UA56166C2 - Use of component в as cicatrizant - Google Patents

Abstract

The present invention relates to the use of Component В as cicatrizant, in particular in the treatment of wounds, ulcers and other traumatic lesions to any of the tissues in the body.

Description

Description of the invention

The present invention relates to the use of Component B as a scarring agent, in particular, in the treatment of wounds, ulcers and other traumatically damaged tissues of the body.

Component B is a protein consisting of 81 amino acids originally excreted in human urine. The human gene was cloned and expressed in CHO cells as recombinant human Component B. The molecule has a molecular weight of about 8.9 kDa and is described in detail in U 94/14259.

Such a protein contains ten cysteine residues and carries a basic element typical for serine protease enzymes. Checking the sequence in the protein database showed some similarities

Component B with known molecules such as CO59, the urokinase receptor! (PRA-K) and some poisonous toxins.

The information obtained by the Applicant from the study of the distribution of Component B in organs and muscle tissues 72 showed that the eye, lung and skin are the areas where RNA is mainly expressed

Component B. It was found that in human tissues, Component B is widely expressed in squamous epithelium and mucosa, for example, in the skin, esophagus, and cervix, as determined by immunohistochemical methods.

Finally, it was found that ESE induces the expression of Component B RNA in A 431 cells of human squamous epidermis.

There are reports that component B has anti-inflammatory, anticoagulant, and antitumor activity, as well as activity as an inhibitor of TOB-o binding to its receptor.

Currently, the applicant has found that Component B is also suitable as a scarring agent, and that therefore it is suitable, in particular, for the treatment of wounds, ulcers and other traumatic damage to any tissue of the body. Gee)

Consequently, the main subject of the present invention is the use of Component B to obtain a pharmaceutical composition suitable as a scarring agent, in particular in the treatment of wounds, ulcers and other traumatic damage to any body tissue.

Another object of this invention is a method of treating wounds, ulcers and other traumatically damaged tissues of the body, which includes the use of an effective amount of Component B together with a pharmaceutically acceptable excipient.

The second aspect of the invention are pharmaceutical compositions obtained as described above. -

As for the method of obtaining Component B and its amino acid sequence, they (Ce) are disclosed in MO 94/14259.

The active ingredient can be administered orally, intravenously, intramuscularly or subcutaneously, or applied topically. This invention also includes other routes of administration, allowing to create the desired level of the corresponding ingredients in the blood.

For the treatment of a person, the preferred dose! make 1mg/kg or less for systemic use and "4mcg/cm? or less for local application. In addition, the invention is illustrated with the help of the following examples, which should not be considered as limiting the present invention. In Examples of tributes! except for the Figures as indicated below.

A brief description of the drawings

Figure 1: shows the effect of intravenous use of Component B in comparison with the effect of betamethasone alone (Vepiepak) in experimental wound healing. In particular, sum up! the result

Experiment 1. The analyzed drug was prescribed daily for 6 consecutive days from 0 (day (22) of wound formation) to day 5 (inclusive). - Figure 2: the effect of intravenous use of Component B (series 0004-0015) is shown in comparison with the effect of betamethasone (Vepiepa) in the experimental wound healing. In particular, the summation of (o) 50 results! Experiment 2. The analyzed drugs were administered daily in the course of successive

F days from 0 (early formation days) to 5 days.

Figure 3: shows the effect of topical application of Component B (series 004-001) in an experimental healed wound. In particular, the summary result of Experiment 3. The analyzed drugs were administered daily for 5 consecutive days from 0 (for the formation of wounds) to 4 days.

Figure 4: the effect of local application of bovine serum albumin in an experimental (F) healed wound is shown. In particular summaries! the result! Experiment 4. The analyzed drugs were administered daily for 5 consecutive days from 0 (the day of wound formation) to day 4.

Figure 5: A sigmoidal dose-response analysis applied to outcomes is presented

Experiment 1. Based on the results of Experiment 1, a statistical evaluation of the effect of intravenous administration of Component B (series 004-001 and 0004-0015, designated as "001" and "0016", respectively) and betamethasone (Vepiepa|v) on experimental healing was carried out wounds

Figure 6: A sigmoidal dose-response analysis in application to outcomes is presented

Experiment 2. Based on the results of Experiment 2, a statistical evaluation of the effect of intravenous administration of Component B (series 0004-0016, marked as "001B") and betamethasone (Vepipai! F) on b5 experimental wound healing was carried out.

Figure 7: presents the analysis of the sigmoidal response to the introduction of a dose applied to the combined results of Experiments 1 and 2. Based on the combined results of Experiments 1 and 2, a statistical assessment of the effect of intravenous administration of Component B (series 0004-0015) on experimental wound healing is performed.

