Drug-induced Liver Fibrosis: Testing Nevirapine in a Viral-like Liver Setting Using Histopathology, MALDI IMS, and Gene Expression.

Nevirapine (NVP) is associated with hepatotoxicity in 1-5% of patients. In rodent studies, NVP has been shown to cause hepatic enzyme induction, centrilobular hypertrophy, and skin rash in various rat strains but not liver toxicity. In an effort to understand whether NVP is metabolized differently in a transiently inflamed liver and whether a heightened immune response alters NVP-induced hepatic responses, female brown Norway rats were dosed with either vehicle or NVP alone (75 mg/kg/day for 15 days) or galactosamine alone (single intraperitoneal [ip] injection on day 7 to mimic viral hepatitis) or a combination of NVP (75/100/150 mg/kg/day for 15 days) and galactosamine (single 750 mg/kg ip on day 7). Livers were collected at necropsy for histopathology, matrix-assisted laser desorption/ionization imaging mass spectrometry and gene expression. Eight days after galactosamine, hepatic fibrosis was noted in rats dosed with the combination of NVP and galactosamine. No fibrosis occurred with NVP alone or galactosamine alone. Gene expression data suggested a viral-like response initiated by galactosamine via RNA sensors leading to apoptosis, toll-like receptor, and dendritic cell responses. These were exacerbated by NVP-induced growth factor, retinol, apoptosis, and periostin effects. This finding supports clinical reports warning against exacerbation of fibrosis by NVP in patients with hepatitis C.

Cardiolipin profiles as a potential biomarker of mitochondrial health in diet-induced obese mice subjected to exercise, diet-restriction and ephedrine treatment.

Cardiolipin (CL) is crucial for mitochondrial energy metabolism and structural integrity. Alterations in CL quantity or CL species have been associated with mitochondrial dysfunction in several pathological conditions and diseases, including mitochondrial dysfunction-related compound attrition and post-market withdrawal of promising drugs. Here we report alterations in the CL profiles in conjunction with morphology of soleus muscle (SM) and brown adipose tissue (BAT) in diet-induced obese (DIO) mice, subjected to ephedrine treatment (EPH: 200 mg kg(-1) day(-1) orally), treadmill exercise (EX: 10 meters per min, 1 h per day), or dietary restriction (DR: 25% less of mean food consumed by the EX group) for 7 days. Mice from the DR and EPH groups had a significant decrease in percent body weight and reduced fat mass compared with DIO controls. Morphologic alterations in the BAT included brown adipocytes with reduced cytoplasmic lipid droplets and increased cytoplasmic eosinophilia in the EX, DR and EPH groups. Increased cytoplasmic eosinophilia in the BAT was ultrastructurally manifested by increased mitochondrial cristae, fenestration of mitochondrial cristae, increased electron density of mitochondrial matrix, and increased complexity of shape and elongation of mitochondria. Mitochondrial ultrastructural alterations in the SM of the EX and DR groups included increased mitochondrial cristae, cup-shaped mitochondria and mitochondrial degeneration. All four CL species (tri-linoleoyl-mono-docosahexaenoyl, tetralinoleoyl, tri-linoleoyl-mono-oleoyl, and di-linoleoyl-di-oleoyl) were increased in the BAT of the DR and EPH groups and in the SM of the EPH and EX groups. In conclusion, cardiolipin profiling supported standard methods for assessing mitochondrial biogenesis and health, and may serve as a potential marker of mitochondrial dysfunction in preclinical toxicity studies.

Species- and dose-specific pancreatic responses and progression in single- and repeat-dose studies with GI181771X: a novel cholecystokinin 1 receptor agonist in mice, rats, and monkeys.

