A method of manufacturing a biologically active preparations of embryonic tissues

 

(57) Abstract:

The invention relates to biochemical technologies for on the basis of embryonic tissues biologically active drugs. The method includes grinding soft embryonic tissues, their homogenization in the dispersion medium on the basis of water to the destruction of cell membranes, the separation of intact cellular elements, fractionation of the supernatant with the separation of the target product with an average mol. m less than 10 kDa, sterilization, filling and capping of the drug, and the target product is isolated and simultaneously sterilize fractionation supernatant molecular weight directly after the separation of intact cellular elements. The technical result is improved stability of the pharmacological properties of the target product and process performance and reduce costs. 5 C.p. f-crystals.

The invention relates to biochemical technologies for on the basis of embryonic tissues biologically active compounds with fixed pharmacological properties, despite the uncertainty of their chemical composition. Technological processes on the basis of the invention can be used in pharmaceutical precorrection and in particular, biostimulators a wide spectrum of action.

The need for drugs of this type is widespread and systematically increasing due to ongoing pollution by xenobiotics (including radionuclides), new (like HIV) infections and a significant weakening of physical activity on most urbanized population.

Therefore, the technological processes of manufacture of such preparations shall meet the complex is hardly compatible requirements. The most important of them are:

suitability for correction of the physiological status of a person in a wide range of possible abnormalities and their causes, at least contraindications to the use of and accessibility to health facilities and population based mainly, firstly, the performance of technological processes and resources for their implementation and, secondly, the stability of the target product and its suitability for long-term storage with preservation of sterility and pharmacological properties.

The first two requirements can be fulfilled with the use of embryonic tissue taken from cos who's substances a wide spectrum of action.

For example, application France 2413912 to a group of inventions "Medicines on the basis of the embryo and the retrieval method" a method of obtaining biologically active agents on the basis of embryonic tissue, which involves grinding and homogenization of the soft tissues of the embryos of animals mixed with saline solution.

Biologically active funds received described a simple method, have been applied to farm animals, thereby increasing their productivity by 7-15% in comparison with the control.

However, burdened with immunosuppressants and macromolecular antigens that are valid when you use these tools in animal husbandry, has not led to a marked immunostimulating effect in humans. In addition, the biochemical composition of the described drug varies significantly from batch to batch and therefore it is unsuitable for long-term storage.

More perfect method of obtaining biologically active agents according to the European application 0249563 for the invention of "Extracts from embryonic tissue of organs of animals, suitable for injection to a person". This method along with grinding and homogenization of the soft tissues of the embryo.

However, this extract does not pass many biologically active substances, which reduces the stimulatory effect when using the target product. In addition, the extract like the above preparation has a biochemical composition, which varies significantly from batch to batch, which is also reflected by the stability of its pharmacological properties during long-term storage.

So often resort to artificial methods of impact on fetal tissue in the hope to improve the efficiency and stability of target products as a means of treatment and prevention of human immune system.

Among analogues of this type of the present invention is closest to a method of manufacturing a biologically active agents on the basis of embryonic tissues, known from the description of an invention to the patent of Ukraine 2164. This method includes:

grinding soft embryonic tissues,

their homogenization in the dispersion medium on the basis of water to the destruction of cell membranes,

Department (for example, by filtration or centrifugation) intact cellular elements,

hydrolysis of the homogenate in the presence of a preservative at a temperature 4-45the processing of the hydrolyzate at a temperature of 60-120oC to denature the ballast substances,

Department denaturirovannykh thermolabile substances and separation of the hydrolysate thermostable target product with an average molecular weight less than 10 kDa by fractionation performed, for example:

- ultrafiltration, or

- centrifugation at acceleration over 1500 g for 20-40 min, or

- dialysis through a semi-permeable film and/or

- gelfiltration,

sterilization, filling and capping of the drug.

The target product is the supernatant, containing the active beginning of a mixture of relatively low molecular weight heat-stable substances of uncertain composition with an average molecular weight in the range from 0.7 to 10.0 kDa.