Figure 8: presents the analysis of the sigmoidal response to the introduction of the dose in application to the cumulative frequency, relative to the combination of Experiments 1 and 2. The effect of Component B (series 004-0016) was evaluated.

Figure 9: presents the analysis of the sigmoidal response to the introduction of the dose applied to the results of 7/0 Experiment 3. Based on the results of Experiment C, a statistical assessment of the effect of local and intravenous administration of Component B (series 004-001) on experimental wound healing is performed.

Figure 10: A sigmoidal dose-response analysis in application to results is presented

Experiment 4. On the basis of Experiment 4, a statistical evaluation of the comparison between the effects produced by the buffer and VZA on wound reduction was carried out.

EXAMPLE

Material

animal

ZRE CO-1 mice of both sexes, purchased from Snagiez Kimeg, Italy (Saiiso, Soto, Italy), were used for experiments after at least a seven-day acclimatization period conducted under

Control of environmental conditions (temperature 22-2°C, humidity 55-1095 and a twelve-hour light/dark cycle).

The analyzed compounds -g-es-th Component B batch 004-001 (sulfated form) and 004-0016 (non-sulfated form) were expressed in CHO cells and obtained, essentially, as described in UMO 94/14259. c - Commercial preparation of betamethasone (Vepiepa!F) from SiIaho (Verona, Italy) - Sodium chloride 0.995 (physiological solution) from Vahieg (Trieste, Italy) o

Bovine serum albumin (B5A), fraction M ot bBidt Spetis! So (St. Louis Mo, USA)

SspPOSOBYI

Experimental wound healing! throughout its thickness Ge)

The used method was proposed by R. Mogiop and M.N. Maiop (Mohiop).R. apa Maiop M.N., Ager. Ips.

Rpagtasodup., 196: 117, 1972), which used this process to evaluate a variety of medicinal products for their wound-healing activity in rats. "-

For the present study of Component B, the original method was modified as follows for the convenience of its use on muscles. eh

A round ink mark (1 cm in diameter) was applied to the dorsal region of male mice (30 - 35 years old, 6 - 7 weeks old), and the skin of the marked area (including the rappis sogpozyv and adjacent tissues) was excised with the help of surgical scissors and surgical forceps. The wound was then blotted dry with a gauze pad until hemostasis occurred. On day 0, that is, on the day of the operation, longitudinal, transverse and two diagonal measurements (relative to the vertebral column) of the diameter of the wounds were made! with an accuracy of 0.1 mm using a caliper. Determining in this way points were marked! indelible ink, 8 s applications on the adjacent skin area. Subsequent measurements of the wounds were carried out every other day except Sunday until the wounds were completely closed. Both the surgical procedure and the "" measurements were performed under light anesthesia of the muscles with zephyr.

The area of each wound! white was obtained by multiplying the square of the average diameter, which was about 0.7854. Then the percentage of wound closure relative to day 0 was calculated. The average values of the percentage of wound closure for each day of measurement were entered in the table for each experimental group and the time of 5095 contractions (ST 50) was interpolated. b Systemic treatment - Two experiments were conducted (Experiment 1 and 2). In the second experiment, on every day of 5 measurements, they were carried out by the same operator who did not know the schemes! treatment In each experiment, the animals were divided into 4 groups and treated according to the following scheme. 42) with o name)

The animals were treated once a day for the following days. The body weight of the animals was measured in br/Over the course of the experiment.

Local processing

In the next experiment (Experiment 3), the effect of local application of various doses of Component B (series 004-001) was studied using the already described methods of wounding with subsequent treatment according to the scheme described in the table below. because Number of groups! Treatment

On the first and second days, the solution of the substance under study was applied (in a volume of 0.005 ml) to the surface of the wounds, while on the following days, when a scab was formed, it was injected under the scab using a syringe equipped with a 250 gauge needle.

Component B administered intravenously at a dose of mg/kg was used as a positive comparative standard.

In order to rule out the possibility of a non-specific effect from the local application of a protein solution, in a parallel experiment the effect from the local application of VZA was investigated in the same 7/5 molar concentrations (8.8 x 10-9M) as Component B in comparison with a phosphate buffer solution (Experiment 4 ).

THE RESULT

Wound healing!

Figure 1 shows the data of the first experiment, in which the activity of two series (004-001 and 004-001B5) of Component B was compared. They both showed the ability to accelerate the scarring process, and their effect was already evident after 1 day of treatment. ST so, that is, the time during which 5,095 closures (area reduction) occurred in the morning, is 3.0 and 3.4 days, respectively, and these values were not statistically different. But in contrast to that, when using betamethasone (Vepiep(f) and physiological solution STbvo was observed on 7.8 and 7 days, respectively (see the paragraph with the heading "Statistical analysis").

