RU2651756C1 - Preparation for glial scars prevention - Google Patents

Abstract

FIELD: pharmacology.

SUBSTANCE: invention is a preparation for glial scars prevention, containing D-asparagine and highly purified water, where 1000 ml of the drug contains 5 g of dry D-asparagine powder, the rest - highly purified water.

EFFECT: significant inhibition of protein synthesis and glial cells proliferation, prevention of glial scarring, and elimination of side effects such as toxicity and mitotic reactions inhibition.

1 ex, 4 dwg

Description

The present invention relates to the field of experimental medicine, namely to neurology and neurosurgery, can be used in complex therapy for acute damage to the spinal cord of various origins to prevent the formation of a glial scar.

The key reason for the poor prognosis in the recovery of neurological deficit in patients with acute spinal cord injury is the formation of a glial scar at the site of the lesion of the spinal cord tissue. Glial scar is both a mechanical and molecular (synthesis of substances inhibiting axon growth) barrier for axon germination distally from the lesion site (Baer M. Neuroprotection. Models, mechanisms, therapy / M. Baer - Moscow. Binom Knowledge Laboratory, 2014. - 436 p.).

The development of drugs that can prevent the formation of a glial scar is a promising area of modern medical science. Traditionally used in the initial period of acute CNS damage, neuroprotective and glioprotective drugs, the main purpose of which is to stimulate the regeneration of the central nervous system, are not able to stimulate effective axon regeneration, remyelination of the CNS pathways and prevent the formation of the glial scar (Rem M. Neuroprotection. Models, mechanisms, therapy / M. Baer - Moscow.Binom Laboratory of Knowledge, 2014 .-- 436 p.).

So far, to solve the problem of neurological deficit, methods have been used to activate axonal growth and restore continuity of the pathways in the damaged spinal cord through implantation of the Sphero® GEL collagen matrix into the break zone, transplantation of various exogenous materials, sections of the peripheral nerve, ganglia, intrathecal transfusion of autologous hematopoietic stem cells (Baer M. Neuroprotection. Models, mechanisms, therapy / M. Baer - Moscow. Bean Laboratory Sign Niy, 2014 .-- 436 p.).

But even with the use of effective stimulants for the regeneration of nerve tissue, the glial scar at the site of the lesion of the spinal cord forms faster than axons regenerate. Thus, the growing axons “abut” against the formed scar and reinnervation of the areas of the spinal cord distal from the focus of damage to the nerve tissue does not occur.

There is no doubt the prevention of glial transformation of the spinal cord after acute damage.

Existing approaches aimed at preventing the formation of a glial scar are divided into 2 groups: 1) the use of agents acting on cells in the area of the lesion of the spinal cord (cytostatics, monoclonal antibodies to cytokines involved in the development of gliosis and their receptors, low-dose x-ray radiation); 2) drugs that modify the composition of the intercellular matrix (bacterial chondroitinase, 2-2'-dipyridine - an iron chelator and an inhibitor of collagen IV deposition, monoclonal antibodies that neutralize the activity of the Nogo-A inhibitory myelin protein) (Rem M. Neuroprotection. Models, mechanisms, therapy / M. Baer - Moscow.Binom Laboratory of Knowledge, 2014 .-- 436 p.).

For the closest analogue, we took drugs from the group of antimetabolites. Basically, preparations of this group contain 0.05 g of the active substance and auxiliary substances: gelatin; potato starch; milk sugar; calcium stearate. Preparations are structural analogues of adenine, hypoxanthine and guanine, which are part of nucleic acids. Their action is aimed at inhibiting the critical processes necessary for DNA replication and cell division. Their main field of application is oncology, but in connection with the pronounced immunosuppressive and anti-inflammatory activity, they are also used in rheumatology, as well as in transplantology.

Each of the approaches listed above, aimed at inhibiting the formation of the glial scar, has certain disadvantages: low effectiveness, low specificity, serious side effects, high cost and low availability.

