear differences in incidence between males and females. Immunomodulatory and anti-inflammatory pharmaceutical drug classes, including Janus kinase inhibitors, increase the incidence of benign thymoma in two-year rat carcinogenicity studies. Potential non-genotoxic mechanisms that might contribute to the pathogenesis of thymoma development in one sex (female) Wistar Han rats include: (1) hormonal differences, (2) high proliferation rate of TECs, (3) delayed physiologic thymic involution, and/or (4) significant level of immunosuppression at high doses of a pharmaceutical drug. Factors to consider in the human cancer risk assessment of pharmaceutical-induced thymoma are: the genotoxicity of the test article, sex and strain of rats, exposure safety margins, and pathophysiologic differences and similarities of thymoma between rats and humans. Totality of weight of evidence approach and available data suggest thymomas observed in carcinogenicity studies of pharmaceutical drugs are not relevant for human risk at clinically relevant therapeutic doses.

Thymic lymphoma detection in RORγ knockout mice using 5-hydroxymethylcytosine profiling of circulating cell-free DNA.

A high incidence of thymic lymphoma has been noted in mice deficient of retinoid-related orphan receptor γ2 (RORγ2), which is required for differentiation of naïve CD4+ T cells into TH17 cells. Using a RORγ homozygous knockout (KO) mouse model of thymic lymphoma, we characterized this tumor progression and investigated the utility of 5-hydroxymethylcytosine (5hmC) signatures as a non-invasive circulating biomarker for early prediction of malignancy. No evidence for malignancy was noted in the wild-type mice, while primary thymic lymphoma with multi-organ metastasis was observed microscopically in 97% of the homozygous RORγ KO mice. The severity of thymic lymphoma was not age-dependent in the KO mice of 2 to 4 months old. Differential enrichment of 5hmC in thymic DNA and plasma cell-free DNA (cfDNA) was compared across different stages of tumor progression. Random forest modeling of plasma cfDNA achieved good predictivity (AUC = 0.74) in distinguishing early non-metastatic thymic lymphoma compared to cancer-free controls, while perfect predictivity was achieved with advanced multi-organ metastatic disease (AUC = 1.00). Lymphoid-specific genes involved in thymocyte selection during T cell development (Themis, Tox) were differentially enriched in both plasma and thymic tissue. This could help in differentiating thymic lymphoma from other tumors commonly detected in rodent carcinogenicity studies used in pharmaceutical drug development to inform human malignancy risk. Overall, these results provide a proof-of-concept for using circulating cfDNA profiles in rodent carcinogenicity studies for early risk assessment of novel pharmaceutical targets.

Kidney Transporters and Drug-Induced Injury in Drug Development.

Influx and efflux kidney tubular transporters are major determinants of the disposition of xenobiotics, including pharmaceutical drugs. On the basolateral membrane of proximal tubular cells, there are influx transporters, such as organic cation transporters. On the apical membrane of proximal tubular cells, there are efflux transporters, such as multidrug and toxin extrusion proteins. The secretion process across the apical membrane into the lumen occurs via efflux transporters which plays an important role in serum creatinine (sCr) elimination in urine. The interference of a pharmaceutical drug with transporters can lead to changes in sCr with no alterations in biomarkers or light microscopic evidence indicative of renal injury. Identification of transporters that influence drug disposition, toxicity, and overall nonclinical safety assessment is important in drug discovery and development programs. This mini review describes some key aspects of kidney tubular transporters and drug-induced renal toxicities in safety risk assessment and drug development.

Opinion on Immune Tolerance Therapeutic Development.

Immune tolerance is defined by an active state of immune system unresponsiveness to foreign and self-antigens. Loss of immune tolerance to self-antigens and the resulting overexpression of autoantibodies can lead to tissue injury and development of various autoimmune diseases. In drug development, the goal of newly emerging immune tolerance therapies is to treat autoimmune disorders by restoring the immunoregulatory capacity of the immune system. Development of immune tolerance targets is initiated with the establishment of pharmacological efficacy in relevant disease animal models, followed by their stepwise translation to humans. This review discusses the major challenges to developing tolerance inducing pharmaceutical drugs, including the selection of appropriate disease models to establish efficacy, adequate, and acceptable in vitro and in vivo safety assessments, relevant biomarkers of human safety and efficacy, and finally, some regulatory guidelines to successfully develop immune tolerance therapeutics. [Box: see text].

