Neoplastic and Non-neoplastic Changes in F-344 Rats Treated with Naveglitazar,a γ-Dominant PPAR α/γ Agonist

Abstract

The carcinogenic potential of naveglitazar, a γ-dominant peroxisome proliferator-activated receptor (PPAR) α/γ dual agonist, was evaluated in a two-year study in F344 rats (0, 0.3, 1.0, or 3.0 mg/kg, males; 0, 0.1, 0.3, or 1.0 mg/kg, females). Increased mortality in male rats of the high-dose group was related to cardiac-associated lesions, neoplasms, and undetermined causes. Degeneration and hypertrophy of the myocardium occurred with dose-responsive increased incidence and severity. Neoplasms with increased incidence included sarcomas in male rats and urinary bladder neoplasms in female rats. Most sarcomas in male rats occurred in the adipose tissue of the subcutis and were diagnosed as fibrosarcomas, with fewer liposarcomas and other histologic types. Non-neoplastic changes in adipose tissue included expansion of adipose tissue in multiple sites, alterations in cytoplasmic vesicular pattern in brown and white fat, increases in stroma and mesenchymal cells, and fibrosis. The severity of chronic progressive nephropathy was decreased in a dose-responsive manner in males, and hyperplasia and neoplasia of the mammary gland were decreased in incidence in females. The adverse effects of cardiotoxicity and increased incidence of neoplasms occurred with dose-responsive incidence and/or severity, and a no-effect level for these effects was not achieved in this study.

Introduction

Peroxisome proliferator-activated receptors (PPARs) play a critical role as regulators of lipid metabolism and insulin sensitization. Synthetic ligands for PPARs have been developed for the treatment of diabetes and dyslipidemia (Berger and Moller 2002). Carcinogenicity has been identified as one of the primary issues in preclinical testing of PPAR γ and α/γ dual agonists (El Hage 2005a, 2005b). Administration of these PPAR agonists to rodents has been associated with the development of hemangiosarcomas in mice, liposarcomas and/or fibrosarcomas in rats, and transitional cell tumors of the urinary bladder and/or renal pelvis in rats. Neoplasms associated with PPAR α agonists, primarily involving the liver, pancreas and testes (Klaunig et al. 2003), have been reported less frequently with PPAR α/γ dual agonists (El Hage 2005a, 2005b).

Mechanistic explanations for the occurrence of neoplasms with PPAR γ agonists are problematic because, with few exceptions, in vitro and in vivo work indicates that PPAR γ agonists generally have antiproliferative effects (Grommes et al. 2004; Na and Surh 2003; Peraza et al. 2006). Peroxisome proliferator-activated receptor γ agonists have been suggested as potential anticancer agents because of their effects to increase cell differentiation and apoptosis and reduce angiogenesis (Panigrahy et al. 2003). Differentiation and reversal of malignant changes in colon cancer have been associated with PPAR γ (Sarraf et al. 1998, 1999). Peroxisome proliferator-activated receptor γ agonists have been associated with inhibition of growth, increased apoptosis, and/or enhancement of terminal differentiation of human prostate cancer and breast cancer cells (Elstner et al. 1998; Kubota et al. 1998; Mueller et al. 1998). Conversely, enhancement of colon carcinogenesis resulting from activation of PPAR γ has been seen in a genetically altered mouse with inherent abnormalities in cell cycle control as well as in normal mice (Lefebvre et al. 1998; Saez et al. 1998; Yang et al. 2005). In addition, mammary gland tumor development was exacerbated in a transgenic mouse model prone to mammary gland cancer and constitutively expressing PPAR γ in the mammary gland (Saez et al. 2004).

The potential for direct effects of PPAR γ for urothelial carcinogenesis has been extensively studied, and most studies suggest that the mechanism is indirect. Peroxisome proliferator-activated receptor γ is normally expressed in the urothelium of the bladder and renal pelvis (Guan et al. 1997), and PPAR γ ligands inhibit the proliferation of normal and neoplastic urothelial cells in vitro (Nakashiro et al. 2001; Yoshimura et al. 2003). With normal urothelial cells in culture, PPAR γ agonists cause terminal differentiation to transitional epithelium, which is maximized when epithelial growth factor receptor (EGFR) is inhibited (Kawakami et al. 2002; Spencer et al. 2004; Varley et al. 2003, 2004). In the rat, the most commonly identified non-genotoxic mode of carcinogenicity of the lower urinary tract is indirect because of increased formation of urinary solids, which leads to chronic irritation, proliferation, and eventually neoplasia (Clayson et al. 1995; Cohen et al. 2002; Cohen 2005). Increased urinary solids were demonstrated to be the cause of urothelial carcinogenesis in rats treated with the PPAR α/γ agonist muraglitazar (Dominick et al. 2006), but this association was not demonstrated with the PPAR α/γ agonist naveglitazar (Long et al. 2008). One potential direct effect of PPAR γ ligands on urothelium is an apparent increase in expression of vascular endothelial growth factor (VEGF) by neoplastic urothelial cells in response to PPAR γ ligands in vitro (Fauconnet et al. 2002).

