Mark A. Dominick

Ketamine prevents ischemic neuronal injury.

The dissociative anesthetic ketamine hydrochloride antagonizes the excitotoxic action of excitatory amino acids in the central nervous system. Proposals that the excitatory amino acid neurotransmitters may become excitotoxic and contribute to the pathophysiology of ischemic brain injury prompted us to examine ketamine in a model of global cerebral ischemia in gerbils. Pretreatment with anesthetic doses of ketamine ameliorated in a dose-dependent manner both behavioral and histopathological assessments of ischemic neuronal injury. These neuroprotective effects are proposed to result from a specific antiexcitotoxic rather than general anticonvulsant drug action. There may be clinical situations in which the neuroprotective actions of ketamine would be of therapeutic importance.

An Analysis of Pharmaceutical Experience with Decades of Rat Carcinogenicity Testing: Support for a Proposal to Modify Current Regulatory Guidelines

Data collected from 182 marketed and nonmarketed pharmaceuticals demonstrate that there is little value gained in conducting a rat two-year carcinogenicity study for compounds that lack: (1) histopathologic risk factors for rat neoplasia in chronic toxicology studies, (2) evidence of hormonal perturbation, and (3) positive genetic toxicology results. Using a single positive result among these three criteria as a test for outcome in the two-year study, fifty-two of sixty-six rat tumorigens were correctly identified, yielding 79% test sensitivity. When all three criteria were negative, sixty-two of seventy-six pharmaceuticals (82%) were correctly predicted to be rat noncarcinogens. The fourteen rat false negatives had two-year study findings of questionable human relevance. Applying these criteria to eighty-six additional chemicals identified by the International Agency for Research on Cancer as likely human carcinogens and to drugs withdrawn from the market for carcinogenicity concerns confirmed their sensitivity for predicting rat carcinogenicity outcome. These analyses support a proposal to refine regulatory criteria for conducting a two-year rat study to be based on assessment of histopathologic findings from a rat six-month study, evidence of hormonal perturbation, genetic toxicology results, and the findings of a six-month transgenic mouse carcinogenicity study. This proposed decision paradigm has the potential to eliminate over 40% of rat two-year testing on new pharmaceuticals without compromise to patient safety.

Urothelial Carcinogenesis in the Urinary Bladder of Male Rats Treated with Muraglitazar, a PPARα/γ Agonist: Evidence for Urolithiasis as the Inciting Event in the Mode of Action

Muraglitazar, a PPARα/γ agonist, dose-dependently increased urinary bladder tumors in male Harlan Sprague–Dawley (HSD) rats administered 5, 30, or 50 mg/kg/day for up to 2 years. To determine the mode of tumor development, male HSD rats were treated daily for up to 21 months at doses of 0, 1, or 50 mg/kg while being fed either a normal or 1% NH4Cl-acidified diet. Muraglitazar-associated, time-dependent changes in urine composition, urothelial mitogenesis and apoptosis, and urothelial morphology were assessed. In control and treated rats fed a normal diet, urine pH was generally ≥6.5, which facilitates formation of calcium- and magnesium-containing solids, particularly in the presence of other prolithogenic changes in rat urine. Urinary citrate, an inhibitor of lithogenesis, and soluble calcium concentrations were dose dependently decreased in association with increased calcium phosphate precipitate, crystals and/or microcalculi; magnesium ammonium phosphate crystals and aggregates; and calcium oxalate-containing thin, rod-like crystals. Morphologically, sustained urothelial cytotoxicity and proliferation with a ventral bladder predilection were noted in treated rats by month 1 and urinary carcinomas with a similar distribution occurred by month 9. Urothelial apoptotic rates were unaffected by muraglitazar treatment or diet. In muraglitazar-treated rats fed an acidified diet, urine pH was invariably < 6.5, which inhibited formation of calcium- and magnesium-containing solids. Moreover, dietary acidification prevented the urothelial cytotoxic, proliferative, and tumorigenic responses. Collectively, these data support an indirect pharmacologic mode of urinary bladder tumor development involving alterations in urine composition that predispose to urolithiasis and associated decreases in urine-soluble calcium concentrations.