Kidney Pathophysiology, Toxicology, and Drug-Induced Injury in Drug Development.

Anatomically, the kidneys are paired, bean-shaped (in most mammals), excretory organs that lie in the retroperitoneum. High blood flow to the kidneys, together with high oxygen consumption, makes them more vulnerable to exposure, via the circulation, and subsequent injury related to high concentrations of xenobiotics and chemicals. In preclinical drug development and safety assessment of new investigational drugs, changes in kidney structure and/or function following drug administration in experimental laboratory animals need to be put in context with interspecies differences in kidney functional anatomy, physiology, spontaneous pathologies, and toxicopathological responses to injury. In addition, translation to human relevance to avoid premature drug termination from development is vital. Thus, detection and characterization of kidney toxicity in preclinical species and human relevance will depend on the preclinical safety testing strategy and collective weight-of-evidence approach including new investigational drug mechanism of action (MOA), preclinical and clinical interspecies differences, and MOA relevance to humans. This review describes kidney macroscopic and microscopic functional anatomy, physiology, pathophysiology, toxicology, and drug-induced kidney toxicities in safety risk assessment and drug development.

Immunopathogenesis of Acute Kidney Injury.

Pathophysiologically, the classification of acute kidney injury (AKI) can be divided into three categories: (1) prerenal, (2) intrinsic, and (3) postrenal. Emerging evidence supports the involvement of renal tubular epithelial cells and the innate and adaptive arms of the immune system in the pathogenesis of intrinsic AKI. Pro-inflammatory damage-associated molecular patterns, pathogen-associated molecular patterns, hypoxia inducible factors, toll-like receptors, complement system, oxidative stress, adhesion molecules, cell death, resident renal dendritic cells, neutrophils, T and B lymphocytes, macrophages, natural killer T cells, cytokines, and secreted chemokines contribute to the immunopathogenesis of AKI. However, other immune cells and pathways such as M2 macrophages, regulatory T cells, progranulin, and autophagy exhibit anti-inflammatory properties and facilitate kidney tissue repair after AKI. Thus, therapies for AKI include agents such as anti-inflammatory (e.g., recombinant alkaline phosphatase), antioxidants (iron chelators), and apoptosis inhibitors. In preclinical toxicity studies, drug-induced kidney injury can be seen after exposure to a nephrotoxicant test article due to immune mechanisms and dysregulation of innate, and/or adaptive cellular immunity. The focus of this review will be on intrinsic AKI, as it relates to the immune and renal systems cross talks focusing on the cellular and pathophysiologic mechanisms of AKI.

Immunologic effects of chronic administration of tofacitinib, a Janus kinase inhibitor, in cynomolgus monkeys and rats - Comparison of juvenile and adult responses.

Tofacitinib, an oral Janus kinase (JAK) inhibitor for treatment of rheumatoid arthritis, targets JAK1, JAK3, and to a lesser extent JAK2 and TYK2. JAK1/3 inhibition impairs gamma common chain cytokine receptor signaling, important in lymphocyte development, homeostasis and function. Adult and juvenile cynomolgus monkey and rat studies were conducted and the impact of tofacitinib on immune parameters (lymphoid tissues and lymphocyte subsets) and function (T-dependent antibody response (TDAR), mitogen-induced T cell proliferation) assessed. Tofacitinib administration decreased circulating T cells and NK cells in juvenile and adult animals of both species. B cell decreases were observed only in rats. These changes and decreased lymphoid tissue cellularity are consistent with the expected pharmacology of tofacitinib. No differences were observed between juvenile and adult animals, either in terms of doses at which effects were observed or differential effects on immune endpoints. Lymphomas were observed in three adult monkeys. Tofacitinib impaired the primary TDAR in juvenile monkeys, although a recall response was generated. Complete or partial reversal of the effects on the immune system was observed.

