Pharmacology of the biological response modifier bropirimine (PNU-54461) on experimental autoimmune encephalomyelitis (EAE) in mice.

In murine severe experimental autoimmune encephalomyelitis (EAE), an animal model for the human disease multiple sclerosis (MS), we tested the efficacy of a 5-halo-6-phenyl pyrimidinone compound, bropirimine (PNU-54461). We observed that the compound is active in suppressing EAE when administered orally, a significant pharmacological advantage compared to some current therapies for the treatment of MS. Furthermore, bropirimine was most efficacious when dosing was begun 5-10 days after injection of myelin basic protein, the protein isolated from the central nervous system and used for inducing EAE in our model. This is a period of time following the initial immunological events leading to the disease, when large-scale leukocyte infiltration into the central nervous system begins. Following oral dosing, bropirimine peaked in the blood within 3 h and was cleared to undetectable concentrations within 16-18 h. Despite the pharmacokinetics in the blood, bropirimine was fully efficacious when dosed orally every two or three days. Surprisingly, bropirimine treatment did not result in a statistically significant decrease in leukocyte infiltration into the lower spinal cord, unless the compound was dosed daily at a high concentration. We also observed the concentration and time course of alpha-interferon in blood following oral dosing of bropirimine. The kinetics of interferon in the blood are similar to, but clearly distinguishable from, the pharmacokinetics of bropirimine in the blood. It is not clear whether or not the induction of interferon plays a key role in the efficacy of bropirimine. Nevertheless, the results using bropirimine in EAE suggest that the compound may be useful for the treatment of multiple sclerosis.

Pharmacokinetic properties, induction of interferon, and efficacy of selected 5-halo-6-phenyl pyrimidinones, bropirimine analogues, in a model of severe experimental autoimmune encephalomyelitis.

We showed previously that a 5-halo-6-phenyl-pyrimidinone, bropirimine (PNU-54461), inhibited progression of severe experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis. In the work presented here, we examined the activity of a group of chemically-related bropirimine analogues. First, the pharmacokinetic properties of the bropirimine analogues were examined in normal mice following oral dosing. After equal oral doses, both PNU-56169 and PNU-63693 were found in the blood of normal mice at equal or higher concentrations than bropirimine, but PNU-54462 and PNU-56359 were present in blood only at very low concentrations. Next, we examined the bropirimine analogues for activity in our model of severe EAE. At a dose of 400 mg/kg administered orally every second day PNU-56169 nearly completely blocked EAE progression, but was ineffective at 100 mg/kg. PNU-63693 was effective in EAE at concentrations of 200 mg/kg, 100 mg/kg, 50 mg/kg, and as low as 25 mg/kg. Histopathology was examined by observing leukocyte infiltration into the lower spinal cords of the mice. Treatment with 400 mg/kg of PNU-56169 and doses of 25, 50, 100, and 200 mg/kg of PNU-63693 significantly inhibited leukocyte infiltration into the lower spinal cord of treated mice in a dose-dependent manner. Orally administered PNU-56169 and PNU-63693 also stimulated significant concentrations of IFNalpha in the serum of treated mice, which may be related to the efficacy of the compounds in EAE. However, the correlation between IFNalpha in the blood and efficacy in treating EAE was not exact. Thus, PNU-56169 and PNU-63693 were delivered to the blood following oral dosing, induced significant concentrations of IFNalpha in the blood, and were equally or more potent than PNU-54461 in inhibiting clinical signs of EAE. The results suggest that 5-halo-6-phenyl-pyrimidinones are an interesting class of compounds to investigate for development in the treatment of multiple sclerosis.

Role of PECAM-1 (CD31) in neutrophil transmigration in murine models of liver and peritoneal inflammation.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is thought to be critical for transendothelial migration of leukocytes, including neutrophils. Because neutrophil-mediated liver injury during endotoxemia is dependent on transmigration, we investigated the role of PECAM-1 in the pathophysiology of endotoxin-induced liver injury. Male C3Heb/FeJ mice were treated with galactosamine (Gal) and endotoxin (ET) (700 mg/kg Gal/100 micrograms/kg ET), and liver sections were stained for PECAM-1 expression. Control livers showed the presence of PECAM-1 on endothelial cells of large vessels but not in sinusoids. Gal/ET treatment did not change the expression pattern of PECAM-1. Gal/ET-induced liver injury (area of necrosis: 38 +/- 3%) was not attenuated by treatment with 3 mg/kg of the antimurine PECAM-1 antibody 2H8. The antibody had no effect on sequestration and transmigration of neutrophils in sinusoids or the margination of neutrophils in large vessels. In contrast, 2H8 inhibited glycogen-induced neutrophil migration into the peritoneum by 74%; this effect correlated with PECAM-1 expression in the intestinal vasculature. Thus PECAM-1 is neither expressed nor inducible in hepatic sinusoids and is consequently not involved in neutrophil transmigration in the liver during endotoxemia. On the other hand, expression of PECAM-1 in mesenteric veins is critical for peritoneal neutrophil accumulation.

