Method for preventing and correcting cutaneous alterations

FIELD: medicine, cosmetology.

SUBSTANCE: one should introduce enzymes-containing liposomes of DNAase activity into skin and/or subcutaneous fiber, moreover, one should apply multi-layer MLV-liposomes at their size ranged 500-2000 nm at temperature of phase transition of liposomal membrane being 30-60 C, moreover, one should deliver and deposit an enzyme into intercellular space, and as enzymes with DNAase activity one should apply endo- and/or exonucleases that destroy single and/or double stranded DNA. The innovation prevents the damage of intracellular DNA of alive skin cells and/or its adnexa and/or subcutaneous fiber.

EFFECT: higher efficiency of prophylaxis and correction.

4 ex, 4 tbl

 

The invention relates to medicine, and cosmetics, in addition, it can also be used in veterinary medicine.

As is known, the most frequent unwanted processes occurring in the skin and subcutaneous fat are infectious diseases of the skin and its appendages. Such processes include, for example, pyoderma, acne, fungal skin, hair, nails, and other degenerative skin condition (thinning, loss of elasticity, turgor and wrinkles with age) and subcutaneous tissue (the so-called "cellulite"). With all these pathologies, and also due to the aging of the basic parts of an unwanted process not occur in the cells of the skin and subcutaneous fat, but mainly in the intercellular space (extracellular matrix).

Known method of correcting changes of the skin by skin application from 0.01 to 1 ml of an aqueous solution deoxyribonuclease (Gnkazy) on 1 cm2the skin, when the solution Gnkazy has a concentration of from 1 to 70% Kunz 1 ml (US 6524578).

The disadvantages of this method:

the method requires a complete drying solution Gnkazy on the skin for a long time (more than an hour), which leads to the formation of a layer of dry Gnkazy on the skin surface, or requires the application of a dry powder Gnkazy, which forms a concentrated solution with usually having the situations on the skin surface, trace water, or requires a large consumption of aqueous solutions of Gnkazy (250 to 500 ml) in the examples; these circumstances cause irrational high consumption of Gnkazy;

- requires subsequent removal Gnkazy with the surface of the skin, which creates certain inconveniences;

- the main part of the enzyme remains on the skin surface and has a negative damaging impact, loosening the keratin plates that leads to a low resistance of the skin to any external influences;

in the area of living cells penetrates only a small part of Gnkazy, which leads to low efficiency of the method;

the method involves the use of Dinkas in aqueous solutions, where Gnkazy unstable, which requires, as a rule, special storage conditions such solutions or prepare the solutions immediately before application to the skin.

Also there is a method of prevention and correction of skin changes by introducing into the skin of enzymes involved in DNA repair and is included in pH-sensitive liposomes (US 5296231). The method involves the correction of damage (double strand breaks) DNA of somatic cells caused by ultraviolet radiation, which increases cell survival after such exposure, the method includes applying enzymes involved in DNA repair your skin with the aim of introducing enzyme alive in the s cells of the skin. Among the enzymes that are available for this purpose (repair of DNA lesions caused by UV-radiation and other effects that lead to changes of nucleotides or nucleotide gap circuit)are the enzymes that may participate in repair of damaged nucleic acids, particularly DNA. These enzymes are6-methylguanine-DNA methyltransferase, photolyase, uracil - and gipoksantin - DNA glycosylase, pyrimidinone/apurinovaya endonuclease, DNA ectonucleoside, damaged-the bases glycosylase currentanalysis and their complexes, as well as other enzymes and enzyme complexes, the activity of which is currently only partially explored, such as the products ERCC-human genes, and RAD genes of yeast.

The method includes shipping inside the skin cells of one such enzyme (endonuclease V of the bacteriophage T4). For the implementation of the known method specially designed pH-sensitive liposomes by mixing a special blend of lipids. That is, at acidic pH the lipid membranes of liposomes protonilus, and membrane lipids unstable, which leads to the release of content. Such acidic conditions occur when antiferromagnet in a cage liposome interacts with lysosomes to the cytoplasm, where the liposome releases its contents. These liposomes are used for DOS is where it is refuelled target enzyme (enzyme, reducing DNA) into the cell. As noted in the description of the patent: "the main limitation is that liposomes should not be toxic to living cells, and that they should deliver their contents into the interior of the cells exposed to therapeutic effects" (column 12, 50 and 55 lines). To achieve this, use a pH-sensitive small single-layer liposomes (SUV) of size less than 250 nm in diameter.

