RU2317818C2 - Wound-healing and antibacterial agent - Google Patents

Abstract

FIELD: medicine, pharmacy.

SUBSTANCE: invention relates to drugs as ointments used in treatment of wounds and ulcerous damages of skin tissue and mucosa envelopes. Proposed medicinal agent comprises active mixture of two medicinal substances represented by thiotrazoline and laevomycetinum taken in the ratio 4:1 or 2:1. Optimal variant of ointment containing thiotriazoline and laevomycetinum comprises 2 g of thiotriazoline and 0.5 g of laevomycetinum in 100 g of ointment. Using the proposed medicinal agent as ointment enhances its wound-healing effect significantly as compared with the known preparations.

EFFECT: enhanced effectiveness of agent.

5 tbl, 5 ex

Description

The invention relates to medicine, in particular to medicines in the form of ointments, suppositories and other forms used to treat wounds and ulcerative lesions of skin tissues and mucous membranes.

Known drug in the form of paste "Solcoseryl", produced by "Solko", Switzerland.

This paste is effective enough to treat wounds.

The disadvantages of the Solkoseril paste are a narrow spectrum of its biological effects, rather high toxicity and the cost of foreign currency funds to purchase it by import.

Known wound healing drug "Ointment methyluracil 10%" (M.D. Mashkovsky. Medicines: in 2 vol. T. 2. - 14th ed., Revised., Rev. And add. - M .: Publishing House New Wave ", 2000. - S.160).

The disadvantage of methyluracil ointment is its increased consumption for treatment, unsatisfactory therapeutic activity in the treatment of infected wounds.

Known wound healing drug "Ointment levomekol" (M.D. Mashkovsky. Medicines: in 2 volumes T. 2. - 14th ed., Revised., Rev. And add. - M .: Novaya Volna Publishing House ", 2000. - P.266).

This ointment in 100 g of mass contains 4 g of methyluracil and 0.75 g of chloramphenicol, excipients up to 100 g as active components.

This ointment is used as a wound healing agent for the treatment of purulent wounds. However, in terms of the effectiveness of the treatment results, the ointment is inferior in some cases to methyluracil ointment.

Known wound healing drug "Thiotriazoline ointment" (Patent of the Russian Federation No. 2210367, CL. A61K 31/41), containing thiotriazoline as an active base.

Thiotriazolin ointment has a wide range of pharmacological effects, is non-toxic, mastered in the production of medicines of Ukraine.

Thiotriazolin ointment as the closest in composition and effect is taken as a prototype.

The disadvantage of thiotriazolin ointment is not quite sufficient therapeutic efficacy in the treatment of infected wounds.

The basis of the invention is the task to develop a wound healing drug, in particular, in the form of ointments, suppositories and other forms with increased therapeutic activity, which reduces the duration of treatment and costs and has a wide range of biological activity.

The solution to this problem provides a wound healing drug containing the active component - thiotriazoline - and auxiliary components for the formation of funds, due to the fact that it additionally contains chloramphenicol as an active component in the ratio of thiotriazoline to chloramphenicol 1: (0.2-0.5). When performing a medicinal product in the form of an ointment, it contains 1.9-2.1% of thiotriazolin and 0.4-1.1% of chloramphenicol, excipients - the rest.

One kilogram of the finished ointment contains 20 g of thiotriazolin, 5-10 g of chloramphenicol, 0.15 g of nipazole, 0.25 g of nipagin, ointment base up to 1 kg.

Advantages of the drug of the invention.

The use of the attached agent significantly (1.4 times) exceeds its wound healing, reparative, antimicrobial and anti-inflammatory effects in comparison with analogues and prototype due to increased therapeutic activity and increased biological activity of the combination of thiotriazoline and chloramphenicol compounds in the above ratios.

This reduces the duration of treatment and, accordingly, the cost of it. The invention is illustrated by the following description of the drug in the form of an ointment and examples of its preclinical studies.

The proposed drug in the form of an ointment contains the active components of thiotriazolin and chloramphenicol in a ratio of 1: (0.2-0.5).

In relation to the total weight of the drug in the form of an ointment, thiotriazolin is 1.9-2.1%, chloramphenicol 0.4-1.1%, the rest are the rest.