Morphogenesis of a Zone-Specific Adrenocortical Cytotoxicity in Guinea Pigs Administered PD 132301-2, an Inhibitor of Acyl-CoA: Cholesterol Acyltransferase

PD 132301-2, a novel inhibitor of acyl-CoA: cholesterol acyltransferase, is adrenotoxic to several laboratory animal species. Morphogenesis of a zona fasciculata-specific cytotoxicity was evaluated in male Hartley guinea pigs administered 100 mg/kg of PD 132301–2 for up to 7 days. Reversibility of adrenal effects was assessed after a 14-day drug withdrawal period (day 21). Serum Cortisol concentrations were determined under basal conditions and after administration of adrenocorticotrophic hormone (ACTH) on days 1, 2, 4, 7, and 21. Isolated foci of cortical cell degeneration and necrosis were apparent in outer zona fasciculata by 2 hr and throughout the zona fasciculata at 6 hr. Early degenerative ultrastructural changes included aggregation of smooth endoplasmic reticulum (SER), variable condensation of mitochondrial matrices and swelling of cristae, partitioning of organelles, autophagosome formation, and disruption of lipid globules. Lesions progressed to locally extensive or diffuse zonal necrosis on days 1 and 2 and near complete ablation of zona fasciculata by day 4. Fasciculata cells remaining on day 4 had reduced numbers and increased size of lipid globules, increased lysosomes, and, occasionally, aggregates of SER and mitochondria. On day 7, SER proliferation and lipid depletion were apparent in remaining cells. ACTH responses were attenuated 24 hr after the first dose, and reduction in basal Cortisol levels were seen by 24 hr after the second dose with both effects maximal on day 7. After a 14-day withdrawal period, ACTH responses and adrenal morphology returned to normal. It was concluded that PD 132301–2 induced rapid, reversible, zone-specific, morphologic, and functional adrenocortical effects. Furthermore, mitochondria and SER represented early subcellular targets of toxicity.

Acute Cardiovascular Toxicity Induced by an Adenosine Agonist-Antihypertensive in Beagles:

An adenosine agonist, designated chemically as (R)-N-(2,3-dihydro-1H-inden-1-yl) adenosine, or CI-947, was administered to 3 male and 3 female beagles in oral doses of 5 mg/kg body weight. Multiple episodes of arrhythmia were recorded electrocardiographically with Holter monitors in 2 males and 2 females monitored up to 48 hr. One male and 1 female were necropsied at 24 hr and the remaining dogs were necropsied at 48 hr post-dosing. At 48 hr, multifocal perivascular epicardial and myocardial hemorrhage was noted grossly in 1 female. Microscopic coronary arterial alterations were present in all treated dogs irrespective of the occurrence of arrhythmias. At 24 hr, proteinic material and red cells were present in the media accompanied by minimal adventitial accumulation of neutrophils. At 48 hr, coronary arterial lesions progressed to media vacuolation, transmural necrosis, and perivascular accumulation of neutrophils. Ultrastructural alterations included: endothelial retraction, subendothelial accumulation of fibrin and platelets, necrosis of smooth muscle cells, and mural infiltration of granulocytes and monocytes. Coronary vascular injury may be due to altered hemodynamics associated with the coronary vasodilator properties of adenosine agonist compounds.

