Revascularization in the rabbit hindlimb: dissociation between capillary sprouting and arteriogenesis.

OBJECTIVE: Animal models of hindlimb ischemia are critical to our understanding of peripheral vascular disease and allow us to evaluate therapeutic strategies aimed to improve peripheral collateral circulation. To further elucidate the processes involved in revascularization following ischemia, we evaluated the temporal association between tissue ischemia, vascular endothelial cell growth factor (VEGF) release, angiogenesis (capillary sprouting), arteriogenesis (growth of the larger muscular arteries), and reserve blood flow (functional collateral flow). METHODS: New Zealand White rabbits (male 3-4 kg) were evaluated at specific days (0, 5, 10, 20 or 40) following femoral artery removal for measurement of hindlimb blood flow, skeletal muscle lactate production and VEGF content, capillary density (a marker of angiogenesis), and angiographic score (a marker of arteriogenesis). RESULTS: Maximal capillary sprouting occurred within 5 days of femoral artery removal and was temporally associated with reduced resting hindlimb blood flow, increased lactate release and detectable levels of skeletal muscle VEGF. The growth of larger angiographically visible collateral vessels occurred after 10 days and was not temporally associated with ischemia or skeletal muscle VEGF content, but did coincide with a large functional improvement in the reserve blood flow capacity of the limb. CONCLUSIONS: Following femoral artery removal in the rabbit, the time course of angiogenesis and arteriogenesis were clearly distinct. Tissue ischemia and/or VEGF may stimulate capillary sprouting, but this response does not translate to a significant improvement in collateral flow. The growth and development of the larger collateral vessels was correlated with a large functional improvement in collateral flow, and occurred at a time when VEGF levels were undetectable.

Idiopathic extramural coronary arteritis in beagle and mongrel dogs.

Hearts obtained from 119 toxicological experiments on dogs during a 21-year period (1965-1986) were evaluated histologically to determine the incidence and characteristics of focal microscopic inflammatory lesions of the extramural coronary arteries. Lesions were encountered in 23% of 119 experiments. The average overall incidence was 2.3% of 1,905 control and chemically treated male and female beagle hearts (3.1% of male and 1.3% of female control beagles, and 1.8% of male and 2.8% of female treated beagles). Lesions were seen in 4.7% of 127 random-source mongrel dogs. Histologically, lesions were characterized by adventitial infiltrates of mononuclear cells, areas of mural necrosis, and inflammatory cell infiltrates in both mural and subendothelial regions. In the latter region, fibrosis developed separating the intima from the internal elastic membrane. Lesions occurred in the right and left coronary arteries and in the dorsal, circumflex, and ventral descending branches of the left coronary artery. Vascular lesions were not present in the intramural coronary vessels or in vessels of other organs in affected animals. More extensive sampling of the extramural coronary arteries revealed an incidence of 5-9%. The lesion of focal extramural coronary arteritis was idiopathic, and not a manifestation of the polyarteritis syndrome of dogs.

Spontaneous extramural coronary arteritis in dogs.

Control and treated beagle and random-source dog hearts obtained from 119 toxicological experiments were evaluated histologically to study the incidence and characteristics of a microscopic inflammatory lesion specific to the extramural coronary arteries. The lesion occurred in 23% of the experiments. The incidence was 2.3% of the control and treated beagle hearts examined. In the control beagles, it was present in 3.1% of males and 1.3% of females and in the treated beagles, in 1.8% males and 2.8% females. It occurred in 4.7% of the random-source animals. While not visible grossly, histologically, the solitary lesions were characterized by adventitial infiltrates of mononuclear cells, occasional areas of necrosis with inflammatory cell infiltrates occurring in the mural and subendothelial regions. The latter resulted in prominent separation of the intima from the media. The lesions occurred in the right and left coronary arteries and in the dorsal, circumflex and ventral descending branches of the left coronary artery. Similar vascular lesions were not found in the intramural coronary vessels or in other organs in the affected animals. Expanded sampling of the extramural coronary arteries revealed an incidence of 5-9%. This lesion of focal extramural coronary arteritis was considered idiopathic, and not a manifestation of recently reported polyarteritis syndromes in dogs.

Prevention of fluvastatin-induced toxicity, mortality, and cardiac myopathy in pregnant rats by mevalonic acid supplementation.

