DOI 10.1007/s10517-014-2620-z Bulletin of Experimental Biology and Medicine, Vol. 157, No. 5, September, 2014
580
PHARMACOLOGY AND TOXICOLOGY Antithrombotic Activity of a New Benzimidazole Derivative in the Thrombosis Model in Mice A. V. Smirnov, A. F. Kucheryavenko*, and A. A. Spasov* Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 157, No. 5, pp. 595-598, May, 2014 Original article submitted April 26, 2013 Antithrombotic activity of a new benzimidazole derivative RU-891, characterized by antiaggregant activity, vs. reference drug acetylsalicylic acid was studied on the epinephrinecollagen thrombosis model in mice. Antithrombotic activity of RU-891 3-fold surpassed that of the reference drug. Histological studies showed that RU-891 significantly reduced the relative and mean areas of thrombi in sections of mouse lungs in comparison with the control. Key Words: antithrombotic activity; thrombosis; platelet aggregation; RU-891; acetylsalicylic acid Arterial thrombosis is an important problem of cardiology, because this condition often becomes the cause of sudden death, myocardial infarction, vascular complications of diabetes mellitus, and often deteriorates the efficiency of surgery for coronary heart disease [7]. The increase in platelet aggregation activity is one of the most important pathogenetic mechanisms of intravascular clotting [4]. Our previous studies have demonstrated that RU-891, a new benzimidazole derivative, suppressed platelet aggregation [2]. Since platelets play the key role in arterial thrombosis, we have studied the antithrombotic effect of RU-891 on the thrombosis model in mice in vivo.
MATERIALS AND METHODS The study was carried out on male and female outbred albino mice (n=40; 20-25 g) kept in a vivarium (22-24oC, 40-50% humidity) at natural illumination on standard fodder (GOST R 50258-92) with due consideration for laboratory practice regulations for preclinical studies in the Russian Federation (GOST Department for Pathological Anatomy, *Department for Pharmacology, Volgograd State Medical University, Russia. Address for correspondence: [email protected]. A. F. Kucheryavenko
Z 51000.3-96 and 1000.4-96) and regulations and International Recommendations of the European Convention for Protection of Vertebrates Used in Experimental Studies (1997). Antithrombotic activity of RU-891 (Research Institute of Physical and Organic Chemistry, South Federal University) was studied in vivo on the thrombosis model reproduced as described previously [5]. Thrombosis was induced with a mixture of collagen and epinephrine (0.5 and 0.06 mg/kg, respectively). The mixture (0.1 ml) was infused in the caudal vein over 10 sec. Acetylsalicylic acid (20 mg/kg) served as the reference drug. The test substance was injected in a dose of 43 mg/kg (equimolal to the dose of the reference drug) 2 h before thrombosis reproduction. Controls were injected with an equivalent volume of the solvent. The drug efficiency was evaluated by the number of survivors in comparison with the control group and the presence of clots in lung vessels. The survivors were observed over 24 h. Dead mice and survivors (after euthanasia with ether overdosage) were autopsied for histological study of the lungs. In controls, histological analysis of the liver, heart, kidneys, and brain was carried out. The protocol of experiment was approved by the Regional Ethic Committee (decision No. 94-2009).
0007-4888/14/15750580 © 2014 Springer Science+Business Media New York
A. V. Smirnov, A. F. Kucheryavenko, and A. A. Spasov
The material was fixed (24 h in 10% neutral buffered formalin, pH 7.4), dehydrated, and embedded in paraffin. Sections (4-6 μ) were sliced from paraffin blocks and stained with hematoxylin and eosin by the standard method [3]. Histological preparations were photographed by an Olympus digital camera (4.0 megapixels) attached to Micros microscope (×10 and ×40 objectives; ×10 ocular). Signs of clotting in the walls of the muscular arterial vessels were evaluated morphologically. The data were processed by basic statistical analysis using Statistica 5.0 software (StatSoft, Inc.). Histological findings were processed using VideoTest-Morpho-4 software and Mann–Whitney test. The significance of the antithrombotic effects of the test substances (difference in the percentage of survivors in the control and experimental groups) was statistically evaluated by Fisher’s F test.
