Regioselective alkylation at the N4 position of a 3-oxo-1,4-benzodiazepine on solid support.

An efficient solid phase regioselective alkylation at the N4 position of a 3-oxo-1,4-benzodiazepine template exemplified by 4-H-2,3,4,5-tetrahydro-7-iodo-3-oxo-1H-1,4-benzodiazepine-2-acetate-polymer ester is described. Further chemical elaboration at position 7, utilizing a modified Heck reaction, allows the incorporation of amides from primary or secondary amines. The two diversity points at positions 4 and 7 were utilized to synthesize a 28-membered, combinatorial array on Sasrin resin in moderate yields and > 80% purity. Having validated the chemistry on solid support, a combine and split approach to prepare a bead-bound combinatorial library is achievable utilizing similar experimental practices and procedures as in the array synthesis.

Discovery of an imidazopyridine-containing 1,4-benzodiazepine nonpeptide vitronectin receptor (alpha v beta 3) antagonist with efficacy in a restenosis model.

In the 3-oxo-1,4-benzodiazepine-2-acetic acid series of vitronectin receptor (alpha v beta 3) antagonists, a compound containing an imidazopyridine arginine mimetic was discovered which had sufficient potency and i.v. pharmacokinetics for demonstration of efficacy in a rat restenosis model.

Chronic toxicity studies of 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione, a potential chemopreventive agent.

The synthetic compound Oltipraz, 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione, is related to the 1,2-dithiolthiones naturally found in cruciferous vegetables, the consumption of which has been epidemiologically associated with reduced frequency of colorectal cancers. Oltipraz has shown chemopreventive efficacy in numerous laboratory epithelial cancer models and is a potential chemopreventive, antimutagenic compound that specifically induces Phase II enzymes. Thirteen-week and 1-year toxicity studies in rats and dogs were performed to characterize the toxicities of the compound at high dosages and to support potential further development as a chemopreventive agent in clinical trials. Administration to rats by gavage for 13 weeks at dosages of 5 and 50 mg/kg/day and for 52 weeks at dosages of 10, 30, and 60 mg/kg/day produced effects on the liver and on clinical chemistry and hematology parameters. Absolute and relative liver weight increases correlated with diffuse hypertrophy in the mid- and high-dose males and centrilobular hypertrophy in the high-dose females. Granularity of hepatocyte cytoplasm was also observed. These anatomical findings were associated with dose-associated slight increases in albumin, total protein, and cholesterol in the males and a moderate increase in cholesterol only in the females. In addition, slight decreases in erythrocyte count, hemoglobin, and hematocrit and reticulocyte elevations occurred. The no effect dose was considered 10 mg/kg/day. Administration by capsule to dogs at dosages of 10 and 100 mg/kg/day for 13 weeks and of 5, 15, and 60 mg/kg/day for 52 weeks also produced effects on the same endpoints noted in the rodent studies. In the 13-week study, precipitate was observed in the bile canaliculi, and gonadal atrophy and increased pituitary weights occurred in the males. Cholesterol and alkaline phosphatase activity were slightly elevated in both studies. Decreased hematology parameters in the 13-week study also occurred. The no effect dose was considered to be 5 mg/kg/day. Oltipraz is being carefully evaluated in clinical trials as a potential antimutagenic compound.

Reduced incidence and severity of collagen-induced arthritis in interleukin-12-deficient mice.

Collagen-induced arthritis (CIA) is an animal model for rheumatoid arthritis. The disease is elicited by immunization of genetically susceptible DBA/1 mice with type II collagen, resulting in a debilitating arthritis characterized by inflammation and involvement of multiple joints. We investigated the role of endogenous interleukin (IL)-12 in the pathogenesis of this disease by undertaking an analysis of IL-12-deficient mice on the DBA/1 genetic background after immunization with type II collagen. Both the incidence and severity of disease were significantly reduced in mice unable to produce biologically active IL-12. Concomitant decreases were observed in serum levels of pathogenic, collagen-specific IgG2a antibodies and collagen-induced secretion of interferon-gamma by immune splenocytes in vitro, consistent with an impaired T helper-1 response. There were, however, a few animals which developed severe disease in a single paw in spite of this highly diminished Th1 response. Taken together, these results demonstrate an important role for IL-12 in the pathogenesis of CIA, although it is not absolutely required for disease development.