The main disadvantages of cytostatic therapy are: high toxicity, inhibition of mitotic reactions in both growing (young) and mature cells. (Antosyuk O.N., Marvin A.M., Marvin N.A. Comparative analysis of the effect of cytostatic drugs on the instability of the Drosophila melanogaster genome // Biomedicine. 2014. No. 3, p. 83-91.)

Tasks:

1) Elimination of adverse side effects (toxicity, inhibition of mitotic reactions, cost reduction, increased effect, prevention of the formation of glial scars);

2) Ensuring the effectiveness and safety of the drug;

3) The exclusion of additional input of various substances as auxiliary therapy.

The proposed drug includes an aqueous solution of D-asparagine for intravenous use. The solution contains the target component AK D-asparagine in the form of a dry powder, per 1000 ml:

- AK D-asparagine - 5 g,

- the rest is highly purified water.

The chemical formula of D-asparagine: C4H8N2O3, which is an odorless white crystalline powder, highly soluble in water, the chemical name is aspartic acid monoamide.

The drug is obtained by dissolving 5 g of a dry powder of D-asparagine in 1000 ml of highly purified water. After dissolution, the drug is left for 1 day in a thermostat at a temperature of 40 ° C, after which it can be used.

The technical characteristic of the action of the drug is: inhibition of cell proliferation by D-asparagine, due to the fact that immature gliocytes show a low enzymatic activity of systems to neutralize D-aspartic acid, which makes it possible to significantly inhibit protein synthesis at a high level of its exogenous intake and, as a result glia cell proliferation (D'Aniello, A. & Giuditta, A. (1977). Identification of D-aspartic acid in the brain of Octopus vulgaris Lam. J. Neurochem. 29, 1053-1057). The antiproliferative properties of D-asparagine are comparable only with the effect when using cytostatic therapy, however, side effects that are caused by its use were not detected during the experiment with D-asparagine.

The experiments were conducted on 60 white non-linear male rats with an average weight of 346 ± 75 g. Characterization of animal groups: group No. 1 (intact) —of 20 rats whose spinal cord was used as a control; group No. 2 (comparisons) - from 20 rats that reproduced focal photothrombosis of blood vessels of the thoracic spinal cord with subsequent removal of a portion of the spinal cord on day 17; group No. 3 (experimental) - of 20 animals that performed a spinal stroke simulation followed by intravenous administration (into the tail veins) of a 0.5% solution of D-asparagine (course dose of 21.7 mg / kg) from 3 to 6 days and withdrawal spinal cord on day 17.

Modeling of spinal stroke was carried out according to a modified method of M. Von Euler, by photothrombosis of the vessels of the thoracic spinal cord with a laser with a wavelength of 514 nm. Erythrosine was used as a photosensitizer, tranexamic acid was used to inhibit fibrinolysis (Von Euler M. Morphological characterization of the evolving rat spinal cord injury after photochemically induced ischemia // Acta Neuropathol. - 1997 Sep; 94 (3). - P. 232-239 ) For surgery, zoletil-xylazine anesthesia was used.

Rat euthanasia was performed on day 17 of the experiment. The spinal cord was drawn by pushing it out with a phosphate buffer injected through a 10 ml syringe cannula inserted into the lumbar spine (Kennedy HS A method for removing the brain and spinal cord as one unit from adult mice and rats // Lab Anim (NY ). - 2011. - Feb; 40 (2): 53-7. Doi: 10.1038 / laban0211-53). After extraction, the spinal cord was placed in zinc-formalin fixative with zinc sulfate (Kierman J. A. Histological and histochemical methods. Theory and practice. - London: Scion Publishing Ltd. - 2008. - 606 p.). The obtained samples were sent through isopropanol-mineral oil, followed by their pouring into paraffin. Paraffin blocks were cut into sections with a thickness of 10 μm on a microtome MPS-2 (USSR). Micropreparations were stained with hematoxylin and eosin. To photograph micropreparations, a Mikmed-5 microscope (Russia) and a Levenhuk-230 ocular camera (USA) were used. For the analysis of photographs of micropreparations, the GIMP2 photo editor was used. Using the grid tool, cells were counted using the cells superimposed on the photo grid, the cells of which were previously calibrated using an object micrometer.