RESULTS In the control group, animal mortality was 95%, which was in line with the results of other scientists [1,6]. Characteristic signs of disorders in the respiratory function of the lungs were noted: higher respiration rate and shallow breathing, manifest exophthalmia and changed color of the iris. Hind limb paresis was observed: mice did not move even in response to pushing, could not withdraw the hind paws if pressed strongly. Tetanic convulsions were observed – the animals were in a characteristic posture with the hind paws straight backward. The mice died from asphyxia within 1-3 min after injection of the thrombotic agents. RU-981 in the studied dose prevented death of 90% animals, their motor activity restored completely
581 TABLE 1. Effects of RU-891 in a Dose Equimolal to the Acetylsalicylic Acid Dose on the Survival of Mice in the Total Systems Collagen-Epinephrine Thrombosis Model Dose, mg/kg
Number of dead animals
Survivors, %
Control (n=20)
–
19
5
RU-891 (n=10)
43
1
90*+
Acetylsalicylic acid (n=10)
20
7
30
Experiment conditions
Note. p≤0.01 in comparison with *control, +animals treated with acetylsalicylic acid (F test).
within 1.0-1.5 min (Table 1). The rest 10% before death were visually more active than controls, motor disorders were less severe, and the manifestations of generalized thrombosis developed slower (within 3-5 min) than in controls. The protective effect of acetylsalicylic acid was negligible. Only 30% animals survived in the group receiving acetylsalicylic acid. LD50 of RU-891 (1260 mg/kg) is high, and we can speak about pronounced antithrombotic activity of this substance manifesting at a dose by several orders of magnitude lower than the toxic dose. According to a previous study [5], the direct cause of mortality in the studied model was massive occlusion of the lung microvessels with platelet aggregations. Histological studies of the liver, heart, kidneys, and brain of animals injected with the thrombotic agents detected virtually no clots in the microcirculatory vessels of these organs, which was in line with the previous reports [1,5].
Fig. 1. Effects of RU-891 on clotting in the lungs of mice induced by collagen and epinephrine. Hematoxylin and eosin staining, ×100. a) control; b) RU-891. Arrows: white clots in microvessels.
Bulletin of Experimental Biology and Medicine, Vol. 157, No. 5, September, 2014 PHARMACOLOGY AND TOXICOLOGY
582
TABLE 2. Effects of RU-891 in a Dose Equimolal to the Acetylsalicylic Acid Dose on Some Morphometric Values of the Clots in Mouse Lung Vessels (M±m) Experiment conditions
Clot area, %
Mean area of a clot in section, μ2
Control (n=5)
3.538±1.982
1423.500±0.328
RU-891 (n=5)
1.047±0.290*
399.8±185.2**
Acetylsalicylic acid (n=5)
1.327±0.152*
1337.7±888.5
Note. *p≤0.01, **p≤0.001 in comparison with the control (Mann– Whitney test).
Microscopic studies showed the predominance of medium-sized alveoli in lung specimens from controls; white microclots were found in an appreciable part of microvessels; these clots adhered to the vascular wall in the vascular lumen (Fig. 1). Manifest dilatation (36-fold) of thrombosed capillaries in the alveolar septa was seen. These changes in some cases were associated with focal lesions in the capillary wall and in the respiratory epithelium. Mixed clots were found in some arterioles. In addition to white clots, marginal stasis of leukocytes was found in some venules. Morphometry of the lung sections from control mice showed that clot area in these sections was 3.538±1.982% and the mean area of a clot in the sections 1423.500±0.238 μ2. Histological studies of the lungs from animals injected with acetylsalicylic acid showed mediumsized alveoli and sites with alternating emphysematous dilated alveoli and smaller alveoli with markedly plethoric capillaries in the alveolar septa. White and mixed clots, sticking to the vascular wall, were detected in some microvessels. Thrombosed capillaries in the alveolar septa were 2-5-fold dilated.
