Chronic effects of the novel glucocorticosteroid RPR 106541 administered to beagle dogs by inhalation.

The preclinical safety of RPR 106541, a novel 17-thiosteroid, was evaluated in young adult and mature dogs by inhalation exposure for 26 weeks and 52 weeks, respectively. A dry powder formulation of RPR 106541 in lactose was administered to young adult dogs (approximately 6 months of age at initiation) at doses of 0 (air and placebo controls), 10, 100, or 1,000 microg/kg/d for 26 weeks. A solution-based aerosol formulation was administered to mature dogs (approximately 10 months at initiation) from a pressurized metered dose inhaler at 0 (air and placebo controls), 10, 50, and 150 microg/kg/d for 52 weeks. Clinical evidence of glucocorticosteroid-induced immunosuppression was observed by weeks 20-26 following relatively high dose exposures (100 microg/kg/d and 1,000 microg/kg/d) in young dogs receiving the dry powder formulation for 26 weeks. Classic glucocorticosteroid effects were observed, including adrenocortical atrophy, reduced bone mass with retention of epiphyseal growth plates in long bones, prominence of stromal adipose tissue in bone marrow, and atrophy of lymphoid tissues. Inhalation administration of RPR 106541 to sexually mature dogs facilitated more definitive characterization of endocrine affects of RPR 106541 as compared with administration in younger, sexually immature animals. Significant effects in female reproductive organs included absence of corpora lutea in association with atresia of vesicular follicles within the ovaries, endometrial hyperplasia, and lobular development of mammary tissue. Discordant development of mammary tissue, accumulation of secretory material within hyperplastic endometrial glands, and hypertrophy of uterine lining epithelium in absence of ovulation were consistent with a secondary progestin effect by a potent glucocorticosteroid.

Toxicologic and carcinogenic effects of the type IV phosphodiesterase inhibitor RP 73401 on the nasal olfactory tissue in rats.

RP 73401, a type IV phosphodiesterase inhibitor, caused toxic effects in the nasal olfactory region of Sprague-Dawley rats when administered by either oral or inhalation exposure. A single oral administration of RP 73401 (at a dose of > or = 50 mg/kg) or 5-day inhalation exposure (1 hr/day) at a dose of approximately 1.0 mg/kg per day caused degeneration and sloughing of the olfactory surface epithelium. Degeneration and loss of Bowman's glands were noted in the underlying lamina propria and submucosa. Electron microscopy of these lesions demonstrated that sustentacular cells and the epithelial cells lining Bowman's glands were the primary target cells in the olfactory mucosa. The earliest ultrastructural changes detected in these cells were dilatation and vesiculation of the endoplasmic reticulum, suggesting that metabolic activation is important for the toxic effects. In repeated-dose studies, 13 wk of oral dosing at 2.0 or 6.0 mg/kg per day resulted in subtle disorganization of the olfactory epithelium, whereas basal cell hyperplasia in the olfactory epithelium was identified in a 6-month inhalation study at a dose of 1.0 mg/kg per day. A 2-yr inhalation carcinogenicity study resulted in tumors of the nasal olfactory region in rats treated at 0.5 and 1.0 mg/kg per day. Most tumors were classified as olfactory neuroblastomas, and immunohistochemistry on selected tumors was consistent with their being of neuroectodermal origin. Of the species studied (rat, mouse, and dog), the olfactory toxicity of RP 73401 was confined to the rat, and the toxicity was likely related to metabolic activation by olfactory epithelial cells rather than the phosphodiesterase activity of the compound.

The toxicity of repeated exposures to rolipram, a type IV phosphodiesterase inhibitor, in rats.

