PL238145B1 - Combination of active substances with antimicrobial properties for topical treatment, in particular of acne, and method of their production and use - Google Patents

Description

Description of the invention

The subject of the invention is a combination of active substances with antibacterial properties for the treatment of local acne and a method of its preparation as well as the medical use of a combination containing retinal and limecycline.

Acne, which is a disease related to the sebaceous apparatus, is considered to be one of the most common dermatological diseases. According to some sources, it may affect up to 100% of the population, occurring in various forms and periods of life. The disease is characterized by a multiform nature of lesions, which may be inflammatory, non-inflammatory and post-inflammatory, located in the seborrheic areas, mainly on the face, chest and back [Adamski Z., Kaszuba A., Dermatologia dla cosmetologów 2008; Janda K., Chwiłkowska M, Common acne - etiology, classification, treatment. Annals of the Pomeranian Medical University in Szczecin 2014, 60 (2), 13].

There are many factors involved in the formation of acne. The main, direct causes are the growth of sebaceous glands and excessive production of their secretions, increased keratinization of the hair follicle and sebaceous gland ducts, increased proliferation of Propionibacterium acnes and the formation of inflammation. Hormonal and immune disorders, genetic predisposition, psychological (stress) and environmental factors, as well as lifestyle (climate, environmental pollution, UV radiation, diet, skin care, hygiene habits, medications used, stimulants, clothes, physical activity) are also important. , a type of job).

The mainstay of acne therapy is to act on the etiopathogenic factors of the disease in a given patient. Treatment should focus primarily on the regulation of sebum secretion, inhibition of Propionibacterium acnes bacterial proliferation, elimination of inflammation, as well as comedolytic and keratolytic activities.

In order to obtain the best possible effects, a combination therapy is usually carried out, consisting in the use of topical and systemic preparations simultaneously and / or combining drugs belonging to different groups with different mechanisms of action in preparations. Local self-therapy is effective in approximately 60% of patients. Therefore, it is often used in conjunction with drugs taken orally [Placek W. et al, Przegl Dermatol 2011, 98, 442].

The most abundant active substances in preparations for external use are: antibiotics, retinoids, azelaic acid, benzoyl peroxide as well as alpha and beta hydroxy acids.

Retinoids are often used in combination therapy with antibiotics. As a result, the effectiveness and penetration of the antibiotic are increased, the treatment period is shorter and the side effects are minimized.

Due to its versatile action against etiopathogenetic factors, benzoyl peroxide is a commonly known and very frequently used anti-acne compound. The drug has antibacterial, anti-seborrhoeic, drying and keratolytic properties. It is characterized by easy penetration into the stratum corneum. Upon contact with the skin, it decomposes into hydrogen peroxide and benzoic acid. In contact with free oxygen radicals, bacterial proteins are oxidized, and thus an oxygen environment is created within the hair follicle.

Conditions are created that are unfavorable for the development of anaerobic bacteria responsible for the development of acne lesions. Due to its strong oxidizing properties, benzoyl peroxide inhibits the multiplication of Propionibacterium acnes bacteria, reducing their number to 90% without producing resistance.

Another drug that has a comprehensive effect on the causes of lesions is azelaic acid. It is produced naturally in the body by the oxidation of polyunsaturated fatty acids. It is also obtained from the Pityrosporum ovale culture. Like benzoyl peroxide, it has antibacterial, anti-seborrhoeic and exfoliating properties. Azelaic acid is active against both anaerobic and aerobic bacteria, and drug resistance is not found with long-term use. The drug is also characterized by strong anti-inflammatory properties related to the anti-radical effect. By inhibiting the synthesis of melanin and the activity of the tyrosinase enzyme, it reduces post-inflammatory discoloration, and long-term use may also contribute to reducing the tendency to scarring.

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In the case of acne, the most frequently selected antibiotics for external use are clindamycin and erythromycin, which, in addition to their antibacterial action, reduce local inflammation by inhibiting the production of pro-inflammatory cytokines, inhibiting the function of macrophages or chemotaxis of neutrophils involved in the inflammatory reaction.

Generally taken medications are indicated for moderate and severe forms of acne. These include retinoids, antibiotics, hormones and corticosteroids. Usually, a combination of oral medications and topical preparations is used.

