Colin J. Dunn

Novel steroid‐based inhibitors of lung inflammation

It is now well-recognized that eosinophil products contribute to the mucosal damage which is a prominent feature of bronchial asthma. Eosinophils migrate to the bronchial epithelium following allergen inhalation in sensitive subjects, and increases in eosinophil numbers are associated with late-phase broncho-conslrictions [I]. The eosinophils capacity to generate lipid mediators [2.3] and the presence of cytotoxic. highly-charged, arginine-rich proteins in the eosinophil granules implicate this cell further as a contributor to mucosal inflammation. An attractive goal in the search for novel anti-asthma drugs would be to discover and develop compounds which inhibit eosinophil activation and/or prevent the influx of eosinophils into the airways. Steroids arc probably the most efficacious drugs currently available for the treatment of asthma, and improvements in symptoms in patients treated with steroids have been associated with a parallel reduction in blood eosinophil counts [4]. Corticosteroids have also been shown to abrogate the development of late-phase asthmatic reactions with a parallel reduction in eosinophil activity as measured by serum eosinophil cationic protein (ECP) levels [5]. Inhaled glucocorticoids which demonstrate few side-effects are increasingly being used worldwide as a prophylactic treatment for the disease. Novel inhaled glucocorticoids which have been designed to break down rapidly after reaching the systemic circulation are currently being developed and are even less likely to suppress the hypothalamo-pituitary adrenal axis. In this paper we have reviewed some of the novel. steroid-based molecules currently in clinical development for treating asthma. In addition we emphasize the change in direction the pharmaceutical industry has taken towards the use of inflammation-based animal models for the screening of novel compounds, and we also describe the activity of some novel, non-glucocorticoid. 21 -aminosteroids which may prove useful in the treatment of asthma.

Acute and chronic inflammatory responses to local administration of recombinant IL-1α, IL-β, TNFα, IL-2 and Ifnγ in mice

The contention that cytokines are important mediators of inflammation prompted the present studies which were designed to compare acute and chronic pathological effects of locally-administered recombinant (r) IL-1α, IL-1β, TNFα, IL-2 and Ifnγ. Acute (6 hr), resolving (48 hr) inflammation was induced by the following, in order of potency: rIL-1α>rIL-1β>rTNFα>rIfnγ=BSA (control) following a single sc. injection. However, only rIL-1β and rIL-2 initiated and maintained chronic granulomatous reactions when delivered locally from a sc. ethylene vinyl acetate (EVA) slow-release polymer. The predominance of macrophages in EVA-rIL-1β lesions contrasted with the proliferative lymphoid granulomata induced by EVA-rIL-2 implants. These “in vivo” observations reinforce, the roles of both IL-1β and IL-2 as potent mediators of chronic immunoinflammatory disease.

Anti-inflammatory/antiarthritic ketonic bisphosphonic acid esters

Bisphosphonate ester 2 is an inhibitor of inflammation, but is devoid of antiarthritic effects. SAR studies on a series of related bisphosphonate esters resulted in compounds 6e, 6i, 6j, and 6m, which exhibited excellent inhibition of an arthritis model, in addition to potent anti-inflammatory effects.

Application of computer-based histomorphometry to the quantitative analysis of methylprednisolone-treated adjuvant arthritis in rats

