RU2613184C2 - Substance with angiogenic activity - Google Patents

Abstract

FIELD: pharmacology.

SUBSTANCE: group of inventions refers to application of low molecular peptide mimetics of the nerve growth factor: hexamethylenediamide bis-(N-monosuccinyl-glutamyl-lysine) - GC-2 compound; hexamethylenediamide bis-(N-acetyl-lysyl-glutamic acid) - GC-4 compound; amide N-aminocaproyl-glycyl-lysine - GC-5 compound; hexamethylenediamide bis-(N-aminocaproyl-glycyl-lysine) - GC-6 compound as compounds with angiogenic activity.

EFFECT: creation of new highly effective means to stimulate neoangiogenesis at different ischemic processes.

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Description

The invention relates to medicine, in particular to pharmacology, and for the use of the low molecular weight peptide mimetics of nerve growth factor: hexamethylenediamide bis- (N-monosuccinyl-glutamyl-lysine) described previously (Seredenin SB, Gudasheva T.A. Patent of the Russian Federation No. 2410392) ) - compounds GK-2; hexamethylene diamide, bis- (N-acetyl-lysyl-glutamic acid) - compounds GK-4; N-aminocaproyl-glycyl-lysine amide - compounds GK-5; hexamethylene diamide bis- (N-aminocaproyl-glycyl-lysine) compounds GK-6, as compounds with angiogenic activity.

The invention relates to the section of Experimental medicine and can be further used to create new original highly effective drugs for stimulating neoangiogenesis in various ischemic processes.

The main field of application of angiogenic drugs is coronary heart disease, including acute myocardial infarction, and chronic lower limb ischemia, especially in cases where the use of surgical methods of treatment, in particular endovascular revascularization, is both impossible and / or not effective enough , and / or is associated with the risk of developing serious complications, as well as in neurology and traumatology. For example, the number of such patients among patients with coronary heart disease, according to various sources, ranges from 30-45% (Cao Y. Discov. Med., 2010, 9 (46): 179-184; Mitsos S. et al. Angogenesis , 2012, 15 (1): 1-22, etc.), and in patients suffering from chronic obliterating endarteritis, amputation is performed in more than 25% of cases because of the impossibility of a full-scale surgical intervention, while mortality in this pathology is 20- 25% (Smith L. Nurs. Times, 2012, 108 (43): 12-14; Ouma GO et al. 2012, 17 (3): 174-192, etc.).

Theoretical approaches justifying the feasibility of pharmacological stimulation of angiogenesis were formulated at the end of the 20th century, and the direction itself was called “therapeutic angiogenesis” or “biological shunting” (Isner JM Clin. Immunol. Immunopathol., 1996, 80 (3 Pt2): S82- 91). Currently, the term “therapeutic angiogenesis” is understood to mean the stimulation of angiogenesis with drugs, mainly biosimilar drugs, with the ability to improve perfusion of ischemic tissues by enhancing natural, but insufficient in critical situations, tissue neovascularization processes, i.e. stimulate the growth and branching (arborization) of blood vessels and / or modulate endothelial function (Mima Y. et al. PLoS One, 2012, 7 (4): e35199; Chu H., Wang Y. Ther. Deliv., 2012, 3 ( 6): 693-714). The most promising in this regard is the development and implementation in clinical practice of exogenous analogues of endogenous growth factors and / or chemical compounds with the ability to activate growth factors, as well as the use of genetically modified progenitor cells (Parfenova E.V., Tkachuk V.A. Cardiological Bulletin , 2007, vol. II (XIV), No. 2, C.5-14; Semeraro F. et al. Chit. Vasc. PharmacoL, 2011, 9 (5): 629-646; Machalinska A. et al. Klin. Oczna, 2012, 114 (1): 63-67; Fariha MM et al. J. Cell. Mol. Med. 2013).

One of the possible approaches to solving this problem is to use the first (compounds GK-4, GK-5, GK-6) and the fourth (compound GK-2) loops of nerve growth (NGF) to stimulate neoangiogenesis of low molecular weight peptide mimetics.