However, the described method

has the lower performance, the lower the temperature of hydrolysis,

associated with significant energy consumption and, most importantly,

not ensures consistent quality of the target product because of the fundamental impossibility of reliable management hydrolysis. Therefore, in the hydrolysate is always a very complex mixture of chemically and biochemically unstable products mainly protein nature. bathrooms proteins, which adversely affect the durability of the target product during storage and which (even with molecular weight less than 10 kDa) can have unintended side effects.

Accordingly, the basis of the invention is by improving methods of preparation of raw materials and Refine its processing to create such a method of manufacture of biologically active preparations from embryonic tissue, which would provide a significant increase in the stability of the pharmacological properties of the target product while increasing productivity and reducing the cost of its implementation.

The problem is solved by the fact that in the method of manufacturing a biologically active preparations from embryonic tissues, including the shredding of soft embryonic tissues, their homogenization in the dispersion medium on the basis of water to the destruction of cell membranes, the separation of intact cellular elements, fractionation of the supernatant with the separation of the target product with an average molecular weight less than 10 kDa, sterilization, filling and capping of the drug, according to the invention, the target product is isolated and simultaneously emitting sterilized fractionation super>The exception hydrolysis and change the order of execution of the remaining operations cause not only a significant improvement of performance and reduction of costs, which positively affects the availability of the target product for the General population of environmentally hazardous areas, but also virtually eliminates the appearance of the target product of uncontrolled impurities caused by hydrolysis of the raw material, and thereby stabilizes its pharmacological properties.

The first additional difference is that the supernatant after separation of intact cellular elements simultaneously fractionary and sterilized by ultrafiltration, which is one of the most effective processes of extraction from complex mixtures of substances with specified maximum value of molecular weight and eliminates bacterial contamination of the target product at its bottling and corking in sterile conditions.

A second difference, which can be used according to the invention independently from the first, and in combination with the first additional difference is that the embryos before chopping soft tissue is frozen and thawed. Would the x tissues of warm-blooded animals contributes to the suppression of their synthesis processes, the preservation of sufficient activity of redox processes and intensification of catabolic processes (see, for example: Tissue therapy/authors/. Ed. by N. A. Puczkowski - Kiev: HEALTH, 1975, S. 5, paragraph 6 above; S. 6, paragraph 5 th top; S. 12). However, we (on the data available to us for the first time) managed to establish experimentally that the freezing of embryos, i.e. their transfer to the solid state, and defrost before separating the soft tissue for subsequent grinding helps, as you can see in the examples below, a marked increase in biological activity of the target product.

The third additional (second) difference is that embryos frozen to a temperature of not lower than -80oC as the freezing to lower temperatures without affecting the increase of biological activity of the target product, substantially lengthen the defrosting and reduces the efficiency of the process.

The fourth additional (third) the difference is that embryos frozen gradually, that is, with a continuous decrease of temperature. It also helps increase the activity of the target product.

Fifth difference, kotoriii, or in combination with any of them or their arbitrary set, is that as the target product secrete substances with an average molecular weight of less than 5.0 kDa. Studies have shown, the target product with the specified characteristic has sufficient (immuno) stimulating activity with virtually no adverse reactions.

Further, the invention is illustrated

description of the proposed method in General,

the list of values and the target values of the controlled parameters of quality of the target product;

a description of the method of comparative tests of biological activity of the target product and

specific examples of the implementation of the inventive concept and the assessment of target products.

The proposed method in General involves:

the selection of embryos of farm animals (usually cows and possibly sheep or pigs in the first half of pregnancy) in slaughter shops of the enterprises of the meat industry,

pre-processing of selected embryos, including

- mandatory processing, i.e. the separation of the placenta, bleeding (usually through neperevyazannymi pupae washed embryos in the refrigerating chamber with the temperature not exceeding 12oC (usually for a period of not more than one day) and

- possible additional processing: freeze (particularly speed) to a temperature below 0oC (but preferably not below -80oC) embryos after bleeding and flushing and thawing;

Department heads, hooves, gall bladder (with caution), stomach and intestines;

the separation of soft tissue from the bones;

grinding of soft tissue (usually with cutters);

homogenization crushed the soft tissues in the dispersion medium on the basis of water (usually in isotonic) to the destruction of cell membranes (using apparatus of the type colloid mill");

