Mudher Albassam

Metabonomic assessment of vasculitis in rats.

The vasculitides are a heterogeneous group of lesions, characterized by inflammation and necrosis of the vascular wall and have proven to be a disconcerting dilemma in the development of several classes, of therapeutics. Metabonomics is an emerging technology having great potential for rapid noninvasive assessment of toxicity in vivo and providing identification of peripheral surrogate markers of toxicity. Metabonomic evaluation of CI-1018, a selective type 4 phosphodiesterase inhibitor associated with vasculitis in rats, was undertaken. Two experiments were performed in which CI-1018 was administered for up to 4 d to groups of male Wistar rats at doses up to 3000 mg/kg. Urine was collected from all animals pretest and daily for metabonomic analysis. Eleven of 38 CI-1018-treatment animals were found to have vascular injury of varying severity at doses ≥750 mg/kg. Principal component analysis produced a clear pattern separation among 8 of 11 animals with lesions and 36 of 37 animals without lesions in samples collected on d 3 or 4. These data demonstrate that the metabonomics approach has significant potential for developing a noninvasive method for identifying, vasculitis in rats. It remains to be seen if urinary analyte patterns identified in this study are reproducible and wheter a biomarker pattern for vasculitis can be established.

Subchronic Toxicity of Atorvastatin, a Hydroxymethylglutaryl-Coenzyme A Reductase Inhibitor, in Beagle Dogs

The toxicity of atorvastatin (AT), an inhibitor of hydroxymethylglutaryl-coenzyme A reductase (HMG), was evaluated in beagle dogs. In 4 studies [2-wk rising dose (daily increasing doses for 1 wk; maintenance for 1 wk), 12-wk rising dose (daily dosing with weekly increases in dose), 2-wk toxicity (daily dosing for 2 wk; 3 dose levels), 13-wk toxicity (daily dosing for 13 wk; 3 dose levels)], dogs received up to 400 mg/kg orally. Doses of 180 mg/kg induced moribundity, necessitating euthanasia. Weight losses up to 26% were seen at doses ≥150 mg/kg. Decreases in cholesterol levels were dose-related. Alanine and/or aspartate aminotransferase were increased at doses ≥80 mg/kg; alkaline phosphatase was increased at doses ≥150 mg/kg. Histopathologic findings were seen at ≥150 mg/kg and included hepatocellular eosinophilia related to increased smooth endoplasmic reticulum and cholangiohepatitis and cholecystitis at 150 mg/kg in the 2-wk toxicity study; hepatocellular degeneration, centrilobular bridging, cholecystitis, hemorrhage in gallbladder and brain, demyelination of optic nerve, and skeletal muscle necrosis at ≥280 mg/kg in the 12-wk rising dose study; and erosion and hemorrhage in large intestine, hepatocellular degeneration and necrosis, and inflammation and necrosis of gallbladder epithelium at 320 mg/kg in the 2-wk rising dose study. Doses up to 80 mg/kg for 13 wk did not induce histopathologic lesions in examined organs. AT effectively lowered serum cholesterol in normal lipidemic dogs. Toxicity of AT in dogs was similar to that with other inhibitors of HMG except that lenticular changes were not seen, significant hepatic, testicular, or neurological toxicity was associated only with high doses of AT, and skeletal muscle changes similar to those described in rats and rabbits were identified.

Differentiating Spontaneous from Drug-Induced Vascular Injury in the Dog

When vascular injury is observed in dogs used in preclinical toxicology studies, careful evaluation of the lesions is warranted, especially when differentiating drug-induced vascular changes from spontaneous findings, such as idiopathic canine polyarteritis. The clinical signs as well as the nature and distribution of lesions can often be distinguishing, as is the case with vasoactive drugs, including vasodilators and/or positive inotropes (hydralazine, minoxidil, endothelin receptor antagonists, and phosphodiesterase III inhibitors). For most types of vasodilator-induced vascular injury, the lesion is often restricted to coronary arteries, whereas in idiopathic canine polyarteritis, arterial lesions not only involve coronary arteries, but also medium to small arteries of other organs. In addition, the nature of the changes in vessels yields important clues. Medial and adventitial hemorrhage is generally associated with vasodilator-induced arterial lesion, whereas hemorrhage is generally absent in idiopathic polyarteritis. Although idiopathic canine polyarteritis can generally be differentiated from vasoactive-induced vascular injury in dogs, there are increasing incidences of this type of polyarteritis in dogs receiving any 1 of a number of unrelated classes of compounds, suggestive of an exacerbation of the spontaneous disease. Therefore, in order to differentiate drug-induced injury from idiopathic canine polyarteritis, it is critical that examination of the vascular pathology be conducted with good understanding of clinical, pharmacological, and mechanistic data associated with the drug.