Mevalonic acid is a product of the enzyme HMG-CoA reductase which is essential for cholesterol biosynthesis. Fluvastatin (Sandoz compound XU 62-320) is a potent inhibitor of this enzyme and, hence, mevalonic acid production. In three separate studies, oral administration of fluvastatin at 12 and 24 mg/kg/day to mated rats from day 15 of gestation through weaning resulted in unanticipated maternal mortality at the time of parturition and during lactation. Microscopic evaluations performed in two studies revealed significant cardiac myopathy in the dying animals. Drug-related clinical signs, significant maternal body weight loss, and an increase in stillborn pups and neonatal mortality were also noted at one or both dose levels. Supplementation of fluvastatin administration with 500 mg/kg b.i.d. of mevalonic acid completely blocked and/or ameliorated the mortality, cardiac myopathy, and other adverse effects. These studies indicate that the adverse maternal effects observed with fluvastatin before or following parturition resulted from exaggerated pharmacologic activity at the dose levels administered, i.e., inhibition of the enzyme HMG-CoA reductase, its immediate product mevalonic acid, and cholesterol biosynthesis.

Naturally occurring intracytoplasmic inclusions in the canine exocrine pancreas.

Bodies similar to acidophilic intracytoplasmic inclusions were found by light microscopy in the pancreatic acinar cells of 56 of 174 (32%) healthy male and female purebred Beagles and 14 of 97 (14%) of healthy male mongrel dogs. The inclusions were ovoid, acidophilic and often granular with basophilic particulates. Many seemed to be enclosed within halos of various widths. Electron microscopically the inclusions consisted of whorls of rough endoplasmic reticulum, vacuoles, and cytoplasmic organelles in various stages of decomposition. These inclusion bodies were interpreted as evidence of focal intracytoplasmic degradation. They appeared similar to the dense ribosomal autophagic vacuoles, hitherto described only in association with various experimental procedures.

Riboflavin 5'-pyrophosphate: a contaminant of commercial FAD, a coenzyme for FAD-dependent oxidases, and an inhibitor of FAD synthetase.

Commercially available preparations of flavin adenine dinucleotide (FAD) have been found to be 94% pure, the remaining 6% being composed of four or five minor contaminants which can be separated from FAD by reverse-phase high-performance liquid chromatography. FAD purified in this manner has been shown to be 100% pure. One of the contaminants has been identified as riboflavin 5'-pyrophosphate (RPP) by spectroscopic and chemical methods of analysis. This compound has been shown to exhibit biological activity as a weak cofactor for two FAD-requiring enzymes. With the apoprotein of porcine D-amino-acid oxidase, values determined for RPP were 8.4 microM for Km and 0.10 for Vmax compared to 0.47 microM and 0.28 (36 U/mg), respectively, for FAD. With fungal glucose apooxidase, values determined for RPP were 474 nM for Km and 0.02 for Vmax and 45 nM and 0.09 (105 U/mg), respectively, for FAD. RPP can also inhibit FAD biosynthesis. For bovine liver FAD synthetase, a Ki value for RPP against FMN was determined to be 9 microM where Km for FMN was 5.5 microM. These studies illustrate the value of riboflavin 5'-pyrophosphate as a flavin analog for use in the study of structure/function relationships within certain flavin-dependent enzymes.

Five-day continuous infusion of 5-fluorouracil for advanced colorectal, gastric, and pancreatic adenocarcinoma.

In a retrospective study, 58 patients with advanced colorectal carcinoma received one or more 5-day infusions of 5-FU at 20 mg/kg per 24 hours followed by weekly IV 5-FU at 15 mg/kg. In 36 patients who received the infusion as first treatment (Group A), the response rate was 23% with a median duration of response of 8.0 months. No improvement in survival was noted. In 22 patients treated with the infusion after relapse on weekly 5-FU (Group B), the response rate was 10.5% with a median duration response of 4.5 months. Stable disease for 3-6 months was seen in 21%. Survival in Group B was improved when compared with a similar group of patients treated by weekly 5-FU without infusion (Group CB), but statistical significance was not obtained. Twenty percent of patients were alive two years after relapsing on weekly 5-FU when given the five-day infusion 5-FU and re-initiated on weekly 5-FU. Toxicity to the five-day infusion of 5-FU was minimal. Three of 69 (4.3%) infusions were not completed because of toxicity. A separate review of procarbazine indicated that it was minimally active in colorectal carcinoma. Other patients with gastric and pancreatic carcinoma were reviewed but no statistical significance for 5-FU infusion was seen for survival. A five day continuous infusion of 5-FU is recommended for colorectal cancer patients who relapse on weekly 5-FU therapy. Further study of the infusion in combination with other chemotherapy is warranted.

Hypolipidemic activity and toxicity studies of a styrl-hexahydroindolinol, 34-250.