Analyses of paw edema and histomorphometry were performed on tibio-talar joints to determine arthritic pathological responses in untreated 28-day adjuvant-induced polyarthritis (AIP) rats, and to determine the drug effect on inhibiting these responses in AIP rats treated with methylprednisolone. Histomorphometric measurements were performed on regions including articular joint space, synovial tissue, articular and epiphyseal cartilage, epiphyseal and metaphyseal bone marrow, and endosteal and periosteal cortical bone surfaces. Analysis of paw edema indicated that paw volume was significantly increased in untreated AIP rats. This increase in paw volume was partially prevented in AIP rats treated with 0.3 mg methylprednisolone/kg per day, and completely prevented in AIP rats treated with the two higher dose levels (1 and 3 mg/kg per day). Histomorphometric analysis of untreated AIP tibio-talar joints showed decreased articular joint space whereas synovial tissue area increased and a minor, but significant, articular cartilage erosion area occurred. Epiphyseal growth cartilage area was decreased. Trabecular bone area in distal tibial epiphyseal and metaphyseal regions was markedly decreased whereas bone marrow area increased involving a large number of macrophages and osteoclasts. Eroded endocortical bone area was increased while cortical bone area decreased. Marked osteophyte proliferation occurred on the periosteal surface. These arthritic pathological changes were inhibited by the treatment of methylprednisolone in a dose-dependent fashion. The animals treated with the highest dose of methylprednisolone complete prevented the development of the AIP-induced pathological changes. These data confirmed qualitative histological evaluation of arthritic changes but did not correlate with the anti-edema effects of methylprednisolone (100% inhibition at 1.0 and 3.0 mg/kg, p.o.). It is suggested that quantitative histomorphometry be used to determine more precisely the AIP rat model and the effects of drugs on different histopathological features in this experimental model of arthritis in preference to paw edema which gives a more limited picture of the arthritic response.

ICAM-1 mediates leukocyte-endothelium adhesive interactions in the reversed passive Arthus reaction.

A murine anti‐rat intercellular adhesion molecule 1 (ICAM‐1) monoclonal antibody (mAb), 1A29, was used to investigate the importance of blood leukocyte‐associated β2‐integrin (CD11/CD18)/vascular endothelmm‐associated ICAM‐1 adhesive interactions in the reversed passive Arthus reaction (RPAR) in rats. An Arthus pleurisy reaction (4 h) was employed in these studies because it permits the accurate quantitation of polymorphonuclear neutrophil (PMN) influx into the pleural space and fluid accumulation. 1A29, which localized within Arthus lung lesions, caused a dose‐dependent (0.5–2.0 mg/kg, i.v.) inhibition of PMN influx (19–56%) and exudate volume (9–55%) in the Arthus pleurisy reaction. P7 (2 mg/kg, i.V.), a murine anti‐human P‐selectin mAb used as an isotype‐matched control tor 1A29, did not localize at the lung lesion site and was inactive. Immunohisto‐chemical analysis of lung tissue from 1A29‐treated rats demonstrated increased granulocyte accumulation in the alveolar capillaries compared with more extensive granulocyte emigration into the lung tissue and pleural space in P7‐treated rats and Arthus control rats; however, quantitative image analysis revealed increased numbers of lung granulocytes in 1A29‐treated rats compared with controls. Neither ICAM‐1 mRNA nor expression, assessed by immunocytochemistry, was increased above control levels in rats during the pleural Arthus reaction. Neutropenia was not observed in either 1A29‐ or P7‐treated rats.

Correlation of leukocyte interleukin-1 production with the stimulation of prostaglandin and tissue factor synthesis by human umbilical vein endothelial cells.

Human leukocyte suspensions (neutrophils 80–85%, monocyte 15–20%) were incubated alone or with cultured human umbilical vein endothelial cells. Leukocytes were either directly added to the endothelial cell cultures or separated from them by a 0.4 micron insert filter. Supernatants or cell lysates were obtained at 0.5, 1, 2, and 4 hours of incubation. Supernatants were assayed for the prostacyclin (PGI2) metabolite 6-keto prostaglandin F1α and prostaglandin E2 (PGE2) by radioimmunoassay and for interleukin-1 (IL-1) by the thymocyte co-mitogen assay. Cell lysates were analyzed for cell-associated procoagulant activity (PCA). Co-incubation of endothelial cells with leukocytes stimulated the synthesis of PGI2, PGE2, and PCA. These biochemical changes correlated partially with the release of IL-1 beta. The results suggest that IL-1 released in monocyte/neutrophil co-cultures can produce prothrombotic (increased PCA expression) and inflammatory changes (increased synthesis of vasodilatory and permeability enhancing PGI2 and PGE2) in endothelial cells. Neutrophils may represent a source of the released IL-1 and/or may act to stimulate monocyte release of this cytokine and thus play an important role in vascular pathology by a mechanism unrelated to their more direct cytotoxic activity.