Apoptosis and Nitrative Stress Associated with Phosphodiesterase Inhibitor-Induced Mesenteric Vasculitis in Rats

Nitric oxide may play a role in phosphodiesterase (PDE) inhibitor-induced rat mesenteric vasculitis. The present study was conducted to identify cellular sources of iNOS, determine the distribution of nitrotyrosine (NT) residues as a footprint of peroxynitrite (ONOO-) production, and evaluate their association with vascular apoptosis. To dissociate primary events from secondary changes associated with the inflammatory response, rats were given the PDE IV inhibitor CI-1018 orally at 750 mg/kg alone or concurrently with dexamethasone (DEX) intraperitoneally at 1 mg/kg for 4—5 days. Neutrophil (PMN) involvement in apoptosis was investigated in CI-1018 treated rats dosed with rabbit anti-rat PMN serum (APS). iNOS expression, NT residues, and caspase-3 were detected by immuno-histochemistry. Apoptosis was evaluated by TUNEL assay. CI-1018 induced vascular lesions were associated with iNOS expression in endothelial cells and inflammatory infiltrates; NT was evident only in the latter. Caspase-3 and TUNEL-positive staining were prominent only in medial smooth muscle cells (SMC) from CI-1018-treated rats and only when associated with active inflammation. iNOS- and NT-positive inflammatory cells were present in close proximity to SMC with caspase-3 staining. Inflammatory infiltrates were absent in rats given DEX with minimal SMC necrosis and hemorrhage remained. DEX eliminated apoptosis and immunoreactivity associated with caspase-3, iNOS, and NT. APS depletion of PMNs decreased the incidence and severity of vasculitis but failed to abolish completely caspase-3 immunoreactivity. Expression patterns for caspase-3, iNOS, and NT demonstrated that nitrative stress is a prominent feature of PDE inhibitor-induced vasculitis, with a possible role in medial SMC apoptosis. Further, medial SMC apoptosis may not be a primary event, but instead may be secondary to the inflammatory response.

Coronary Arteriopathy in Monkeys Following Administration of CI-1020, an Endothelin A Receptor Antagonist

A selective non-peptide endothelin A (ETA) receptor antagonist, CI-1020, was administered to cynomolgus monkeys intravenously (iv) for 2 or 4 wk and orally for 4 wk. Groups consisting of 3 animals of each sex received CI-1020 at 1, 5, and 10 mg/kg/hr (iv) or orally at 250, 500, and 750 mg/kg body weight for 4 wk. Control animals received the vehicle only. In a separate experiment, 1 male was infused with 10 mg/kg/hr for 2 wk, and Monastral blue dye was administered iv to facilitate localization of lesions to the vascular walls. One female was administered saline and the dye and served as a control. One female at 1 mg/kg/hr was found dead at week 2, and 1 female at 5 mg/kg/hr was euthanatized during week 4 as a result of severe thigh swelling at the catheter site. Macroscopically, extramural coronary arteries appeared thickened and nodular in the 4-wk iv study in the female found dead at 1 mg/kg/hr, in 1 male and 1 female at 5 mg/kg/hr, and in 2 females at 10 mg/kg/hr. Histologically, Monastral blue pigment trapped in the walls of coronary arteries with arteriopathy was observed in the male treated with CI-1020 at 10 mg/kg/hr for 2 wk. Extramural coronary arteriopathy occurred at all doses in the 4-wk iv study, with higher incidence occurring in females than in males (7 of 9 treated females compared with 3 of 9 treated males). In the oral study, 1 female at 500 mg/kg/day and 1 male and 2 females at 750 mg/kg/day had coronary arteriopathy. Histological changes after 2 wk of treatment were characterized by intimal thickening, fragmentation of the internal elastic lamina, necrosis and edema of the media, and mixed inflammatory-cell infiltrates in the intima, media, and adventitia. After 4 wk of iv administration, arteriopathy was characterized by segmental disruption of the elastic lamina and intimal and medial fibrosis with complete replacement of smooth muscle with fibrous tissue. The adventitia was thickened as a result of fibrosis and mixed or mononuclear inflammatory-cell infiltrates. CI-1020 concentrations were higher in males (1.57 to 29 μg/ml) than in females (0.974 to 24.4 μg/ml) in the iv study. Transient systemic exposure with high maximum plasma concentration (Cmax) (120-352 μg/ml) in the oral study was insufficient to provoke arterial changes of the same magnitude as those noted with continuous iv administration. The regeneration of the media by fibrous tissue and the disruption of the elastic lamina may weaken the arterial wall and increase the susceptibility of the artery to the development of aneurysm.