Renal and Hematologic Comparative Effects of Dissociated Agonist of the Glucocorticoid Receptor and Prednisone in Dogs With and Without Food Restriction.

Glomerulopathy and body weight gain were noted after chronic oral administration of a novel nonsteroidal dissociated agonist of the glucocorticoid receptor compound, fosdagrocorat, to beagle dogs fed an ad libitum diet. To further investigate the role of diet and treatment with either fosdagrocorat or the glucocorticoid comparator, prednisone, on renal safety, a 13-week investigative study was conducted in beagle dogs. Renal histopathology, clinical chemistry, urinalysis, glomerular filtration rate (GFR), body weight, heart rate, blood pressure (BP), and hematology were investigated in restricted- and ad libitum-fed dogs administered prednisone (2.2 mg/kg/d), fosdagrocorat (5 mg/kg/d), or vehicle for 13 weeks. Glomerulopathy was primarily observed in fosdagrocorat- and prednisone-treated ad libitum but not in feed-restricted or ad libitum vehicle-treated dogs. Kidneys in dogs from the prednisone-treated ad libitum had the greatest incidence and severity of tubular degenerative changes. Increased urine volume and decreased urine-specific gravity were present in prednisone- and fosdagrocorat-treated dogs, regardless of diet. These changes were not associated with consistent changes in GFR. Fosdagrocorat or prednisone treatment ad libitum dogs had the greatest increase in body weight gain. Sporadic changes in systolic and diastolic BP were noted in fosdagrocorat- and prednisone-treated groups. Significant reductions in serum cortisol and absolute eosinophils were noted in both ad libitum- and restriction-fed prednisone- and fosdagrocorat-treated dogs. In conclusion, prednisone-treated dogs fed ad libitum had greater glucocorticoid-induced renal effects than those dosed with fosdagrocorat.

Cellular and functional actions of tofacitinib related to the pathophysiology of hibernoma development.

Tofacitinib is an oral JAK inhibitor for the treatment of rheumatoid arthritis. In the 2-year carcinogenicity study with tofacitinib, increased incidence of hibernoma (a neoplasm of brown adipose tissue [BAT]) was noted in female rats at ≥30 mg/kg/day (≥41x human exposure multiples). Thus, signaling pathways within BAT were investigated by measuring BAT: weight, cell proliferation biomarkers, content of basal and prolactin-induced phosphorylated Signal Transducer and Activator of Transcription (STAT), and uncoupling protein 1 (UCP-1). The relationship between cardiovascular hemodynamics and plasma norepinephrine (NE) levels was also investigated. Tofacitinib administered to female rats at doses of 10, 30, or 75 mg/kg/day for 14 days increased BAT weight at 75 mg/kg/day and cell proliferation at ≥30 mg/kg/day. JAK inhibition, observed as lower pSTAT3 and pSTAT5 in BAT, was noted at ≥10 mg/kg/day, while lower activity of BAT was observed as lower UCP-1 protein at ≥30 mg/kg/day. In cultured brown adipocytes, prolactin-induced increase in pSTAT5 and pSTAT3 were inhibited by tofacitinib in a concentration-dependent manner. Tofacitinib lowered blood pressure, increased heart rate, and resulted in dose-dependent increases in circulating NE. Thus, JAK/STAT inhibition in BAT and sympathetic stimulation are two factors which might contribute to the genesis of hibernomas by tofacitinib in rats.

Gastric parietal cell atrophy and depletion after administration of a sphingosine-1-phosphate 1 inhibitor.

