Bulletin of Experimental Biology and Medicine, Vol. 157, No. 1, May, 2014
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GENERAL PATHOLOGY AND PATHOPHYSIOLOGY Antifibrotic Activity of Conjugates Based on Amphiphilic Pluronic F68 and Hydrophobic Pluronic L31 with Hyaluronate-Endo--N-Acetylhexosaminidase in Pulmonary Fibrosis E. G. Skurikhin, A. M. Reztsova, N. N. Ermakova, V. A. Krupin, O. V. Pershina, E. S. Khmelevskaya, A. V. Artamonov*, A. A. Bekarev*, P. G. Madonov*, D. N. Kinsht*, D. V. Reykhard, V. E. Goldberg, and A. M. Dygai
Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 157, No. 1, pp. 9-13, January, 2014 Original article submitted October 8, 2013 Antifibrotic activity of intranasally administered conjugates of pluronics L31 and F68 with hyaluronate-endo-β-N-acetylhexosaminidase was studied in C57Bl/6 mice under conditions of single and repeated bleomycin-induced injury to the alveolar epithelium. Conjugates were prepared using the technique of protein immobilization with ionizing radiation. We demonstrate that in cases of single and repeated injuries to the alveolar epithelium, the conjugates administered during phases of inflammation or deposition of fibrotic masses prevent the development of pulmonary fibrosis. The conjugates demonstrated more pronounced antifibrotic activity than hyaluronate-endo-β-N-acetylhexosaminidase. The conjugate based on hydrophobic pluronic L31 showed higher effectiveness in comparison with the conjugate based on amphiphilic pluronic F68. Key Words: lung fibrosis; polimerosome; hyaluronate-endo-β-N-acetylhexosaminidase; pluronic The antifibrotic effect of hyaluronate-endo-β-N-acetylhexosaminidase (HN), an enzyme of the hyaluronidase family, was reported by many researchers [1,3]. HN prevents death of alveolar epithelium cells and development of pulmonary fibrosis (PF) in mice. Specific activity of HN is mediated by cleavage of hyaluronic Department of Pathophysiology and Regenerative Medicine Research Institute of Pharmacology, Siberian Branch of the Russian Academy of Medical Sciences, Tomsk; *Siberian Center of Pharmacology and Biotechnology, Novosibirsk, Russia. Address for correspondence:
[email protected]. O. V. Pershina
acid into low-molecular weight fragments. However, due to enzymatic hydrolysis, the concentration of HN in tissues, including lungs, decreases rapidly. This leads to restitution of profibrotic viscosity of the hyaluronan matrix and, as a consequence, to progression of PF. Different carriers (microemulsions, liposomes, polyethylene glycol, etc.) are used for the delivery of pharmacologically active molecules to their targets and prevention of their hydrolysis in aggressive environments. Pluronics, block-copolymers of polyoxyethy-
0007-4888/14/15710005 © 2014 Springer Science+Business Media New York
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Bulletin of Experimental Biology and Medicine, Vol. 157, No. 1, May, 2014 GENERAL PATHOLOGY AND PATHOPHYSIOLOGY
lene and polyoxypropylene, are proposed as new and safer carriers [12]. Polymeric nanoparticles are synthesized chemically, and form nanoaggregates (micelles) under certain conditions. A micelle can be composed of a combination of two pluronics or of polyethylene glycol and a pluronic [8]. Basically, these structures are coming into use in the targeted therapy [5,8,9,14]. Polymeric micelle Itraconazole for treatment of pulmonary fungal infections in immunosuppressed patients has been synthesized and experimentally tested [11]. Polymeric micelles can cross the blood-brain barrier and enter the brain, which opens up new prospects in the treatment of neurodegenerative diseases [15]. Here we studied antifibrotic properties of poloxamer-HN based on pluronics L31 and F68 under conditions of bleomycin-induced PF.