The optimal variant of the content of components g / kg ointment, confirmed by the following examples of preclinical studies, is as follows: the active base is thiotriazolin 20 g and chloramphenicol 5 g; preservatives - nipazole - 0.15 g and nipagin 0.25 g, ointment base up to 1 kg. For other forms of the proposed drug, the composition and amount of excipients will be different, but the ratio of the active components of thiotriazoline to chloramphenicol remains.

In this case, two drugs with different contents of chloramphenicol in% were studied:

The drug 1 composition: The drug 2 composition: Thiotriazoline 2.0 Thiotriazoline 2.0 Chloramphenicol 0.5 Chloramphenicol 1,0 Nipazole 0.015 Nipazole 0.015 Nipagin 0,025 Nipagin 0,025 Ointment base up to 100.0 Ointment base up to 100.0

The study of antimicrobial action.

The drug No. 1 and the drug No. 2 proposed as medicines have a high antimicrobial effect, significantly superior to that in the reference drug (see table 1).

When studying in vivo antagonistic activity in white mice, the symptoms of local inflammation (hyperemia, tissue swelling, skin tightening, pus presence at autopsy) were clearly expressed, which developed in mice 2–3 days after infection and persisted for 10–13 days.

The degree of manifestation of local symptoms of inflammation was systematically monitored, and after 3-5 days in several animals, for the purpose of bacteriological control, 10% yolk-saline (JSA) and blood agar detached from the lesion were sown and blood agar was detected for staphylococci.

The studied drugs were applied to the inflamed areas for 7 days, after which they were seeded with 10% WAS. In the control group of mice, a persistent manifestation of the inflammatory process was observed, in the group of mice treated with the comparison drug and the studied drugs No. 1 and No. 2, improvement was observed, and after 10 days complete healing.

The survival of animals can also serve as an indicator of the resistance of animals to staphylococcal infections during treatment with various ointments. Thus, by the 10th day of the experiment, only 20% of the animals remained alive in the control group of mice. In the group of mice treated with thiotriazolin ointment and No. 1 preparation, 40% and 50% of the animals remained by day 10, respectively (Table 2).

Thus, the healing effect of all ointments is present, but purulent wounds heal better under the influence of the drug No. 1. In this case, the systemic effect of this ointment on the animal organism has a higher rating.

Assessment of reparative activity of the claimed drugs.

Studies have shown that after damage to the skin in the initial state, the wound was a moist, bleeding and secreting area. In control rats, the wound surface remained so for 18 hours after surgery. In animals that were applied with reference preparations (titriazoline, methyluracil ointment, levomekol), the damaged area of the skin dried out by 8 hours, was covered with a thin crust, i.e. the regeneration process began. In the case of using the studied preparations No. 1 and No. 2, the formation of a crust on the surface of the wound and cleansing took place already 6 hours after application. Animals of this group looked tidier, calmer and more active. Obviously, in this case, the pain irritations emanating from the wound decreased and the animal's behavioral activity was restored faster.

By the 5th day of the experiment, in 2 and 3 groups of rats to which the studied ointments were applied, the wound surface area decreased by 62.29% and 53.76%, respectively (p 0.05) (Table 3), in group 1 (comparison drug - thiotriazoline ointment) - by 45.78% (p 0.05). As can be seen from the data in table 3, in animals of groups 2 and 3 a more intense reparative activity was observed.

By the 10th day of the experiment, the intensity of wound healing in animals of all groups except the control group is leveled. But a higher reparative activity is observed in the studied drug No. 1 (group 2).

We have also studied the treatment of burn wounds on the gums of rabbits with ointment of composition 1 and ointment of thiotriazoline.

It was found that the use of an ointment of thiotriazoline with chloramphenicol (drug 1), as well as an ointment of thiotriazoline, leads to a decrease in heperemia, swelling of the mucosa, which disappear on the 7th day. In the control group, these phenomena begin to decrease only by 7 days.

The wound cleans faster after using the drug 1: on the 5th day, the wound clears of plaque, the size of the wound decreases and on the 7th day of observation the wound is not detected at all. In control, the wound is healed only on the 14th day.

