RU2302237C2 - Method for obtaining biologically active fraction s-1-10, a tremor-decreasing pharmaceutical composition at parkinsonism and convulsive activity-decreasing at epilepsy - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: the present innovation deals with obtaining a biologically active peptide fraction out of poultry blood serum being useful at some human and animal diseases and disorders. It is necessary to carry out electrostimulation of a bird's head at the mode of about 70-80 V for about 2-3 sec to sample blood and incubate it at 4-8°C for about 18-24 h, then the serum sampled should be irradiated with C0 60 at the mode of 25±5.0 kGy and extracted after irradiation with 0.5 mM solution of phenylmethane sulfonyl fluoride in distilled water at the ratio of 1:10 to be mixed at 5°C for 1 h and centrifuged at 12000 rot./min for 30 min, then the residue should be repeatedly centrifuged under the same conditions, supernatant should be filtered successively through the membrane at pore's diameter being 0.45 mcm and 10 kDa, and a target product being a peptide fraction at molecular weight up to 10 kDa should be lyophilized and kept at about 4-8°C. The suggested pharmaceutical composition is characterized by the fact that as an active substance it contains efficient quantity of peptides of molecular weight up to 10 kDa obtained to a certain technique and a pharmaceutically acceptable carrier or filler. The innovation enables to obtain new biologically active peptide fraction up to 10 kDa and develop its medicinal form.

EFFECT: higher efficiency.

2 cl, 2 ex, 3 tbl

Description

FIELD OF THE INVENTION

The present invention relates to medicine, can be used to obtain a biologically active peptide fraction from avian blood serum, useful in a number of diseases and disorders in humans and animals (physical performance, coordination of movements, parkinsonism, epilepsy, stress, etc.)

State of the art

Widely known methods for producing biologically active blood serum, based on the collection of blood from donors and animals, incubation, separation, followed by canning. The methods include obtaining blood serum that increases the body's resistance to such exogenous and endogenous factors as atmospheric pressure, temperature, gravity, light, etc., as well as hunger, thirst, sleep and sexual needs, etc. (Japanese Patent No. 2123287, EP 0542303 A2, Russian Patents No. 2096041, 2120301).

Serum is obtained from the blood of a donor previously introduced into a certain functional state, and, depending on the nature of the effect, serums with various biological activity are obtained: myogenic, somnogenic, ophthalmic, audioactive, thermoactive, dietetic, sex active, antihypoxic, anti-alcohol and anti-nicotine.

Serum from animals is subjected to gamma processing in order to enhance biological activity (patent PCT / RU00 / 00073 - Russia and EP).

The objective of the invention was to obtain a biologically active peptide fraction of up to 10 kDa from electroshock blood serum of chickens.

Summary of the invention

It turned out that if the electroshock blood serum of birds was irradiated in the range of 20-30 kGy, filtered through 0.45 mm and 10 kDa membranes, it was possible to obtain a low molecular weight biologically active peptide fraction with a number of activities (which will be described below), which are useful for some unfavorable patient conditions (to be disclosed below).

Thus, the aim of the present invention is to develop a new method for producing a biologically active peptide fraction S-1-10.

Another objective of the present invention is the development of a dosage form of a new biologically active serum. The invention involves various dosage forms: including for oral, parenteral, nasal, buccal, in the form of suppositories, etc. administration.

The invention provides for the use of suitable physiologically acceptable carriers (such as distillate water, physiol. Solution), excipients (e.g. cocoa butter, witepsol), a biologically active peptide fraction. The electroshock biologically active peptide fraction can be used both independently and in combination with other therapeutic agents.

An additional objective of the invention is a pharmaceutical composition in which the active principle is the peptide fraction according to the present invention. In this case, the pharmaceutical composition must contain the active ingredient in an amount sufficient to provide a beneficial effect on the patient's body, i.e. keep it in an effective amount.

Another objective of the invention is to determine the effective dose of the peptide fraction according to this invention. It is believed that this dose is in the range from 0.1 to 50 mg / kg of patient weight. Obviously, the specific dose will be determined by the attending physician depending on the condition of the patient, his age, weight and course of treatment.