This method is adopted for the prototype of the present invention.

The disadvantages of this method are:

narrow scope: method allows to prevent damage or to repair damaged cellular DNA; DNA in the intercellular space of the impact is not as liposomes, which is based prototype method, captured by endocytosis living cells or merge with them;

according to the method prototype enzymes deliver inside a living cell, which can cause damage to cellular DNA of the cells of the skin and subcutaneous tissue;

- the basis of the prototype method required the use of liposomes, which is not enough rigidly at high temperatures, which leads to the destruction of liposomes and/or leakage from liposomes enzyme.

The present invention is based on a solution of the following tasks:

- ensure their prevention and correction of changes in the skin and subcutaneous fat, associated with high levels of extracellular DNA in the intercellular space;

- prevent damage to cellular DNA of living cells of the skin.

According to the invention in a method of prevention and correction of skin changes, and/or its appendages, and/or subcutaneous tissue, comprising introducing into the skin and/or subcutaneous tissue of liposomes containing enzymes with Dnesday activity, use of multilayer MLV liposomes ranging in size from 500 to 2000 nm with the temperature of the phase transition of the membrane of liposomes from 30 to 65°With carry out the delivery and deposition of the enzyme in the intercellular space.

The applicant found that the introduction and deposition in the intercellular space of the enzymes that destroy DNA, leads to the prevention of unwanted, including infectious, processes, and their reverse development. In the process of the invention it was found that this introduction of these enzymes unexpectedly led to the prevention and/or partial reduction of skin changes, naturally occurring in the aging process of the skin, its appendages and subcutaneous fat. The study of possible mechanisms of this effect showed that targeted nucleases, purposefully introduced into the extracellular space with the creation of the depot, is extracellular DNA, including the DNA of dead keratinous the tov, DNA produced by the infectious agent, as well as DNA of pathogens - bacteria and fungi. In this case there is damage to the DNA of living cells of the skin and subcutaneous fat.

In the applicant proposed method, liposomes do not penetrate into living cells by endocytosis or pinocytosis, resistant to merge with living cells and their contents are not proniknout in the living cell and does not damage the DNA of this cell; used liposomes to take care of enzymatic activity in the intercellular matrix in a wide range of temperature and pH, as with the development of undesirable (in particular, infectious processes in the skin and subcutaneous tissue there is a significant fluctuation of these parameters.

MLV liposomes ranging in size from 500 to 2000 nm with the temperature of the phase transition of the membrane of liposomes from 30 to 65°To provide high efficiency activate nucleases, as the ratio of the volume of the aqueous phase/lipids high enough, nucleases, as a rule, lipophobia, and a multilayer structure such liposomes prevents nucleases to quickly flow from the liposomes.

When used for design of liposomes mixture of lipids with the resulting temperature of the phase transition their membranes less than 30°With part of the liposome fuses with the cell, its content goes live in somatic cells. This leads to the damage is the establishment of nuclear and non-nuclear cell DNA by nucleases.

The use of liposomes with the temperature of the phase transition of lipid membranes above 65°leads to a significant reduction of enzyme activity is included in liposomes enzymes, as well as partial loss of enzyme activity caused by thermal denaturation of enzymes in the preparation of such liposomes.

As the enzyme (enzymes) with Dnesday activity is preferable to use non-specific nucleases, destroying any (single-stranded and/or Dunaeva) DNA regardless of its origin (cell type) or specific properties of the DNA. The most suitable are non-specific endo - and/or ectonucleoside. The use of specific nucleases results in insufficient efficiency of the method or limit the spectrum korregiruet unwanted processes of the skin and subcutaneous tissue. As such endo - and/or economies, non-splitting the DNA can be used, for example, bovine pancreatic Tnkase (as obtained from animal products, and recombinant), human Tnkase I (as derived from human liquids and recombinant), mutant Gnkazy with enhanced enzymatic activity, as well as Gnkazy microbial origin.