Compound-induced pancreatic injury is a serious liability in preclinical toxicity studies. However, its relevance to humans should be cautiously evaluated because of interspecies variations. To highlight such variations, we evaluated the species- and dose-specific pancreatic responses and progression caused by GI181771X, a novel cholecystokinin 1 receptor agonist investigated by GlaxoSmithKline for the treatment of obesity. Acute (up to 2,000 mg/kg GI181771X, as single dose) and repeat-dose studies in mice and/or rats (0.25-250 mg/kg/day for 7 days to 26 weeks) showed wide-ranging morphological changes in the pancreas that were dose and duration dependent, including necrotizing pancreatitis, acinar cell hypertrophy/atrophy, zymogen degranulation, focal acinar cell hyperplasia, and interstitial inflammation. In contrast to rodents, pancreatic changes were not observed in cynomolgus monkeys given GI181771X (1-500 mg/kg/day with higher systemic exposure than rats) for up to 52 weeks. Similarly, no GI181771X treatment-associated abnormalities in pancreatic structure were noted in a 24-week clinical trial with obese patients (body mass index >30 or >27 kg/m(2)) as assessed by abdominal ultrasound or by magnetic resonance imaging. Mechanisms for interspecies variations in the pancreatic response to CCK among rodents, monkeys, and humans and their relevance to human risk are discussed.

Use of animal models of human disease for nonclinical safety assessment of novel pharmaceuticals.

Animal models of human disease are commonly utilized to gain insight into the potential efficacy and mode of action of novel pharmaceuticals. However, conventional (healthy) rodent and nonrodent models are generally utilized in nonclinical safety testing. Animal models of human disease may be helpful in understanding safety risks of compounds in nonclinical or clinical development, with their greatest value being in targeted or hypothesis-driven studies to help understand the mechanism of a particular toxicity. Limitations of animal models of disease in nonclinical safety testing include a lack of historical control, heterogeneity in disease expression, a limited life span, and confounding effects of the disease. In most instances, animal models of human disease should not be utilized to supplant testing in conventional animal models. While of potential benefit, testing in an animal model of human disease should only be taken after adequate consideration of relevance along with benefits and limitations of the proposed model.

Responses of brown adipose tissue to diet-induced obesity, exercise, dietary restriction and ephedrine treatment.

Drug-induced weight loss in humans has been associated with undesirable side effects not present in weight loss from lifestyle interventions (caloric restriction or exercise). To investigate the mechanistic differences of weight loss by drug-induced and lifestyle interventions, we examined the gene expression (mRNA) in brown adipose tissue (BAT) and conducted histopathologic assessments in diet-induced obese (DIO) mice given ephedrine (18 mg/kg/day orally), treadmill exercise (10 m/min, 1-h/day), and dietary restriction (DR: 26% dietary restriction) for 7 days. Exercise and DR mice lost more body weight than controls and both ephedrine and exercise reduced percent body fat. All treatments reduced BAT and liver lipid accumulation (i.e., cytoplasmic lipids in brown adipocytes and hepatocytes) and increased oxygen consumption (VO2 ml/kg/h) compared with controls. Mitochondrial biogenesis/function-related genes (TFAM, NRF1 and GABPA) were up-regulated in the BAT of all groups. UCP-1 was up-regulated in exercise and ephedrine groups, whereas MFSD2A was up-regulated in ephedrine and DR groups. PGC-1α up-regulation was observed in exercise and DR groups but not in ephedrine group. In all experimental groups, except for ephedrine, fatty acid transport and metabolism genes were up-regulated, but the magnitude of change was higher in the DR group. PRKAA1 was up-regulated in all groups but not significantly in the ephedrine group. ADRß3 was slightly up-regulated in the DR group only, whereas ESRRA remained unchanged in all groups. Although our data suggest a common pathway of BAT activation elicited by ephedrine treatment, exercise or DR, mRNA changes were indicative of additional nutrient-sensing pathways in exercise and DR.

Drug-induced valvulopathy: an update.

Drug-induced valvulopathy is a serious liability for certain compound classes in development and for some marketed drugs intended for human use. Reports of valvulopathy led to the withdrawal of fenfluramines (anorexigens) and pergolide (antiparkinson drug) from the United States market in 1997 and 2007, respectively. The mechanism responsible for the pathogenesis of valvulopathy by these drugs is likely a result of an "off-target" effect via activation of 5-hydroxytryptamine (5-HT) 2B receptor (5-HT2BR) expressed on heart valve leaflets. Microscopically, the affected valve leaflets showed plaques of proliferative myofibroblasts in an abundant extracellular matrix, composed primarily of glycosaminoglycans. However, the valvular effects caused by fenfluramines and pergolide were not initially predicted from routine preclinical toxicity studies, and to date there are no specific validated animal models or preclinical/toxicologic screens to accurately predict drug-induced valvulopathy. This review covers the structure and function of heart valves and highlights major advances toward understanding the 5-HT2BR-mediated pathogenesis of the lesion and subsequently, development of appropriate animal models using novel techniques/experiments, use of functional screens against 5-HT2BR, and more consistent sampling and pathologic evaluation of valves in preclinical studies that will aid in avoidance of future drug-induced valvulopathy in humans.