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.

The effects of cyclosporin A on leucocyte infiltration and procoagulant activity in the mouse delayed hypersensitivity response in vivo

A model of delayed type hypersensitivity (DTH) to methylated bovine serum albumin was established and characterised in the mouse pleural cavity. Fluid exudate formation was delayed, peaking around 24 h and diminishing by 48-72 h. Exudate leucocyte accumulation was also delayed, rising by 24 h and reaching maximal levels between 24-72 h. Significant PMN leucocyte infiltration was observed early on, although macrophages dominated the reaction from 24 h onwards. Small numbers of lymphoid cells were also observed. Treatment with cyclosporin A (CsA) at 100 mg/kg p.o. around the time of antigen challenge significantly reduced fluid exudate volume but consistently failed to reduce either leucocyte infiltration or the PMN: mononuclear cell ratio throughout the reaction. CsA treatment enhanced DTH exudate leucocyte procoagulant activity (PCA) although leucocyte morphology remained unaltered. The development of carrageenan edema in the hind paws of mice was unaffected by CsA treatment (50-200 mg/kg p.o.). These results suggest that (1) CsA selectively suppresses DTH vascular permeability responses without affecting leucocyte accumulation and (2) CsA does not affect the lymphokine-mediated induction of leucocyte PCA associated with DTH reactions in vivo.

New Anti-Inflammatory/Anti-Arthritic Heterocyclic Bisphosphonates

Abstract. In the course of research toward a safe and effective treatment for rheumatoid arthritis, we identified new pyrazolo[1,5-a]pyrimidine and 4-pyrimidinone bisphosphonate esters, which are potent inhibitors of a murine model of chronic, cutaneous inflammation (delayed type hypersensitivity granuloma) and a murine antigen induced arthritis model. 9a has EC30 values of 0.01 and 0.005 mg/kg respectively and represents a new class of antiinflammatory/antiarthritic bisphosphonate ester.

The inflammatory potential of IL-2: local induction of a specific chronic granulomatous lesion in mice

Subcutaneous injection of recombinant human interleukin‐2 (rhuIL‐2) at 102‐104 U/mouse induced delayed (48 h) accumulation of mononuclear leukocytes with diffuse granulocytes, including eosinophils. Subcutaneous local infusion of rhuIL‐2 or recombinant murine IL‐2 (102–104 U/mouse) via implanted Alzet miniosmotic pumps in mice induced chronic inflammatory lesions characterized by infiltration of large vacuolated mononuclear leukocytes, lymphoid cells, and eosinophil foci; neovascularization, with high endothelial‐like cells, was prominent, exhibiting intravascular trapping and migration of large mononuclear leukocytes. Leukocyte infiltrates comprised T lymphocytes (CD4+; CD8+), B lymphocytes, and macrophages. Control infusions of bovine serum albumin (BSA) induced weak fibrotic lesions with sparse macrophage infiltration and minimal accumulation of lymphocytes; VLA4+ and ICAM‐1+ leukocyte infiltrates were significantly greater in IL‐2‐induced lesions compared with BSA‐induced lesions. Quantitative image analysis showed significantly increased lesion size in the IL‐2‐induced lesions compared with those induced by BSA infusion. The vascularity of IL‐2‐induced lesions assessed by immunostaining for platelet‐endothelial cell adhesion molecule was increased compared with control, BSA‐induced lesions mainly due to neovascularization. ICAM‐1 and VCAM‐1 expression was significantly enhanced in IL‐2 lesions. No systemic pathological changes were observed following IL‐2 infusion. We conclude that local slow‐release of IL‐2 causes the evolution and maintenance of a specific chronic inflammatory lesion.