Alteration of adipose tissues is another prominent effect in rats treated with PPAR γ agonists and α/γ dual agonists. Fat deposition is increased in sites of normal fat deposition as well as in areas with normally scant adipose tissue (Hellmold et al. 2007; Tannehill-Gregg 2006). Morphologic changes in fat associated with PPAR agonists include microvesiculation in white adipose cells, macrovesiculation in brown adipose cells, and changes in size of adipose cells (de Souza et al. 2001; Okuno et al. 1998; Tannehill-Gregg et al. 2007). Altered adipose tissues in rats treated with PPAR γ agonists may have increased proliferation of mesenchymal cells (Hellmold et al. 2007), which may be related to later development of sarcomas in these tissues.

Peroxisome proliferator-activated receptor γ agonists have also been associated with adverse cardiovascular effects in laboratory animal species and humans (El Hage 2005a, 2005b). Plasma volume expansion and associated cardiac overload are generally believed to contribute to the increased incidence of congestive heart failure in animals and humans treated with PPAR γ agonists (Arakawa et al. 2004; Nesto 2003). Treatment of rats and monkeys with the PPAR α/γ dual agonist muraglitazar resulted in dose- and time-dependent effects on the cardiovascular system, including edema, increased heart weight, and myocardial hypertrophy (Waites et al. 2007). Other PPAR γ agonists have been recognized to have human clinical safety issues associated with fluid accumulation and increased incidence of congestive heart failure (El Hage 2006).

Naveglitazar is a non-thiazolidinedione (non-TZD), γ-dominant PPAR α/γ dual agonist. In vitro, naveglitazar binds selectively to PPAR γ with high affinity (IC50 = 0.024 μM, Ki = 0.022 μM) and to PPAR α with lower affinity (IC50 = 1.71 μM, Ki = 1.66 μM) (Reifel-Miller et al. 2003). Two-year rodent carcinogenicity studies were conducted to support the development of naveglitazar as a chronic use therapeutic agent. Results of the carcinogenicity study of naveglitazar in rats showed findings within the spectrum of those expected with a PPAR α/γ dual agonist in this species. Differences were generally related to differences in dose responsiveness of the incidence and/or severity of specific changes.

Materials and Methods

Test Animals

F344/Ntac rats were obtained from Taconic Farms (Germantown, NY). Rats were housed individually in stainless steel, wire-bottom cages. Environmental controls for the animal rooms were set to maintain a temperature of 19°C to 25°C, a relative humidity of 30% to 70%, and a twelve–hour light/dark cycle. The animals were offered certified rodent pellets (#5002C, PMI, Richmond, IN) and tap water ad libitum. Rats were 54 to 60 days of age at initiation of treatment. Males weighed 168–239 g; females weighed 116–155 g.

Test Substance

Naveglitazar is described chemically as benzenepropanoic acid, α-methoxy-4-[3-(4-phenoxyphenoxy)propoxy],(α-S)-(Figure 1). The test article potency was maintained throughout the live phase of the studies. The control article (vehicle) was 1.0% (w/v) sodium carboxymethylcellulose, 0.5% (w/v) sodium lauryl sulfate, 0.085% (w/v) povidone, and 0.05% (v/v) Dow Corning Antifoam 1510-US in reverse osmosis water. Naveglitazar was stable in vehicle over the concentration range used for fourteen days when stored at 20°C to 80°C. Concentrations of test article in vehicle were verified at intervals of seven to sixteen weeks during the study.

Experimental Design

The study was conducted at Covance Laboratories, Inc., Madison, WI and was compliant with U.S. FDA Good Laboratory Practice Regulations. Animal use was in accordance with the Guide for the Care and Use of Laboratory Animals.

Rats were randomly assigned to treatment groups based on weight. Rats (60/sex/group) were assigned to groups receiving 0, 0.3, 1.0, or 3.0 mg naveglitazar/kg body weight/day (males) or 0, 0.1, 0.3, or 1.0 mg naveglitazar/kg body weight/day (females) by oral gavage. Doses were selected on the basis of increases in heart weights in previously conducted three and six-month studies of naveglitazar in rats and were targeted at an approximate 25% increase in heart weight at the high dose. All doses were administered at a dose volume of 1 mL/kg in vehicle (control article). Dosing was conducted in the morning, generally within four hours of the beginning of the light cycle.