Sphingosine-1-phosphate (S1P) is a major bioactive phospholipid, which binds to and activates a family of five G-protein-coupled receptors designated as S1P 1 (S1P1) through S1P5. The S1P1 receptor subtype, expressed primarily on lymphocytes, is known to play a critical role in the regulation of lymphocyte trafficking. S1P1 inhibitors result in the inhibition of lymphoid cell trafficking and are of interest to treat various inflammatory conditions. In this study, we describe a gastric finding associated with oral gavage administration of a small molecule S1P1 inhibitor to Sprague-Dawley rats. Rats were administered an S1P1 inhibitor once daily for 4 weeks and necropsies were conducted at the end of the dosing phase, and clinical pathology and histopathologic examination were performed. Lymphopenia and changes in lymphoid tissues were noted and were consistent with the pharmacodynamic effects for S1P1 inhibitory action. Histopathologic examination of the stomach revealed atrophy and depletion of gastric parietal cells in the glandular portion of the stomach. There are no literature data to suggest that this gastric effect is related to S1P1 pharmacology. Therefore, the mechanism of the observed gastric lesion is likely chemotype mediated.

Reversible effects on female rat fertility with abrocitinib, a Janus kinase 1 inhibitor.

BACKGROUND: Abrocitinib is a Janus kinase (JAK) 1 selective inhibitor approved for the treatment of atopic dermatitis. Female reproductive tissues were unaffected in general toxicity studies, but an initial female rat fertility study resulted in adverse effects at all doses evaluated. A second rat fertility study was conducted to evaluate lower doses and potential for recovery. METHODS: This second study had 4 groups of 20 females each administered abrocitinib (0, 3, 10, or 70 mg/kg/day) 2 weeks prior to cohabitation through gestation day (GD) 7. In addition, 2 groups of 20 rats (0 or 70 mg/kg/day) were dosed for 3 weeks followed by a 4-week recovery period before mating. All mated females were evaluated on GD 14. RESULTS: No effects were observed at ≤10 mg/kg/day. At 70 mg/kg/day (29x human exposure), decreased pregnancy rate, implantation sites, and viable embryos were observed. All these effects reversed 4 weeks after the last dose. CONCLUSIONS: Based on these data and literature on the potential role of JAK signaling in implantation, we hypothesize that these effects may be related to JAK1 inhibition and, generally, that peri-implantation effects such as these, in the absence of cycling or microscopic changes in nonpregnant female reproductive tissues, are anticipated to be reversible.

Qualified kidney injury biomarkers demonstrate value during early clinical drug development.

Drug-induced kidney injury (DIKI) is of significant concern, both during drug development and in clinical practice. We report a patient-centric approach for clinical implementation of the FDA-qualified kidney safety biomarker panel, highlighting Phase 1 and 2 trials for candidate therapeutics in Pfizer's portfolio (PFE-1 and PFE-2, respectively) that induced kidney tubular injury in rat toxicity studies. Clusterin (CLU), cystatin-C (CysC), kidney injury molecule-1 (KIM-1), N-acetyl-beta-d-glucosaminidase (NAG), neutrophil gelatinase-associated lipocalin (NGAL), and osteopontin (OPN) were measured in urine samples from (i) Phase 1 healthy volunteers (HVs; n = 12) dosed with PFE-1, (ii) Phase 2 rheumatoid arthritis (RA) patients (n = 266) dosed with PFE-2, (iii) lupus patients on standard-of-care therapies (n = 121), and (iv) healthy volunteers (n = 60). The FDA-defined composite measure (CM), calculated as the geometric mean response across the 6 biomarkers, was increased ∼30% in HVs administered 100 mg PFE-1 relative to placebo, providing evidence of DIKI. In contrast, the CM for RA patients dosed with PFE-2 was comparable to placebo controls, helping to de-risk the concern for DIKI at clinically relevant doses. Comparing individual biomarker concentrations across disease states revealed that CLU, KIM-1, NAG, NGAL, and OPN are elevated in the urine of RA and lupus patients (those without severe active proliferative lupus nephritis) relative to HVs. Overall, these case studies demonstrate the value of using the FDA-qualified kidney biomarker panel to guide risk assessment, dose selection, and clinical decision making for novel therapeutics, both in HVs and patient populations.