The implementation of the present invention is as follows.

Manelmellado (single) liposomes typically have a spherical shape and a minimum ratio of surface area/volume", while multilayer liposomes are extruded cylinders and/or spheroids.

MLV liposomes are produced by a method of hydration of the dry film of lipids. The formation of liposomes, widely varying in size, with a diameter of approximately 0.1 to 0.5 μm. To obtain fractions of liposomes with a more uniform size of the liposomes is shared by various methods, for example by centrifugation in a gradient of ficoll or gel filtration. Choosing the fractionation conditions, you can select one or another faction of the multilayer (multilamellar) liposomes with a more uniform size. For example, were allocated to the so-called "large" multilamellar liposomes (with a diameter of approximately 1.0-0.3 microns) and "small" multilamellar liposomes (with a diameter of approximately 0.3 to 0.2 μm).

Properties of liposomes as carriers of biologically active substances depend on many parameters, the main ones are the properties of the lipids forming the liposomes, the size of the liposomes and the structure of its wall (manelmellado or a mul is lamellare). The most important properties of the lipids forming the liposomes include transition temperature and the charge of the lipid.

The temperature of the phase transition determines the fluidity of the lipids constituting the liposome, the rigidity of the liposome, its ability to interact with biological membranes both in vitro and in vivo stability of the liposomes in a certain temperature range, the effectiveness of the inclusion of biologically active substances in liposomes.

The lipid component of biological membranes, including artificial liposomes, including), at certain temperatures, you may experience the so-called phase transition of the first kind. At lower temperatures there is a transition of the lipids from the liquid state to the gel state, sometimes called cordocentesis. This happens when the temperature drops below the critical (Tcrit - temperature phase transition of the gel - liquid crystal"). This temperature is different for each membrane and depends on the chemical composition of the lipids forming the membrane. The transition temperature may range from -20°With (for membranes of unsaturated lipids) to +60°With (for membranes from saturated lipids). For example, in belayneh membranes of pure phosphatidylcholine transition temperature is 23 °C. the Degree of ordering of the lipid molecules is in the gel state increases dramatically. Hydrophobic "tails" of the lipid in the gel phase are parallel to each other (are transconformation). The thickness of the bilayer in the gel phase is significantly higher than in the liquid crystal phase. The implementation of all functions of the membranes of living cells is possible only in the liquid crystal phase. Liposomes, the lipids are transferred into the gel phase, become completely rigidly, the membrane loses its fluidity and becomes quite similar to a living membrane.

Enzymes are protein molecules that are included in liposomes to deliver them into cells to compensate for enzyme deficiency or for any other purpose. With this enabled, the following objectives are pursued: the direction of the enzyme (by infusion into the bloodstream) in those tissues where it accumulates substrate, preventing premature interaction of the enzyme with substrates and other proteins, overcoming the blood-brain barrier, reducing the potential antigenicity of the enzyme.

For enzymes associated with membranes, rigidity (fluidity) of the membranes is a critical moment in the exercise of their functions.

For preparation necessary for the implementation of this method MLV liposomes ranging in size from 200 to 500 nm and the temperature of phase transition membranes from 30 to 65°used With cholesterol and phospholipids with varying degrees of us is the security of hydrophobic tails, including high temperature phase transition, in particular, such as dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidyl, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine. Cholesterol and phospholipids were mixed in various molar ratios. By changing the molar ratios of mixtures of cholesterol with lipid with high and low temperature phase transition, received liposomes with varying degrees of phase transition of lipid membranes. Cholesterol and phospholipids were mixed in various molar ratios. The resulting mixture was dissolved in chloroform (10 ml chloroform per 100 mg of phospholipid). Chloroform drove on a rotary evaporator in a round bottom flask to obtain on the walls of the lipid film at temperatures up to 65°C. Drugs Dnes was dissolved in phosphate buffer. For Dinkas dependent on the presence of ions dwuhvalentnykh metals, the buffer was pre-entered from 0.5 to 5 mM per liter dwuhvalentnykh cations magnesium (as sulfate). Multilamellar liposomes formed in phosphate buffer with dissolved enzyme (about 2 mg of phospholipid per 10 ml buffer) by stirring for 20 minutes. In order to wash the preparation of liposomes from free enzyme, three times (2.5 hours each) conducted dialysis against 50-fold excess of phosphate buffer. Lipase is s divided by size into fractions by gel filtration or by centrifugation in a gradient piccola.