Department of intact cellular elements and fibrous tissue (e.g. by centrifugation at 1-80 thousand rpm, or by filtration, typically by vacuum suction filter);

the simultaneous selection of the target product with an average molecular mass of less than 10 and preferably less than 5) kDa and sterilization fractionation of the supernatant with the use of funds, employees bacterial filters (preferably using fiber ultrafiltration plants);

bottling and capping of the target product (usually in water as a dispersion medium to prepare the homogenate to a target product before bottling add sodium chloride to achieve isotonic point.

In the target product, put on long storage, can be added a suitable preservative, such as chinosol, sufficient to achieve an antiseptic effect and pharmacologically acceptable amount, in particular about 0.8-1.0 g/L. in Addition, such product may be further sterilized by any known method.

In the target product in accordance with the approved in 1994 Pharmacopoeia Committee of the Ministry of health of Ukraine Time pharmacopoeial article VFS U "Erbisol for injection (Erbisolum pro injectionibus)" control methods provided by "the State Pharmacopoeia" and referred to the global Fund:

transparency (the drug must be able to withstand comparison with the reference solution 1; GF XI, vol. 1, S. 198);

chroma (color of the drug should not be more intensively reference 3A; GF XI, vol. 1, S. 194), and the drug without preservatives practically colorless, and when using chinosol has a yellow-brown color;

pH from 6.5 to 7.5 (potentiometric determination; GF XI, vol. 1, S. 113);

mechanical incorporation (the drug must be able to withstand the requirements specified in the Temporary instructions for the control of injection solutions in mechanical inclusions And 42-3-8. , S. 187);

toxicity (drug should be non-toxic in vivo when administered intravenously laboratory animals at a dose of 0.5 ml/kg and observed for 48 hours; GF XI, vol. 2, S. 182);

progenote (the drug should be aerogenes in vivo when administered intravenously laboratory animals at a dose of 0.5 ml/kg of a mixture, prepared in a ratio by volume 1/1 from the test sample and 0.9% isotonic sodium chloride for injection, when observed within 48 hours; GF XI, vol. 2, S. 183);

the protein content (when processing 1 ml 1 ml 5% solution of trichloroacetic acid and stirring the solution should be transparent, indicating the absence of protein);

the content of peptide in 1 ml of the drug from 1.0 to 3.0 mg/ml (biuret reaction);

the dry residue from 14 to 20 mg/ml (when drying to constant mass 10 ml at a temperature of from 100 to 105oC);

biological activity, which in the determination method described below should not be lower than 3 units.

The biological activity of the obtained substances determined by the absorption of the dye (nicrosini tetrazole) by macrophages under the influence of the target product. To do this, from the abdominal cavity of mice or rats wash f is ü contribute vials made of glass with a flat bottom and incubated in a thermostat at 37oC for at least 1 hour. After that, the bottles three times washed with saline or buffer solution to remove unattached cells.

Next, make one part of vials portion of the liquid, consisting respectively of 0,5/0,5/1 parts saline solution, the target product and a 0.1% solution of the dye, and in another part of the bottles - the portion of the liquid, consisting respectively of 1/1 parts of saline and 0.1% solution of the dye. Cells with the indicated experimental and control mixtures incubated for 1 hour, then washed three times to remove residual dye. Vials with washed cells add dimethylsulfoxide and 2M caustic alkali in the ratio of 1:1. After 10-15 min to measure the optical density of the solution at a wavelength of 630 nm.

Biological activity was assessed by the ratio of optical densities in the experiment and the control.

Example 1.

To obtain the target product were derived embryos of pigs and cattle (hereafter CRS).

After the mandatory pre-processing: separation of the placenta, bleeding, flushing with running water to remove traces of amniotic fluid and blood and daily withstand crashes-auto St the hoof, gallbladder, part of the gastrointestinal tract and soft tissue from the bones.