Involvement of intercellular adhesion molecule-1 in the antigen-induced infiltration of eosinophils and lymphocytes into the airways in a murine model of pulmonary inflammation.

We investigated the effects of in vivo intraperitoneal treatment with the rat monoclonal antibody (mAb), YN1.7.4 (YN1) against intercellular adhesion molecule-1 (ICAM-1) on the ovalbumin (OA)-inhalation-induced infiltration of leukocytes into the airways of OA-sensitized mice. YN1 (100 to 400 microg) given over a period of 72 h dose-dependently reduced the influx of lymphocytes and eosinophils into the bronchial lumen by > 60% and > or = 70%, respectively, when compared with saline or purified rat IgG-treated controls. Alveolar macrophages (AM) in the bronchoalveolar lavage fluid (BALF) were also decreased by > 50%. Lung tissue inflammation as determined by histopathologic examination was reduced. The number of neutrophils in the blood of OA-sensitized mice 3 days after challenge was significantly increased by treatment with YN1. However, at 24 h and 72 h after OA-challenge, the numbers of eosinophils and mononuclear cells in the bone marrow were reduced by YN1 treatment. Additionally, at 72 h after OA-challenge, the numbers of bone-marrow neutrophils were depressed. BALF levels of interleukin-5 (IL-5) and of IgA were lower for YN1-treated mice than for controls. With increasing doses of YN1, the levels of anti-ICAM-1 mAb in the plasma were proportionally increased. To correlate these results with YN1 treatment, blood and BALF T cells and BALF eosinophils were examined with flow cytometry. Blood T cells from YN1-treated mice were unable to bind phycoerythrin (PE)-labeled anti-ICAM- mAb ex vivo. These results implied that ICAM-1 on these cells was bound (occupied) by YN1 administered in vivo. Dose-related decreases were observed in the percentage and mean channel fluorescence (MCF) values of ICAM-1+ BALF T cells and eosinophils. The percentages of CD11a+ or CD49d+ eosinophils were also suppressed. Our data suggest that ICAM-1 is an important molecule involved in the recruitment of leukocytes into the airways of sensitized mice after pulmonary challenge.

Role of very late activation antigen-4 in the antigen-induced accumulation of eosinophils and lymphocytes in the lungs and airway lumen of sensitized brown Norway rats.

We used flow cytometry and treatment in vivo with a monoclonal antibody (mAb), TA-2, to the alpha 4 integrin to investigate the role of alpha 4 beta 1, CD49d/CD29 (VLA-4) in antigen-induced lung inflammation in Brown Norway (BN) rats. Ovalbumin (OVA) inhalation induced an accumulation of eosinophils and lymphocytes in the lungs and bronchoalveolar lavage (BAL) fluid of sensitized BN rats at 24 h after challenge. Phenotypic analyses demonstrated that the percentages of T cells expressing detectable alpha 4 and CD25 in the bronchial lumen after antigen challenge were dramatically increased compared with blood and lymph node T cells. The mean channel fluorescence values of alpha 4 expression were also increased on BAL T cells compared with blood or lymph node T cells. Treatment of OVA-sensitized rats in vivo with total cumulative doses of 0.75 to 6 mg/kg TA-2 mAb intraperitoneally produced dose-related increases in circulating TA-2 and a peripheral blood lymphocytosis, basophilia, and eosinophilia. Flow cytometric analysis of the peripheral blood T cells after in vivo TA-2 mAb administration showed decreases in detectable alpha 4 when these cells were examined ex vivo. Treatment with TA-2, but not an isotype-matched control mouse immunoglobulin G1 mAb, markedly inhibited the OVA-induced recruitment of lymphocytes and eosinophils into the airway lumen. Very few CD3+CD49d+ cells migrated into BAL fluid following anti-alpha 4 mAb treatment in vivo. Treatment with TA-2 also significantly attenuated OVA-induced inflammatory histopathology. We conclude that VLA-4 is a critically important adhesion molecule involved in antigen-specific lung inflammation in sensitized BN rats.