At present, quite convincing literary material has been accumulated indicating that, in addition to the central nervous system, nerve growth factor is synthesized and excreted by vascular endothelial and smooth muscle cells, and NGF TrkA-specific receptors are presented on their cell membrane (Meloni M. et al. Circ. Res., 2010, 106 (7): 1275-1284; Saygili E. et al. J. Mol. Cell. Cardiol. 2010, 49 (1): 79-87 et al.), Through interaction with which NGF realizes their angiogenic effects. NGF-mediated activation of TrkA receptors located on the cell membranes of vascular endothelial and smooth muscle cells entails the activation of PI3K-Akt, Ras-MAPK and PLCγ1-IP3 intracellular signaling pathways that initiate neoangiogenesis (Caporali A., Emanueli C., 2009, 80 : 279-308 and others.). This phenomenon is described for both normal and pathologically altered tissues. In vascular endothelial cells, the final step in NGF-mediated activation of TrkA receptors is the activation of matrix metalloproteinase-2 (MMP-2), which belongs to the family of zinc-dependent endoproteinases. NGF-mediated activation of MMP-2, in turn, initiates activation of the AP-2 gene transcription factor (Park M.-J. et al. J. Biol. Chem., 2007, 282 (42): 30485-30496), which translocates into the cell nucleus, where it triggers a cascade of reactions that stimulate the proliferation, migration and invasion of endothelial cells. In addition, there is evidence that this process can also be initiated through the activation of NGF vascular endothelial growth factor - VEGF-A (Lazarovici P. et al. Endothelium, 2006, 13 (1): 51-59). In vascular smooth muscle cells, activation of the Ras-MAPK signaling pathway involves activation of metalloproteinase-9 (MMP-9), which, unlike MMP-2, only activates cell migration and invasion (Lucchesi PA et al. Circulation, 2004, 110: 3587-3593). Migration of vascular smooth muscle cells can also be initiated by NGF-mediated activation of PI3K-Akt and PLCγ1-IP3 of intracellular signaling cascades (Kraemer R. et al. Arterioscler. Thromb. Vasc. Biol., 1999.19: 1041-1050).

As a result of many years of basic research at the V.V. Research Institute of Pharmacology Zakusova RAMS synthesized a number of low molecular weight peptide mimetics NGF with agonist properties (compounds GK-2, GK-4, GK-5, GK-6) TrkA receptors (Gudasheva T.A. et al. Reports of the Academy of Sciences, 2010, 434 (4 ): 549-551) and their angiogenic activity was revealed.

The following examples illustrate the angiogenic activity of bis- (N-monosuccinyl-glutamyl-lysine) hexamethylene diamide - compound GK-2; bis (N-acetyl-lysyl-glutamic acid) hexamethylene diamide - compounds GK-4; N-aminocaproyl-glycyl-lysine amide - compounds GK-5; bis- (N-aminocaproyl-glycyl-lysine) hexamethylenediamide - compounds GK-6.

Example 1. The effect of compounds GK-2, GK-4, GK-5, GK-6 on the length of capillaries in the ischemic limb in rats.

The experiments were performed on outbred male rats weighing 180-200 g. Animals were randomized into 5 groups: 1st (n = 23) - control and 2nd - n = 15, 3rd - n = 8, 4th - n = 10 and 5th - n = 14, receiving compounds GK-2, GK-4, GK-5 and GK-6, respectively. Hind limb ischemia in anesthetized rats (sodium thiopental, 50 mg / kg, ip) was caused by simultaneous resection of the femoral artery, after which the wound was sutured in layers. The studied compounds (1 mg / kg) were administered intraperitoneally for 14 days from the moment of resection of the femoral artery. The first injection was carried out 1 hour after the end of the operation. Control animals in a similar pattern were injected intraperitoneally with 0.3 ml of physiological saline. One day after the last injection, the animals were sacrificed and the calf muscle was removed from the ischemic limb. To visualize the capillaries, a 1% Evans blue solution was used, which was introduced 5 minutes before the animals were slaughtered. Using light microscopy, the total length of capillaries in 1 mm ^{2 of} ischemic tissue was evaluated, which made it possible to judge the intensity of vascular growth under the influence of the studied compounds. The significance of the differences was determined using one-way analysis of variance with further processing by the method of multiple comparisons according to Dunnet.

An analysis of the results indicates that the studied compounds in one way or another show angiogenic activity (Table 1). For example, in animals treated with compound GK-6, the total length of the capillary bed was 32% longer than in the control (p = 0.008). Compound GK-2 showed the greatest angiogenic activity: in animals treated with this compound, the indicator of the total capillary length was more than 50% higher than in control animals — 21024 ± 1344 and 13607 ± 715 μm / mm ² , respectively (p <0.001) .