Renal pathophysiologic role of cortical tubular inclusion bodies.

Renal tubular inclusion bodies are rarely associated with drug administration. The authors describe the finding of renal cortical tubular intranuclear and intracytoplasmic inclusion bodies associated with the oral administration of a norepinephrine/serotonin reuptake inhibitor (NSRI) test article in Sprague-Dawley (SD) rats. Rats were given an NSRI daily for 4 weeks, and kidney histopathologic, ultrastructural pathology, and immunohistochemical examinations were performed. Round eosinophilic intranuclear inclusion bodies were observed histologically in the tubular epithelial cells of the renal cortex in male and female SD rats given the NSRI compound. No evidence of degeneration or necrosis was noted in the inclusion-containing renal cells. By ultrastructural pathology, inclusion bodies consisted of finely granular, amorphous, and uniformly stained nonmembrane-bound material. By immunohistochemistry, inclusion bodies stained positive for d-amino acid oxidase (DAO) protein. In addition, similar inclusion bodies were noted in the cytoplasmic tubular epithelial compartment by ultrastructural and immunohistochemical examination.  This is the first description of these renal inclusion bodies after an NSRI test article administration in SD rats. Such drug-induced renal inclusion bodies are rat-specific, do not represent an expression of nephrotoxicity, represent altered metabolism of d-amino acids, and are not relevant to human safety risk assessment.

JAK inhibition with tofacitinib suppresses arthritic joint structural damage through decreased RANKL production.

OBJECTIVE: The mechanistic link between Janus kinase (JAK) signaling and structural damage to arthritic joints in rheumatoid arthritis (RA) is poorly understood. This study was undertaken to investigate how selective inhibition of JAK with tofacitinib (CP-690,550) affects osteoclast-mediated bone resorption in a rat adjuvant-induced arthritis (AIA) model, as well as human T lymphocyte RANKL production and human osteoclast differentiation and function. METHODS: Hind paw edema, inflammatory cell infiltration, and osteoclast-mediated bone resorption in rat AIA were assessed using plethysmography, histopathologic analysis, and immunohistochemistry; plasma and hind paw tissue levels of cytokines and chemokines (including RANKL) were also assessed. In vitro RANKL production by activated human T lymphocytes was evaluated by immunoassay, while human osteoclast differentiation and function were assessed via quantitative tartrate-resistant acid phosphatase staining and degradation of human bone collagen, respectively. RESULTS: Edema, inflammation, and osteoclast-mediated bone resorption in rats with AIA were dramatically reduced after 7 days of treatment with the JAK inhibitor, which correlated with reduced numbers of CD68/ED-1+, CD3+, and RANKL+ cells in the paws; interleukin-6 (transcript and protein) levels were rapidly reduced in paw tissue within 4 hours of the first dose, whereas it took 4-7 days of therapy for RANKL levels to decrease. Tofacitinib did not impact human osteoclast differentiation or function, but did decrease human T lymphocyte RANKL production in a concentration-dependent manner. CONCLUSION: These results suggest that the JAK inhibitor tofacitinib suppresses osteoclast-mediated structural damage to arthritic joints, and this effect is secondary to decreased RANKL production.

Modulation of innate and adaptive immune responses by tofacitinib (CP-690,550).