Acetylsalicylic acid led to a significant (62.5%) shrinkage of the clot area in a lung section and a slight (6%) reduction of the mean area of a clot in a section in comparison with the control (Table 2). Microscopic examinations in the survivors after RU-891 treatment showed predominant circulatory disorders: plethoric capillaries in the alveolar septa and other microvessels. The alveolar septa were thickened because of plethora and edema; erythrocyte diapedesis in the alveolar septa, in the lumens of some alveoli, stasis and small focal hemorrhages were detected. Slight lymphoid infiltration was detected in the perivascular compartments and in the small bronchial walls. The area of the clots in a lung section reduced by 70.5%, of the mean area of a clot in a section by 72% (Table 2). Changes in the morphometric values in our study in general corresponded to qualitative structural changes in the lung vessels and indicated more pronounced antithrombotic activity of compound RU-891 in comparison with acetylsalicylic acid.
REFERENCES 1. M. A. Murina, O. D. Fesenko, V. I. Sergienko, et al., Bull. Exp. Biol. Med., 134, No. 1, 36-38 (2002). 2. A. A. Spasov, V. A. Anisimova, V. I. Petrov, et al., Patent RF No. 2453312. Substance with Antithrombogenic Activity, Bull. No. 17, June 20, 2012. 3. D. S. Sarkisov and Yu. L. Perov, Microscopic Technology [in Russian], Moscow (1996). 4. K. Broos, H. B. Feys, S. F. De Meyer, et al., Blood Rev., 25, No. 4, 155-167 (2011). 5. G. DiMinno and M. J. Silver, J. Pharmacol. Exp. Ther., 225, No. 1, 57-60 (1983). 6. B. Furie and B. C. Furie, Pathophysiol. Haemost. Thromb., 35, Nos. 1-2, 1-4 (2006). 7. I. Hagedorn, T. Vogtle, and B. Nieswandt, Haemostaseologie, 30, No. 3, 127-135 (2010).
580
PHARMACOLOGY AND TOXICOLOGY Antithrombotic Activity of a New Benzimidazole Derivative in the Thrombosis Model in Mice A. V. Smirnov, A. F. Kucheryavenko*, and A. A. Spasov* Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 157, No. 5, pp. 595-598, May, 2014 Original article submitted April 26, 2013 Antithrombotic activity of a new benzimidazole derivative RU-891, characterized by antiaggregant activity, vs. reference drug acetylsalicylic acid was studied on the epinephrinecollagen thrombosis model in mice. Antithrombotic activity of RU-891 3-fold surpassed that of the reference drug. Histological studies showed that RU-891 significantly reduced the relative and mean areas of thrombi in sections of mouse lungs in comparison with the control. Key Words: antithrombotic activity; thrombosis; platelet aggregation; RU-891; acetylsalicylic acid Arterial thrombosis is an important problem of cardiology, because this condition often becomes the cause of sudden death, myocardial infarction, vascular complications of diabetes mellitus, and often deteriorates the efficiency of surgery for coronary heart disease [7]. The increase in platelet aggregation activity is one of the most important pathogenetic mechanisms of intravascular clotting [4]. Our previous studies have demonstrated that RU-891, a new benzimidazole derivative, suppressed platelet aggregation [2]. Since platelets play the key role in arterial thrombosis, we have studied the antithrombotic effect of RU-891 on the thrombosis model in mice in vivo.