In fact, two days earlier granulations appear (7 days) in rabbits receiving drug 1, which leads to faster wound epithelization by 14 days in all animals. In the control, there was no similar effect by the seventh day of observation, and the wound epithelialization in the control group of rabbits was sluggish and not completely completed by the 14th day (table 4).

Biochemical studies have shown less pronounced hypoproteinemia on days 7-21 in animals treated with drug 1 and thiotrialoline ointment, due to the ability of thiotriazoline to stimulate protein synthesis. The detoxifying effect of ointment No. 1 on the levels of liver damage markers AlAT and AsAT was established, starting from 3 days 1.5 times lower than in the control, as well as low toxicity of alkaline phosphatase at 3-14 days of observation compared with the control.

Thus, the use of ointment No. 1 for the treatment of burn wounds allows you to more quickly eliminate the effects of inflammation, enhance regeneration processes and faster to achieve epithelization and wound healing.

The study of anti-inflammatory activity of drugs

During the first three hours after the administration of carrageenin, we observed an increase in the volume of paw edema in both experimental and control animals. The results of the effect of the applications of the studied drugs and the comparison drug on the intensity of carrageenan edema are presented in table 5.

After a day, there is a significant improvement in the state of the paw in experimental groups of animals (groups No. 1, No. 2, No. 3). Here, the magnitude of the edema decreased. In the control group of rats that were treated with boiled water in the form of a compress, almost no changes were observed compared to the volume of the edematous paw of the previous day. After 5 days, paw swelling in the experimental groups was practically not observed. A more pronounced decrease in the inflammatory process during the observed period was observed in 2 groups of experimental animals (applications of the studied combined ointment of thiotriazolin and a lower dose of chloramphenicol).

Thus, we can conclude that the studied drugs (with course applications) have a pronounced anti-inflammatory effect, especially the studied drug No. 1.

To illustrate the following examples.

Example 1. The study of antimicrobial action.

The antagonistic effect of the studied drugs in vitro was tested on Petri dishes with meat peptide (MPA) and blood agar. The studied preparations were applied to plates with MPA and blood agar; after 24 hours of incubation, 1 billion suspension of various test cultures was seeded with a perpendicular line. Analysis was carried out after 24 hours of incubation at 37 ° C. The results are presented in table 1

Table 1 Antagonistic activity of the studied drugs No. p / p Test culture strains Growth Inhibition Area (mm) Thiotriazolin Ointment Drug 1 Drug 2 one PE 0151 2 16 16 2 Y.pseudot 5 19 twenty 3 Vibrio I gr. X 10 twenty 21 four S.enteritidis 0 17 twenty 5 PE 025 0 eighteen 22 6 S.banana 0 eighteen 21 7 S.sonnei II K. 2 17 twenty 8 S.derby one 16 twenty 9 S.agona one 16 eighteen 10 S.tiphimurium 0 19 twenty eleven Str.faecalis 3 16 twenty 12 Str.piogenes 3 16 21 13 St.aureus one 2 6 fourteen St. Epidermidis 2 19 twenty fifteen C.albicans 0 0 one 16 E.coli one fourteen fifteen 17 P.vulgaris one 13 fourteen

Example 2. The study of the effect of drugs on chronic staphylococcal infection.

To test the antagonistic effect of the drug in vivo on staphylococci, a model of chronic staphylococcal infection in white mice was used. 3 hours before infection, 40 mice weighing 18-19 grams were removed using depilatory hair on one side. A silk surgical thread 2 cm long was placed in suspension (1 billion / ml) of the standard virulent strain St.aureus 209, obtained from the Museum of the State Scientific Research Institute for Standardization and Control of Medical Biological Preparations named after L.A. Tarasevich, for 30 minutes at 37 ° C, and then using a surgical needle was injected under the skin so that there remained a piece 1 cm long. The puncture sites were sealed with collodion.

Every day for 7 days, thiotriazoline ointment (group 1), preparation No. 1 (group 2) and preparation No. 2 (group 3) were applied to the affected area, the control group of animals did not receive treatment (Table 2).