Inhibitors of the JAK family of nonreceptor tyrosine kinases have demonstrated clinical efficacy in rheumatoid arthritis and other inflammatory disorders; however, the precise mechanisms by which JAK inhibition improves inflammatory immune responses remain unclear. In this study, we examined the mode of action of tofacitinib (CP-690,550) on JAK/STAT signaling pathways involved in adaptive and innate immune responses. To determine the extent of inhibition of specific JAK/STAT-dependent pathways, we analyzed cytokine stimulation of mouse and human T cells in vitro. We also investigated the consequences of CP-690,550 treatment on Th cell differentiation of naive murine CD4(+) T cells. CP-690,550 inhibited IL-4-dependent Th2 cell differentiation and interestingly also interfered with Th17 cell differentiation. Expression of IL-23 receptor and the Th17 cytokines IL-17A, IL-17F, and IL-22 were blocked when naive Th cells were stimulated with IL-6 and IL-23. In contrast, IL-17A production was enhanced when Th17 cells were differentiated in the presence of TGF-ß. Moreover, CP-690,550 also prevented the activation of STAT1, induction of T-bet, and subsequent generation of Th1 cells. In a model of established arthritis, CP-690,550 rapidly improved disease by inhibiting the production of inflammatory mediators and suppressing STAT1-dependent genes in joint tissue. Furthermore, efficacy in this disease model correlated with the inhibition of both JAK1 and JAK3 signaling pathways. CP-690,550 also modulated innate responses to LPS in vivo through a mechanism likely involving the inhibition of STAT1 signaling. Thus, CP-690,550 may improve autoimmune diseases and prevent transplant rejection by suppressing the differentiation of pathogenic Th1 and Th17 cells as well as innate immune cell signaling.

Animal models of inflammatory bowel disease: novel experiments for revealing pathogenesis of colitis, fibrosis, and colitis-associated colon cancer.

Inflammatory bowel disease (IBD), comprising Crohn's disease and ulcerative colitis, is a lifelong disease that manifests with chronic intestinal inflammation, sequential fibrosis, and an increased risk of colitis-associated colon cancer (CAC). The combined effects of genetic, immunological, environmental, and microbial factors render it difficult to determine the specific mechanism underlying the induction and perpetuation of IBD. Various animal models of IBD have contributed enormously to the understanding of IBD pathogenesis in terms of genomics, transcriptomics, proteomics, microbiome, and drug development of novel therapeutics. Although comprehensive research on IBD has been enabled by advanced technologies, such as genetically engineered models, there is a great need to develop relevant in vivo models of colitis and fibrosis. Here, we review 4 categories of animal models of acute and chronic intestinal inflammation, fibrosis, and CAC: chemically induced, genetically engineered, T cell transfer, and spontaneous gene mutation models.

Increased serum enzyme levels associated with kupffer cell reduction with no signs of hepatic or skeletal muscle injury.

Macrophage colony-stimulating factor (M-CSF) is a hematopoietic growth factor that is responsible for the survival and proliferation of monocytes and the differentiation of monocytes into macrophages, including Kupffer cells (KCs) in the liver. KCs play an important role in the clearance of several serum enzymes, including aspartate aminotransferase and creatine kinase, that are typically elevated as a result of liver or skeletal muscle injury. We used three distinct animal models to investigate the hypothesis that increases in the levels of serum enzymes can be the result of decreases in KCs in the apparent absence of hepatic or skeletal muscle injury. Specifically, neutralizing M-CSF activity via a novel human monoclonal antibody reduced the CD14(+)CD16(+) monocyte population, depleted KCs, and increased aspartate aminotransferase and creatine kinase serum enzyme levels in cynomolgus macaques. In addition, the treatment of rats with clodronate liposomes depleted KCs and led to increased serum enzyme levels, again without evidence of tissue injury. Finally, in the osteopetrotic (Csf1(op)/Csf1(op)) mice lacking functional M-CSF and having reduced levels of KCs, the levels of serum enzymes are higher than in wild-type littermates. Together, these findings support a mechanism for increases in serum enzyme levels through M-CSF regulation of tissue macrophage homeostasis without concomitant histopathological changes in either the hepatic or skeletal system.

Human safety risk assessment of lymph node angiomas observed in 2-year carcinogenicity studies in rats.