MATERIALS AND METHODS The study was carried out on male and female outbred albino mice (n=40; 20-25 g) kept in a vivarium (22-24oC, 40-50% humidity) at natural illumination on standard fodder (GOST R 50258-92) with due consideration for laboratory practice regulations for preclinical studies in the Russian Federation (GOST Department for Pathological Anatomy, *Department for Pharmacology, Volgograd State Medical University, Russia. Address for correspondence: [email protected]. A. F. Kucheryavenko
Z 51000.3-96 and 1000.4-96) and regulations and International Recommendations of the European Convention for Protection of Vertebrates Used in Experimental Studies (1997). Antithrombotic activity of RU-891 (Research Institute of Physical and Organic Chemistry, South Federal University) was studied in vivo on the thrombosis model reproduced as described previously [5]. Thrombosis was induced with a mixture of collagen and epinephrine (0.5 and 0.06 mg/kg, respectively). The mixture (0.1 ml) was infused in the caudal vein over 10 sec. Acetylsalicylic acid (20 mg/kg) served as the reference drug. The test substance was injected in a dose of 43 mg/kg (equimolal to the dose of the reference drug) 2 h before thrombosis reproduction. Controls were injected with an equivalent volume of the solvent. The drug efficiency was evaluated by the number of survivors in comparison with the control group and the presence of clots in lung vessels. The survivors were observed over 24 h. Dead mice and survivors (after euthanasia with ether overdosage) were autopsied for histological study of the lungs. In controls, histological analysis of the liver, heart, kidneys, and brain was carried out. The protocol of experiment was approved by the Regional Ethic Committee (decision No. 94-2009).
0007-4888/14/15750580 © 2014 Springer Science+Business Media New York
A. V. Smirnov, A. F. Kucheryavenko, and A. A. Spasov
The material was fixed (24 h in 10% neutral buffered formalin, pH 7.4), dehydrated, and embedded in paraffin. Sections (4-6 μ) were sliced from paraffin blocks and stained with hematoxylin and eosin by the standard method [3]. Histological preparations were photographed by an Olympus digital camera (4.0 megapixels) attached to Micros microscope (×10 and ×40 objectives; ×10 ocular). Signs of clotting in the walls of the muscular arterial vessels were evaluated morphologically. The data were processed by basic statistical analysis using Statistica 5.0 software (StatSoft, Inc.). Histological findings were processed using VideoTest-Morpho-4 software and Mann–Whitney test. The significance of the antithrombotic effects of the test substances (difference in the percentage of survivors in the control and experimental groups) was statistically evaluated by Fisher’s F test.
RESULTS In the control group, animal mortality was 95%, which was in line with the results of other scientists [1,6]. Characteristic signs of disorders in the respiratory function of the lungs were noted: higher respiration rate and shallow breathing, manifest exophthalmia and changed color of the iris. Hind limb paresis was observed: mice did not move even in response to pushing, could not withdraw the hind paws if pressed strongly. Tetanic convulsions were observed – the animals were in a characteristic posture with the hind paws straight backward. The mice died from asphyxia within 1-3 min after injection of the thrombotic agents. RU-981 in the studied dose prevented death of 90% animals, their motor activity restored completely
581 TABLE 1. Effects of RU-891 in a Dose Equimolal to the Acetylsalicylic Acid Dose on the Survival of Mice in the Total Systems Collagen-Epinephrine Thrombosis Model Dose, mg/kg
Number of dead animals
Survivors, %
Control (n=20)
–
19
5
RU-891 (n=10)
43
1
90*+
Acetylsalicylic acid (n=10)
20
7
30
Experiment conditions
Note. p≤0.01 in comparison with *control, +animals treated with acetylsalicylic acid (F test).
within 1.0-1.5 min (Table 1). The rest 10% before death were visually more active than controls, motor disorders were less severe, and the manifestations of generalized thrombosis developed slower (within 3-5 min) than in controls. The protective effect of acetylsalicylic acid was negligible. Only 30% animals survived in the group receiving acetylsalicylic acid. LD50 of RU-891 (1260 mg/kg) is high, and we can speak about pronounced antithrombotic activity of this substance manifesting at a dose by several orders of magnitude lower than the toxic dose. According to a previous study [5], the direct cause of mortality in the studied model was massive occlusion of the lung microvessels with platelet aggregations. Histological studies of the liver, heart, kidneys, and brain of animals injected with the thrombotic agents detected virtually no clots in the microcirculatory vessels of these organs, which was in line with the previous reports [1,5].