The degree of manifestation of local symptoms of inflammation was systematically monitored, and after 3-5 days in several animals, for the purpose of bacteriological control, 10% yolk-saline (JSA) and blood agar detached from the lesion were sown and blood agar was detected for staphylococci.

table 2 The effectiveness of the treatment of purulent wounds Drug Wound healing 1 day 2 days 3 days 4 days 5 days 6 day 7 days 10 day (% survival) Thiotriazolin Ointment - - - - - - ± 40 Drug No. 1 - - - - ± + + fifty Drug No. 2 - - - - - ± + twenty The control - - - - - - - twenty Note: "-" - the presence of a wound; "+" - absence of wounds.

Example 3. Determination of wound healing activity

Studies were conducted on 36 white mongrel rats of both sexes weighing 160-200 g.

Wound healing activity was studied by the method of Efimov E.A. In rats, under light ether anesthesia, a small area of skin was excised. For 15 days every day (2 times a day), the studied drugs were applied on the wound surface - No. 1 and No. 2, comparison preparations - thiotriazoline ointment, methyluracil ointment and levomekol ointment. At the same time, the rats were in plexiglass houses for half an hour. We evaluated the reparative activity by reducing the area of the wound surface in the initial state, as well as after 5, 10 and 15 days.

In the experiment, animals were divided into 6 groups:

Group I - thiotriazoline ointment;

Group II - drug No. 1;

Group III - drug No. 2;

Group IV - control (water);

V group - ointment "levomekol";

Group VI - methyluracil ointment.

In each group, 6 animals were observed. The results of the experiments were subjected to statistical processing using Student's criterion; at p <0.05, the data were considered statistically significant.

To assess the speed of wound healing, the test of L.N. Popova was used, based on the measurement of the area of the wound in dynamics. A sterile sheet of cellophane was applied to the wound (1-5-10-15 days of treatment), onto which the contours of the wound were applied in ink. Then a cellophane received loop placed on graph paper and the wound area was determined by counting the number of square millimeters inside the contour (S = 3,14 · d ^2/4). The percentage reduction of the wound was determined by the deviation from the initial background.

The results are presented in table 3.

Table 3 Wound healing activity of the studied drugs Test drug Duration of observation, days Source 5 days 10 days 15 days dcp dcp % decrease to the outcome. dcp % decrease to the outcome. dcp % decrease to the outcome. Scp Scp Scp Scp Group 1 (thiotriazoline ointment) 13.75 ± 1.30 10.13 ± 1.27 -45.87 * 6.63 ± 1.67 -76.62 * 3.88 ± 0.60 -92.06 * 148.41 80.47 34.70 11.79 Group 2 (drug No. 1) 14.25 ± 1.75 8.75 ± 0.85 -62.29 * 5.88 ± 0.44 -82.90 * 4.13 ± 0.18 -91.62 * 159.40 60.10 27.10 13.36 Group 3 (drug No. 2) 15.63 ± 0.97 10.62 ± 1.76 -53.76 * 8.25 ± 1.89 -72.14 * 5.13 ± 0.60 -89.24 * 191.65 88.62 53.40 20.62 Group 4 (control) 15.00 ± 0.76 11.83 ± 0.64 -37.77 * 9.66 ± 0.88 -58.47 * 6.67 ± 0.34 -81.25 * 176.63 109.92 73.30 34.89 Group 5 (ointment "levomekol") 14.75 ± 1.89 12.25 ± 1.60 -31.00 * 7.50 ± 1.22 -74.14 * 5.88 ± 0.6 -84.00 * 170.78 117.79 44.16 27.09 Group 6 (methyluracil ointment) 11.00 ± 1.10 9.75 ± 1.31 -21.40 6.25 ± 0, b1 -67.72 * 4.75 ± 0, b1 -81.35 * 94.98 74.62 30.66 17.71 Note: * changes are significant in relation to the initial background (p <0.05); Scp is the average area of wounds in rats in mm ² , dcp is the average diameter of the wound in mm.

Example 4. The study of wound healing activity on the gingival mucosa

To study the wound healing effect of the ointment (preparation No. 1), an experimental ulcer model was created. The ulcer arose as a result of the application of the tip (square - 5 mm ² ) of a steel rod of an electric soldering iron heated to 100 ° C to the mucous membrane of the gums of the lower front rabbit incisors for 1 second (Sulim Yu.V., 1991). Burn injury was applied after preliminary anesthesia of the gum mucosa with a solution of lidocaine hydrochloride 10%. All animals were divided into 3 groups, in each group of 6 animals. In the first group (control), the wound was left untreated, in the second - thiotriazoline ointment was used, and in the third group, the wound was treated with thiotriazoline ointment with chloramphenicol (preparation No. 1).