All animals were checked twice daily for mortality and signs of pain or distress. Detailed observations were done on all animals once prior to initiation of treatment, weekly during the study, and on the day of scheduled sacrifice. The time of onset, location, size, appearance, and progression of each grossly visible or palpable mass were recorded for each animal. Body weights were taken on all animals prior to initiation of treatment, weekly for Weeks 1 to 14, and once every two weeks thereafter. Food consumption was measured for all animals weekly for Weeks 1 through 13 and every two weeks thereafter.

A necropsy was conducted on all rats that died or were sacrificed at an unscheduled interval. All surviving rats were sacrificed after at least 104 weeks of treatment and were necropsied. Protocol-specified tissues were collected from each animal and preserved in 10% neutral buffered formalin. All tissues from each rat were embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Selected tissues with an initial diagnosis of sarcoma, were sectioned and immunolabeled for S-100, using a primary rabbit polyclonal antibody to S-100 and a Universal DAB detection system (Ventana Medical Systems Inc, Tucson, AZ). All tissues from all animals were examined by light microscopy using established diagnostic criteria. Severity scores of nonproliferative lesions were assigned on a five-point grading scale. Sections of all proliferative lesions and nonproliferative, compound-related findings were peer reviewed.

Statistical Analysis

Body weights, body weight change, and food consumption were analyzed for each time point for each sex. Levene’s test (Levene 1960) was performed to test for homogeneity. Homogeneous data were tested by linear regression of untransformed data (Draper and Smith 1966) at the .05 significance level. Heterogeneous data were tested for trend by the Terpstra-Jonckheere test (Thakur 1984) at the .05 significance level. Significant data were further analyzed by deleting the highest dose group and repeating the trend analysis until no significant trend was found. Nonsignificant data were tested by analysis of variance (ANOVA) (Witner 1971) at the .01 significance level on nontransformed data (homogeneous data) or RANK-transformed data (heterogeneous data). Significant data at this step were further tested by Dunnett’s test at the .05 significance level.

Survival data were adjusted for identified, non-compound–related causes (e.g., related to gavage procedures, trauma). Adjusted data were evaluated for trend and heterogeneity using the Cox-Tarone binary regression on life tables with graphical visualization of Kaplan-Meier product limit estimates using the National Cancer Institute Life Table Package (Thomas et al. 1977). Two sets of survival analyses were performed for males: through Week 99 for all groups and through Week 105 (end of study) for groups receiving 0, 0.3, or 1.0 mg naveglitazar/kg body weight/day (excluding the high-dose group, 3.0 mg/kg). One-sided probabilities for positive trends were evaluated at <5.0% significance level, and negative trends were evaluated with two-sided probabilities.

Neoplastic lesions were analyzed by the interval-based methods described in the International Agency for Cancer Research document (Peto et al. 1980), using the tumor as the cause of death information. Significance levels were based on relative historical frequency of specific neoplasms (Lin 1997; Lin and Rahman 1998). Exact probabilities were used for evaluation of these lesions when the tables were sparse (fewer than ten). Otherwise, asymptotic normal probabilities were used for this purpose. Benign and malignant lesions were analyzed individually and combined, as deemed appropriate (McConnell et al. 1986). Palpable neoplastic lesions, such as those in the skin/subcutis, were analyzed by the same methods as the survival data using the first clinical observation time of histopathologically verified lesions as the onset time.

Results

Survival (adjusted) of carcinogenicity study rats to Week 104 is shown in Figure 2. Males in the high-dose group (3.0 mg/kg) had significantly decreased survival. Survival of males in other naveglitazar-treated groups and of females in all naveglitazar-treated groups was not significantly different from the respective controls. Common causes of death (greater than five total instances in the study) are shown in Table 1. Increased mortality for males given 3.0 mg/kg was related to an increased incidence of neoplasms of the skin/subcutis, heart failure, and undetermined cause of death. Many of the rats with undetermined specific cause of death were euthanized because of general debilitation. Of the normally common causes of death for F344 rats, death ascribed to chronic progressive nephropathy was markedly decreased in compound-treated males. Death associated with hematopoietic neoplasia (primarily large granular lymphocytic leukemia) was unchanged by naveglitazar administration. Death associated with neoplasms of the pituitary was decreased in males and unchanged in females.

Dose-responsive increased body weights, compared to controls, were present in naveglitazar-treated rats for most of the study. Differences in body weights at one year and at term are shown in Table 2. Increased body weights were likely related to increased deposition of adipose tissue, but increases in plasma volume, evidenced by edema, may have been contributory. Increases in food consumption, compared to controls, were observed during most of the study for males of the 3.0-mg/kg group and females of the 1.0-mg/kg group. Food consumption in these groups during Week 51 was approximately 5% and 11% greater than respective controls. Increased food consumption was noted in other compound-treated groups less frequently during the study.