Isotretinoin (13-cis-retinoic acid) is considered to be the most effective of the drugs used in the treatment of the disease, administered in severe forms or resistant to other preparations, in the case of constantly recurring lesions, as well as in people with scarring and severe seborrhea. . Isotretinoin is the only substance that has an effect on all of the four basic pathogenetic mechanisms of acne, while not requiring simultaneous therapy with topical preparations. It has properties that inhibit the growth of bacteria, including Propionibacterium acnes, anti-inflammatory, normalizing keratinization within the capillary units and anti-seborrhoea, reducing the size of the sebaceous glands and the amount of secretions produced by them up to 90%. Despite its excellent results and ease of administration, caution should be exercised and its use should be considered due to a number of serious side effects, including teratogenic effects and high sensitivity to light.

Antibiotics with bactericidal, bacteriostatic and anti-inflammatory properties are used to stop the multiplication of Propionibacterium acnes, control increased bacterial infection and prevent re-infection. Antibiotic therapy is combined with topical agents, usually retinoids or benzoyl peroxide. Tetracyclines are usually the first line antibiotics for systemic treatment of acne. They are generally well-tolerated, safe, quickly penetrating into tissues and effective substances. The mechanism of action of tetracyclines is to inhibit protein biosynthesis and disrupt energy processes in bacterial cells. Synthetic tetracyclines, which include doxycycline, minocycline or limecykin, are characterized by better absorption from the gastrointestinal tract and a longer half-life compared to natural tetracyclines. Tetracyclines are quick and effective in action, however, their use may be accompanied by side effects, including swelling, rashes and skin, inflammation of the esophagus and conjunctivitis, gastrointestinal disorders, kidney damage, discoloration of tooth enamel, yeasts, impaired bone growth and fractures. The use of tetracyclines makes the skin more sensitive to UV rays, therefore it is necessary to avoid the sun and other sources of radiation and to use photoprotection. An alternative to tetracyclines is erythromycin, a drug from the macrolide group. In recent years, however, there has been a decline in its effectiveness associated with the acquisition of resistance by Propionibacterium acnes. When acne is caused by hormonal disorders, or when other therapy attempts have failed, hormonal drugs are introduced: androgen receptor blockers, which include spironolactone, flutamide, cyproterone acetate and blockers of the production of ovarian or adrenal androgens, including oral contraceptives and glucocorticosteroids [Szepietowski J. et al., Przegl Dermatol 2012, 99, 649; Gliński W., Glińska O., Medical Standards 2006, 3 (2), 113; Dziekan G., Olechowska M, Pediatric Clinic 2010, 18 (3), 333; Kulczycka-Siennicka L, Medicine after the Diploma 2013, 22 (9), 40].

In order to reduce the toxicity and increase the stability, a new hybrid derivative of retinol - retinyl gallate was developed, showing a strong antioxidant and whitening effect [Kim S. et al. Bioorganic & Medicinal Chemistry Letters, 2009, 19, 508].

Another proposed hybrid is bis-retinamide-methylpentane, characterized by greater chemical stability, biological activity, safety profile and efficiency compared to free retinol [Ki Hyun Kim et al., International Journal of Pharmaceutics, 2015, 495 (1), 93],

For the local, controlled release of retinoic acid, it has also been proposed to covalently link retinoic acid with polyvinyl alcohol via an ester moiety showing a slight inflammatory response of the resulting derivative compared to free acid, and an extension of the release time to 6 days [Castleberry S. et al., Journal of Controlled Release, 2017, 262, 1]. Increasing the stability of retinoids was achieved by synthesizing retinyl retinate [Kim H. et al., Skin Research and Technology, 2012, 18 (1), 70]

The combination of active substances from WO2013096516 for the treatment of retinal acne and amoxycycline as antibacterial agent is known. There is also known from WO2007103555 a composition for application to the skin containing retinal and amoxycycline. In turn, from the description of WO201419587,

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KR20160015335, US2008038316, TW201238609, the combination of retinal and sprafloxacin is known.