Dnazol enzyme activity included in the liposomes was evaluated in samples of drug obtained liposomes after the destruction of liposomes by detergent (Triton X100) and removal of lipids. Activity Gnkazy was determined by the method Kunz (1950) when the pH optimum for the specific enzyme.

Examples of implementation of the invention

Example 1. Bovine pancreatic to Tenkasu (manufactured by MP Biomedicals with specific activity of at least 2500 units Kunz 1 mg of enzyme) were introduced in the MLV liposomes size of 500 nm and the temperature of the phase transition 30°C.

The experiments were carried out on rats "Wistar" average weight of 200-250 g Purulent inflammation of the skin was modeled by intradermal injection of a suspension of bacteria in the area of 1.5 cm2back in the interscapular area, where previously shaved off my hair. Introduced daily suspended agar culture of Staphylococcus aureus strain 394 in the total number of 100 CFU per 1 animal. The introduction was carried out by 10 microinjection, distributed evenly on the specified area of the skin. After 5 days was formed purulent inflammation of the skin. Animals were divided into 5 groups. Treatment was started on the first day of the formation of purulent inflammation of the skin and held for 10 days. Estimated area purulent lesions and microbial colonization.

Group 1 were microinjection suspension MLV liposomes loaded with enzyme from the calc is and 0.2, 2 and 20 units Kunz on 1 cm2affected pyoderma skin surface (3 groups - a, b, c, respectively).

Group 2 were microinjection suspension of liposomes loaded with the same enzyme, obtained as specified for the prototype, at the same rate of enzyme activity per unit area of the surface of the skin, as in group 1 with the same subgroups of 0.2, 2 and 20 units Kunz on 1 cm2affected pyoderma skin surface (3 groups - a, b, c, respectively).

Group 3 received treatment similar to the treatment received by the first group, with the difference that Tnkase was not introduced into the solution in the preparation of liposomes, that is, the animals received empty MLV liposomes in the same amount (lipid) and the introduction of a suspension of liposomes, as in group 1.

Group 4 animals appliciable 10% tetracycline eye ointment in a volume of 0.2 ml on 1 cm2the surface of the affected area (positive control group).

Group 5 animals served to provide a negative control.

All impacts was performed 1 time per day.

The results of the experiment are presented in table 1.

Table 1
Group12345
SubgroupAndInAndInAndIn--
The number of animals1010101010101010101010
Dose Gnkazy (units Kunz on 1 cm2the surface of the affected skin)
0,22200,2220-----
Microbial colonization (CFU/g), average
1 day7,57,87,7,7,47,67.47,47,67,37,87,8
4 days6,45,03,27,17,57,07,27,47,16,36,9
8 days5,03,21,56,76,6 6,56,76,86,94,86,5
16 day4,12,104,85,25,0the 4.75,1a 4.93,05,0
The area of purulent lesions, % of initial average
1 day100100100100100100100100100100100
4 days7761559496939496937997
8 days5855468386838386835080
16 day191082425232425221527

From the data presented in table 1, it follows that no "empty" MLV liposomes, either way-the prototype does not have a measurable action is I on the development of infection, the claimed method is comparable in efficiency with the use of one of the known antibacterial agents.

Similar results were obtained when comparing the claimed method and the prototype method on this model in the preventive purposes. Introduction to preventive mode was performed for 2 hours before inoculation of the infectious agent into the skin. The prototype had no preventive actions. Implementation of the invention using MLV liposomes, the size of 500 nm with a transition temperature of 30°loaded with recombinant bovine Dnazol (manufactured by Sigma)was exceeded on preventive effect of applying a 10% ointment tetracycline (ointment "Tetracycline" 10%, manufactured by OAO Nizhpharm).