Noteworthy clinical observations related to naveglitazar treatment included an increased incidence of tissue masses and swelling in the axillary and maxillary areas. The incidence of lateral tissue masses was increased in a dose-responsive manner in males of all compound-treated groups and females given 0.3 or 1.0 mg/kg (Table 3). Most of the lateral tissue masses were bilateral and correlated with increased deposition of adipose tissue. Swelling in the axillary and maxillary areas was present, with a dose-responsive incidence, in males. This finding was generally correlated with subcutaneous edema.

Morphologic changes in adipose tissue were common in all groups that were given naveglitazar (Table 4). Numbers of adipose cells were increased in areas of normal deposition of both white and brown adipose tissue. Increased adipose cells were also present in areas that normally have few adipose cells. The increase in adipose cells in areas of normal deposition, including the subcutis, was often associated with apparent lobular formation of those adipose tissues. These increases in adipose tissues were associated with the clinically observed lateral masses. Numbers of adipose cells were increased in bone marrow, diagnosed as infiltration of adipocytes, and displaced hematopoietic tissue. This change was similar in the femur and sternum. Increased adipose cells in areas that normally have few adipose cells, diagnosed as infiltration of adipocytes, occurred at greater incidence and/or severity in the choroid and sclera of the eye, diaphragm, and glandular stomach of males and females, interlobular septae of the pancreas in males, and muscles of the tongue and quadriceps in females. The adipocyte infiltrate of the choroid did not have any apparent relationship to the incidental retinal atrophy/degeneration that occurred with sporadic incidence in all groups.

Histologic alterations were common in both brown and white fat and were characterized primarily as increased vesicle size in the cytoplasm of brown fat (macrovesiculation) (Figure 3a) and decreased vesicle size in the cytoplasm of white fat (microvesiculation) (Figure 3b). Stromal change, seen in many sections of fat, was characterized by an increased number of undifferentiated mesenchymal cells within an increased intercellular and interlobular matrix or ground substance (Figure 3b). Within brown fat and extra sections of adipose tissue of males given 3.0 mg/kg, the stromal change occasionally progressed to fibroplasia/fibrosis (Figures 3c and 3d). Interlobular stroma was often increased in areas of increased deposition of white fat. Increases in the incidence and severity of vacuolation within the smooth muscle of blood vessels also occurred in the brown fat (Figure 3a). A frequent microscopic finding in the subcutaneous tissues of naveglitazar-treated males was edema and dilation of lymphatics and/or venules. This finding correlated with the occasional macroscopic finding of edema and with in-life observations of skin–swelling, usually in the head area, for some of the compound-treated males.

Males had a dose-responsive increased incidence of soft tissue sarcomas (Table 5). Statistical analysis of sarcomas was completed for the incidence of specific sarcoma types and by combining the soft tissue sarcomas. The incidences of skin fibrosarcoma and the combined sarcomas in skin/subcutis/adipose tissue and in multiple organs occurred with statistical significance (significance level of .005 for a common tumor) in males of the 3.0 mg/kg group. Fibrosarcomas of the skin/subcutis were significantly increased at the .01 level in males of the 1.0 mg/kg group. The numerical trends for related sarcomas across all sites were increased in all compound-treated male groups. By morphological evaluation, the cell of origin varied, leading to diagnoses including fibrosarcoma, liposarcoma, and undifferentiated sarcoma (Figures 3e and 3f). Many of the sarcomas appeared to be arising within altered adipose tissue, and it was sometimes difficult to distinguish pre-existing entrapped altered adipocytes from adipocyte differentiation in the neoplastic cells. Ten of the sarcomas with unclear differentiation (seven in males and three in females) were immunolabeled for S-100 to identify adipocytes (Cocchia et al. 1983; Hashimoto et al. 1984). Five of these ten were subsequently diagnosed as liposarcomas, based in part on labeling of membranes of intracellular vacuoles in neoplastic cells, similar to the labeling of membranes of normal adipocytes. Sarcomas were not increased in any naveglitazar-treated female groups.

Hyperplasia of the urothelium of the bladder occurred in males and females of the high-dose groups (Table 6). Most affected rats had simple hyperplasia, but nodular or papillary hyperplasia (diagnostic criteria of Frith et al. 1995) occurred in one to three males in each naveglitazar-treated group and in nine females of the high-dose (1.0 mg/kg) group (Figure 4a). Neoplasms (carcinomas plus papillomas) of the urothelium were increased significantly only in females of the high-dose (1.0 mg/kg) group (Figures 4a and 4b). Transitional cell carcinomas also occurred in two of sixty females given 0.3 mg/kg. Urinary bladder neoplasms were grossly observable masses in one female in the mid-dose (0.3 mg/kg) group and six females in the high dose (1.0 mg/kg) group. Inflammatory changes were present in few rats in the compound-treated groups. Of the five females in the high-dose (1.0 mg/kg) group with chronic active inflammation in the bladder, two had urothelial carcinomas, two had papillomas, and one had papillary hyperplasia. There were no neoplasms of the transitional epithelium of the renal pelvis in rats of any dose group.