Detailed Disclosure of Invention

Further, the invention is disclosed in detail in preferred embodiments of a specific implementation, while the examples of the active peptide fraction, pharmaceutical compositions, dosage forms should not become the basis for limiting claims, but are intended solely to demonstrate the feasibility of the invention and the implementation of the specified (s) destination (s). Each person skilled in the art will surely be convinced that numerous modifications of the cited embodiments of the invention may be proposed, which fall within the scope of the claims set forth in the claims.

1. Obtaining a biologically active peptide fraction S-1-10.

To obtain a biologically active peptide fraction S-1-10, the blood of birds (chickens) was used, taken 3-4 seconds after electrical stimulation of the head for 2-3 with a voltage of 70-80 V. Blood was collected by gravity into plastic bottles after transection of the carotid arteries and veins and incubated at a temperature of 4-8 ° C for 18-24 hours, the serum was aspirated and irradiated With ₀ ⁶⁰ in the mode of 25 ± 5.0 kGy.

The treated serum was extracted with distilled water containing 0.5 mM phenylmethane sulfonyl fluoride (PMSF) in a ratio of 1:10, stirred for 1 h at a temperature of 5 ° C. The resulting suspension was centrifuged at 12,000 rpm for 30 minutes. The pellet was again extracted with PMSF water and centrifuged again at 12,000 rpm for 30 minutes. The supernatant was filtered through a membrane with a pore diameter of 0.45 μm, and then the filtrate was again filtered through 10 kDa membranes. The final filtrate (peptide fraction) was lyophilized by the conventional method and stored at 4-8 ° C.

2. Evidence of the biological activity of the peptide fraction S-1-10.

2.1. Physical endurance of animals

Physical endurance was determined by the influence of the peptide fraction on the duration of swimming of Wistar rats at 25 ° C, with a weight based on the tail weighing 10% by weight of the animal. In the experiment, 40 rats weighing 180-200 were used. Rats were divided into 4 groups of 10 animals in each group. Group 1 - control, rats were injected with 1.0 ml of physiological saline, groups II, III and IV were administered the peptide fraction in doses of 0.1; 1.0; 50.0 mg / kg of weight dissolved in 1.0 ml of physiological saline.

All test solutions were administered intraperitoneally 30 minutes before testing.

The results are presented in table 1.

Table 1 The effect of the peptide fraction on the physical endurance of animals (M + m) Statist. indicators The control The dose of the peptide fraction (mg / kg weight) 0.1 1,0 fifty M + m 3′31 ± 24 ′ ′ 4′23 ± 26 ′ ′ 4′36 ′ ′ ± 29 ′ ′ 5′27 ± 36 ′ ′ R > 0.05 > 0.05 <0.05

Thus, the peptide fraction at a dose of 50 mg / kg of body weight increases physical endurance by 54% of the control.

2.2. Parkinson's disease

When modeling Parkinson's disease, an analog of 6-HD was used - oxytremorin, which caused changes in animals typical of parkinsonism: tremor of the extremities, the latent period of the onset of general tremor (whole body), periods of absence of tremor, and termination of tremor.

Oxytremorin was administered intraperitoneally at a dose of 1.0 mg / kg body weight in saline, taken in a volume of 1.0 ml. The peptide fraction was administered in doses of 0.1; 1.0; 10.0; and 50 mg / kg weight, also dissolved in 1.0 ml of physiological saline. The fraction was introduced 45 minutes before the use of oxytremorine. In the experiment, 49 Wistar rats weighing 200-220 g were used.