None of the proposed compositions contain a covalent association of retinal as a vitamin A derivative with the antimicrobial drug (antibiotic) limecycline active against Propionibacterium acnes as the major pathogen in the development of acne.

The aim of the invention was to obtain a combination of vitamin A derivative - retinal (retinaldehyde, vitamin A aldehyde; an intermediate metabolite in the conversion of retinol into retinoic acid in human keratinocytes) and an antibacterial agent with an amino group, especially active against P. acnes bacteria, dedicated to the treatment of local infections - acne lesions.

The combination according to the invention comprises retinal and an antimicrobial agent with an amino group in the form of limecycline linked by a covalent bond, the active substances being used in a 1: 1 ratio.

The method of obtaining hybrid compounds according to the invention consists in reacting an alcoholic retinal solution with an antimicrobial agent, which is a substance with an amino group in the form of limecycline, whereby this substance is introduced, depending on its solubility, into an aqueous medium, preferably with an acidic reaction. or organic and the reaction mixture obtained is evaporated to dryness, the optionally precipitated precipitate is isolated by filtration or centrifugation, or a precipitate is precipitated from the mixture, which is isolated by filtration or centrifugation, the active substances in the form of retinal and antimicrobial agent with the group amine are present in relation to each other in a 1: 1 ratio.

Preferably, dilute acids are used as the acidic aqueous medium, preferably dilute hydrochloric acid or buffer solutions, preferably with a pH of 3-6.

Preferably, ethanol, methanol, DMSO, ethyl acetate are used as the organic medium.

Preferably, a methanolic or ethanolic retinal solution is used as the alcoholic retinal solution.

Preferably the combination (hybrid) is obtained at room temperature.

Preferably, an organic solvent is used to dissolve the hybrid, e.g. methanol, ethanol, DMSO, acetone, ethyl acetate.

Combination containing active substances in the form of an antibacterial agent with an amino group, such as limecycline and retinal, linked by a covalent bond, for use in the treatment of acne caused by aerobic and anaerobic Gram + bacteria and to a lesser extent aerobic Gram- bacteria.

Retinal is the least known and tested form of vitamin A, showing favorable biological properties and relatively low side effects compared to retinoids routinely used in the treatment of acne lesions.

A beneficial effect of the heterodimeric hybrid compounds obtained according to the invention is the combination of two active substances with different action directions, one with antibacterial properties and the other with anti-seborrheic, comedolytic, keratinization-regulating and anti-inflammatory properties. The obtained hybrid structure potentially shows multidirectional activities, much greater microbiological activity compared to the "non-hybrid" precursors, especially against the Propionibacterium acnes bacterium, which is assigned an important role in the pathogenesis of acne. Due to the modification of the antimicrobial substance in the hybrid structure, the obtained compound may prevent the progression of resistance. After topical application, the effect of the obtained hybrid is concentrated mainly at the site of application, gradually penetrating into the body, and thus showing relatively low side effects compared to routine therapy in which limcycline is administered systemically. Moreover, the binding of the vitamin A derivative in the hybrid structure significantly extends its microbiological stability in relation to the unbound form, and thus its activity. Due to the possibility of hydrolysis of the hybrid link, the synthesized compounds can be treated as carriers for the slow release of "non-hybrid" precursors, thus prolonging their biological activity. An additional advantage of the obtained hybrid is its pleasant smell in relation to the offensive smell, e.g. free limecycline, which seems to be of great importance for dermatological and cosmetic applications.

The created combination of substances (hybrid) can be incorporated into the structure of the matrix, preferably gel, in order to obtain antibacterial, anti-inflammatory and anti-acne pharmaceutical forms dedicated to local therapy.

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Example 1. 10% hydrochloric acid was used to extract limecycline from Tetralysal capsules (1 capsule contains 408 mg of limecycline). 301.47 mg of capsule weight (corresponding to 250 mg of limecycline) were weighed into a 25 ml volumetric flask, 20 ml of 10% hydrochloric acid were added, shaken for 1 min, then extracted for 10 min in an ultrasonic bath, made up to the mark with the same acid and filtered. to obtain a solution with a concentration of 10 mg / ml. The solution should be used immediately after receipt.