The gross observation of a large heart occurred in all groups of males given the compound, with the highest incidence (eleven of sixty) in males given 3.0 mg/kg (Table 7). Microscopic lesions in the heart were diagnosed as specific histopathologic lesions (e.g., degeneration/necrosis, fibrosis, lymphohistiocytic infiltrate, chronic inflammation, mineralization) rather than as a general diagnosis of cardiomyopathy. Common heart lesions of degeneration/necrosis and fibrosis affected almost all males and the majority of females. The incidence and severity of these lesions were unchanged by treatment, on a group basis, without adjusting for duration based on survival. Microscopic findings in the heart with increased incidence and/or severity increased by treatment included dilation of the atria, hypertrophy, and vacuolation. The hypertrophy, primarily in the atria, was increased in all compound-treated groups and was characterized by karyomegaly and cytomegaly of myocardial cells (Figure 4c). Vacuolation of myocardial cells occurred primarily in the atria and was increased in incidence in males of all compound-treated groups. The heart lesions were occasionally associated with changes secondary to cardiac failure, including centrilobular necrosis and congestion in the liver, pulmonary alveolar macrophages with cytoplasmic pigment consistent with hemosiderin, and congested lymph nodes. These secondary lesions were most common in males of the 3.0 mg/kg group that died with heart failure as the identified cause of death.

Cytoplasmic eosinophilia of centrilobular hepatocytes occurred with dose-related incidence and severity in males of all compound-treated groups and females of the mid- and high-dose groups (Table 7). Mildly increased pigment in Kupffer cells occurred with increased incidence in rats of all compound-treated groups, but it did not have an apparent dose-response relationship. Brown pigment in kidney tubular epithelial cells was also increased in incidence in males and females of the mid- and high-dose groups. The incidence and severity of chronic progressive nephropathy (CPN) was decreased compared to controls in a dose-responsive pattern in males and was decreased slightly in severity in females. Conditions likely secondary to severe chronic progressive nephropathy, namely, mineralization of vessels, myocardium, and stomach mucosa, and decreases in the incidence of thyroid C-cell hyperplasia, parathyroid gland hyperplasia, and fibrous osteodystrophy were also decreased in incidence and/or severity. A decreased incidence and severity of thickened trabeculae was observed in bone of naveglitazar-treated females.

In the reproductive tract of males, there was an increase in the incidence and severity of decreased secretion in prostate and seminal vesicle tissues, atrophy/degeneration in testis tissues, and epididymal aspermia in all groups given the compound (Table 8). Decreased secretion and atrophy/degeneration in these tissues are common findings with aging laboratory rats, particularly with the high background incidence of testicular interstitial cell tumors in the F344 strain; however, the incidence of testicular interstitial cell tumors was very similar across all groups in this study. In the reproductive tract of females, there was an increased incidence of endometrial cysts in the uterus in all naveglitazar-treated groups and an increased incidence and severity of endometrial cystic hyperplasia occurred in animals given 1.0 mg/kg. The incidence of proliferative lesions of the mammary gland was decreased in naveglitazar-treated males and females (Table 8). Hyperplasia of the mammary gland was decreased in incidence in males and females, and fibroadenoma, carcinoma, and combined fibroadenoma and adenoma were significantly decreased in females of the 0.3- and 1.0-mg/kg groups. Pituitary gland neoplasms were decreased in incidence in males of the mid- and high-dose groups. This finding was associated with increased incidence of focal hyperplasia of the pituitary.