5-hydroxytryptamine (5HT)-induced valvulopathy: compositional valvular alterations are associated with 5HT2B receptor and 5HT transporter transcript changes in Sprague-Dawley rats.

Several drugs have been linked to valvulopathy in humans, including therapeutic agents for obesity, Parkinson's disease and migraine. There is increasing evidence that the 5-hydroxytryptamine 2B receptor (5HT2BR) activation and/or increased circulating 5HT (5-hydroxytryptamine) may play a significant role in the pathogenesis of drug-induced valvulopathy. In the present study, we investigated whether 7-day 5HT subcutaneous injections led to structural and compositional abnormalities in conjunction with transcriptomic modulation of 5HT2BR and 5HT transporter (5HTT) genes in the aortic and mitral valves of Sprague-Dawley (SD) rats. Subcutaneous injections of 5HT for 7 days resulted in thickening and compositional alteration of aortic and mitral valves in SD rats. More specifically, valve-leaflets from 5HT-treated rats had greater valve thickness, a higher amount of glycosaminoglycans (GAGs) and a lower amount of collagen. The compositional alteration was associated with up-regulation and down-regulation of 5HT2BR and 5HTT genes, respectively. The present study strongly suggests that the activation of 5HT2BR and inhibition of 5HTT played a significant role in the pathogenesis of 5HT-induced valvulopathy in SD rats. Thus, these findings further highlight the necessity and/or utilization of animal models to screen potential valvular effects of serotonergic compounds.

Evaluation of glycosaminoglycans content and 5-hydroxytryptamine 2B receptor in the heart valves of Sprague-Dawley rats with spontaneous mitral valvulopathy--a possible exacerbation by dl-amphetamine sulfate in Fischer 344 rats?

Spontaneous valvulopathy has been described as nodular or segmental thickenings composed of fibromyxoid tissue in the subendocardium of various valve-leaflets in aging rats, but its pathogenesis and significance are incompletely understood. In this study, we examined the 5-hydroxytryptamine 2B receptor (5HT2BR) expression and characterization of extracellular matrix (ECM) components, and related these to the presence of valvulopathy in the mitral valve-leaflet (spontaneous mitral valvulopathy, SMV) of Sprague-Dawley (SD) rats. We also examined hearts from Fischer 344 (F344) rats treated with dl-amphetamine sulfate for 103 weeks to further explore the potential for drug-induced exacerbation of SMV. In SD rats, valve-leaflets with SMV exhibited a greater valve thickness, a higher amount of glycosaminoglycans, a lower amount of collagen and increased number of 5HT2BR-positive cells. Our data on morphology and ECM changes showed a striking similarity between SMV in SD rats and anorexigen-associated valvulopathy in humans, and increased 5HT2BR-positive cells in SMV implies that 5HT2BR may play a role in pathogenesis. Further, increased incidence and severity of SMV in F344 rats by treatment with dl-amphetamine suggest that a drug-induced exacerbation of SMV may exist in rats. However, additional research is needed to confirm a role for 5HT2BR in the pathogenesis of SMV in SD rats, and to further characterize the relationship between dl-amphetamine treatment and exacerbation of SMV in F344 rats.

5-Hydroxytryptamine (5HT) receptors in the heart valves of cynomolgus monkeys and Sprague-Dawley rats.