5.0 ml of the extract (50 mg of limecycline, equivalent to 0.083 mmol) were taken, transferred to a beaker, a magnetic stir bar was introduced and mixed gently on a magnetic stirrer while 2.4 ml of 10 mg / ml methanolic retinal solution (24 mg of retinal, corresponding to 0.083 mmol). The reaction mixture was allowed to stand 0.5 h. at room temperature, then placed in a shaking incubator, gently stirred at 40 ± 5 ° C by evaporating the solvent and allowed to dry at room temperature. The process of obtaining the compound was carried out by protecting the solutions from light. Yield: 95.8%.

Example 2. 5% hydrochloric acid was used to extract limecycline from capsules (1 capsule contains 408 mg of limecycline). 301.47 mg of capsule weight (corresponding to 250 mg of limecycline) were weighed into a 25 ml volumetric flask, 20 ml of 5% hydrochloric acid were added, shaken for 1 min, then extracted for 10 min in an ultrasonic bath, made up to the mark with the same acid and filtered. to obtain a solution with a concentration of 10 mg / ml.

6.4 ml of the extract (64 mg of limecycline, equivalent to 0.105 mmol) was taken, transferred to a beaker, a magnetic stirrer was introduced and mixed gently on a magnetic stirrer while 3 ml of 10 mg / ml methanolic retinal solution (30 mg of retinal, corresponding to 0.105 mmol). The reaction mixture was left for 0.5-1 h. The precipitate was filtered, washed with 2 portions of water, 2 ml each, then the precipitate was dissolved by washing with 3 portions of methanol (96%), 2 ml each, and collecting the filtrate in a glass vessel. The solution was evaporated to dryness at room temperature. The process of obtaining the compound was carried out by protecting the solutions from light. Yield: 74.2%.

Example 3 The mass of Tetralysal capsules corresponding to 250 mg of limecycline was weighed into a 10 ml volumetric flask, 7 ml of methanolic hydrochloric acid (5 ml of 20% HCl made up with methanol to 25 ml) were added, shaken for 2 min, then extracted for 10 min. in an ultrasonic bath, made up to 10 ml with the same solution and filtered to give a solution with a concentration of 25 mg / ml.

2 ml of the extract (50 mg of limecycline, equivalent to 0.083 mmol) were taken, transferred to a beaker, a magnetic stir bar was inserted and mixed gently on a magnetic stirrer, adding dropwise 1.2 ml of 20 mg / ml methanolic retinal solution (24 mg of retinal, corresponding to 0.083 mmol). The reaction mixture was left for 0.5 h at room temperature, then placed in a shaking incubator, gently stirred at 37 ± 5 ° C while evaporating the solvent to dryness. The process of obtaining the compound was carried out by protecting the solutions from light. Yield 98.8%.

Example 4. 30 mg of limecycline (0.050 mmol) was transferred to a beaker and dissolved in 2 ml of 10% hydrochloric acid or methanolic hydrochloric acid (5 ml of 20% HCl + 20 ml of MeOH). A magnetic stirrer was introduced to the solution and stirred gently on a magnetic stirrer while 1.4 ml of 10 mg / ml methanolic retinal solution (14 mg of retinal, corresponding to 0.050 mmol) was added dropwise. The reaction mixture was allowed to stand 0.5 h. at room temperature, then placed in a shaking incubator, gently stirred at 37 ± 5 ° C by evaporating the solvent and allowed to dry at room temperature. The process of obtaining the compound was carried out by protecting the solutions from light. Yield: 75.1%.

The structure, spectrum of antimicrobial activity and microbial stability for the selected hybrid obtained from retinal and limecycline were determined on the basis of FTIR instrumental analysis and microbiological tests, respectively.

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Scheme 1. Structure of the LIM-RET hybrid determined on the basis of FTIR analysis

Analysis of the LIM-RET hybrid (combination) by FTIR infrared absorption spectrophotometry using the attenuated total internal reflection (ATR) technique showed the presence of a new imine moiety: -N = CH- (Scheme 1 above) confirming the covalent bond formed between the amino group of the antibiotic (LIM) and RET retinal).