The next day after applying the burn was considered the first day of the experiment. Ointments were applied once a day, ointment was applied with a dental spatula to the wound for 10-15 minutes. For this, the animals were fixed in an individual mobilized machine, an expander was used. The procedures were performed daily after feeding the animals until the ulcers completely healed. The studies were carried out on the 1-3-5-7-14-21th day of the experiment (table 4).

The research material was gum tissue and blood of experimental animals. Blood for hematological and biological studies was taken with a sterile syringe from the extreme vein of the ear. Gum tissue in the area of injury for morphological studies was cut with an acute surgical scalpel after infiltration anesthesia with 2% lidocaine hydrochloride solution.

In all animals, the day after the burn was applied, a picture of the acute inflammatory process was macroscopically observed. The surface of the wound is covered with a grayish-yellow coating, the gum mucosa is sharply hyperemic and swollen, the ulcer is framed by a bright red nimbus.

On the 3rd day of the experiment, no significant changes were visually observed in all groups of animals. All signs of inflammation are preserved, the ulcer is covered with a grayish-yellow coating.

On the 5th day of research in animals of the second and third groups clinically revealed a decrease in the inflammatory process. The gums become less hyperemic, in some places the swelling decreases, the ulcer clears of plaque, decreases in size. In the control group, all signs of inflammation persist.

On the 7th day of the experiment in animals of the second and especially third group, a significant improvement in the state of the gums is observed. Swelling of the mucous membrane of the gums is practically absent against the background of slight hyperemia. Ulcers are covered with young granulation tissue, completely cleaned of plaque. In the control group, all signs of a burn remained.

On the 14th day of the experiment, macroscopic signs of the pathological process were practically not detected. The gum defect is completely epithelized in the second and third groups. The gums are pale pink, dense, do not bleed when touched. In the control group, the completion of the pathological process was much slower; ulcer epithelization did not occur equally in animals of this group.

On the 21st day, the pathological process in the gums of all three experimental groups of animals was visually completely eliminated.

Biochemical studies have shown less pronounced hypoproteinemia on days 7-21 in animals treated with drug No. 1 or thiotriazoline ointment, due to the ability of thiotriazoline to stimulate protein synthesis. The detoxifying effect of ointment No. 1 and thiotriazoline ointment was established, therefore, the levels of liver damage markers AlAT and AsAT, starting from 3 days, are 1.5 times lower than in the control, and lower alkaline phosphatase activity was revealed on the 3-14 days of observation compared to with control.

Table 4 The dynamics of signs of inflammation, the appearance of granulations and epithelization of a burn wound Signs A drug Observation days 3 5 7 fourteen 21 Edema The control +++ +++ ++ ± - Thiotriazolin Ointment +++ ++ ± - - Drug No. 1 +++ ++ ± - - Plaque The control +++ +++ ++ / + ± - Thiotriazolin Ointment +++ ++ ± - - Drug No. 1 +++ ++ - - - The size The control ++++ ++++ +++ + - Thiotriazolin Ointment ++++ +++ ++ - - Drug No. 1 ++++ +++ + - - Manifestation of granulation The control - - - + / ++ +++ Thiotriazolin Ointment - ± ++ +++ ++++ Drug No. 1 - ± ++ ++++ ++++ Wound epithelization The control - - - + + Thiotriazolin Ointment - - + + + Drug No. 1 - - + + +

Example 5. The study of anti-inflammatory activity,

The dynamics of the formation of the focus of inflammation includes several stages associated with changes in neuro-humoral regulation and the appearance of physiologically active substances in the focus of inflammation. To study anti-inflammatory drugs, the possibility of experimental isolation of individual phases of inflammation, which characterize a complex process with a predominance of alteration, exudation or proliferation, is widely used. We used a model of exudative inflammation. In the mechanism of the anti-inflammatory effect - the phlogogenic agent - carrageenin, kinins play an important role in the first 3-4 hours, and proteolytic enzymes and prostaglandins play an important role in a later period.