Thus, the results of screening studies presented indicate that the compounds in question stimulate angiogenesis to one degree or another. In this case, the compound GK-2 has the greatest angiogenic activity.

Example 2. Angiogenic and anti-ischemic activity of the low molecular weight peptide mimetic NGF - compounds GK-2.

The experiments were performed on outbred male rats weighing 180-200 g. Animals were randomized into 2 groups: control (n = 18) and main (n = 17). In this series of experiments performed on the rat hind limb ischemia model (the experimental protocol is similar to that described in Example 1), not only the angiogenic, but also anti-ischemic effects of the GK-2 compound were evaluated. For this purpose, the effect of the GK-2 compound on the intensity of vascularization and necrobiotic processes in the ischemic calf muscle was studied. To assess the intensity of necrobiotic processes occurring in the ischemic gastrocnemius muscle, light microscopy was used, for which the muscle was fixed in a 10% neutral formalin solution, 5 μm thick sections were prepared using a freezing microtome and stained with hematoxylin-eosin according to standard methods. To visualize the capillaries, Evans blue was used. For each field of view, the total length of the vessels and their number in 1 mm ^{2 were} calculated. The degree of vascularization was judged by the magnitude of the vascularization index, which was taken as the product of the length of the capillaries and their number in 1 mm ^{2 of the} ischemic tissue. The statistical significance of the changes caused by the GK-2 compound was determined using the Mann-Whitney test. The significance of the differences between the series of experiments was determined using the Fisher exact probability criterion.

The results obtained indicate that in animals treated with GK-2, the total length of the capillary bed is statistically significantly greater (p <0.003) than in control animals - 19531 (16085 ÷ 24511) and 14420 (10901 ÷ 17404) μm / mm ^2, respectively (Fig. 1. The effect of the compound GK-2 (ip, 1 mg / kg / day, 14 days) on the total length of blood vessels in 1 mm of tissue under conditions of rat hind limb ischemia). The number of vessels in 1 mm ^{2 of} ischemic tissue in animals treated with the compound GK-2 was also greater (p <0.001): 71 (71 ÷ 78) and 53 (50 ÷ 57), respectively (Fig. 2. The effect of the compound GK-2 (iv, 1 mg / kg / day, 14 days) for the number of vessels in 1 mm ² tissue under conditions of rat hind limb ischemia). A mathematical analysis of the results indicates that the intensity of vascularization of ischemic tissue in animals treated with GK-2 is almost 2 times higher than in the control - 27794 (25218 ÷ 35941) and 14725 (9030 ÷ 19630), respectively (p <0.001 )

According to light microscopy data in control animals, the basis of the tissue is necrobiotic muscle with areas of waxy necrosis. Sarcoplasm is brightly colored, homogeneous, transverse striation is absent (Fig. 3a. Morphological picture of the posterior gastrocnemius muscle of the rat (x 10)). A large number of inflammatory infiltrates are noted. The nuclei of striated muscles are small, hyperchromic or absent. The vessels are full-blooded, the perivascular edema is well defined, the capillaries are convoluted, small, thin, and poorly distinguishable. Thus, as a result of the removal of the femoral artery in the gastrocnemius muscle, pronounced necrotic and necrobiotic changes are observed, accompanied by an inflammatory reaction and circulatory disorder. In animals treated with compound GK-2, the microscopic picture of the gastrocnemius muscle differs significantly from that in the control - the intensity of the alternative processes in the muscle of these rats is less pronounced, the number and size of sections of waxy necrosis is significantly less than in control rats. The area of inflammatory infiltrates is negligible. In contrast to control animals, vascular endomysium is pronounced. The transverse striation of skeletal muscle is preserved. The capillary network is clearly distinguishable, the capillaries go straight and are located along the muscle fibers (figb).

Thus, the example indicates that the GK-2 compound exhibits not only pronounced angiogenic, but also anti-ischemic activity, which, most likely, is associated with the ability of the compound to stimulate neoangiogenesis in damaged calf muscle.

Example 3. The effect of compounds GK-2 on the formation of tubular structures (tubulogenesis) in the culture of human endothelial cells HUVEC.

