http://bankpatentov.ru/node/331967
The invention relates to the field of pharmacology and biotechnology can be used to obtain funds for the treatment of acute stroke and acute periods of its development.
Closest to the claimed invention is an agent for the treatment of hemorrhagic stroke or ishimicheskogo "Cere" firm Ebewe (Austria), which is a product of the brain processing of intact pigs. The structure of "Cere" includes amino acids (~ 85%), peptin (~ 15%), minerals and water. Trophic effect of the drug at the expense of specific peptides and amino acids dominant defining properties of the drug, are lysine, leucine and alanine.Drug prototype intended for intravenous administration (Reference of Clinical Pharmacology. Eds. I. S.Chekmana, A.P.Poleschuka OA dimes. Kyiv, "3dorov" I ", 1987).
"Cere 'has a low concentration of the active components, thus introduced into the body of the patient in large doses for a long time.
The disadvantage of this drug is a significant duration of treatment, a short period of remission, its low activity, which generally characterizes him as insufficiently effective.
Closest to the claimed method is a method of getting "Cere" (Mashkovskii MD Drugs, M., "Medicine" 1986, v. 2, p. 99).
In the prototype method feedstock used intact porcine brain, it is subjected to fragmentation, homogenization, and the resulting homogenate is subjected to further processing, which is the basic stage of hydrolysis.
Known method allows to obtain remedy for the treatment of stroke characteristics mentioned above. The method is simple to implement, easy to use perdpolagaet available raw materials.
However, the drawback of this method is that the totality of its mode of action and is such that no sufficiently high activity provides a means for treating stroke.
The object of the present invention is to provide an agent for treating a stroke of new quality, which is achieved and cutting operations characteristic of the manufacturing method thereof, whereby the method for acquiring the resulting high activity, increased treatment efficiency, and moreover, extends the existing arsenal of drugs aimed at the treatment of stroke .
The object is achieved in that the means for treating stroke, isolated from porcine brain and containing a predominant number of amino acids according to the invention is characterized by the presence of specific peptides travmotroficheskih M.W. 400 - 1500 A and several dominant amino acid is Asp, glutamine, alanine, glycine and serine.
The resulting tool is an amino acid-peptide complex, which feature determines its specificity and activity. Among travmotroficheskih peptides that best define the properties of the claimed means are neurotrophins.
The present inventors have determined that the specific travmotroficheskih molecular weight peptides is in the range of 400-1500 A, and the amino acid composition of the proposed drug is the 18 amino acids of which are dominant, and therefore greatly affect its properties are aspartic, glutamic, alanine, glycine and serine. Table. 1 shows the amino acid composition of the proposed drug "Cerebrate" and the famous "Cere" (in mM%).
High activity of the proposed drug and its sufficient concentration constitute the feature that allows you to enter it as nose drops, which is very convenient and helps achieve rapid therapeutic effect because the drug enters the brain, bypassing the blood-brain barrier.
To determine the potency and efficacy of the proposed drug tests were carried out on 71 cats of both sexes polozrelyh who previously modeled bilateral hemorrhagic stroke (a. s. USSR N 1767518 from 08.06.1992 y.)
Stroke severity was similar and controlled by the neurological examination (hemi and tetraparesis plegia, anesthesia, nystagmus, etc.).Animals were divided into 3 groups - control (n = 25) experienced and 2 which respectively receive "Cere" (n = 22) and "Cerebrate" (SNC-TTR) (n = 24). Three days after the restoration of hemorrhagic stroke animals that survived, one-time received appropriate treatment: animals in the control group - saline (0.85% NaCl solution in / br), and experimental - respectively "Cere" (1 ml / kg in / br) or "Cerebrate" (SNC-TTR) (1 ml of a 2.5% solution, w / br). Based on the 14 days. ECoG study various areas of the neocortex, behavioral recovery, and finally data biopsinnogo study sensorimotor cortex was concluded that the effectiveness of protivoinsultnaya "cerebrate" 2-3 times higher than that often, long and high doses of the drug "Cere" which is assigned to the treatment and hemorrhagic stroke in patients ishimicheskogo (Table 2). The high efficiency protivoinsultnoy "cerebrate" compared to "Cere" indicate voltage ECoG recovery and normalization of its rhythms already 4-6 days after stroke and simulation, respectively, at 8-10 days after use "Cere". Compared with controls, 2.6 times faster recovery independent walking, eating and Mangus-reflex (1.6 times and the introduction of "Cere").
Even more impressively, these differences manifest themselves in the study of animal survival, while "Cerebrate" is actually 2 times the "Cere" for these indicators. Table. 3 This conclusion is detailed and updated. After treatment of animals with the third day of the disease mortality animals administered "Cerebrate" reduced 2-fold compared with animals that received "Cere", and three times as compared with the control.
Said therapeutic agent has an extremely low toxicity, full compatibility with other types of therapy does not cause allergic reactions and side effects, contraindications have been identified.
Technique shows expressive effect neotropic tsitoprotektornoe effect, has a regulating effect on brain activity.
Besides, the task set is solved by a method for production of an agent for treating a stroke, which comprises a split pig brain, homogenization, and further processing the homogenate obtained according to the invention, porcine brain crushed previously undergone bilateral modeling hemorrhagic stroke, from the resulting homogenate at pH 7.3-7.6 to a concentration of the crushed parts of the brain do not emit more than 20% insoluble fragments, after which the remaining liquid mixture after adding it hexane 1: (3-5) was extracted lipid components are isolated and are filtered extract the resultant mixture to achieve its lightening, then - sterilizing filtration, and frozen at -30 o C the filtrate obtained is lyophilized.