MATERIALS AND METHODS Experiments were carried out on 7-8-week-old C57Bl/6 mice (n=290), first category animals, conventional mouse strain, obtained from the breeding nursery of Research Institute of Pharmacology of Siberian Branch of Russian Academy of Medical Sciences (certificate is available). Toxic alveolar epithelial injury, partially reversible pulmonary fibrosis, was simulated by single intratracheal administration of 80 μg bleomycin (Bleotsin, Nippon Kayaku Co, Ltd.) to mice in 30 μl saline [13]. Recurrent toxic alveolar epithelial injury was simulated by single intratracheal administration of bleomycin (80 μg per mouse in 30 μl saline) on experimental day 0 followed (on days 7, 14, 20) by intranasal administration of the drug in a dose of 80 μg per mouse in 15 μl of saline (irreversible PF). The day of intratracheal bleomycin administration was taken as experimental day 0. Specific effects of conjugates of polymeric carriers pluronics L31 (predominance of hydrophobic properties) and F68 (amphiphilic properties) with HN were estimated and compared to those of HN enzyme (all substances produced by Siberian Center for Pharmacology and Biotechnology). The conjugates were prepared using the technique of protein immobilization with ionizing radiation. The conjugates and the enzyme were injected during the inflammatory (preventive mode, experimental days 1-5) and fibrotic (therapeutic mode, experimental days 10-20) phases of PF development. The daily dose of HN in conjugates and pure enzyme was 16 U. Three experimental series were performed. In series I, we studied antifibrotic activity of intranasally administered conjugates of pluronic L31 with HN (L31-HN) and pluronic F68 with HN (F68-HN) under conditions of partially reversible PF. The animals with
partially reversible PF without treatment were used as the control (group 1); groups 2, 3, and 4 consisted of mice with partially reversible PF treated with L31HN, F68-HN, or HN-1, respectively on day 5 of the experiment (inflammatory phase of the disease, preventive administration). In addition, specific activity of L31-HN (group 5) and F68-HN (group 6), as well as activity of the HN enzyme (group 7), administered on days 10-20 (fibrotic phase of disease; therapeutic mode of administration), were estimated in the model of single bleomycin-induced alveolar epithelial injury. Specific activity of L31-HN, the most effective conjugate (based on results of experimental series I), when administered intravenously (group 8) and intragastrically (group 10) in the therapeutic mode, was studied and compared to that of HN in the model of partially reversible PF. Experimental series III was focused on the influence of L31-HN, administered intranasally in therapeutic mode on experimental days 10-20, on development of irreversible PF (group 12). Additionally, we evaluated antifibrotic activity of L31-HN in case of administration on days 10-15 (group 13). Animals with irreversible PF without treatment were in group 14. Intact animals were used in all sets of experiment as background (intact control). Animal grouping is presented in Table 1. On experimental day 21, the mice were sacrificed with CO2 overdose, and lung morphology was analyzed. For histological examination, paraffin blocks containing specimen of the middle lobe of the right lung were prepared by standard methods. Sections were made from each block and stained by van Gieson [2]. Mathematical processing of results was performed using standard analysis of variance. Significance of differences was assessed using parametric Student’s t test or nonparametric Mann–Whitney U test.
RESULTS Experimental series I showed that intranasal administration of conjugates L31-HN and F68-HN prevented collagen deposition in mouse lungs under conditions of partially reversible PF (Fig. 1). Staining after van Gieson showed ~2-fold reduction (p=0.0063) of the area of fibrous masses deposition in the lungs of diseased animals treated with F68-HN in preventive mode in comparison with the pathological control (group 1). However, despite pronounced shifts, the area occupied by the connective tissue did not reach the level of intact control. L31-HN conjugate was more effective in PF prevention than F68-HN. The area of collagen fibers in group 2 was reduced to the level of intact control (Fig. 2).