Discussion

Chronic treatment of rats with naveglitazar was associated with the class-specific effects in rats of increased incidence and severity of cardiac lesions and increased incidences of sarcomas (fibrosarcoma/liposarcoma) and urinary bladder neoplasms (El Hage 2005a, 2005b). Cardiac toxicity was dose limiting in both male and female rats with naveglitazar and was associated with heart failure–related mortality in the high-dose male group. Cardiac effects in rats treated with naveglitazar were similar to those reported for other PPAR γ agonists and α/γ dual agonists. Issues related to cardiac events in both pre-clinical and clinical studies with PPAR γ agonists have been responsible for the discontinuation of more compounds than rodent carcinogenicity issues (El Hage 2006). Fluid accumulation, weight gain, and cardiac hypertrophy can be observed with short latency (e.g., one to three months). Heart failure appears with longer duration, and the no-effect levels for cardiac events decrease with increased treatment duration (El Hage 2006). Treatment of rats for two years with troglitazone (Herman et al. 2002) resulted in increased mortality associated with myocardial lesions in males and females in the high-dose group (800 mg/kg). Cardiac changes observed in both the mid-and high-dose groups included increased heart weights, increased incidence and severity of ventricular dilation, myocardial fibrosis, and karyomegaly of atrial myocytes, and increased incidence of atrial thrombosis. Rats treated with tesaglitazar (4.1 mg/kg) had increased mortality associated with heart failure by Week 76 of a two-year study (Hellmold et al. 2007). Heart weights were significantly increased in this dose group, and myocardial hypertrophy occurred in males and females of this dose group and males of the next lower dose group (1.2 mg/kg). In contrast, cardiac effects were not reported in rats treated with muraglitazar for two years (Tannehill-Gregg et al. 2007). However, muraglitazar was associated with cardiac effects, including increased heart weights and myocardial cell hypertrophy, in a six-month study in rats (Waites et al. 2007) conducted with a higher dose (300 mg/kg) than that employed in the two-year rat study (50 mg/kg).

Peroxisome proliferator-activated receptor γ agonists and α/γ dual agonists have been associated with multiple types of neoplasms in two-year studies in rodents. The most consistently reported neoplasms with these compounds have been hemangiosarcomas in mice, urothelial neoplasms in rats, and sarcomas (fibrosarcomas and liposarcomas) in rats (El Hage 2005b; Hardisty et al. 2007). Treatment of rats with naveglitazar was associated with an increased incidence of sarcomas in males and urothelial tumors in females. As with other PPAR agonists, the sarcomas in rats treated with naveglitazar were associated with morphologic alterations in the adipose tissue, but the histogenesis of the neoplasms was not clear. In many of the sarcomas, it was difficult to distinguish vacuolation from entrapped altered adipocytes from lipid vacuolation within neoplastic cells. Immunolabeling with S-100 was used in a number of individual cases to aid in this differentiation. Labeling for S-100 has been used to differentiate liposarcomas from fibrosarcomas, fibrous histiocytomas, and undifferentiated sarcomas (Cocchia et al. 1983; Hashimoto et al. 1984). For the naveglitazar associated sarcomas, the labeling for S-100 was supportive, but not definitive, in the diagnosis of liposarcoma versus fibrosarcoma or undifferentiated sarcoma. This lack of a clear histologic differentiation in some sarcomas associated with PPAR agonists is consistent with the results of a Pathology Working Group (PWG) that reviewed rodent sarcomas associated with PPAR agonist treatment (Hardisty et al. 2007). The recommendations for nomenclature and diagnostic criteria of these neoplasms by the PWG were slight modifications to the previously published recommendations by the Society of Toxicologic Pathology (STP) (Greaves et al. 1992). The diagnosis of sarcomas in the naveglitazar study generally followed the recommendations of the STP (Greaves et al. 1992). Because of the lack of clear histogenic differentiation in some sarcomas, and because many of the sarcomas were associated with adipose tissue of the subcutis or elsewhere, the incidence of sarcomas in the naveglitazar study was analyzed by specific histologic type and tissue and by combining the soft tissue sarcomas. Sarcomas were increased in incidence in naveglitazar-treated male rats by both methods of analysis, suggesting that the sarcomas may represent a common histogenic origin, in spite of subtle differences in histologic differentiation.

Increased deposition and histologic changes of adipose tissue in naveglitazar-treated rats were similar to findings in rats treated with other PPAR γ agonists and α/γ dual agonists. The increased deposition of adipose tissue included grossly overt increases in the amount of subcutaneous fat, including subcutaneous fatty masses (Hellmold et al. 2007). Increased amounts of fat are also reported in sites such as the bone marrow and stroma of various organs (Hellmold et al. 2007; Tannehill-Gregg 2006). Beyond increased amounts of adipose tissue, morphologic alterations in adipose tissues of rats treated with PPAR γ agonists and α/γ dual agonists include microvesiculation in white fat and macrovesiculation in brown fat (Tannehill-Gregg et al. 2007). Treatment of Zucker diabetic rats with troglitazone (Okuno et al. 1998) also suggested a change in adipocyte recruitment and/or differentiation. The total weight of white adipose tissue was not altered in the troglitazone-treated rats, but the number of small adipocytes was increased, the number of large adipocytes was decreased, and the percentage of apoptotic nuclei was increased. Since the study was in genetically obese rats, the effects were associated with “normalization” of metabolism in the adipose tissue; levels of tumor necrosis factor (TNF)-α and leptin in the adipose tissues of the troglitazone treated obese rats were decreased toward those of normal rats. The PPARγ agonist pioglitazone had a similar effect on adipocytes in Zucker rats, causing an increase in the number of small adipocytes, resulting from both the appearance of new adipocytes and the reduction in size and/or number of larger adipocytes (de Souza et al. 2001).

