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Antifibrotic Effect of Combined Treatment with Neuroleptic Drug and Immobilized Hyaluronidase in Pulmonary Fibrosis A. M. Dygai, E. G. Skurikhin, N. N. Ermakova, O. V. Pershina, V. A. Krupin, A. M. Reztsova, L. A. Ermolaeva, E. S. Khmelevskaya, I. E. Stepanova, A. V. Artamonov*, A. A. Bekarev*, P. G. Madonov*, D. N. Kinsht*, V. E. Goldberg, and T. A. Semiglazova
Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 154, No. 9, pp. 312-316, September, 2012 Original article submitted April 4, 2011 Using the model of lung fibrosis induced by intratracheal administration of bleomycin we studied anti-fibrotic activity of combined treatment with neuroleptic haloperidol and hyaluronidase immobilized on polyethylene oxide using electron-beam synthesis. It was shown that successive administration of immobilized hyaluronidase and the neuroleptic drug inhibits deposition of collagen fibers in the bleomycin-treated lungs. Combined treatment with the test compounds reduced swelling of the alveolar epithelium, exudation and infiltration of the alveolar interstitium and alveolar passages by inflammatory cells, and desquamation of alveolocytes into alveolar lumen, so that the alveolar-capillary membrane function was preserved. Key Words: bleomycin; pulmonary fibrosis; neuroleptic drug; hyaluronidase immobilized on polyethylene oxide In the last 30 years, the anti-inflammatory and antifibrotic properties of more than 240 compounds have been described in various models of pulmonary fibrosis [6,12]. However, none of the compounds showed antifibrotic activity under clinical conditions comparable to that in experimental studies. Remodulation of extracellular matrix is a possible approach to reduce the fibrogenesis. According to modern ideas, radiationand bleomycin-induced fibrosis is characterized by not only damage to endothelium, epithelial structures, and pulmonary interstitium, inflammation, increased content of fibrogenic cells, deposition of pathologic collagen [13], but also increased concentration of hyaluronic acid in the parenchyma [6,11]. In the experiment, intranasal inoculation of hyaluronidase (HA) blocked the development of bleomycin-induced pulmonary fibrosis [6]. These data allow Institute of Pharmacology, Siberian Division of the Russian Academy of Medical Sciences, Tomsk; *Scientific Future Management Company, Novosibirsk, Russia. Address for correspondence:
[email protected]. O. V. Pershina
us to consider HA as a component of antifibrotic therapy for the treatment of pulmonary fibrosis. However, in view of rapid degradation of HA, its pegylated form is preferable, because HA conjugated with polyethylene glycol has high pharmacological activity, greater physical stability, and along with that, lower solubility, reduced sensitivity to proteolytic enzymes, and lower immunogenicity (polyethylene glycol and polyethylene oxide “close” the antigenic epitopes of the protein), than the native analogue [1,14]. The antifibrotic effect of HA is associated with inhibition of collagen deposition and production of profibrotic transforming growth factor β1 (TGF-β1), whereas antiserotonin drugs directly affect collagen synthesis [4,8,10]. However, administration of compounds simultaneously reducing activity of several aminergic systems is more promising in the treatment of lung fibrosis. Thus, bleomycin-induced fibrotic changes in the lungs were less pronounced during blockade of central D2, D3, D4 dopaminergic receptors and postsynaptically located serotonin 5-HT2A
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Bulletin of Experimental Biology and Medicine, Vol. 154, No. 3, January, 2013 PHARMACOLOGY AND TOXICOLOGY
and lung morphology was studied. For histological analysis, paraffin blocks were prepared by the standard technique from the middle part of the right lung. The sections were obtained from each block and stained with hematoxylin and eosin to evaluate the content of lymphocytes, macrophages, neutrophils, and plasma cells and by the method of van Gieson (for connective tissue) [2,3]. The results were analyzed by the standard methods of variation statistics. The significance of differences was evaluated by parametric Student’s t test and nonparametric Mann–Whitney U test.
receptors with neuroleptic drug haloperidol than after administration of antiserotonin preparation [2,4]. A question arises whether combined administration of neuroleptic drug and pegylated HA is feasible under conditions of lung fibrosis. Here we studied the effects of neuroleptic drug during combined treatment with HA, immobilized on polyethylene oxide, on the development of bleomycininduced pulmonary fibrosis.
MATERIALS AND METHODS Experiments were performed on 7-8-week-old C57Bl/6 mice (n=156). The mice (certified class I conventional strain) were obtained from the nursery of the Institute of Pharmacology. Lung fibrosis was induced by intratracheal administration of bleomycin (Bleomycetin, Lensfarm Company) in a single dose of 80 μg in 30 μl saline. We studied anti-inflammatory and antifibrotic activities of individual and combined administration of immobilized HA (pegHA) modified with activated polyethylene oxide (molecular weight 1500 Da) using electronbeam synthesis (both Scientific Future Management) and neuroleptic haloperidol (Gedeon Richter A.O.). The mice were divided into 4 groups. In group 1, 30 μl bleomycin was injected intratracheally. In group 2, neuroleptic haloperidol in a dose of 1.5 mg/kg was injected intraperitoneally 3 h and 1, 3, 6, 7, 13-21 days after intratracheal administration of the cytostatic. In group 3, pegHA (800 U/kg) was introduced intranasally 2 h and 1, 3, 7 days after intratracheal administration of bleomycin. In group 4, pegHA (800 U/kg) was administered 2 h and 1, 3, 7 days after the administration of the cytostatic and the neuroleptic (1.5 mg/kg, intraperitoneally) after 3 h and on experimental days 1, 3, 6, 7, 13-21. In this case, haloperidol was injected 1 h after pegHA administration on days 1, 3, and 7. Intact animals (intact controls) served as a background. On days 7, 14 and 21 after bleomycin administration, the mice were sacrificed by CO2 overdose
RESULTS The neuroleptic haloperidol and pegHA administered individually significantly changed the morphology of murine lung in pulmonary fibrosis. Thus, neuroleptic reduced the severity of the inflammatory response in the lung against the background of bleomycin administration (group 3). Swelling of the alveolar epithelium, exudation, and infiltration of the walls and alveolar lumens with inflammatory cells were less pronounced. The inflammatory infiltrate contained less neutrophils compared to bleomycin controls (group 1). At the same time, neuroleptic decreased the severity of alveolocyte desquamation into the alveolar lumen, thus preventing alveolar obliteration, preserving the function of the alveolar-capillary membrane, and inhibiting cytostaticinduced destruction of the lung tissue (Fig. 1). Inflammatory infiltration in the bleomycin-treated lungs in mice receiving pegHA (group 2) persisted throughout the observation period. In contrast to the lungs of the animals treated with bleomycin, where infiltration was localized mainly at the root of the lung, diffuse infiltration was observed in the lungs of mice with pegHA. In addition, the number of alveolar macrophages was increased in the infiltrate in animals with HA compared to that in group 1 (Fig. 1). Staining by the method of van Gieson revealed extensive deposition of collagen fibers in the lung tis-
TABLE 1. Content of the Connective Tissue in the Lungs of C57Bl/6 Mice after Intratracheal Administration of Bleomycin (% of Collagen Fibers from Total Lung Tissue Area) Group Tome points, days 1
2
3
7
3.07±0.07*
2.09±0.15*
2.22±0.12*+
-
14
3.91±0.07*
2.75±0.12*+
2.12±0.12*+
1.22±0.14+
21
4.24±0.16*
2.12±0.13*+
2.24±0.18*+
1.4±0.2+
Note. p