Fig. 1. Effects of RU-891 on clotting in the lungs of mice induced by collagen and epinephrine. Hematoxylin and eosin staining, ×100. a) control; b) RU-891. Arrows: white clots in microvessels.
Bulletin of Experimental Biology and Medicine, Vol. 157, No. 5, September, 2014 PHARMACOLOGY AND TOXICOLOGY
582
TABLE 2. Effects of RU-891 in a Dose Equimolal to the Acetylsalicylic Acid Dose on Some Morphometric Values of the Clots in Mouse Lung Vessels (M±m) Experiment conditions
Clot area, %
Mean area of a clot in section, μ2
Control (n=5)
3.538±1.982
1423.500±0.328
RU-891 (n=5)
1.047±0.290*
399.8±185.2**
Acetylsalicylic acid (n=5)
1.327±0.152*
1337.7±888.5
Note. *p≤0.01, **p≤0.001 in comparison with the control (Mann– Whitney test).
Microscopic studies showed the predominance of medium-sized alveoli in lung specimens from controls; white microclots were found in an appreciable part of microvessels; these clots adhered to the vascular wall in the vascular lumen (Fig. 1). Manifest dilatation (36-fold) of thrombosed capillaries in the alveolar septa was seen. These changes in some cases were associated with focal lesions in the capillary wall and in the respiratory epithelium. Mixed clots were found in some arterioles. In addition to white clots, marginal stasis of leukocytes was found in some venules. Morphometry of the lung sections from control mice showed that clot area in these sections was 3.538±1.982% and the mean area of a clot in the sections 1423.500±0.238 μ2. Histological studies of the lungs from animals injected with acetylsalicylic acid showed mediumsized alveoli and sites with alternating emphysematous dilated alveoli and smaller alveoli with markedly plethoric capillaries in the alveolar septa. White and mixed clots, sticking to the vascular wall, were detected in some microvessels. Thrombosed capillaries in the alveolar septa were 2-5-fold dilated.
Acetylsalicylic acid led to a significant (62.5%) shrinkage of the clot area in a lung section and a slight (6%) reduction of the mean area of a clot in a section in comparison with the control (Table 2). Microscopic examinations in the survivors after RU-891 treatment showed predominant circulatory disorders: plethoric capillaries in the alveolar septa and other microvessels. The alveolar septa were thickened because of plethora and edema; erythrocyte diapedesis in the alveolar septa, in the lumens of some alveoli, stasis and small focal hemorrhages were detected. Slight lymphoid infiltration was detected in the perivascular compartments and in the small bronchial walls. The area of the clots in a lung section reduced by 70.5%, of the mean area of a clot in a section by 72% (Table 2). Changes in the morphometric values in our study in general corresponded to qualitative structural changes in the lung vessels and indicated more pronounced antithrombotic activity of compound RU-891 in comparison with acetylsalicylic acid.
REFERENCES 1. M. A. Murina, O. D. Fesenko, V. I. Sergienko, et al., Bull. Exp. Biol. Med., 134, No. 1, 36-38 (2002). 2. A. A. Spasov, V. A. Anisimova, V. I. Petrov, et al., Patent RF No. 2453312. Substance with Antithrombogenic Activity, Bull. No. 17, June 20, 2012. 3. D. S. Sarkisov and Yu. L. Perov, Microscopic Technology [in Russian], Moscow (1996). 4. K. Broos, H. B. Feys, S. F. De Meyer, et al., Blood Rev., 25, No. 4, 155-167 (2011). 5. G. DiMinno and M. J. Silver, J. Pharmacol. Exp. Ther., 225, No. 1, 57-60 (1983). 6. B. Furie and B. C. Furie, Pathophysiol. Haemost. Thromb., 35, Nos. 1-2, 1-4 (2006). 7. I. Hagedorn, T. Vogtle, and B. Nieswandt, Haemostaseologie, 30, No. 3, 127-135 (2010).