The occurrence of mesenteric lymph node angiomas (benign vascular neoplasms including lymphangioma and hemangioma) in untreated control rats in 2-year carcinogenicity studies can range from rare to common depending on the strain used. This lesion is most common in male rats. Factors and conditions that may contribute to the etiopathogenesis of lymph node angiomas in rats include: (1) genetic drift, (2) congenital/developmental malformation, (3) sinus vascular transformation/venous obstruction of outflow, (4) "inflammatory" pseudo-tumors, and/or (5) defects of endothelial lymphatic vascular secretion/permeability. Lymph node angiomas in humans are extremely rare, not reported in mesenteric lymph nodes, and more common in females than males. The evaluation of increased mesenteric lymph node angiomas in rats for overall human safety risk assessment of novel pharmaceutical therapeutics should consider: genotoxicity of the test article, occurrence of vascular neoplasms in other locations in rats and in mice, occurrence of proliferative vascular lesions in nonclinical toxicology studies in non-rodent species, dose/exposure response, and pathophysiologic/morphologic differences and similarities of lymph node angiomas between rats and humans. Angiomas are independent lesions from angiosarcomas and are not precursors for angiosarcomas in either humans or animals. Mesenteric lymph node angiomas in rats are unlikely to be relevant for human risk assessment of pharmaceutical agents.

Pharmacologic evaluation of sulfasalazine, FTY720, and anti-IL-12/23p40 in a TNBS-induced Crohn's disease model.

BACKGROUND: 2,4,6-Trinitrobenzenesulfonic acid (TNBS)-induced colitis has been used as an inflammatory bowel disease (IBD), Crohn's disease (CD), preclinical model. However, published data on pharmacologic and therapeutic efficacy testing of this model are limited. FTY720 inhibits lymphoid cell trafficking in inflammatory conditions and is of interest to treat IBD. AIM: We investigated the pharmacologic therapeutic efficacy of sulfasalazine, FTY720, and anti-IL-12/23p40, in a TNBS CD model. METHODS: Female, 7-week-old, BALB/c mice were given sulfasalazine orally (PO) and intraperitoneally (IP) at 10 mg/kg, FTY720 at 3 mg/kg PO, and mouse anti-IL-12/23p40 at 25 mg/kg IP. Vehicle groups given PO either phosphate-buffered saline/water or 40% ethanol served as controls. Pharmacologic efficacy was assessed using body weight loss, clinical scores of diarrhea and intestinal gross pathology, and colon weight parameters. RESULTS: Sulfasalazine and FTY720 treatment did not prevent body weight loss or reduce clinical scores of diarrhea or intestinal gross pathology, when compared with vehicle treatment. However, anti-IL-12/23p40 treatment showed significant efficacy by preventing body weight loss, reducing clinical scores of diarrhea, and reducing intestinal gross pathologic lesions, when compared with vehicle-treated animals. Sulfasalazine, anti-IL-12/23p40, and FTY720 were not effective in reducing colon weight. CONCLUSION: With the exception of anti-IL-12/23p40, sulfasalazine, and FTY720 did not demonstrate full pharmacologic efficacy in our TNBS CD model.

Bilateral dystrophic calcinosis circumscripta in a cynomolgus macaque (Macaca fascicularis).

The authors describe a case in which well-circumscribed, expansile, and nonencapsulated nodular masses with missing digits were detected on the right and left feet in a 6-year-old female cynomolgus macaque from a routine toxicology study. Grossly, these masses were composed of variably sized and firm nodules containing white, chalklike material in the subcutaneous tissue on cross section. Microscopically, the nodules were composed of irregular lobules containing amorphous to granular, light to dark basophilic material that was surrounded by macrophages and multinucleated giant cells and separated by fibrous connective tissue. The nodule's contents were von Kossa and Alizarin red S positive. Serum calcium and phosphorus levels of this monkey were within normal ranges. Based on the gross pathology, histopathology, serum chemistry, and histochemistry, a diagnosis of dystrophic calcinosis circumscripta was made. Dystrophic calcinosis circumscripta is an uncommon syndrome of mineralization that occurs following tissue damage, without abnormalities in calcium and phosphorus homeostasis, and it is characterized by deposition of calcium salts in soft tissues. To the best of the authors' knowledge, this is the first report of dystrophic calcinosis circumscripta in a cynomolgus macaque.