RU2661613C1 - Method for treatment of human diseases caused by gram-positive bacteria - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to the field of medicine and is intended for the treatment of human diseases caused by gram-positive bacteria. As an antibacterial agent, eremomycin is used, or its pharmaceutically acceptable salts and solvates. Dose is 20 to 2,000 mg per day, while the administered eremomycin contains, as an impurity, dezaremozaminil-eremomycin not more than 3 % by weight.

EFFECT: group of inventions allows expanding the arsenal of therapeutic agents.

25 cl, 11 ex, 9 tbl

Description

FIELD OF THE INVENTION

The invention relates to medicine and relates to a method for treating human diseases caused by gram-positive bacteria, in which eremomycin or its pharmaceutically acceptable salts and solvates are used as an active principle. This treatment method can be implemented in various regimens and dosage forms for the administration of eremomycin, or its pharmaceutically acceptable salts and solvates, to humans. The claimed method of treatment is characterized by increased effectiveness, reduced number of side effects, low relapse rate, suppression of resistant bacterial strains, which was not observed for other polycyclic glycopeptides, for example, vancomycin, teicoplanin, ristomycin, dalbavancin and others. The invention also provides pharmaceutical compositions of eremomycin used in the claimed method of treatment.

State of the art

Gram-positive bacteria (Gram (+) bacteria) - bacteria that, unlike gram-negative bacteria, retain their color, do not discolor when washed using the stain of microorganisms according to the Gram method.

Most Gram (+) bacteria have a single-layer cell membrane, without the outer membrane inherent in gram-negative bacteria.

Most of the pathogens for humans microorganisms are gram-positive. Six genera of gram-positive organisms (Bacillus, Listeria, Staphylococcus, Streptococcus, Enterococcus and Clostridium) are typical human pathogens. Two of them, streptococci and staphylococci, are cocci (spherical bacteria). The rest are rod-shaped and further divide as far as possible to form spores. Non-spore forming: Corynebacterium and Listeria; spore-forming: Bacilli and Clostridia. Spore-forming can be divided into facultative anaerobes of Bacillus and obligate anaerobes of Clostridium.

Gram-positive bacteria cause a wide variety of human diseases, including endocarditis, sepsis, respiratory tract infections (pneumonia, lung abscess, otitis media, sinusitis, epiglottitis), CNS infections (meningitis), various colitis (staphylococcal enterocolitis, pseudomembranous colitis associated with Clostridium diff ), as well as all kinds of infections of the skin, soft tissues, bones and joints (cellulite, erysipelas, impetigo, diabetic foot ulcers, infections after human bites, folliculitis, boils, carbuncles, purulent hydradenitis, pa onychia, pyomyositis, purulent tenosynovitis, decubitus ulcers, infectious arthritis, osteomyelitis, acute bursitis, postoperative wound infections).

Among the vast array of diseases caused by gram-positive bacteria, clostridial infection, more commonly known as clostridial pseudomembranous colitis or pseudomembranous colitis associated with Clostridium difficile, is most dangerous. The danger of this disease lies in the fact that it is caused by antibiotic therapy, including antibiotics, which can suppress Clostridium difficile.

Pseudomembranous or Clostridium difficile - associated colitis is represented by a wide range of clinical manifestations from moderate diarrhea to fulminant form with a possible fatal outcome. In the "pre-antibiotic" era, pseudomembranous colitis was caused by various pathogens (for example, Staphylococcus); Currently, the most typical reason is the excessive reproduction of the symbiont anaerobic Clostridium difficile against the background of antibiotic therapy or chemotherapy for cancer patients.

Pathophysiology of the disease: the death of normal colon bacteria caused by antibiotics (from several days to 10 weeks after taking antibiotics) leads to the colonization of toxin-producing Clostridium difficile (oral-fecal route of infection). The release of toxins A and B leads to mucosal death and inflammation. The various degrees of manifestation of the disease are based on: 1) host immune factors and 2) virulent factors of microorganisms.

Antibiotics associated with clostridial colitis: any antibiotics (even those used in the treatment of clostridial colitis - metronidazole and vancomycin); the incidence of colitis is largely a reflection of the widespread use of appropriate drugs: penicillins, cephalosporins, fluoroquinolones, clindamycin, and even metronidazole (despite the fact that the latter is used to treat Clostridium difficile).

Clostridial colitis is treated from conservative (including antibiotics vancomycin and metronidazole) to colectomy, which can save the patient’s life.

Clostridium difficile is the most common nosocomial gastrointestinal infection. There is an epidemic increase in incidence from 6-7 cases per 1000 hospitalizations in 1990 to 23 cases per 1000 hospitalizations in 2004; > 450 thousand cases a year in the USA,> 60 out of 100,000 patients are discharged with an initial diagnosis of “clostridial colitis” after a short-term hospitalization.