Proliferation of mesenchymal cells and fibrosis in adipose tissue, such as occurred in the naveglitazar study, has been reported with other PPAR γ agonists and α/γ dual agonists, but the relationship to development of sarcomas is not clear. Treatment of rats with troglitazone was associated with increased fibrosis and/or fibroplasia in the adipose tissue, but not with increased incidence of sarcomas (Herman et al. 2002). Angiolipoma and liposarcoma of skin in males occurred at low incidence in troglitazone-treated groups, but did not reach statistical significance. In females, fibrosarcoma and liposarcoma of the skin were marginally increased in incidence in the high-dose group. The incidence of fibrosarcoma in females was slightly higher than that in historic controls. Increased mesenchymal cells and/or fibrosis were not reported in the two-year study with muraglitazar (Tannehill-Greg et al. 2007) but were reported in the six-month study at a dose (30 mg/kg) that overlapped the high doses in the two-year study (Waites et al. 2007). An increased incidence of liposarcomas in males and lipomas in females of the high-dose group (50 mg/kg) was reported in the muraglitazar carcinogenicity study. Combined mesenchymal neoplasms of subcutaneous tissue in female rats were increased numerically in the 30 and 50 mg/kg groups (three and four of sixty-five, respectively), but were not statistically different from the incidence in the control group (one of sixty-five).

Peroxisome proliferator-activated receptor γ is expressed in adipose tissues (Braissart et al. 1996; Tontonoz et al. 1994), and it is also expressed at high levels in human liposarcomas. Activation of PPAR γ in fibroblasts or undifferentiated mesenchymal cells is associated with differentiation of these cells into adipocytes (Rosen and Spiegelman 2000; Tontonoz et al. 1994, 1995). Primary human liposarcoma cells can be forced to terminal differentiation by PPARγ agonists (Tontonoz et al. 1997). The relationship of PPAR agonist effects on adipose tissue and proliferation of mesenchymal cells was investigated with the PPAR α/γ dual agonist tesaglitazar. Treatment of rats with tesaglitazar in short-term studies resulted in increased mesenchymal cells in the adipose tissue (Hellmold et al. 2007). The mesenchymal cells were proliferating, as indicated by increased label for BrdU. The labeling for PPAR α and γ in adipose tissue did not change with tesaglitazar treatment and the BrdU labeled cells did not label for PPAR γ, suggesting that the effect for mesenchymal cell proliferation was not a direct effect of the PPAR γ agonism. This finding suggests that indirect effects such as alterations in cytokines or tissue growth factors in the adipose tissue may have led to the proliferation of mesenchymal cells. An alternative or contributory factor could be that metabolic and/or degenerative changes in the adipose tissue could lead to increased levels of oxidative tissue damage, and this chronic tissue damage could be related to chronic cellular proliferation.

The increased incidence of neoplasms in the rat urinary bladder associated with naveglitazar treatment occurred only in females, with an incidence of 23% in the high-dose group. In contrast, the incidence of urinary bladder tumors in male rats treated with muraglitazar was significantly increased in three of four compound-treated groups and was 58% in the high-dose group (Tannehill-Gregg et al. 2006). The incidence of urothelial tumors in naveglitazar-treated female rats in the mid-dose group (two of sixty) was not significantly different from controls. However, these tumors are normally rare in female F344 rats, with background incidences < 0.2% for papillomas and < 0.1% for carcinomas (Goodman et al. 1979; Haseman et al. 1990), and the urothelial tumors in the mid-dose females may also have been compound related. Hyperplasia of the urothelium was increased in incidence in both males and females in the naveglitazar study. In the rat, increased formation of urinary solids has been commonly associated with urothelial carcinogenesis, and this mechanism has been suggested as the cause of increased urinary bladder tumors in rats with PPAR γ agonists (Cohen 2005). Alterations in the composition of urine were shown to be related to increased urolithiasis and associated increased incidence of urinary bladder neoplasms in male rats treated with muraglitazar (Dominick et al. 2006). In contrast, the urinary bladder neoplasms in female rats treated with naveglitazar could not be associated with urolithiasis or inflammation in the urinary bladder (Long et al. 2008). An increased incidence of urinary calculi was not observed in the two-year study with naveglitazar and chronic-active inflammation was present at only a low incidence in naveglitazar-treated rats. The five high-dose females with chronic-active inflammation in the bladder had advanced proliferative changes, therefore a potential cause–effect relationship of inflammation and proliferative changes could not be determined.