Rolipram is a selective inhibitor of Type IV phosphodiesterase isozymes (PDE IV) which is often used as a baseline comparator for compounds in this class. To document the toxicological effects of rolipram, it was administered to female rats at 0, 10, 30 or 100 mg/kg/day orally for up to 2 weeks. One treatment-related death in the 100 mg/kg/day dose group was observed on day 3, and all rats at this dose level were considered moribund and euthanatized on day 5. Several clinical signs were observed in treated rats, including increased salivation, slight distention of the abdomen, emaciated appearance, and ataxia. After 14 days of treatment, the rats were necropsied and tissues examined microscopically. A number of compound-related histopathological changes were observed in rats receiving 30 or 100 mg/kg/day. Myocardial degeneration and necrosis, endocardial fibrosis, epicarditis, and arteritis/periarteritis of intramural and extramural coronary arteries were observed in the heart. A necrotizing vasculitis and inflammation were observed in the mesentery and interstitial areas of the liver, affecting medium-sized portal arteries and veins. Focal necrosis was also observed in the glandular mucosa of the stomach at these 2 dose levels. Other treatment-related effects included squamous hyperplasia and hyperkeratosis with or without ulceration in the nonglandular stomach of at least one animal from all treatment groups. Enlarged salivary glands were noted at necropsy in animals treated with 100 mg/kg/day, and this finding correlated microscopically with dilatation and degeneration of ducts and acini in the sublingual gland with secondary inflammation and edema. The results of this study demonstrate that rolipram, a selective inhibitor of the type IV class of PDE, can cause effects on the heart and vasculature of rats which heretofore have been ascribed only to selective inhibitors of the PDE III class of isozymes. Therefore, these organs should be closely examined in studies with other PDE IV inhibitors. In addition, the gastrointestinal tract and salivary glands were sites for rolipram-induced toxicity and may be targets of other PDE IV inhibitors.

Hemochromatosis in Salers cattle.

Two 2-year-old Salers cattle from different herds raised on pasture were evaluated for retarded growth and diarrhea. Increase of liver enzyme activities and prolonged sulfobromophothalein (BSP) half life (T1/2) indicated liver disease with impaired liver function. Histopathologic examination of liver biopsies revealed a micronodular cirrhosis with marked deposition of hemosiderin in hepatocytes, Kupffer cells, and arterioles. Transferrin saturation (TS) and liver iron content were markedly increased, consistent with a diagnosis of hemochromatosis. Both animals were euthanatized due to deterioration in their condition. Necropsy findings included hepatomegaly and hemosiderin accumulation in the liver, lymph nodes, pancreas, spleen, thyroid, kidney, brain and other glandular tissue. Continued surveillance of the second herd (serum iron, total iron binding capacity [TIBC], unsaturated iron binding capacity [UIBC], and TS), identified a heifer as a hemochromatosis suspect in a subsequent generation. Liver biopsies from that animal revealed the same histopathologic changes as the previous 2 animals, and similar increases in liver iron content (8,700 ppm, normal range 45 to 300 ppm). The 3 affected cattle were all products of line breeding programs and shared a common ancestor. The absence of dietary iron loading in conjunction with the histopathologic and metabolic findings were consistent with a diagnosis of primary hemochromatosis. The reported disease is similar to idiopathic hemochromatosis in human beings in which there is a hereditary defect in iron metabolism.

Effects of ozone and neutrophils on function and morphology of the isolated rat lung.

Since whole-animal studies suggest that neutrophils play a role in ozone-induced impairment of pulmonary function and increases in airway reactivity, this study was designed to study more precisely the interaction of neutrophils and ozone using the isolated perfused rat lung. Sprague-Dawley rat lungs (n = 60) were ventilated for 3 h with 95% air and 5% CO2 alone or mixed with 1 ppm ozone and perfused with buffer alone or with neutrophils (8 x 10(7)). RL, Cdyn, Ppa, airway reactivity to methacholine, lung/body weight, bronchoalveolar lavage fluid (BALF) protein concentration, and airway lesions were analyzed using a two-way GLM or Kruskal-Wallis test (p < or = 0.05 significant). Both ozone and neutrophils increased RL and decreased Cdyn. Ozone but not neutrophils increased airway reactivity to methacholine. Neutrophils but not ozone increased lung weight/body weight and BALF protein concentration. Ozone damaged airway epithelium. In distal bronchioles, neutrophils enhanced this damage. We conclude that (1) ozone impaired pulmonary function, increased airway reactivity, and damaged airway epithelium without causing measurable microvascular leak; (2) neutrophils impaired pulmonary function, probably a result of microvascular leak, but did not change airway reactivity or damage airway epithelium; and (3) neutrophils had no effect on ozone-induced airway reactivity but had an additive effect on ozone-induced pulmonary function impairment and a synergistic effect on ozone-induced airway epithelial injury.

Effect of PAF receptor antagonism on cardiopulmonary alterations during coinfusion of TNF-alpha and IL-1 alpha in pigs.