Then a soft cloth to grind the cutter with the holes in the grill with a diameter of 3 mm and stuffing missed twice through a colloid mill to homogenize using pyrogen-free distilled water as a dispersion medium.

The homogenate was ofcentrifugal for 15 min at 3 thousand rpm to separate intact cellular elements and fibrous tissue, sediment was transferred for recycling, and the supernatant was added sodium chloride to a final concentration of 0.9% and the mixture was subjected to sterilizing ultrafiltration to isolate a target product with an average molecular weight less than 10 kDa.

Next, the above-described method defined biological activity, which for a product derived from porcine embryos were 5.0 times, and from embryos of cattle - 4.0 times higher than the control.

After controlling transparency, color, pH, particulate matter, sterility, toxicity, progenote and content, proteins, peptides, and the dry residue, which is in compliance with the above-mentioned Temporary monograph VFS U "Erbisol for injection", target Holocene of the target product were taken fresh and frozen embryos of cattle.

Target product from fresh embryos (CPSA) was obtained mainly as described in example 1 with the difference that before the separation of the soft tissues they are not kept in the refrigerator, and when sterilizing ultrafiltration as the target product were isolated substances with an average molecular weight of up to 5 kDa.

To obtain the target product from frozen embryos (CPSA) the method described in example 1, was supplemented by operations such as freezing drained and washed embryos in the freezing chamber to a temperature of about -40oC, holding at this temperature for 48 hours and their spontaneous thawing at room temperature. As in example 1, when sterilizing ultrafiltration as the target product were isolated substances with an average molecular mass of 10 kDa.

The biological activity of CPSA was 7.8, and CPSA - 10.8 times higher than the control.

Example 3.

To obtain the target product were derived embryos of cattle, gradually (when the temperature drops with the speed -2oC/hour) frozen to -80oC in the freezer, equipped with a temperature controller, and then thawed at room Tempel injection of 0.9% isotonic sodium chloride.

The biological activity of CPSA was 11.0 times higher than the control.

Example 4.

To obtain the target product were derived embryos of cattle, gradually with decreasing temperature with a rate of-2.5oC/hour) frozen to -90oC in the freezer, equipped with a temperature controller, and then thawed at room temperature.

CPSA was obtained as described in example 2, using homogenization suitable for injection with 0.9% isotonic sodium chloride.

The biological activity of CPSA was 10.8 times higher than the control.

Example 5.

To obtain the target product were derived embryos of cattle, of which the above described method of the prototype using hydrolysis homogenate in the presence of a preservative (hinote) at a temperature of 40oC for three days, heat treatment of the hydrolyzate at a temperature of 120oC to denature the ballast substances, Department denaturirovannykh thermolabile substances and ultrafiltration of the hydrolyzate obtained target product with an average molecular weight less than 10 kDa.

Its biological activity was 2.3 times higher than the control.

Industrial primenimosti, which in all cases is more than 4 times higher than the control), and reduction of energy consumption on average by 15%, and ability to perform the entire production cycle is not more than 60 hours (usually at night).

1. A method of manufacturing a biologically active preparations from embryonic tissues, including the shredding of soft embryonic tissues, their homogenization in the dispersion medium on the basis of water to the destruction of cell membranes, the separation of intact cellular elements, fractionation of the supernatant with the separation of the target product with an average mol. m less than 10 kDa, sterilization, filling and capping of the drug, wherein the target product is isolated and simultaneously emitting sterilized by fractionation of the supernatant molecular weight directly after the separation of intact cellular elements.

2. The method according to p. 1, characterized in that the supernatant after separation of intact cellular elements simultaneously fractionary and sterilized by ultrafiltration.

3. The method according to p. 1, characterized in that the embryos before chopping soft tissue is frozen and thawed.

4. The method according to p. 3, characterized in that the embryos samarjit gradually.

6. The method according to p. 1, characterized in that as the target product secrete substances with an average mol. m less than 5.0 kDa.