The results are presented in table 2.

table 2 The effect of the peptide fraction on tremor in parkinsonism (M ± m) Tremor indicators The control Dose fraction (mg / kg weight) 0.1 1,0 10.0 50,0 1. The latent period of the onset of tremor of the limbs (s) 64.4 ± 3.8 85.0 ± 7.1
> 0.05 92.8 ± 7.3
<0.05 126.4 ± 17.7
<0.01 157.1 ± 26.0
<0.01 2. The latent period of the onset of general tremor of animals (s) 216.4 ± 21.7 294.4 ± 19.9 <0.05 Absent Absent Absent 3. The appearance of periods of absence of general tremor (min) 7.4 ± 0.5 6.7 ± 1.0
> 0.05 Absent Absent Absent 4. Termination of tremor of the extremities (min) 32.1 ± 5.7 21.3 ± 2.5
<0.05 21.2 ± 2.3
<0.05 23.1 ± 3.1
<0.05 14.9 ± 4.0
<0.01

Thus, the peptide fraction introduced 45 minutes before the simulation of Parkinson's disease, already at a dose of 0.1 mg / kg of body weight, prolongs the onset of general tremor (tremor of the extremities and trunk) by 31% and reduces the extinction time of tremor of the extremities by 50% of the control . Doses in the range from 1.0 to 50 mg of weight are most effective, not only by changing these parameters 2 times (at a dose of 50 mg / kg), but they also completely prevent the general tremor of animals (doses: 1.0; 10.0 and 50, 0 mg / kg).

2.3. Epilepsy

We studied the effect of various concentrations of peptide fractions on the onset of tonic and clonic seizures in the simulation of epilepsy by intraperitoneal administration of 0.5 ml. 20% camphor solution.

Lyophilized samples of the peptide fraction, dissolved in 0.5 ml of physiological saline, were also administered intraperitoneally 45 minutes before the introduction of camphor.

We studied 4 samples of the peptide fraction in doses of 0.1; 1.0; 10.0 and 50.0 mg / kg of animal weight.

In total, 50 Wistar rats weighing 220-230 g were used.

The results are presented in table 3.

Table 3 The effect of the peptide fraction on convulsive activity in epilepsy (M + t) Convulsive Activity Parameters Control (camphor) Dose fraction (mg / kg weight) 0.1 1,0 10.0 50,0 1. The beginning of tonic convulsions (min) 6.7 ± 0.8 7.1 ± 1.1
> 0.05 9.0 ± 1.0
<0.05 10.4 ± 1.1
<0.01 11.0 ± 1.4
<0.01 2. The beginning of clonic seizures (min) 14.3 ± 1.0 15.7 + 1.6 18.7 + 1.4 20.0 ± 1.9 20.6 ± 1.0 > 0.05 <0.05 <0.01 <0.01

Thus, the peptide fraction introduced 45 minutes before the simulation of epilepsy, taken in doses from 1.0 to 50 mg / kg of animal weight, lengthens the manifestation time of tonic and clonic seizures by 30-50%, respectively, i.e. reduce the convulsive manifestation of epileptic disease.

Claims (2)

1. A method of obtaining a biologically active peptide fraction that reduces tremor in Parkinson's disease, convulsive activity in epilepsy, characterized in that the head of the bird is electrostimulated in the mode of 70-80 V for 2-3 s, blood is taken, incubated at 4-8 ° C for 18-24 hours, then the selected blood serum is irradiated with C ₀ ⁶⁰ in the mode of 25 ± 5.0 kGy, extracted after irradiation with a 0.5 mM solution of phenylmethane sulfonyl fluoride in distilled water at a ratio of 1:10, stirred at 5 ° C for 1 h, centrifuged at 12,000 rpm for 30 minutes, then the precipitate is re-extracted with a solution of phenylmethane sulfonyl fluoride, centrifuged again under the same conditions, the supernatant is filtered sequentially through a membrane with a pore diameter of 0.45 μm and 10 kDa, and the target product, which is a peptide fraction with a molecular weight of up to 10 kDa, is lyophilized and stored at 4-8 ° C. 2. A pharmaceutical composition that reduces tremor in Parkinson's disease, convulsive activity in epilepsy, characterized in that the active principle contains an effective amount of peptides with a molecular weight of up to 10 kDa obtained according to claim 1 and a pharmaceutically acceptable carrier or excipient.