We examined the possibility that platelet-activating factor (PAF) might be a mediator of cardiopulmonary alterations induced by a 6-h coinfusion of human recombinant tumor necrosis factor (TNF-alpha) and interleukin-1 alpha (IL-1 alpha) in anesthetized pigs. Our hypothesis was tested by pretreating TNF-alpha + IL-1 alpha-infused pigs with WEB 2086 (3 mg/kg from -0.5 to 0 h + 0.75 mg.kg-1.h-1 from 0-6 h), a specific PAF receptor antagonist. Each cytokine was infused intravenously at 0.5 microgram/kg from 0-0.5 h + 5 ng.kg-1.min-1 from 0.5-6 h. WEB 2086 attenuated the early (0.25 h) cytokine-induced increases in mean pulmonary arterial pressure and pulmonary vascular resistance and blocked or markedly attenuated the later occurring (4-6 h) systemic hypertension and increased systemic vascular resistance. WEB 2086 lessened the severity of TNF-alpha + IL-1 alpha-induced hemoconcentration and airway constriction, but did not modify leukopenia, granulocytopenia, or the cytokine-induced increases in plasma concentrations of thromboxane B2, prostaglandin F2 alpha, and 6-ketoprostaglandin F1 alpha. Microscopically, WEB 2086 did not modify the increased number of granulocytes present in lung tissue derived from pigs infused with TNF-alpha + IL-1 alpha. We conclude that PAF occupies a physiological role in modulating TNF-alpha + IL-1 alpha-induced hemoconcentration, the early changes in pulmonary hemodynamics, and the later alterations in systemic hemodynamics.

Acute ozone-induced lung injury in neutrophil-depleted rats.

To test the hypothesis that neutrophils contribute to acute, ozone-induced epithelial damage in the lung, rats were depleted of their circulating neutrophils by intraperitoneal injection of a rabbit anti-rat neutrophil serum (ANS) 12 hr prior to an 8-hr exposure to 1.0 ppm ozone. Additional rats were given an injection of normal rabbit serum (NRS) prior to ozone exposure. Exposures were followed by postexposure periods in filtered air for 0, 4, or 16 hr. Control rats were given either ANS or NRS and then exposed only to filtered air. Analysis of bronchoalveolar lavage fluid (BALF) from NRS-treated rats revealed a significant increase in total neutrophils above that of controls at the 4- and 16-hr postexposure times, with a peak increase at 4 hr postexposure. In contrast, there was almost total ablation of the BALF neutrophil response in the ANS-treated rats at all times. Ozone caused an increase in BALF protein, fibronectin, and interleukin-6 above those in controls in both the NRS- and ANS-treated rats, but the only significant difference between the two groups was a level of fibronectin in the neutrophil-depleted animals higher than that in the neutrophil-sufficient animals at the 0-hr postexposure time. Electron microscopic morphometry on lungs fixed by intravascular perfusion demonstrated no significant differences in the volume per surface area epithelial basal lamina (Vs) of necrotic and degenerating epithelial cells in central acini between the neutrophil-depleted and neutrophil-sufficient animals. From these results, we concluded that neutrophils do not play a detectable role in contributing to the early epithelial damage in the lung caused by an acute exposure to ozone.

Functional and morphologic changes caused by acute ozone exposure in the isolated and perfused rat lung.

Ozone has been shown to increase airway resistance and/or airway reactivity in vivo in animals and humans. Because of the complexities inherent in studying this phenomenon in whole animals, we developed a model of ozone-induced effects on airway physiology using the isolated perfused rat lung. Rat lungs were suspended in an airtight chamber and perfused via the pulmonary circulation with a modified Krebs-Henseleit buffer containing 4.5% bovine albumin. Ventilation of the lungs was achieved by generating a fluctuating negative pressure within the chamber (-2 to -7 cm H2O) at a rate of 60 breaths/min. The lungs were ventilated with humidified 95% air and 5% CO2 alone (control condition) or mixed with ozone at 1.0 or 2.0 ppm. Transpulmonary pressure, flow rate, and tidal volume were recorded at 0, 1, 2, and 3 hours, and pulmonary resistance (RL) and dynamic compliance (Cdyn) were calculated. There was no significant difference in lung weight/total body weight ratios between the three groups at the end of the 3-h period. RL increased and Cdyn decreased in a time- and dose-dependent manner with ozone exposure. The percent increase above baseline in RL +/- SEM at 3 h was 9.4 +/- 4.1% for control lungs, 21.0 +/- 3.2% for 1.0 ppm ozone-exposed lungs, and 63.6 +/- 13.5% for 2.0 ppm ozone-exposed lungs. The percent decrease below baseline in Cdyn +/- SEM at 3 h was 27.4 +/- 2.1% for control lungs, 37.1 +/- 2.7% for 1.0 ppm ozone-exposed lungs, and 55.2 +/- 7.3% for 2.0 ppm ozone-exposed lungs.(ABSTRACT TRUNCATED AT 250 WORDS)