5-Hydroxytryptamine-2B receptor (5HT2BR) stimulation is known to cause fibroblast mitogenesis, and the mitogenic effect has been proposed to trigger valvular heart disease in humans. In this study, we used real-time polymerase chain reaction (TaqMan) to quantify transcript levels of 5HT2B, 5HT2C, and 5HT1B receptors and immunohistochemistry (IHC) to detect the tissue localization of these receptors in the normal heart valves of cynomolgus (CM) monkeys and Sprague-Dawley (S-D) rats. In both species, positive immunostaining was noted for 5HT1B and 5HT2B receptors in mitral, tricuspid, aortic, and pulmonary valves, and the cell types showing positive staining were interstitial cells and endothelial cells lining the valve leaflet. In CM monkeys, 5HT2CR was expressed only in the endothelial cells lining the leaflet, whereas S-D valves were negative for this receptor. IHC results were correlated with 5HT2B and 5HT1B receptor transcripts for all four valves. However, 5HT2C receptor transcripts were lower than 5HT2B or 5HT1B in all CM monkey valves, whereas 5HT2C transcripts were below the level of detection in any of the S-D rat valves. Our data showed the expression of 5HT2B, 5HT1B, and 5HT2C receptors in the normal heart valves of CM monkeys and S-D rats, and IHC and TaqMan techniques may be used to study the potential mechanism of compounds with 5HT2BR agonist activity.

Vascular effects of GI262570X (PPAR-gamma agonist) in the brown adipose tissue of Han Wistar rats: a review of 1-month, 13-week, 27-week and 2-year oral toxicity studies.

We describe and discuss microscopic findings in the brown adipose tissue (BAT) blood vessels of Han Wistar rats treated with GI262570X, a peroxisome proliferator-activated receptor-gamma agonist (PPAR-gamma agonist) by oral gavage for 28 days, 13 weeks, 27 weeks, and 2 years. Review of these studies revealed a consistent vascular change, consisting of multifocal fatty infiltration in the BAT of treated rats. A similar vascular change was not seen in other vessels or organs. Microscopically, fatty infiltration was characterized primarily by round, clear vacuoles within the tunica media and/or tunica adventitia of small and medium-sized arteries and arterioles. Occasionally, these vacuoles had peripherally located nuclei and morphologically resembled adipocytes, suggesting a well-characterized PPAR effect (ie, differentiation of stem cells or preadipocytes into mature adipocytes). However, administration of GI262570X up to 2 years failed to induce more severe or progressive lesions in the blood vessels of rat BAT and, in particular, did not result in induction of any atherosclerotic-like lesions or foam cell infiltration. At the longer exposure, there was an apparent reduction of severity and/or incidence, indicating a possible adaptive response. These results suggest that the possibility of generating atherosclerotic-like lesions through prolonged treatment of GI262570X (PPAR-gamma agonist) is highly unlikely in rats.

Endocardial myxomatous change in Harlan Sprague-Dawley rats (Hsd:S-D) and CD-1 mice: its microscopic resemblance to drug-induced valvulopathy in humans.

A full assessment of all heart valves in rats and mice is often impractical and is usually not performed in routine toxicity studies, largely due to an inevitable inconsistency of histological sampling. The majority of reported heart valve changes involve the examination of a single, semirandom section through the heart and the valvulopathy occurring with age or induced by xenobiotics may have been generally underestimated in mice and rats. Here we describe the incidence and microscopic features of endocardial myxomatous change (EMC) in Hsd:S-D rats and CD-1 mice. EMC was common and widespread in both CD-1 mice and Hsd:S-D rats (188 of 220 rats and 96 of 215 mice were affected by EMC). Microscopically, EMC consisted of focal or segmental thickening of valves, primarily due to the presence of fibromyxoid tissue in the subendocardium. Occasionally, fibrin or thrombi deposits and collection of neutrophils or mononuclear cells were observed. These microscopic features were similar to those seen in valvular disease in humans induced by fenfluramine-phentermine (fen-phen), ergot alkaloids (ergotamine, methysergide), and carcinoid syndrome. The mitral valve in rats and pulmonary valve in mice were most frequently affected. An association between murine progressive cardiomyopathy (MPC) and EMC was noted only in rats, suggesting that there may be a possible relationship between MPC and EMC. However, additional research is needed to confirm a relationship between EMC and MPC in rats and/or mice.