In the second experiment (Figure 2), two doses of Component B (batch 0004-0015) were investigated. At the highest i) doses, mg/kg, STvo was 3.7 days, while at the lowest doses (0.1 mg/kg) it was 6.6 days. The groups treated with physiological solution and betamethasone showed ST2 as 91 and 10 days, respectively (see the paragraph entitled "Statistical analysis"). "I have a positive effect of Component B on wound healing! It is also confirmed by the second indicator, namely ETvo, which indicates the time when the wound completely closes in 50,956 animals! (see paragraph titled "Statistical analysis"). -

The result! of an experiment in which Component B (batch 004-001) is applied topically to the surface of wounds! (Experiment 3), shown in Figure 3. The compound was tested in doses of 1.2 and 4 μg/day for 5 h

For 5 consecutive days. All studied doses demonstrated the ability to enhance the wound healing process compared to the control. In particular, at doses of 2 to 4 μg, the value of ST5o was 38 and 44, respectively, which is comparable to the result of Sto (3.9) when using Component B in a dose of mg/kg administered intravenously. At the lowest doses (1 μg), the value of STro was 5.3 days, which is more than that! CT obtained by the two second doses when applied locally, but still significantly differs from the control "(see the paragraph entitled "Statistical analyses"). These data allow us to think that a dose of 2 μg when applied locally to a wound gives the maximum effect, and that a dose of 1 μg still remains effective in accelerating the scarring process. In order to check whether the positive effect of Component B on the wound healing process is a specific characteristic of the product, a parallel experiment was conducted in which the effect of B5A in the same molar concentrations as Component B was compared with the effect of using a phosphate buffer (Experiment 4). This data is presented in Figure 4. Steo of 9.9 and 7.9 days is marked in white with VZA and phosphate buffer, respectively. The above values are not significantly different (see the section "Statistical analysis"), thus indicating that a standard protein solution, such as B5A, does not affect the tightening of skin wounds.

Specific data from experiments are presented in tables TA - 4B. ia STATISTICAL ANALYSIS

F Statistical strategy

The purpose of the statistical analysis is to compare the effect shown after a certain period of time from the use of two preparations of Component B (Comp.B) as compared to the physiological solution, and two drugs for comparison - Vepiepa.

In addition, an evaluation of the effectiveness of the systemic and local application of one (F, component B) drug was also carried out. In accordance with the treatment schedule, the effect of the drug under study was studied taking into account the entire observation period. The wound healing experiment was repeated twice in order to confirm the influence of the component .V at different doses.

Statistical test

In the statistical test, we used Analysis of response to the introduction of sigmoidal doses! to estimate STvo (that is, the time when the area of the wound decreases by 50965) (see Rippeu 0..), Vioteiigisv, 32, pp. 721-40, 1976). 65 Statistical indicators 1) Reduction of wounds (STvo): The average percentage is deviated from the average (main) level of values.

2) Cumulative frequency (ET 50): cumulative frequency of animals that showed complete wound closure! at any time.

Group treatments (Zksp. Mo1) 1 - Physiological solution - 1 Oml/kg/day, intraperitoneally for 6 days. 2 - Vepiyepai - 1mg/kg/day, intraperitoneally for 6 days.

Z - Comp.B 004-001 - 1mg/kg/day, intravenously for 6 days. 4 - Comp.B 004-0016 - 1mg/kg/day, intravenously for 6 days.

Group treatment (Zksp. Mo2) 70 1 - Physiological solution - 1Oml/kg/day, intravenously for 6 days. 2 - Vepiepai - Tmg/kg/day, intravenously for 6 days.

Z - Comp.B 004-001 - O, 1mg/kg/day, intravenously for 6 days. 4 - Comp.B 004-0016 - 1mg/kg/day, intravenously for 6 days.

Group treatments (Zksp. MoZ) 1 - Phosphate buffer - 5Omkl/day, locally, for 5 days. 2 - Comp. B 004-001 - Imkg/day, locally, for 5 days.

Z - Comp.B 004-001 - 2mcg/day, locally, for 5 days. 4 - Comp. B 004-001 - 4mcg/day, locally, for 5 days. 5 - Comp. B 004-001 - 1 mg/kg/day, intravenously, for 5 days.

Group treatments (Exp. Mo 4) 1 - Phosphate buffer - 5Omkg/day, locally, for 5 days. 2 - Bnichy serum albumin (B5A) - 5Omkl/day, (8.8 x 109M), locally, for 5 days.