Pulmonary inflammation and epithelial injury in response to acute ozone exposure in the rat.

To document the time course of the inflammatory response and epithelial injury in the lung following an acute ozone exposure, rats were exposed to 1.0 ppm ozone for periods between 4 and 24 hr. Some of the exposures were followed by postexposure periods in filtered air for up to 20 hr. Bronchoalveolar lavage fluid (BALF) analysis and electron microscopic morphometry on centriacinar regions of lungs fixed by intravascular perfusion were used to assess the degree of pulmonary inflammation and epithelial cell necrosis. Total protein and numbers of neutrophils and epithelial cells in BALF increased as the duration of ozone exposure increased, while BALF macrophages decreased. Quantitation of the neutrophil response in centriacinar lung regions (capillary, interstitial, and epithelial/luminal compartments of the terminal bronchiole and proximal alveolar duct) by morphometry generally correlated with the BALF analysis, and revealed a greater volume per surface area epithelial basal lamina (Vs) of neutrophils in the terminal bronchiole compartments compared to proximal alveoli. Necrosis of epithelial cells in terminal bronchioles, primarily ciliated cells, occurred as early as 4 hr after initiation of ozone exposure, before marked neutrophil migration, and continued during periods of maximal neutrophil influx. We concluded that the early epithelial necrosis in terminal bronchioles during the first few hours of ozone exposure was primarily due to direct ozone toxicity, but could not rule out the possibility of neutrophils contributing to the injury at later time points, especially between 8 and 12 hr of exposure (during periods of maximal neutrophil migration).

89Strontium-induced bone marrow depression suppresses the early inflammatory response and fibrosis caused by intratracheal bleomycin.

To investigate the effect of bone marrow depression on the development of bleomycin-induced lung injury, F-344/Crl rats were given an intraperitoneal (IP) injection of 89SrCl2 (2 mCi/kg body weight) 7 days prior to the intratracheal (IT) instillation of 7.0 U/kg body weight bleomycin (Sr-bleomycin group). A second group of rats was given an IP injection of saline followed 7 days later by IT bleomycin (bleomycin group). Additional rats were given 89Sr IP and saline IT (Sr group) or saline IP and saline IT (saline group). Rats were sacrificed at 0, 3, 10, 21, and 30 days after the intratracheal instillations. 89Sr administration resulted in significantly lower numbers of circulating blood neutrophils and monocytes in the Sr-bleomycin group compared with the bleomycin group through at least the first 21 days following the IT instillations. Lymphocyte numbers were also depressed in the Sr-bleomycin group at days 3 and 21. Analysis of bronchoalveolar lavage fluid (BALF) revealed significantly reduced protein and lymphocyte numbers in the BALF from the 89Sr-bleomycin group compared with the bleomycin group at day 3, but not at later time points. Neutrophils in BALF were also lower (though not significantly) in the 89Sr-Bleomycin group at day 3. There was no difference in the number of BALF macrophages between the Sr-bleomycin and bleomycin groups at any time point throughout the study. Histology and morphometry showed the same trends as the BALF data with much less severe lesions in the 89SR-Bleomycin group compared with the bleomycin group at day 3, but not at later time points. At day 10, hydroxyproline values were significantly higher in the bleomycin group (47% increase above saline group) than the Sr-bleomycin group (only 18% increase above Sr group), but by day 21, there was no longer a significant difference between these two groups. These results demonstrate that bone marrow depression significantly suppresses the early inflammatory response and collagen deposition caused by a single IT dose of bleomycin, but has little effect on the resolution of bleomycin-induced injury.