34-250 evoked hypocholesterolemic activity in the rat (14, 25, 31, 52, 112 mg/kg, po), dog (10, 20, 40 mg/kg, po), and monkey (30 mg/kg, po). Serum triglycerides were lowered in the rat and dog but not in the monkey. 34-250 increased [14C]acetate incorporation into liver cholesterol, but incorporation of 14C-labeled acetate into serum cholesterol was decreased. Desmosterol or 7-dehydro-cholesterol did not accumulate in serum of the three species, suggesting that inhibition of cholesterol biosynthesis by 34-250 possibly does not occur at a late stage. Normal fecal bile acid excretion was observed in rats, suggesting that cholesterol catabolism probably was not enhanced by 34-250. Compound 34-250-induced hypocholesterolemia may result from inhibition of hepatic release of this sterol into blood. The reversible hepatic lipidosis observed in rats is also possibly related to decreased hepatic transport and/or secretion of triglycerides. 34-250 did not cause a proliferation of hepatic microbodies; the lack of an increase in this fatty acid oxidizing organelle suggests that it may also have had a role in increased hepatic lipidosis. In dogs, a high incidence of severe cataracts with an early onset was induced by 20 and 40 mg/kg, po of 34-250 despite the lack of desmosterol or 7-dehydro-cholesterol in serum. The absence of these late stage intermediates of cholesterol biosynthesis in the serum of a test species does not preclude the occurrence of ocular toxicity.

Generalized phospholipidosis induced by an amphiphilic cationic psychotropic drug.

Numerous amphiphilic cationic drugs cause generalized phospholipidosis in animals; one of these drugs is the Sandoz compound 200-125, a psychotropic agent. During a 6-month toxicity study in Charles River CD rats, a dramatic increase in foamy macrophages was seen in the lungs. A follow-up experiment was done to study the pathologic basis of these changes including a reversibility phase. Generalized phospholipidosis was induced after 4 weeks of 500 mg/kg/day of 200-125 by gavage. Characteristic pulmonary lesions consisted of extensive accumulations of large pale foamy macrophages as well as granular eosinophilic extracellular material. Lipid analyses of lungs showed marked increases in phospholipids (144%) and cholesterol esters (110%) in rats treated with 200-125. Drug metabolism studies employing 14C-labeled 200-125 showed an affinity for the drug to concentrate in the lungs and lymphoreticular system (spleen, lymph nodes) as well as in the adrenals, liver, and kidney. Reversibility of the phospholipidosis was nearly complete 4 weeks after drug withdrawal. The tissue changes were characterized by transmission and scanning electron microscopy. The potential pulmonary toxicity in humans with the amphiphiles is discussed.

The safety evaluation of fluvastatin, an HMG-CoA reductase inhibitor, in beagle dogs and rhesus monkeys.

Fluvastatin is a potent synthetic competitive inhibitor of beta-hydroxy-beta-methyl-glutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in the biosynthetic pathway for hepatic cholesterol synthesis. The therapeutic indication is reduction of elevated total and low-density lipoprotein cholesterol levels. Results from four toxicity studies in beagle dogs and one study in rhesus monkeys following oral administration of fluvastatin are reported. In two 26-week dog studies, doses were 0, 1, 8, or 48 mg/kg/day (reduced to 36 mg/kg/day in Week 7) and 0, 6, 24, or 36 mg/kg/day (reduced to 30 mg/kg/day in Week 2). In a 2-year dog study, doses were 0, 1, 8, or 16 mg/kg/day. Dose levels in the 26-week monkey study were 0, 0.6, 12, and 48 mg/kg/day (raised to 84 mg/kg/day in Week 17 and to 108 mg/kg/day in Week 22). In these studies, evaluations included clinical and physical examinations, body weight and food consumption, electrocardiography, ophthalmoscopy, hematology and clinical chemistries, urinalysis, blood drug concentration, and macroscopic and microscopic examinations of observed lesions and representative tissues. In the 26- and 52-week dog studies and the monkey study, lenticular biochemistry, the HMG-CoA reductase activity of liver microsomes, and serum lipid concentrations were investigated. The fourth dog study was a single-dose toxicokinetic study in which 48 mg/kg [3H]-fluvastatin was monitored for up to 2 weeks. Sampling was limited to ocular tissues for enzyme analysis. Doses of > or = 24 mg/kg/day were lethal in dogs. At lethal doses, ataxia, convulsions, fecal blood, multifocal congestion and hemorrhage, isolated foci of malacia in the medulla oblongata, and liver necrosis were observed. Reduced weight gain, emesis, cataracts, elevated liver enzymes, reduced cholesterol, and gallbladder inflammation with mucosal hyperplasia occurred at > or = 8 mg/kg/day. In contrast to other HMG-CoA reductase inhibitors, fluvastatin did not cause significant central nervous system hemorrhage or testicular changes in dogs. Monkeys tolerated exposure to fluvastatin well with only mild gallbladder changes observed. Reduced serum cholesterol and slight hyperplasia of the gallbladder mucosa occurred in the 12 and 48/84/108 mg/kg/day groups.