Example 2. In MLV liposomes with a size of 1000 nm and the temperature of the phase transition of the membrane 50°introduced Serratia marcescens endonuclease, having received it from the culture of bacteria.

The claimed method was used for the treatment of experimental fungal infection caused Microporum canis, with applications to the surface of the affected skin and its appendages.

The treatment according to the present method was compared with exposure in accordance with the method of the prototype. As a positive control was used 1% cream terbinafine (drug lamisil", manufactured by Novartis).

Used Guinea pigs of both sexes weighing 600-700 g and W the AMM Microporum canis VT 999, isolated from the patient. Colony Microporum canis were cultured in the medium Micosel (Difco) and on potato agar for 2-3 weeks at a temperature of 25°C. Colonies were transferred from the surface of the agar in a test tube with saline solution and triturated in a plastic homogenizer for about 1 min to obtain a homogeneous suspension. Cell viability was assessed by the ability to include fluorescence dye. Spores were counted in the camera Goryaeva. The lower part of the back of the Guinea pig shaved and on the surface scarification skin inflicted 0.4 ml of a suspension containing 1 million spores Microporum canis 1 ml. Place inoculation covered with polyethylene film (4 cm × 4 cm) and recorded its elastic bandage for 24 hours. Edema, erythema and peeling at the site of inoculation of the fungus developed in animals for 5-6 days. Successful infection was confirmed by microbiological method. In the experiment consisted of animals with developed infection, confirmed by the microbiological method. The sowing material from the inoculation was performed every 3 days after inoculation.

The animals were divided into 5 groups. The drugs were starting to take on day 7 after inoculation. Animals of groups 1, 2, 3 preparations containing enzymes, was applied at a rate of 0.1; 1.0, and 10.0% Kunz on 1 cm2the affected surface, respectively.

Group 1 received the nuclease of Serratia marcescens coz the ACLs claimed method.

Group 2 received Tnkase Serratia marcescens according to the method prototype.

Group 3 received applications solution nuclease of Serratia marcescens in phosphate buffer.

Group 4 received the application of sterile phosphate buffer solution and was used for control.

Group 5 received MLV liposomes of the same composition and dimensions as for group 1, but not loaded with enzyme ("empty" MLV liposomes).

Group 6 received appliques cream 1% terbinafine (drug lamisil", manufactured by Novartis) (0.05 g per cm2surface) and served as a positive control group.

Best premature drying and draining applied to the skin of the drugs prevented the imposition of a polyethylene film with sticky ends and fixing bandage. All drugs were applied 1 time per day. Estimated date of start of hair growth, the deadline for the complete resolution of lesions and duration of microbiological purification of the inoculation (sowing material from the inoculation). The results are shown in table 2.

Table 2
no group

(number of animals per group)
The dose of enzyme units Kunz on 1 cm2Commencement of hair growth, days (mean)The time resolution of the hearth, days (mean)The term micro is biologicheskogo purification of the hearth, days (average)
1(10)The inventive method0,1233442
1193039
10162735
2(10)The placeholder0,1334252
1314353
10324250
3(10)Applique solution nucleases in phosphate buffer0,1314350
1294049
10294153
4(10)Applique sterile phosphate buffer solution (negative control)-314252
5(10)Applique mist "empty" MLV liposomes (control MLV liposomes)-324452
6(10)1% cream terbinafine (positive control)-223642

When the analysis of the results shows that the inventive method is much more efficient than the prototype, and compare the results or exceeds the impact of known antimycotic drug.

Similar results were obtained when comparing the claimed method and the prototype for this model in the preventive mode of administration. Introduction to preventive mode was performed for 2 hours before inoculation infektsionnogo agent into the skin. The prototype method is not provided preventive actions, the claimed method was superior to the preventive effect of application of a 1% cream terbinafine.

Example 3. When implementing the method the human recombinant Tnkase I (the production company "Genentech", Pulmozyme) were introduced in the MLV liposome size 2000 nm and the temperature of the phase transition 65°C.