The LIM-RET spectrum shows the disappearance of the bands of stretching vibrations of the aldehyde group at 2861.04 and 2827.46 cm ¹ in the retinal molecule and the appearance of the band of stretching vibrations of the -CH = N- moieties at 1623.73 cm ¹ in the molecule of the LIM-RET hybrid ( Figures 1 and 2) The observed changes indicate the reaction of the aldehyde group of the retinal with the amino group of the limecycline with the formation of the azomethine group (-CH = N-).

The FTIR spectrum of the LIM-RET hybrid shows 28.44% of the standard substance of limecycline, and 20.05% of the standard substance of retinal (Figure 3) Significant incompatibility of the FTIR spectrum of the hybrid with FTIR spectra of substances used for its synthesis proves the creation of a new structure .

Tests of microbiological activity on the example of the LIM-RET hybrid

The hybrid (LIM-RET) synthesized according to the above-mentioned examples with the use of the antibiotic ulimecycline (LIM) and the vitamin A derivative - retinal (RET) was assessed for antibacterial activity against selected microbial strains:

Anaerobic, Gram-positive:

- Propionibacterium acnes PCM 2334,

- Propionibacterium acnes PCM 2400,

Gram-positive aerobes:

- Staphylococcus aureus ATCC 25923,

- Staphylococcus epidermidis ATCC 12228,

Gram-negative aerobes:

- Escherichia coli ATCC 25992,

- Pseudomonas aeruginosa ATTC 27853

Assessment of bacterial sensitivity to the test compound by diffusion into a solid medium.

Solutions of the test substances with a concentration of 10 mg / ml were prepared: LIM-RET and RET in DMSO and a water extract of LIM from the Tetralysal preparation. Then Mueller-Hinton media was prepared, which was poured in the amount of about 20 ml on Petry plates and allowed to solidify at room temperature, and a suspension of bacterial cells with a concentration of 0.5 McFarland (1.5 CFU), inoculating them by spreading them on the surface of the solidified agar medium. Solutions of the test compounds with a final concentration of 100 µg were applied to the prepared plates. The plates were incubated under conditions suitable for optimal bacterial growth:

- aerobic bacteria at 37 ° C for 24 hours in aerobic conditions,

- anaerobic bacteria at 37 ° C for 48 hours in anaerobic conditions.

After the incubation period, the resulting zones of inhibition of bacterial growth around the test compounds were measured. The results of the experiment are presented in Table 1 below.

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Table 1. Zones of growth inhibition [mm] of bacteria against the test substances

A strain of bacteria Tested substances LIM-RET LIM RET Gram + aerobic bacteria S. aureus 22 26 15 S. epidermidis 17 4 15 Gram-aerobic bacteria P. aeruginosa 5 8 4 E. coli 15 17 4 Gram + anaerobic bacteria P. acnes PCM 2400 21 13 14 P. acnes PCM 2334 23 8 12

The presented numerical data indicate the intensification of the antibacterial activity of the obtained hybrid in relation to the compounds used in its synthesis, against Gram + Propionibacterium acnes anaerobic bacteria, which are assigned a major role in the pathogenesis of acne (Contassot E., French L, Journal of Investigative Dermatology, 2014, 134). , 310)

Assessment of the minimum bacterial growth inhibitory concentration (MIC).

The MIC value (pg / ml) of the hybrid was determined by duplicate microdilution in 96-well plates in appropriate bacterial media.

For this purpose, solutions of the test substances were prepared at a concentration of 50 mg / ml in a suitable solvent, showing no inhibitory effect on the test bacteria (DMSO or H2O), and a bacterial inoculum with a concentration of 0.5 McFarland of the aerobic bacteria (S. aureus, S. epidermidis, E .coli, P.aeruginosa) and anaerobes (P. acnes PCM 2334, P. acnes PCM 2400).

382 µl of the appropriate growth medium was poured into the first row of wells of the plate and 200 µl into the remaining wells. Then, 8.16 pl of the test substances solutions were added to the first upper wells of A by mixing, and the final concentrations were obtained by double microdilution: 1000 pg / ml, 500 pg / ml, 250 pg / ml, 125 pg / ml, 62.5 pg / ml , 31.2 µg / ml, 15.6 µg / ml, 7.8 µg / ml, 3.9 µg / ml, 1.95 µg / ml. In the next stage, each well was inoculated with a given bacterium in the amount of 2 pl, except for the control wells: reagent control - substance adequately diluted with the medium (made to eliminate the error caused by color), positive control - broth inoculated with a given bacterium (it proves proper bacterial growth) and control negative - pure broth (proving the sterility of the experiment). The finished plates were incubated under appropriate conditions

- aerobic bacteria for 24 hours at 37 ° C, aerobic conditions

- anaerobic bacteria for 48 hours at 37 ° C, anaerobic conditions.