We do not have an explanation for the apparent sex predilection of neoplasms associated with naveglitazar treatment of rats. No sex-specific alterations in urinary parameters that could be associated with carcinogenesis were identified in naveglitazar-treated rats (Long et al. 2008). Because of the normally greater severity of chronic progressive nephropathy (CPN) in aging male rats compared to female rats, changes in urinary parameters would be expected to occur with greater incidence and/or severity in males. Potential alterations in urinary parameters in male rats treated with naveglitazar may have been ameliorated by the concomitant decreased severity of CPN. Decreased severity of CPN in rats associated with a PPARγ agonist was reported previously in Zucker fatty rats treated with rosiglitazone (Buckingham et al. 1998). It was not determined if the decreased severity of CPN in the rosiglitazone-treated rats was associated with normalization of metabolic parameters or with direct activity of the PPAR agonist on renal tubular cells. In the naveglitazar-treated male rats, the decreased severity of CPN may have been at least partly a result of the decreased age at death of the high-dose (3 mg/kg) group. However, the severity of CPN was also reduced in the 1-mg/kg group, in which survival was similar to that of controls. The reduced severity of CPN may also have been related to the apparent endocrine changes, as evidenced by the changes in the pituitary, testes, prostate, and seminal vesicles, but the underlying endocrine alteration was not identified.

Effects of naveglitazar in the liver were limited to increased cytoplasmic eosinophilia of hepatocytes, likely a change indicative of mild hepatocellular hypertrophy. This effect was minimal, and not associated with an increased incidence of proliferative changes in hepatocytes, which is consistent with the relatively low affinity of naveglitazar for PPARα. Neoplasms of the rodent liver, a common finding with PPAR α agonists (Klaunig et al. 2003) have been reported with only a few of the PPAR α/γ agonists tested in rats (El Hage 2005a, 2005b). The relationship of the occurrence of liver tumors to the relative PPAR α and PPARγ affinities of PPAR agonists is not clear. An increased incidence of liver tumors was reported in female rats treated with tesaglitazar (Hellmold et al. 2007) but did not occur with naveglitazar. Both tesaglitazar and naveglitazar are γ-dominant PPAR α/γ agonists, with similar relative affinities for α and γ receptors in in vitro transactivation assays (Hellmold, et al. 2007; Reifel-Miller et al. 2003).

A decreased incidence of proliferative lesions in endocrine tissues and alterations in reproductive tissues in naveglitazar-treated rats were consistent with multiple indirect endocrine perturbations, but direct causative factors were not identified. The decreased incidence of proliferative lesions of thyroid C-cells and parathyroid glands in males were likely secondary to the decreased severity of chronic nephropathy and decreased incidence of renal secondary hyperparathyroidism. The decreased incidence of pituitary neoplasms in males, associated with the increased incidence of pituitary hyperplasia, suggests a decreased rate of progression from hyperplasia to neoplasia. This apparent decrease in the progression of proliferative changes in the pituitary may be at least partly a result of the decreased age at death of the high-dose group (3 mg/kg) males, but it does not explain the finding in the mid-dose group (1 mg/kg) males. A specific endocrine alteration underlying the change in the pituitary was not identified, but the apparently decreased secretory function in the prostate and seminal vesicles and degenerative changes in the testes suggest some alteration of endocrine function. These changes may be associated with testicular interstitial cell tumors in rats, but the incidence of these tumors was not changed by naveglitazar treatment. However, the incidence of this neoplasm is normally very high in F344 rats (Goodman et al. 1979; Haseman et al. 1990) and differences in incidence associated with endocrine alterations may not manifest in relation to the high background rate. The changes in the endometrium and decreased incidence of mammary gland neoplasms in female rats treated with naveglitazar could also be reflective of changes in endocrine function, but they could be related to the antineoplastic effects of PPAR γ agonists that are demonstrated in some models of mammary carcinogenesis (Elstner et al. 1998; Mueller et al. 1998; Panigrahy et al. 2003). Increased incidence of mammary tumors with other PPAR γ agonists (El Hage 2005a) may be suggestive of endocrine effects of the class, but they were not as common as neoplastic changes in other organ systems and were not further characterized.

The changes in rats treated chronically with naveglitazar were generally similar to those reported for other PPAR γ and PPAR α/γ agonists. The important adverse events in naveglitazar-treated rats were increased incidence of neoplasms and cardiotoxicity that occurred with a dose-responsive incidence and/or severity. Although the relationship to human clinical risk of increased neoplasms in rodents treated with PPAR γ and PPAR α/γ agonists is not entirely clear, cardiotoxicity appears to be an important dose-limiting toxicity in both rodents and humans.