The study included 30 women aged 41 to 55 years. Before the study the women were assigned to groups as follows. In group 1 suspension MLV liposomes in 4-6 ml of buffer solution was applied on the skin in the volume of 4-6 ml at the rate of 0.01% Kunz on 1 cm2the surface of the skin in accordance with the claimed method. In group 2 the same Tnkase applicable in liposomes, described in the prototype. In group 3 applicable suspension MLV liposomes obtained as well as for group 1, but not loaded with enzyme ("empty" liposomes) and in the same volume of buffer solution.

All applications were made 2 times a day.

Before the study and after its completion was measured and evaluated: the average depth of wrinkles (using the silicone replicas), the elastic index (using apparatus Dermaflex And firm "Cortex Ltd, Denmark), the thickness of the epidermis and dermis (using the Dermascan machine company "Cortex Ltd, company Denmark"). The effects were evaluated at 0 (before impact), 60 and 120 day study.

The results of the study are shown in table 3.

& P<0,01 compared with group 2.

From the analysis results, it follows that the inventive method is more efficient than the method-prototype addresses associated with aging changes in all indicators cosmetic healthy skin.

The use of the claimed method in a preventive mode (courses for 15 days every 2 months for 2 years) showed that the development of skin changes associated with aging, is delayed for a period of 5-6 months. That is, corresponding changes occur in women receiving exposure in accordance with the claimed method, 5-6 months later, than in women not using the claimed method.

Example 4. The use of the claimed method locally in the mode of mesotherapy in the middle layers of the dermis to reduce the manifestations of "cellulite" (dystrophic changes p decornoy fiber).

Bovine pancreatic to Tenkasu (drug, "Desoksiribonukleaza" production LLC Samson-med, St. Petersburg) were introduced in the MLV liposomes with a size of 1500 nm and a temperature of the phase transition of the membrane 45°C.

The study was conducted on 42 women aged 45 to 65 years. Women randomly distributed into three groups of 9-12 people.

In each group were exposed to the same skin problem areas.

The first group of women suspended MLV liposomes in accordance with the claimed method were injected subcutaneously with mezoinzhektor PISTOR-4 to a depth of 1-1 .5 mm, injections were made at a distance of 2-3 mm from each other. Introduction conducted sessions 1 time per day for 15 days with breaks between courses of 10 days. The exposure was carried out for 3 months.

The second group of the same liposomes was injected through without needle system Medi-Jector VISION in the same manner and amounts as in the first group.

The third group received the enzymes according to the method prototype. Liposomes were injected in the same volume of buffer solution as in the first group.

In all three of these groups had the same number of enzymatic activity Gnkazy per 1 cm2skin.

The fourth group (placebo group) were injected suspension "empty" MLV liposomes, is not loaded with the enzyme, in the same volume of buffer and in the same way as Yves group 1.

All drugs were prepared in aseptic conditions.

The rate of exposure consisted of 15 sessions held through the day. Estimated percent reduction for the entire group as a whole square dystrophic changes of the subcutaneous tissue (the presence of characteristic symptoms of "lemon peel")

The results are presented in table 4.

Table 4
GroupThe number of observationsThe percentage reduction of area per group
11055
2964
3120
4115

From the data presented in table 4, it follows that the inventive method allows to weaken undesirable dystrophic processes in the subcutaneous tissue ("cellulite"), with the prototype method is practically no effect on this process.

Additional studies have shown that women 35-40 years of age, who MLV liposomes obtained as described in example 4 was administered in a preventive mode described above mesotherapeutic method (10 daily courses with an interval of 30 days) in the area of the buttocks and thigh area phenomena "cellulite" in the zone entered what I need. The use of the prototype method (introduction in a similar way) had a similar prophylactic effect.

The method of prevention and correction of skin changes, and/or its appendages, and/or subcutaneous tissue by injecting into the skin and/or subcutaneous tissue of liposomes containing enzymes with Dnesday activity, characterized in that use multilayer MLV liposomes ranging in size from 500 to 2000 nm with the temperature of the phase transition of the membrane of liposomes from 30 to 65°With carry out the delivery and deposition of the enzyme in the extracellular space, as well as enzymes with Dnesday activity using endo - and/or ectonucleoside, destructive single-stranded and/or DNA Dunaeva.



 

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