Arteriopathy Induced by an Adenosine Agonist-Antihypertensive in Monkeys

An adenosine agonist, designated chemically as (R)-N-(2,3-dihydro-lH-inden-1-yl) adenosine or CI-947, was administered orally to 2 males and 2 female cynomolgus monkeys each at 5, 10, 20, and 50 mg/kg of body weight for 2 wk. One male and 1 female given 50 mg/kg were euthanatized on days 10 and 8, respectively, because of poor clinical condition. Emesis was present at 10, 20, and 50 mg/kg. Decreased heart rate and QT prolongation were present at 50 mg/kg. Extramural coronary arterial lesions consisting of medial necrosis with cellular debris and mixed inflammatory cell response in the intima, media, and adventitia were present in 1 male at 20 mg/kg and 1 male at 50 mg/kg at study termination. Similar arterial lesions were present in the small and large intestines and testis of the male at 50 mg/kg. Colonic mucosal erosions with mixed inflammatory cell infiltrates in the lamina propria were seen in this male and in all CI-947 treated females at 10, 20, and 50 mg/kg. Myocardial degeneration and necrosis of myocardial fibers with mononuclear cell infiltrates in the interstitium were noted in the left ventricle of 1 female at 20 mg/kg and in all animals at 50 mg/kg. Renal cortical tubular dilatation with increases in serum creatinine and/or blood urea nitrogen were noted in a control female and animals at 10 and 50 mg/kg. Plasma CI-947 concentration increased with increasing dose. Coronary vascular injury in the monkey was similar to the arterial lesion in CI-947-treated dogs and may relate to the pharmacologic/hemodynamic effects induced by CI-947. When compared with the dog, the monkey appears to be less sensitive to development of arteriopathy, as indicated by lower incidence, at similar systemic exposure levels.

Studies on Coronary Arteriopathy in Dogs Following Administration of CI-1020, an Endothelin A Receptor Antagonist

A selective nonpeptide endothelin A (ETA) receptor antagonist, CI-1020, was administered to beagle dogs intravenously (iv) for 4 hours to 4 weeks. One animal/sex received CI-1020 at 1 mg/kg/hr intravenously for 4, 8, or 24 hours to investigate onset of arteriopathy. Control animals (1/sex) received the vehicle only. To determine reversibility of arteriopathy, 8 dogs/sex were given CI-1020 at 1 mg/kg/hr for 4 days. Two dogs/sex were sacrifi ced 1, 3, 8, and 29 days following cessation of infusion. Lesion development with prolonged exposure was investigated in 1 male dog. It was given CI-1020 by iv bolus at 120 mg/kg/day for 4 weeks and Monastral blue dye was administered iv to facilitate localization of vascular lesions. Coronary blood fl ow was determined in 4 dogs infused with CI-1020 at 0.3, 3, and 30 mg/kg for one hour at each dose. Macroscopically, hemorrhage or blue discoloration of Monastral blue was noted in the extramural coronary arteries along the coronary groove and atrium. Histologically, the earliest coronary changes were noted in animals sacrifi ced after 24 hours of treatment and characterized by medial hemorrhage and necrosi s with a few infi ltrating neutrophils. In the reversibility study, incidence and severity of arteriopathy was dependent on time of sacrifi ce following cessation of infusion. Acute necrotizing infl ammation of arteries was present in all animals (n = 4) on day 1 postinfusion, whereas on day 8 postinfusion, lesions characterized by medial small pockets of trapped red cells, cell debris, and adventitial thickening were seen in 1 dog/sex. By day 29 postinfusion, coronary arteries were similar to controls. In the dog given daily IV bolus injections of CI-1020 for 4 weeks, arterial infl ammatory lesions varied from acute to chronic, although most lesions were considered chronic active. Monastral blue pigments were noted in the wall of most arteries with chronic or chronic active lesions. Acute lesions were similar to those noted in day 1 postinfusion of the reversibility study. Medial smooth muscle necrosis and/or fi brosis with mixed infl ammatory cell infi ltrates characterized chronic or chronic active lesions. Smooth muscle proliferation and migration into the intima were also noted. There were no signifi cant changes in coronary blood fl ow, coronary vascular resistance, or mean arterial blood pressure following CI-1020 infusion for 3 hours. In the 24-hour infusion study, plasma endothelin 1 (ET-1) levels were mildly elevated (1.5—4 fold) during CI-1020 infusion when compared to either pretest or control values. These results indicate that administration of endothelin antagonist (CI-1020) to dogs was associated with development of coronary arteriopathy, which was completely resolved within 29 days following cessation of treatment. With prolonged (4-week) CI-1020 treatment, arterial lesions at varying stages of development (acute, chronic active, chronic) were seen, suggesting that tolerance to treatment (up to 4 weeks) does not occur.