 

Same patents:
The invention relates to medicine, namely neurosurgery, neurology and psychotria and can be used for examination of patients with suspected epilepsy
The invention relates to medicine, in particular to pulmonology and can be used in the treatment of diseases of the lungs with the formation of residual cavities
The invention relates to medicine and can be used in the treatment of diseases of the Central nervous system

The invention relates to biotechnology and can be used in pharmacology and medicine
The invention relates to biotechnology and can be used in pharmacology and medicine
The invention relates to biotechnology and can be used in Oncohematology and neurooncology
Candles // 2128049
The invention relates to medicine, the pharmaceutical industry and relates to the creation of new dosage forms containing hyaluronidase
The invention relates to medicine, in particular to pulmonology

The invention relates to medicine, in particular for cell therapy and can be used for the treatment of diseases caused by disorders of the immune system, for example, diabetes
The invention relates to medicine, in particular to a method of local treatment of burns

FIELD: medicine, otorhinolaryngological surgery.

SUBSTANCE: one should apply thin layer of "Solcoseryl" gel onto osseous facial walls of frontal and maxillary sinuses at the border with trepanation opening after removing pathological content out of them and before applying a transplant out of flat bone of human fetal cranial arch that exceeds the diameter of trepanation opening by 3-4 mm. Then, one should additionally fix the transplant by affecting with distal edge part of a light guide of semi-conductor laser "ATKUS-15" with contact-type technique at output power of laser radiation being 8 W at constant mode. The method enables to increase fixation density of allobrefobone to osseous walls of sinus along its whole diameter.

EFFECT: higher efficiency of fixation.

1 ex

FIELD: medicine, surgery.

SUBSTANCE: the present innovation deals with local treatment of erosive-ulcerous defects of skin and mucosa, inflammatory intestinal diseases and prolongly non healing wounds, among them. The method includes the use of diploid embryonic fibroblasts cultivated beyond the body, moreover, the fibroblasts should be pre-affected with dexametason at concentration of 10-4 M/l under culture conditions for 1 h followed by washing it from preparation and changing the medium. Then one should apply the developed supernatant in the form of local application onto affected area once daily. The method enables to decrease inflammatory manifestations in wounds and erosive-ulcerous defects of skin and mucosa, stimulates regenerative processes at minimal restrictions while applying the present innovation from the side of lesion section and decreases financial expenses.

EFFECT: higher efficiency of local treatment.

7 dwg, 2 ex, 6 tbl

FIELD: veterinary science.

SUBSTANCE: one should apply a honey-tissue preparation that contain emulsion out of the walls of pregnant uterus and ovaries without cow's yellow bodies 6.0 ml, natural bee honey 5.0 g, isotonic sodium chloride solution 2.0 ml sodium benzoate caffeine 1.5 g to be introduced into dorsal lumbar muscles both from the right and from the left per a half of the dosage being equal to 0.03-0.04 ml/kg either once or twice at 6-7-d-long interval. The present innovation enables to improve metabolism, normalize the work of hormonal and nervous systems, normalize functions of uterine muscles at hypo- and atonias, at delayed afterbirth and restore affected ovarian functions.

EFFECT: higher efficiency of therapy.

FIELD: veterinary medicine.

SUBSTANCE: the present innovation deals with methods to activate the activity of protective mechanisms and organs of hormonal regulations. One should introduce a tissue preparation for an animal, moreover, to obtain it is necessary to apply 2.5 g pregnant or postpartum uterus, per 1.5 g thyroid, parathyroid and thymus glands, 2.0 g pancreas, 2.5 g liver all purified against spare tissues from clinically and hematologically healthy animals from cattle group up to 4-6-yr-aged period. The organs mentioned should be reduced, then tissue mixture obtained should be mixed with fresh running water at 1:2 ratio to obtain emulsion to be thermally treated in water bath at 58-59 C. After cooling emulsion should be supplemented with formalin, ACD f-2 and natural bee honey to obtain the following ratio of components in ready-to-use product: tissue emulsion 10 g, 68.7%; ACD f-2 1.5 g, 10.3%; formalin 0.006 g, 0.4%; natural bee honey 3.0 g, 20.6%. Before being introduced for an animal one should carefully mix preparation to detect its dosage at 0.02-0.5 ml/kg animal body weight to be then introduced per Ѕ parts from the right and from the left into dorsal lumbar muscles either once or twice at interval of 7-9 d. The method enables to perform complex biostimulating and hormonal impact upon animal body due to keeping active substances of tissue preparation being obtained due to above-mentioned technique.