Pathologic features of abdominal and thoracic paragangliomas in F344/N rats.

Seventeen paragangliomas were identified in a retrospective review of 200 NTP/NCI carcinogenicity studies in F344/N rats that served either as control or treated animals. Most tumors were grossly visible and located in the retroperitoneum adjacent to the vertebrae and aorta near the kidneys. Three microscopically detected paragangliomas were found at the base of the heart. Microscopically, neoplastic cells were in nests separated by reticulin fibers and capillaries. Argyrophil granules were in the cytoplasm of the retroperitoneal and mediastinal paravertebral tumors. Dense granules were found in the one tumor examined ultrastructurally. Some tumors had areas of necrosis and tumor emboli were present in the lumen of the abdominal aorta and vena cava adjacent to the tumor with metastases present in pulmonary vessels. The incidence of retroperitoneal neoplasms was 3 times more frequent in male than in female F344/N rats.

Effect of methyl isocyanate (MIC) gas on the eyes of Fischer 344 rats.

The accidental release of methyl isocyanate gas in Bhopal, India, was reported to cause temporary blindness and other eye injuries in many of the exposed people. Methyl isocyanate (MIC) is known to be corrosive and to irritate intact skin and mucous membranes, but little is known about the extent of ocular damage incurred during exposure to its vapors. The eyes of male and female Fischer 344 rats were evaluated immediately after a 2-hr exposure to 0, 3, 10, or 30 ppm of MIC, and periodically thereafter during a 91-day recovery period. During exposure to 10 ppm and higher concentrations, rats kept their eyes partially closed. Copious lacrimation and occasional frothy nasal discharge were evident. Eyes were examined under ultraviolet light after topical application of sodium fluorescein, and histopathologic examination included lids, cornea, lens, retina, optic nerve, and Harderian gland. There was no significant gross or microscopic evidence of epithelial erosion or ulceration of the cornea, or of adjacent tissues immediately after, or at any time following exposures. No skin irritation was noted. It would appear that the natural protective mechanisms of the eye of rats were adequate to prevent ocular damage at these exposure levels.

Two-hour methyl isocyanate inhalation exposure and 91-day recovery: a preliminary description of pathologic changes in F344 rats.

The accidental release of methyl isocyanate (MIC) in Bhopal, India, was reportedly responsible for the deaths of more than 2,000 people. To study the pathology of acute inhalation exposure to MIC, the tissues of male and female Fischer 344 rats were evaluated immediately after a single 2-hr exposure to 0, 3, 10, or 30 ppm MIC, and through day 91. Early gross pathologic changes in the 30 ppm-exposed rats included a reddish white encrustation around the mouth and nose, a small thymus, and distension of the gastrointestinal tract with gas. Lungs (middle and median lobes) showed consolidation and hemorrhage and failed to deflate when the chest cavity was opened. Microscopic changes in the upper respiratory tract 3 hr after exposure included marked erosion and separation of olfactory and respiratory epithelia from the basement membrane with accumulation of serofibrinous fluid. On day 1, acute inflammation and fibrinopurulent exudate partially blocked the nasal passages. Epithelial cells had sloughed from the nasopharynx, trachea, bronchi, and major bronchioles, leaving the basement membrane covered with fibrin and exudate. Granulomatous inflammation and intraluminal fibrosis of the airways were observed by day 3, with increased intraluminal fibrosis by day 7. Lower airways became blocked by exfoliated cells, mucous plugs, and/or intraluminal fibrosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Reproductive toxicology of methyldopa in male F344/N rats.

Methyldopa, a widely used antihypertensive drug, was administered to male Fischer 344/N rats by gavage 5 days/week for 65 days at dosages of 0, 50, 100, 200, or 400 mg/kg. Decreased body weight was seen in treated animals. After mating to untreated female Fischer 344/N rats on days 57-61, the male rats were necropsied (days 65-67) and reproductive toxicity was measured by sperm count, sperm motility, organ weights, hormone levels and histologic evaluation of the testis. Decreased fertility was seen in males dosed with 200 or 400 mg/kg methyldopa. Decreases were also seen in sperm count, sperm motility, apparent number of late spermatids, and plasma testosterone levels in males in the 200 and 400 mg/kg groups. This alternation of reproductive function in male rats was found to be reversible after a 13-week recovery period without dosing. The marked decrease in circulating testosterone levels following methyldopa treatment at 200 or 400 mg/kg may have contributed to the reproductive toxicity of this drug.