The readings were made in an automatic microplate reader with dedicated software at a wavelength of 600 nm. The minimum concentration that inhibits bacterial growth is considered to be the concentration at which no bacterial growth (i.e. changes in absorbance) is observed. The determined MIC values for the tested compounds against selected bacteria are presented in Table 2 below.

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Table 2. MIC values [pg / ml] of test substances against selected bacteria

A strain of bacteria Tested substances LIM-RET LIM RET Gram + aerobic bacteria S. aureus 3.9 31.25 31.25 S. epidermidis 3.9 250 62.5 Gram-aerobic bacteria P. aeruginosa 1000 > 1000 > 1000 E. coli 3.9 62.5 > 1000 Gram + anaerobic bacteria P. acnes PCM 2400 1.95 250 1000 P. acnes PCM 2334 1.95 500 1000

Liquid media MIC tests confirmed the results obtained in the solid diffusion pilot test. The hybrid LIM-RET has the strongest antibacterial properties, achieving a MIC in the range of 1.95-3.9 pg / ml, with the exception of the P. aeruginosa strain. The MIC values in the mean activity range of 31.25 to 500 pg / ml were also obtained by limecycline, and in this case no activity against P. aeruginosa was noted. The derivative of vitamin A showed the weakest activity, and only against gram-positive strains. The creation of a hybrid connection significantly intensifies the activity against unbound precursor substances.

Assessment of the minimum bactericidal concentration (MBC) at which 99.9% of bacteria die. The MBC value (pg / ml) was determined against the hybrid using 96-well plates in which the MIC values for specific compounds were previously determined for the selected bacterial strains. From the wells where no bacterial growth was noted, 10 µl of medium containing declining concentration of the test compound were removed and transferred to clean Petry plates with a suitable agar medium. The plates were then incubated at temp. 37 ° C to assess the sterility of the culture or possible growth of bacterial colonies. After incubation, possible bacterial growth was assessed. Plates containing the medium with the active substance concentration, where no colonies appeared, were taken as the bactericidal concentration (MBC). The calculated MBC / MIC values determining the efficacy of the tested compounds against the tested bacteria are presented in Table 3 below.

T a b e I a 3. MBC / MIC values of tested substances against selected bacteria

A strain of bacteria Tested substances LIM-RET LIM RET Gram + aerobic bacteria S. aureus 1 2 16 S. epidermidis 2 4 8 Gram-aerobic bacteria P. aeruginosa 16 nb nb E. coli 8 4 nb Gram + anaerobic bacteria P. acnes PCM 2400 1 2 8 P. acnes PCM 2334 1 2 16

nb - not tested due to lack of antibacterial activity.

Hybrid LIMRET and limecycline showed bactericidal nature against acne strains of Gram +.

Application:

The LIM-RET hybrid shows significant bactericidal activity against the Propionibacterum acne bacteria, as an essential factor activating the pro-inflammatory cascade in the development of acne; activity

PL238 145 Β1 significantly greater compared to limecycline and especially retinal as "non-hybrid" precursors. Due to the fact that a hybrid is a novel compound, it may be much more effective than limecycline against which bacteria have developed resistance mechanisms.