EFFECT: higher efficiency of prophylaxis and therapy.

FIELD: biopharmacology, medicine.

SUBSTANCE: the suggested biotransplant contains an active component: the culture of genetically unmodified neuronal stem cells (NSC) obtained out of anterior cerebral tissue of human embryos of the 1st trimester of pregnancy or periventricular cerebral area of 15-20 wk gestation and selectively multiplied under cultivating conditions up to the quantity of 10 (7) - 10 (9) pluripotent undifferentiated cells in composition of neurospheres, at the content of NSC being 50-500 mln., and 2 ml physiological solution. The method for treating ischemic insult deals with introduction of 50-500 mln. NSC in 2 ml physiological solution. The suggested biotransplant and method for treating ischemic insult enable to quickly restore and improve cerebral functions.

EFFECT: shortened terms of therapy.

5 cl, 2 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: biotransplant has active ingredient like culture of genetically unmodified neuron trunk cells taken from anterior brain of the first pregnancy trimester human embryo or periventricular region of brain of 15-20 gestation weeks and selectively reproduced under cultivation conditions to the amount of 107-109 pluripotent non-differentiated cells in neurospheres having 50-500 mln of neuron trunk cells and 2 ml of physiologic saline. Method involves introducing 50-500 mln of neuron trunk cells and 2 ml of physiologic saline in one stage in endolumbal way.

EFFECT: enhanced effectiveness of treatment; accelerated repair and improvement of injured brain functions; stable treatment results.

5 cl, 1 tbl

FIELD: medicine, gerontology.

SUBSTANCE: beforehand one should detect human biological age and the age of human immune system to compare them with human calendar age: in case of predominance of at least one of them against calendar age one should once introduce the suspension of human fetal tissues into subcutaneous fiber of anterior abdominal wall. For this purpose one should apply fetal tissues being heterogeneous by their histogenesis and, also, preparations of placental origin. The innovation provides normalization of immunocytogram values and, thus, similarity of calendar and biological age for 1 yr.

EFFECT: higher efficiency of correction.

FIELD: medicine; medical engineering.

SUBSTANCE: biotransplant has genetically unmodified mesenchyma stem cell culture as active component obtained from fetal donor autologous material. The tissue is subjected to disaggregation and the produced cell suspension is resuspended and cultivated on growth medium containing transferrin, insulin, fibroblast growth factor and heparin to accumulate mature stroma in cell culture. Method involves intravenously dropping mesenchyma stem cell culture in the amount of 50 to 500 mln in 50-100 ml of physiologic saline.

EFFECT: accelerated recovery of bone tissue; positive biochemical factors dynamics; improved patient locomotor activity.

6 cl

FIELD: medicine; medical engineering.

SUBSTANCE: biotransplant has genetically unmodified mesenchyma stem cell culture as active component obtained from fetal donor autologous material and genetically unmodified fetal myoblast culture. Method involves intravenously dropping mesenchyma stem cells in the amount of 50 to 500 mln in 50-100 ml of physiologic saline. The fetal myoblast culture is intramuscularly introduced at a dose of 100 mln of cells per 10 kg.

EFFECT: enhanced effectiveness in repairing incretory function of pancreas and reducing resistance to insulin; reduced risk of nephropathy and other complications.

9 cl

FIELD: biopharmacology, medicine.

SUBSTANCE: the present innovation deals with preparing the culture of genetically unmodified chondroprogenitor cells and method for treating parodontium diseases. Biotransplants, methods for their obtaining and method for treating parodontium diseases deal with introducing the suspension of mesenchymal stem and chondroprogenitor cells or their combination that contains 1-50 mln. cells/ml, intraligamentally from lateral and medial sides of every tooth and subperiosteally, into mandibular and maxillary parodontal parts.

EFFECT: higher efficiency of therapy.

13 cl, 1 ex

Up!