α2u-Globulin nephropathy without nephrocarcinogenesis in male Wistar rats administered 1-(aminomethyl)cyclohexaneacetic acid

Alpha 2u-Globulin (alpha 2u) nephropathy is a male rat-specific condition caused by a diverse group of xenobiotics. Features of this nephropathy include hyaline droplet accumulation in proximal tubules, tubular epithelial necrosis and regeneration, exacerbation of spontaneous renal disease, and induction of renal epithelial tumors. Nephrocarcinogenicity of compounds that cause this nephropathy may be a consequence of increased proximal tubular proliferation resulting from cell injury. These studies document alpha 2u nephropathy without primary renal epithelial tumors in male Wistar rats administered 1-(aminomethyl)cyclohexaneacetic acid (gabapentin), a therapeutic agent with antiepileptic/anticonvulsant properties. In a series of preclinical studies gabapentin was administered to rats at the following doses and durations: 50 and 2000 mg/kg for 2 weeks; 250, 1000, 2000, and 3000 mg/kg for 13 weeks; 250, 1000, and 2000 mg/kg for 52 and 104 weeks. Renal effects were evaluated by biochemical, immunocytochemical, histopathologic, and ultrastructural techniques. Reversible increases in size and distribution of hyaline droplets within proximal tubular epithelium occurred through 1 year of treatment at a severity that was dose-dependent. In males given 2000 mg/kg, alpha 2u accumulation, degeneration, and necrosis of the P2 segment and intraluminal cellular casts were seen after 2 days of treatment. In the 2-week study, the size and number of phagolysosomes containing alpha 2u and the renal tissue alpha 2u increased with increasing dose and time. By Day 7, polymorphic crystalline inclusions were abundant in phagolysosomes of 2000 mg/kg males. In subchronic and chronic studies, spontaneous glomerulonephrosis was exacerbated in males given 2000 mg/kg, and, interestingly, no drug-related effect on renal tumor incidence was observed. To the best of our knowledge, this is the first documentation of the absence of nephrocarcinogenic effect in male rats treated for up to 104 weeks with a compound that causes acute and chronic lesions of alpha 2u nephropathy.

Toxicologic Effects of a Novel Acyl-CoA: Cholesterol Acyltransferase Inhibitor in Cynomolgus Monkeys

PD 132301-2, an acyl-CoA: cholesterol acyltransferase (ACAT) inhibitor, was administered orally to cynomolgus monkeys for 2 wk at doses of 25, 50, 100, and 200 mg/kg to assess potential subacute toxicity. Sporadic episodes of soft feces and diarrhea increased in incidence from 100 to 200 mg/kg. Histopathologic alterations in adrenocortical cells of treated monkeys consisted of a dose-related decrease in cytoplasmic fine vacuolation and an increase in cytoplasmic eosinophilia most conspicuous in the zona fasciculata and reticularis. At 50, 100, and 200 mg/kg, a narrow discontinuous zone of cytotoxic cortical cell degeneration occurred in the outer zona fasciculata. Decreased fine vacuolation of cortical cells correlated ultrastructurally with reduced size and number of intracellular lipid vacuoles and biochemically with a dose-related decrease in adrenal total cholesterol (from 56 to 13% of control) and cholesteryl ester (from 51 to 3% of control) concentrations. Other ultrastructural changes noted in zona fasciculata cortical cells at all doses were an apparent increase in both smooth endoplasmic reticulum and variably sized autophagic vacuoles. Ovarian corpora lutea in some females at all doses had increased coarse vacuolation of luteal cells, foci of cellular degeneration, increased numbers of cholesterol clefts, and slight infiltrates of mononuclear cells. Sebaceous glands were atrophic in all treated monkeys due largely to a reduction in size and number of differentiated foam cells. Sebaceous gland reserve cells were hypertrophic and hyperplastic. Toxicity data from this study indicated that PD 132301-2 at 25-200 mg/kg targeted cholesterol-rich cells of the adrenals, ovaries, and skin adnexa.

Renal structure and function in rats after suprapharmacologic doses of quinapril, an angiotensin-converting enzyme inhibitor.