Evaluation of the angiogenic activity of compound GK-2 in in vitro experiments was performed on a culture of isolated human endothelial cells HUVEC. This approach is due to the fact that, according to published data, the formation of tubular structures (tubulogenesis) is the initial stage of angiogenesis. NGF was used as a reference.

Endothelial cells were seeded in DMEM containing 20 mM Hepes, 2 mM L-glutamine, heparin (5 U / ml), ECGF (20 μg / ml), 10% FBS with a density of 3.5 thousand per 96-well plates, coated with polylysine. GK-2 (10 ^-6 M) and NGF (10 ^-9 M) were added 30 min after scattering of the cells on the plates and then every 48 hours (total 3 applications). Then, to assess the effect of the GK-2 and NGF compounds on the formation of tubular structures, endothelial cells were photographed using a Canon Nikon Eclipse TS-100F inverted microscope camera. ImageJ was used to calculate tubulogenesis. The length of microtubules was measured in 5 fields of view of each well.

The results obtained are summarized in table 2, from which it follows that the length of the microtubules in the endothelial cell culture into which the GK-2 compound was added is statistically significantly (p <0.001) longer than in the control and comparable to that when NGF was added.

Thus, the results of in vitro experiments indicate that GK-2, like NGF, activates tubulogenesis, the initial stage of angiogenesis.

Description of drawings

Figure 1. The effect of the compound GK-2 (ip, 1 mg / kg / day, 14 days) on the total length of blood vessels in 1 mm ² tissue under conditions of rat hind limb ischemia.

The ordinate axis is the length of the vessels in microns / mm. The abscissa shows groups of animals.

From FIG. 1 shows that in animals treated with compound GK-2, the total vessel length in 1 mm of ischemic limb tissue is statistically significantly greater (P = 0.003) than in the control.

Figure 2. The effect of the compound GK-2 (ip, 1 mg / kg / day, 14 days) on the number of vessels in 1 mm ² tissue under conditions of rat hind limb ischemia.

The y-axis is the number of vessels in 1 mm. The abscissa shows groups of animals.

From FIG. 2 shows that in animals treated with the compound GK-2, the number of vessels in 1 mm ^{2 of} tissue of the ischemic limb is statistically significantly greater (P <0.001) than in the control.

Figure 3. Morphological picture of the posterior calf muscle of the rat (x 10).

The upper panel is the control (a), the lower one is GK-2 (ip, 1 mg / kg / day, 14 days) (b).

ЗВ - inflammation zone, НМВ - necrotic muscle fibers; MB - muscle fibers, FS - a fragment of the vessel; K - capillaries.

Table 1 The effect of compounds GK-2, GK-4, GK-5, GK-6 (1 mg / kg, ip) on the density of the vascular bed in the ischemic muscle of rats (arithmetic means, their standard errors and 95% confidence intervals). Indicator Connections Control, n = 23 GK-2, n = 15 GK-4, n = 8 GK-5 n = 10 GK-6, n = 14 The density of the vascular bed, μm / mm ² 13607 ± 715 12123 ÷ 15091 21024 ± 1344 18142 ÷ 23906 P <0.001 14363 ± 812 10443 ÷ 15284 p≈0.822 15201 ± 1411 11216 ÷ 16002 p≈0.057 17937 ± 1185 15376 ÷ 20497 p≈0.008

table 2 The effect of the compound GK-2 (10 ^-6 ) and NGF (10 ^-9 M) on the formation of microtubules in the culture of isolated human endothelial cells-HUVEC (medians, lower and upper quartiles are indicated). Indicator Experimental groups Control, n = 92 NGF, n = 78 GK-2, n = 54 The total length of microtubules, microns twenty 36 38 14 ÷ 29 26 ÷ 49 27 ÷ 53 p <0.001 P <0.001

Claims (4)

1. The use of hexamethylene diamide bis- (N-monosuccinyl-glutamyl-lysine) as an agent with angiogenic activity. 2. The use of bis- (N-acetyl-lysyl-glutamic acid) hexamethylene diamide as an agent with angiogenic activity. 3. The use of the amide N-aminocaproyl-glycyl-lysine as an agent with angiogenic activity. 4. The use of bis- (N-aminocaproyl-glycyl-lysine) hexamethylene diamide as an agent with angiogenic activity.

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