E. G. Skurikhin, A. M. Reztsova, et al.
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TABLE 1. Distribution of Animals in Experimental Series Series
Group
Conjugate
Time of conjugate administration
Mode of conjugate administration
Partially reversible PF I
II
1
–
–
–
2
L31–HN
days 1-5 (inflammation phase)
Intranasally
3
F68-HN
4
HN
5
L31-HN
6
F68-HN
7
HN
8
L31-HN
9
HN
10
L31-HN
11
HN
12
L31-HN
days 10-20 (phase of fibrotic masses deposition)
13
L31-HN
days 10-15 (phase of fibrotic masses deposition)
14
–
–
days 10-20 (phase of fibrotic masses deposition)
days 10-20 (phase of fibrotic masses deposition)
Intravenously
Intragastrically
Irreversible PF III
Antifibrotic activity of conjugates was also observed after intranasal administration during the fibrotic phase of the disease development: the connective tissue content the lungs after administration of L31-HN and F68-HN decreased by 2.25 (p=0.0037) and 1.7 times (p=0.0047; Figs. 1 and 2), respectively, in comparison with untreated partially reversible PF. Thus, in case of a single bleomycin-induced alveolar epithelial injury intranasal administration of conjugates L31-HN and F68-HN in the preventive mode inhibited the formation of fibrotic masses in the lungs. Administration of conjugates in the therapeutic mode promoted resorption of previously formed fibrosis foci. Antifibrotic activity of conjugates was vastly superior to the ability of HN to reduce the content of connective tissue in lungs. Effect of the conjugate based on hydrophobic pluronic L31 was more pronounced than that of the conjugate based on amphiphilic pluronic F68. In search of the best administration route (series II), we studied specific activity of conjugates L31HN and F68-HN after intravenous and intragastric administration in the model of partially reversible PF and compared it to that of HN enzyme. It was found that intravenous and intragastric administration of conjugates and enzyme in the preventive and therapeutic modes had no effect on lung histology in mice with PF.
Intranasally
–
In clinical practice, the idiopathic PF at the stage of inflammation is extremely rarely diagnosed and the development of the disease is impossible to predict. In this connection, the drugs preventing the progression of pulmonary fibrosis are most important for clinical application [1]. It is believed that compounds effective during the most severe phase of the disease (progressive deposition of collagen fibers) can be considered as “true” antifibrotic factors [4]. In the model of repeated bleomycin-induced injury to the alveolar epithelium (experimental series III) we demonstrated that intranasal administration of L31-HN on experimental days 10-20 prevented lung damage and blocked fibrogenesis (Fig. 3). Survival rate among animals treated with the substance was significantly higher. Under these conditions, the target enzyme did not substantially affect fibrogenesis. The positive effect of L31-HN remained practically the same after reduction of the treatment from 10 days (group 12) to 6 days (group 13). The conjugate based on L31-HN was effective in irreversible PF during the phase of collagen fibers deposition. This suggests that the L31-HN can be regarded as a new and promising tool for PF treatment. This pharmacological construct is very interesting, because polymerosome as the carrier of the enzyme allows varying the duration of treatment without impairment of treatment efficiency.
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Bulletin of Experimental Biology and Medicine, Vol. 157, No. 1, May, 2014 GENERAL PATHOLOGY AND PATHOPHYSIOLOGY
Fig. 1. Lung morphology in C57Bl/6 mice with partially reversible PF on experimental day 21. a) Intact control; b) partially reversible PF; c) L31-HN administration on days 1-5; d) F68-HN administration on days 1-5; e) L31-HN administration on days 10-20; f) F68-HN administration on days 10-20. Picrofuchsin staining after van Gieson, 100.
The mechanisms underlying the effects of polymerosomes are intensively discussed. According to some reports, polymerosomes are adsorbed on the basal and cell membranes [10], which is associated with marked changes in physicochemical properties of the
membranes (viscosity, permeability) [6], followed by degradation of the polymerosome [7]. On this background, the action of the drugs is implemented. The conjugates studied here (~100 nm in diameter, so-called “respirable fraction”) produce the maximum pharma-
E. G. Skurikhin, A. M. Reztsova, et al.
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Fig. 2. Connective tissue content in the lungs of C57Bl/6 mice with partially reversible PF on experimental day 21. 1) Intact control; 2) bleomycin control; 3) L31-HN on days 1-5; 4) F68-HN on days 1-5; 5) HN on days 1-5; 6) L31-HN on days 10-20; 7) F68-HN on days 10-20; 8) HN on days 10-20. Ordinate: content of collagen fibers in the lungs. p