Summary Angiotensin-converting enzyme (ACE) inhibitors have adverse effects on renal function in some hypertensive patients, and some of them produce renal tubular lesions in animals at high doses. To assess the effect of quinapril on renal function and structure, a 4-week time-course study was conducted in male Wistar rats with daily oral gavage doses of 0, 10, 100, or 400 mg/kg. Glomerular filtration rate (GFR) estimated as creatinine clearance and fractional electrolyte excretion values were derived from urinalysis and blood chemistry data obtained at days 1, 7, 14, and 28. Renal sections were collected on these days for histopathologic evaluation, and cortical slices were obtained to assess organic ion transport in vitro. Expected pharmacologic effects of an ACE inhibitor were observed at all doses and included increased urine output, increased water consumption, decreased serum aldosterone (65 or 25% of control at 10 or 400 mg/kg, respectively, on day 28), increased plasma renin activity (PRA, up to two- to threefold higher than controls at day 28), and hypertrophy of the juxtaglomerular apparatus. Despite these expected class effects, quinapril administration to male rats for 28 days produced no functional alterations or renal tubular lesions suggestive of renal toxicity at doses up to 400-fold higher than the effective antihypertensive dose in rats.

Ultrastructural Juxtaglomerular Cell Changes in Normotensive Rats Treated with Quinapril, an Inhibitor of Angiotensin-Converting Enzyme:

Sequential histologic and ultrastructural changes in juxtaglomerular apparatus (JGA) were defined in male rats treated with quinapril, an inhibitor of angiotensin-converting enzyme (ACE). Doses of 0, 10, 100, and 400 mg/kg were administered daily by gavage for up to 4 weeks. Granular juxtaglomerular (JG) cells were normal or hypogranular on Day 1 at all doses and were hypergranular by Day 7 in rats given 100 and 400 mg/kg relative to age-matched controls. Histologically, JGA hypertrophy was apparent by Day 7 at all doses and was most pronounced by Day 14 in intermediate and deep cortical zones of rats given 100 and 400 mg/kg. Ultrastructurally, hypertrophic JG cells had abundant rough endoplasmic reticulum and free ribosomes, and prominent Golgi complexes associated with numerous cytoplasmic coated vesicles. Dose-dependent increases in numbers of protogranules, altered granules, and cytoplasmic vacuoles occurred in association with decreased size and increased pleomorphism of mature secretory granules. Granule alterations included vesicular to lamellar membranous matrical inclusions, irregular patterns of osmiophilia, matrical vacuolation, and flocculent to coarsely granular matrix of greater density than mature granules. We concluded that JG cell hypertrophy and hyperplasia occurred rapidly in response to subchronic ACE inhibition. Further, ultra-structural changes in JG cells were indicative of stimulated renin synthesis by a regulated pathway, renin secretion by exocytosis and cytoplasmic solubilization of granules, and autophagy of granules as a mechanism whereby JG cells regulate levels of stored renin under conditions of excessive stimulation.

Proliferative Bone Lesions in Rats Given Anticancer Compounds

Proliferative endosteal lesions were observed in metaphysis and diaphysis of femur and sternebra of Wistar (CRL:[WI]BR) rats administered 3 chemically-distinct anticancer compounds with dissimilar mechanisms of action: trimetrexate glucuronate, an antifolate; pentostatin, an adenosine deaminase inhibitor; and CI-980, a mitotic inhibitor. Islands of woven bone, often circumscribed by conspicuous myelostromal proliferation, were seen on Days 8-28 in rats given trimetrexate glucuronate daily by gavage, and on Day 4 but not Day 29 in rats given a single intravenous dose of pentostatin. Intravenous administration of CI-980 for 1 or 5 days resulted in marrow necrosis, marked centripetal new bone formation, and myelostromal proliferation on Days 4 and 8, respectively. These lesions were not present at the termination of these latter studies (Days 29 and 35, respectively). In conclusion, anticancer compounds induced local bone marrow injury and the release of local inflammatory mediators which may have provided the stimulus for bone formation and myelostromal proliferation.