Testicular changes observed in boars following experimental inoculation with pseudorabies (Aujeszky's) virus.

Four boars were inoculated intranasally with pseudorabies virus to determine if microscopic testicular changes occurred as a result of infection. Testicular biopsies and semen samples were taken at two, four and six weeks postinoculation and the boars were castrated immediately after the last sample collection. Testicular samples and semen were cultured to determine if the virus was present. Pseudorabies virus was not isolated from the semen or testicular tissue. Virus was isolated from trigeminal ganglia at necropsy and from nasal swabs taken one day after castration. Consequently, a time of high risk for shed of the virus from clinically normal carrier animals is immediately following castration. Gross changes were not observed in testicular tissues and microscopic changes in the testicles were the result of biopsy. Lesions consistent with pseudorabies virus infection were observed in the central nervous system of all inoculated boars. Temporary lowered fertility may result from the effects of elevated body temperature on spermatogenesis during acute clinical disease. However, it appears that the strain of pseudorabies virus used, lacked the ability to infect and/or replicate in the boars' reproductive tracts.

The effect of pseudorabies (Aujeszky's) virus infection on young mature boars and boar fertility.

This study was designed to determine the effects of experimental inoculation with pseudorabies virus on the reproductive tracts of young adult boars. Pseudorabies virus was inoculated intranasally into 12 boars and intrapreputially into four boars. All animals seroconverted after nasal or preputial inoculation. Semen abnormalities were observed 21 days postinoculation with partial recovery by 50 days postinoculation. Virus was isolated from the preputial sheath of two intrapreputially inoculated boars 12 days postinoculation. It was concluded that pseudorabies virus infection can be established via preputial inoculation and that decreased spermatogenesis and infertility can result.

Aminoglycoside antibiotics inhibit lysosomal phospholipase A and C from rat liver in vitro.

Accumulation of aminoglycoside antibiotics and phospholipids in lysosomes is a prominent feature of aminoglycoside nephrotoxicity, suggesting the possibility that these agents may inhibit the activity of lysosomal phospholipases. We examined the effect of four aminoglycoside antibiotics, amikacin, dibekacin gentamicin and tobramycin, on the hydrolysis of [3H]dioleoylphosphatidylcholine by a lysosomal protein fraction obtained from rat liver which contains phospholipase A and C. Phospholipase A was inhibited strongly by these agents. Phospholipase C was also inhibited, especially by amikacin and tobramycin. These results suggest that the accumulation of phospholipids in the kidney cortex in aminoglycoside nephrotoxicity may be due to inhibition of lysosomal phospholipase action.

Inhibition of kidney lysosomal phospholipases A and C by aminoglycoside antibiotics: possible mechanism of aminoglycoside toxicity.

Nephrotoxicity is an important side effect of aminoglycoside antibiotics, which are used to control infections caused by Gram-negative bacteria. Accumulation of aminoglycosides and phospholipids in the lysosomes is a prominent and early feature of aminoglycoside nephrotoxicity and is characterized histologically by the presence of numerous multilamellar bodies in kidney proximal tubule cells. Previous studies have shown that the drug-induced phospholipid fatty liver in man and animals is due to concentration of certain cationic amphiphilic drugs in lysosomes with inhibition of lysosomal phospholipases. It seemed possible that this mechanism might also explain the elevated levels of phospholipid and increased numbers of multilamellar bodies reported in the kidney cortex in aminoglycoside nephrotoxicity. In this study, subcellular localization of acid phospholipases A and C has been shown to be lysosomal in rat kidney cortex. A soluble lysosomal protein fraction was isolated and found to contain both phospholipase A and phospholipase C activity. Streptomycin did not inhibit the release of fatty acids from [3H]dioleoylphosphatidylcholine. However, amikacin, dibekacin, gentamicin, and tobramycin inhibited both phospholipase A and phospholipase C. Our results suggest that the accumulation of phospholipids in lysosomes of kidney cortex, an early and pervasive feature of acute aminoglycoside nephrotoxicity, is due to inhibition of lysosomal phospholipases.