Stability assessment on the example of a LIM-RET hybrid

The experiment consisted in the daily control of antibacterial activity in the test on the solid support (zone) of the LIM-RET hybrid with respect to the vitamin A derivative (RET) and limecycline (LIM), stored in visible light at room temperature (RT). The zones of inhibition of bacterial growth, formed around 100 pg of solutions of the tested compounds at a concentration of 50 mg / ml, were examined for a period of 16 days (Table 4 below):

LIM-RET - solution of the LIM-RET hybrid in DMSO

LIM- limecycline water extract from Tetralysal

RET - solution of vitamin A derivative in DMSO

Table 4. Zones of growth inhibition [mm] of selected bacteria against the tested solutions

A strain of bacteria Test compound Experience day 1 2 3 4 5 8 16 5. aureus LIM-RET 22 21 18 15 15 13 5 LIM 27 15 10 5 0 0 0 RET 14 10 5 0 0 0 0 S. epidermidis LIM-RET 17 15 13 12 13 12 3 LIM 0 0 0 0 0 0 0 RET 15 10 5 0 0 0 0 P. aeruginosa LIM-RET 4 0 0 0 0 0 0 LIM 6 0 0 0 0 0 0 RET 4 0 0 0 0 0 0 E. coli LIM-RET 18 18 16 15 10 4 0 LIM 8 0 0 0 0 0 0 RET 8 0 0 0 0 0 0 P. acnes PCM 2400 LIM-RET 23 18 14 13 14 14 4 LIM 9 0 0 0 0 0 0 RET 10 4 0 0 0 0 0 P. acnes PCM 2334 LIM-RET 21 21 18 15 15 12 4 LIM 13 4 0 0 0 0 0 RET 7 0 0 0 0 0 0

The results indicate that the LIM-RET hybrid was characterized by the highest stability for 16 days when stored in visible light at room temperature. On the other hand, the "non-hybrid" substances limecycline (LIM) and retinal (RET) lost the fastest activity against Gram negative bacteria (1 day) and against pathogens causing acne P: acnes: 1-2 days as shown in the figure, where Fig. 1 shows a comparison of the spectrum standard substance retinal (RET) and LIM-RET hybrid in the wavelength range 4000-600 cm-1. 4 and 5), Fig. 2 comparison of the spectra of the retinal of a standard substance (RET), limecycline of a standard substance (LIM) and a hybrid (LIM-RET) in the range of 2000 600 cm-1, Fig. 3 analysis of the matching of the spectrum of a hybrid LIM-RET to a standard substance LIM Fig. 4 graphical interpretation of the stability of the LIM-RET hybrid (LIM-RET composition), assessed by its microbiological activity against Propionibacterium acnes PCM 2400 against free (non-hybrid) active substances: limecycline (LIM) and retinal (RET), and Fig. 5 a graphical interpretation of the stability of a LIM-RET hybrid (LIM-RET composition), assessed by its microbial activity against Propionibacterium acnes PCM 2334 against the free (no hybrid) active substances: limecycline (LIM) and retinal (RET).

Claims (7)

Patent claims A combination of active substances with antimicrobial properties based on retinal characterized in that, in addition to retinal, it comprises an antibacterial agent with an amino group in the form of limecycline, the active substances used in a ratio of 1: 1 being linked by a covalent bond. A method for obtaining a combination of active substances with antibacterial properties for the treatment of topical retinal acne, characterized in that the alcoholic retinal solution is reacted with an antibacterial agent which is a substance with an amino group in the form of limecycline, the substance being incorporated into depending on its solubility in an aqueous medium, preferably acidic or organic, and the resulting reaction mixture is evaporated to dryness, optionally the precipitate is isolated by filtration or centrifugation, or a precipitate is formed from the mixture, which is isolated by filtration or centrifugation, the active substances in the form of retinal and the antibacterial agent with an amino group are present in a ratio of 1: 1 to each other. 3. The method of preparation according to claim 1 A process according to claim 2, characterized in that dilute acids, preferably dilute hydrochloric acid, or buffer solutions, preferably pH 3-6, are used as the acidic aqueous medium. 4. The method of preparation according to claim 1 A process according to claim 3, characterized in that the organic medium is ethanol, methanol, DMSO, ethyl acetate. 5. The method of preparation according to claim 1 The process of claim 3, wherein the alcoholic retinal solution is a methanolic or ethanolic retinal solution. The method of preparation according to claim 1 A method according to claim 3, characterized in that the obtained precipitate is purified by rinsing with water and allowed to dry or dissolved in alcohol and evaporated to dryness. 7. A combination of active substances with antibacterial properties, containing retinal and limecycline linked by a covalent bond for use in the local treatment of acne.