Characterization of Xenobiotic-Induced Hepatocellular Enzyme Induction in Rats: Anticipated Thyroid Effects and Unique Pituitary Gland Findings

During routine safety evaluation of RO2910, a non-nucleoside reverse transcriptase inhibitor for HIV infection, histopathology findings concurrent with robust hepatocellular induction occurred in multiple organs, including a unique, albeit related, finding in the pituitary gland. For fourteen days, male and female rats were administered, by oral gavage vehicle, 100, 300, or 1000 mg/kg/day of RO2910. Treated groups had elevated serum thyroid-stimulating hormone and decreased total thyroxine, and hypertrophy in the liver, thyroid gland, and pituitary pars distalis. These were considered consequences of hepatocellular induction and often were dose dependent and more pronounced in males than in females. Hepatocellular centrilobular hypertrophy corresponded with increased expression of cytochrome P450s 2B1/2, 3A1, and 3A2 and UGT 2B1. Bilateral thyroid follicular cell hypertrophy occurred concurrent to increased mitotic activity and sometimes colloid depletion, which were attributed to changes in thyroid hormone levels. Males had hypertrophy of thyroid-stimulating hormone–producing cells (thyrotrophs) in the pituitary pars distalis. All findings were consistent with the well-established adaptive physiologic response of rodents to xenobiotic-induced hepatocellular microsomal enzyme induction. Although the effects on the pituitary gland following hepatic enzyme induction-mediated hypothyroidism have not been reported previously, other models of stress and thyroid depletion leading to pituitary stimulation support such a shared pathogenesis.

Spontaneous Cardiomyopathy in Cynomolgus Monkeys (Macaca Fascicularis)

A previously undescribed spontaneous cardiomyopathy was identified by routine light microscopic examination of the heart from four clinically healthy purpose-bred cynomolgus monkeys that ranged from four to nine years of age and included 2 males and 2 females. Special stains of Sirius red, Masson’s trichrome, and Mallory’s phosphotungstic acid hematoxylin (PTAH); and immunohistochemistry using anti-CD68, troponin-I, and desmin antibodies were used to facilitate lesion characterization and assess cardiomyocyte viability. Microscopically, the apical to mid-ventricular myocardium to subendocardium had foci of cardiomyocyte disarray with cytoplasmic pallor to stippling and karyomegaly, vacuolization of the perimyseal connective tissue, a meshwork of fibrous tissue that concentrated around medium-sized blood vessels and dissected between or less often replaced affected cardiomyocytes; and a minimal, predominantly macrophage infiltrate. The disrupted cardiomyocytes were immunoreactive to desmin and troponin-I antibodies and had a normal cross-striation pattern by PTAH, indicating the chronic cardiomyopathy was not associated with active cardiomyocyte damage. The consistent distribution and morphology of the cardiomyopathy suggested a common etiology and pathogenesis. The features were reminiscent of chronic catecholamine-induced experimental cardiomyopathy and stress cardiomyopathy in monkeys and humans, respectively. This report documents another spontaneous heart lesion in clinically healthy monkeys for consideration during interpretation of toxicology studies.

Hyaline glomerulopathy in B6C3F1 mice

Hyaline glomerulopathy is a spontaneous disease of undetermined etiology that occurs sporadically in various strains of aging mice. In our laboratory, this disease was observed with unusual ultrastructural features as an incidental finding in 2 female B6C3F1 mice from 2 carcinogenicity bioassays. Microscopically, renal lesions were characterized by marked diffuse enlargement and prominent hyalinization of the glomeruli, equally affecting both kidneys. Affected glomeruli were PAS positive, but were negative for amyloid by the Congo red method. Immunocytochemical staining revealed weakly positive glomerular deposits with polyclonal anti-mouse IgG-IgM-IgA cocktail. Ultrastructurally, there were characteristic subendothelial osmio-philic deposits composed of loosely-packed linear structures in the glomeruli. Lamellae, which appeared as fibrils in perpendicular sections, were relatively uniform, measured 6.1-17.01 nm in diameter, and formed single or double-layered structures. The ultrastructural and immunocytochemical characteristics are suggestive of a spontaneous immune-mediated mechanism in a strain of mouse commonly used in toxicology studies.