An experimental study was performed on 40 white Wistar rats weighing 160 ± 50 g. The animals were divided into 4 groups of 10 pieces: group 1 - a compress of thiotriazoline ointment (comparison drug) was applied to the edematous paw; Group 2 received a compress with the test drug 1 on the edematous paw; 3 group - study drug 2; 4 group - control - applied a compress with boiled water.

We used a model of exudative inflammation. Acute aseptic inflammation in animals was reproduced by subplanar administration of 0.1 ml of a 1% solution of carrageenin in the sole of the hind paw.

The effect in rats was taken into account after 3 hours and 1 and 5 days after the administration of carrageenin by the volume of the paw, which was determined by immersion of the limb in a graduated tube, i.e. by the amount of water displaced. Additionally, measurements were made of the circumference of the legs (table No. 5).

The activity of the test substance was determined by its ability to reduce the development of edema in comparison with the initial background and the resulting inflammation, expressed as a percentage that showed how these drugs depressed the development of carrageenan edema. In all cases, the volume of displaced fluid was expressed in ml, and the circumference of the legs in mm.

Table 5. The effect of applications of the studied drugs on the intensity of carrageenan edema in rats Registration time Test indicator Group of animals Group 1 (thiotriazoline ointment) Group 2 (drug No. 1) Group 3 (drug No. 2) Group 4 (control group) one 2 3 four 5 6 Volume pushed 1.25 ± 0.23 1.05 ± 0.14 1.00 ± 0.16 1.10 ± 0.30 The initial state liquid ml Length circles 24.75 ± 1.46 21.75 ± 1.11 21.25 ± 1.21 21.50 ± 0.65 paws, mm After 3 hours Volume of ejected liquid, ml; 1.90 ± 0.19 1.98 ± 0.41 1.80 ± 0.23 1,9010.16 % off from ref. + 52.0 ** + 88.1 ** + 80.0 ** + 72.7 ** The circumference of the foot, mm; 33.75 ± 1.87 35.00 ± 1.95 34.25 ± 2.40 35.00 ± 1.83 % off from ref. + 36.4 * + 60.9 * + 61.1 * + 62.8 * After 1 day Volume of ejected liquid, ml; 1.55 ± 0.27 1.35 ± 0.47 1.35 ± 0.25 1.80 ± 0.13 % off from images. inflammation -18.40 -31.65 * -25.00 * -5.26 The circumference of the foot, mm; 29.50 ± 1.52 28.25 ± 1.62 28.50 ± 1.78 34.00 ± 1.29 % off from images. inflammation -12.60 -19.30 -16.80 -2.85 After 5 days Volume of ejected liquid, ml; 1.33 ± 0.17 1.13 ± 0.35 1.18 ± 0.24 1.40 ± 0.10 % off from images. inflammation -30.26 * -43.04 * -34.72 * -26.30 * The circumference of the foot, mm; 25.75 ± 0.69 24.5 ± 0.85 25.50 ± 1.87 27.00 ± 0.81 % off from images. inflammation -23.70 * -30.00 * -25.55 * -22.85 * Note: ** - the difference is significant in relation to the initial background ; • - significantly in relation to the resulting inflammation (P <0.05).

Thus, we consider the combination of thiotriazolin with chloramphenicol in a ratio of 4: 1 or 2: 1 rational and safe, and further studies in the form of ointments, pastes, tablets, solutions are rational and reasonable.

Claims (3)

1. A wound healing and antimicrobial drug containing the active component - thiotriazolin - and excipients for shaping, characterized in that the active component additionally contains chloramphenicol in the ratio of thiotriazoline to chloramphenicol 1: (0.2-0.5). 2. The wound healing and antimicrobial drug according to claim 1, characterized in that when performed in the form of an ointment, it contains thiotriazoline 1.9-2.1% and chloramphenicol 0.4-1.1% of the total mass and excipients - the rest . 3. The wound healing and antimicrobial medicine according to claim 2, characterized in that it contains nipagin, nipazole, water-soluble methylcellulose, glycerin and purified water as excipients in the following ratio of ingredients, g / kg (± 10%): Thiotriazoline twenty Chloramphenicol 5-10 Nipazole 0.15 Nipagin 0.25 Cellulose thirty Glycerol one hundred Water Up to 1 kg