Statistical processing of the obtained data was carried out using the Statistica 10 version software of Stat Soft Inc., using the parameters: median (Me), percentiles (p25 and p75), confidence interval 95 (CI 95). To test the hypothesis of a Gaussian distribution of indicators in the studied groups, the Shapiro-Wilk test was used. Due to the fact that the distribution of the values of the studied parameters in the groups differed from the normal one, their comparison was carried out according to the non-parametric Kolmogorov-Smirnov criterion, with the establishment of a significance level of * p≤0.05.

On micropreparations of the spinal cord obtained from rats from group No. 2 (without the use of D-asparagine), the phenomena of pronounced glial transformation of the spinal cord are observed. In the area of the pathways, there is a large accumulation of glia cells (presumably astrocytes) that form cellular structures, which indicates the formation of a glial scar in the focal area of photothrombosis of the vessels of the spinal cord (see Figure 1 with an increase in X100, Figure 2, with an increase in X400).

On spinal cord micropreparations obtained from rats from group No. 3 (using D-asparagine), a small focus of gliosis is observed, which slightly disrupts the path of the pathways. At the same time, in the area of the focus of photothrombosis of the vessels of the thoracic spinal cord, in comparison with micropreparations from group No. 2 (without the use of D-asparagine), a significantly smaller number of glia cells are visible, individual axons are traced through the area of the forming glial scar (see Figure 3 , with an increase in X100, 4, with an increase in X400).

When comparing the number of cells on micropreparations in the area of the focus of photothrombosis of the spinal cord obtained from rats from group No. 2 (D-asparagine was not used), and on micropreparations of the spinal cord obtained from rats from group No. 3 (D-asparagine was used in a course dose of 21 , 7 mg / kg), significant differences are visible. The distribution density of glia cells in the focus of photothrombosis on micropreparations of the spinal cord in rats from group No. 3 (received D-asparagine) is three times lower than that in rats from group No. 2 (without D-asparagine), (p≤0, 01). Thus, the density distribution of cells in the area of the focus of photothrombosis of the spinal cord on micropreparations from group No. 3 (received D-asparagine) approaches the value obtained in group No. 1 (control), a statistically significant difference in the number of cells between group No. 1 (control) and group No. 3 (received D-asparagine) was not detected (p≥0.01).

Example. A nonlinear male rat weighing 246 g was operated on using a laser with a wavelength of 514 nm to simulate photothrombosis of the vessels of the thoracic spinal cord. For surgery, zoletil-xylazine anesthesia was used. From 3 to 6 days after surgery, a 0.5% solution of D-asparagine was injected intravenously into the veins of the tail. Euthanasia was performed on day 17, after which, using a phosphate buffer inserted through a cannula of a syringe inserted into the lumbar region, the spinal cord was removed. Then the sample was drawn through isopropanol-mineral oil, the resulting micropreparation was stained, photographed using a Levenhuk-230 camera (USA), and the results were statistically processed using the Statistica 10 version program. The experimental results are confirmed by Fig. 3-4, where fig. Figure 3 shows the region of the emerging glial scar, the lower density of glia cells and the partially preserved axons passing through the region of the emerging glial scar under magnification of XI00 (hematoxylin-eosin staining). In fig. 4, axons passing through the scar region are visible under X400 magnification (hematoxylin-eosin staining).

Thus, the intravenous administration of a 0.5% solution of D-asparagine in a course dose of 21.7 mg / kg from 3 to 6 days from the beginning of the experimental spinal stroke, according to the histological examination of the spinal cord conducted on the 17th day of the pathology, is significantly less the severity of the processes of gliosis transformation, in comparison with rats that did not receive D-asparagine.

Claims (2)

A preparation for preventing the formation of glial scars containing the target component D-asparagine in dry powder per 1000 ml: D-asparagine 5 g rest highly purified water

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