In carrying out the method claimed in feedstock used pig brain which had previously been subjected to bilateral hemorrhagic stroke simulation according to a conventional technique. Porcine brain predominantly take it tserebrokorteks (neocortex), as this part of the brain has the maximum number of neurotrophic factors and cytoprotective. Selection porcine brain feedstock due to the fact that these animals are the most similar to humans, are more resistant to disease and, in addition, more readily available and economically viable industrial release formulation.
During the simulation, hemorrhagic stroke in animals develop appropriate response, in which brain cells secrete growth factors (neyrotofiny) providing a protective effect on them, causing accelerated pharmacotherapeutic effect.
All operations and modes of fashion related and this relationship allows us to give the proposed method for the treatment of stroke desired properties.
Pigs with an experienced brain hemorrhagic stroke is crushed and then homogenized in a certain way in the selected medium at pH 7,3-7,6, when choosing environment guided by the maximum preservation of brain cells. At this stage, to establish substantially the crushed parts of the brain concentration of not more than 20% because at higher concentrations there are technological difficulties. The concentration of the homogenate is one of the factors which affects the properties of the resulting therapeutic agent.
From the obtained homogenate is necessary to select the destroyed cell fragments, which impede further concentration of active amino acid-peptide complex. This operation is carried out, for example, centrifugation.
The resulting liquid mixture after separation of insoluble fragments were mixed with hexane at a ratio of 1: (3-5) which corresponds to the maximum possible the extraction of the lipid components. Selection hexane as to extract the lipid components due to its additional advantages, namely, it has low toxicity, which is essential, since the obtained treating agent, in addition it acts as a preservative, i.e. possess antimicrobial activity.
After separating the extract containing the lipid products, filtration of the remaining liquid is carried out (antireflection filtering), which is designed to remove large molecules, lipoproteins, bacteria, etc., and the remaining lipids
And finally, sterilizing filtration is needed to remove viruses, bacteria, molecular aggregates.
All previous steps of the claimed method to allocate the maximum allowed components that reduce the activity of the resulting funds.
The resulting filtrate was frozen at -30 o C, in these conditions, structural changes occur resulting product, thereby optimizing the subsequent freeze-drying process, i.e. maximum preservation of the biological activity of amino acid-lipid complex.
The invention is illustrated a concrete example of its implementation.
Example. After experienced industrial slaughter pigs - females aged 1-1.5 years weighing 100-150 kg produced beheading, removed the brain, it was dried blood, and then the cold isolated tserebrokorteks, crushed him to a state of stuffing.
To prepare a 10% homogenate was taken and the corresponding amount of minced meat was mixed with homogenization medium, representing a solution of 0.34 M tsukrozy and 0.005 M Tris - HCl - buffer (pH 7.4) and the mixture was homogenized with a homogenizer PT 2 at ( -3 o C) - (-4 o C) for 3 - 4 minutes.
Thereafter, the resulting mixture was centrifuged at 780 g for 20 minutes, the precipitate is separated, and the supernatant was mixed in a separating funnel and hexane in a ratio of 1:3. Extraction was performed twice, the extract was separated.
Then, after separation of the liquid extract was filtered by a filter with a pore size of 0.48 microns for 2 hours, resulting in the clarification of the liquid.
Then, a sterilizing filtration using filters with a pore size of 0.22 microns. The filtrate was dispensed into 2 ml vials of 5 ml, the contents were frozen at -30 o C and kept under these conditions for at least 20 hours, frozen lyophilized product.
Lightening and sterilizing filtration, lyophilization vials and sealing performed under sterile conditions.
The resulting product is a magnificent white powder, soluble in water and alcohol. Other features of the method the resulting "Cerebrate" given above.
Thus, the claimed agent has protivoinsultnuyu expressive activity.
The experiments conducted on animals suggest that "Cerebrate" is able to provide high pharmacological effects on stroke patients in acute and acute periods of its development. Nature derived method determines not only its use for the treatment of stroke, but also for other diseases associated with brain damage, such as: cerebral palsy, multiple sclerosis, etc.
Furthermore, the inventive method with the above properties is prepared in such a concentration of active components, which defines a simple and easily accessible for the patient way of its application.
The proposed method of obtaining funds is simple to implement and suitable for large-scale introduction.
Moreover, an advantage of the proposed solution is an extension of the existing arsenal protivoinsultnyh remedies.
CLAIMS
1. Agent for treating stroke, isolated from porcine brain and containing a predominant number of amino acids, characterized in that it is isolated from porcine brain, previously subjected to bilateral modeling hemorrhagic stroke, and is characterized by the presence of specific peptides travmotroficheskih M.W. 400 - 1500 W and several dominant amino acids: aspartic, glutamic, alanine, glycine and serine.
2. Method for producing an agent for treating a stroke, which comprises a split pig brain, homogenization and subsequent treatment of the homogenate, characterized in that the pig brain crushed previously undergone bilateral modeling hemorrhagic stroke, from the resulting homogenate at pH 7.3 - 7 6 with a concentration of the crushed parts of the brain do not emit more than 20% insoluble fragments, whereupon the liquid mixture from remaining in it after the addition of hexane at a ratio of 1: (3 - 5) was extracted lipid components separated are filtered and extract the resultant mixture to achieve its lightening, then - sterilizing filtration, and frozen at -30 o C the filtrate obtained is lyophilized.