Treatment scheme (for experiments 1, 2, Zy 4)

Phase 2: Days of re-introduction in accordance with the above-mentioned treatment description for groups. with

Phase 2: Observation period up to the moment of complete healing of the wounds.

RESULTS OF STATISTICAL ANALYSIS i)

Chart! (analysis of the sigmoidal dependence "effect-dose" when introduced), the readings in Figure 5 - 10, summarize the effect of the drug under study, using the area of wounds! as variable values. Gee)

Experiment 1

A link is given to figure 5. Fr

The results of the analysis of the sigmoidal dependence of the effect on the dose when administered (СТсо) in the applied area of the wound, related to experiment 1, are presented! in the following table. communication / eizolochesyuryastor 72 5283096, « - with Experiment 2

A link is given to figure 6. ;" The results of the analysis of the sigmoidal dependence of the effect on the dose when injected (STvo) into the area of the wound, related to experiment 2, are presented! in the following table. with

F 7 Veleyanmyu 00000000 zv 04 ovo, in 1856. 55 ovk (22)

Ф Combination of Experiments You 2

A link is given to figure 7

The results obtained by combining the data for the treatment groups, common to both of Experiments 1 and 2, that is, physiological solution relative to Comp.B - 004-001r μg/kg, are summarized.

In addition, the frequency of animals showing fullness after the passage of time (F. zatyagianye rani! (analysis of the dependence of the effect (response) on the dose when sigmoidal is introduced) from the association was also estimated

GI of these Experiments 1 and 2.

The results of the analysis of the sigmoidal dependence of the effect on the dose when introduced (STvo) into the area of the wound applied to the area of the wound, relating to the combination of experiments 1 and 2, are presented! in the following table. because For the cumulative frequency! a link to figure 8 is given.

The result of the analysis of the sigmoidal dependence of the effect-dose upon the introduction of (ETvo) into the applied to the cumulative frequency, relating to the combination of experiments 1 and 2, representations! in the following table. 1 komvoolostvimyu 171 lem ovo

In conclusion, a comparison of the values of SToo. and among the values EToo is a good confirmation of the effect of each researched drug on the experimental model. It was found that Comp.B-001 (mg/kg, intravenously) and Comp.B-0015 (dose level O, mg/kg and mg/kg, intravenously) statistically differ from physiological solution and Vepiepa drug! in experiments 1 and 2. The results of the combination of treatment groups common to experiments 1 and 2 confirm the effect of the intravenous route of administration of Comp.B mg/kg.

Experiment Z

A link is given to figure 9.

An additional stage of the experiment was conducted, in which the product was applied topically. The intravenous route of administration was used as a positive standard for comparison. The data were analyzed using the statistical model described above.

The result! analysis of the sigmoidal dependence of the effect-dose when introduced (STeo) in applied to the area of the wound, related to experiment 3, representations! in the following table. 1 eoratnyavyuur 0830 7395096 sia o

In conclusion, the local application of Koml.B-0016 showed that at all investigated doses, the reduction of wounds (STvo) significantly differs from that obtained when using a phosphate buffer. (Se)

Experiment 4

A link to figure 10 is given -

The diagram shows a comparison between the effect of local application of phosphate buffer and VZA on (Ce) wound reduction in order to rule out the possibility of a non-specific effect of Component B.

The result! analysis of the sigmoidal dependence of effect-dose! when introduced (STevo) in the applied to the area about rania, concerning experiment 4, representations! in the following table. "- with 7 vvA777777111ve 111 v5-mya bob z" The above results do not show any difference between the local application of phosphate buffer and VZA.

FINDINGS FROM THE WHOLE RESEARCH

An interesting result of this study is the activity of Component B in the process of wound healing, both with intravenous and local application. The experimental model used in Vol

F studies, directly related to the modeling of injuries! человека and to the instructions for the use of Component B in the healing of traumatic skin damage and during plastic and reconstructive operations on the mucosa and mucous membranes. b 50

F

Claims (5)

The formula of the invention 1. Application of Component B as a scarring agent. 99 2. Application of Component B as an agent for obtaining a medicinal product suitable for the treatment of wounds, ulcers and other traumatically damaged tissues of the body, the healing of which is accompanied by scarring. at 3. Application according to claims 1 or 2, where the drug is suitable for the treatment of surgical wounds. 4. A pharmaceutical composition suitable as a scarring agent and in a suitable form for use as a scarring agent, containing Component B as an active ingredient together with a pharmaceutically acceptable excipient. 5. A method of treating wounds, ulcers and other traumatically damaged body tissues, the healing of which is accompanied by scarring, including the introduction of an effective amount of Component B together with a pharmaceutically acceptable excipient. b5