Asymptomatic colonization of Clostridium difficile:

-> 50% of healthy newborns;

- 1-3% of healthy adults;

- 25% of adults recently given antibiotics => an important hidden reservoir of Clostridium difficile;

- 20-25% of hospitalized patients undergo colonization of Clostridium difficile during a hospital stay.

In addition, a group of especially virulent strains (8% of isolates) has been identified that determine the severity of C. difficile infection: toxinotype III, CD binary toxin, ribotype 027. These strains are highly resistant to antibiotic treatment (vancomycin and others).

The high prevalence of diseases caused by gram positive and bacteria, as well as the emergence of new, more resistant strains of bacteria (The American Journal of Medicine, 2006, V. 119 (6A), S11-19; Antimicrobial Agents and Chemotherapy, 2012, V. 56, No. 6, P. 2929-2932) leads to the need to develop new antibiotics, as well as eliminate the disadvantages of already known antibiotics.

The antibiotic eremomycin (the structural formula is presented below) belongs to the group of polycyclic glycopeptides (dalgapeptides), which includes the antibiotics ristomycin (ristocetin), actinoidin, vancomycin, teicoplanin, dalbavancin (Drugs, 2004, V. 64, P. 913-936).

Eremomycin is a hydrophilic amphoteric compound, soluble in aqueous solutions of acids and alkalis, insoluble in ordinary organic solvents. From concentrated aqueous solutions during a slightly alkaline reaction of the medium (pH = 8.1), the antibiotic drops out in the form of a base. Acidification of an aqueous solution of the antibiotic with acid (up to pH 6.5-6.7) leads to the formation of the corresponding salt. For example, when using sulfuric acid, eremomycin sulfate of the following formula is precipitated:

The composition of eremomycin includes amino acids - actinoidic acid, dechlororicinic acid, asparagine, N-methyl-leucine and sugars - eremozamine (2 residues) and glucose. The gross formula of eremomycin is C ₇₃ H ₈₉ N ₁₀ ClO _{26. The} relative molecular weight is 1558.

Eremomycin gives positive reactions to free amino groups (with ninhydrin), to carbohydrates (Molish and Feling reaction), to phenolic hydroxyls (with ferric trichloride and Pauli reaction).

It has a characteristic UV spectrum with λ _max at 280 nm (water) and λ _min at 263 nm and a specific absorption of 41.

The IR spectrum (KBr) has strong absorption bands in the region (ν _max ): 3400-3200, 2900, 1670, 1600, 1200-1000 cm ^-1 .

Eremomycin has a higher chemotherapeutic activity than the known antibiotics of dalgapeptides. Eremomycin is 2-3 times more effective against various strains of staphylococcus, streptococcus, bacillus. In addition, eremomycin proved to be a less toxic antibiotic than vancomycin and other representatives of dalhapeptides (Antibiotics and Medical Biotechnology, 1987, XXXII, No. 8, pp. 571-575; J. Antibiotics, 1989, V. 42, P. 1790-1799 )

Clinical trials of eremomycin (J. Antibiotics, 1989, V. 42, P. 1790-1799) were also mentioned, but they were unsuccessful due to the extremely strong allergic reaction when the eremomycin was administered intravenously (severe anaphylactic shock). Later, information appeared confirming the strong allergenic properties of eremomycin when administered intravenously in an animal experiment (J. Antibiotics. 1996. v. 49 (2), p. 194-198).

They tried to correct the failure of eremomycin with intravenous administration due to its oral use (eremomycin is practically not absorbed orally) in the treatment of clostridial infection in animals. A comparison of eremomycin and vancomycin in the treatment of clostridial infections in the same doses (100 mg / kg) in the Golden Hamster model showed superior efficacy in vancomycin (Antibiotics and Chemotherapy, 1992, v. 37, No. 11, pp. 32-35).

Despite the good data on the effectiveness of eremomycin in vitro, in vivo studies have shown its ineffectiveness in the treatment of diseases caused by gram-positive bacteria, due to the strong allergic effect when administered intravenously, and also significantly lower activity when administered orally / intraperitoneally, compared with vancomycin in the treatment of clostridial infection on a model of a golden hamster.

The authors of the present invention unexpectedly found that allergic and anaphylactic reactions in humans to eremomycin were caused by the presence (more than 3% of the mass.) In the eremomycin preparation of deseremosaminyl-eremomycin, the main impurity of the breakdown of eremomycin. The structural formula of deseremosaminyl-eremomycin is presented below:

Deseremosaminyl-eremomycin was first obtained in 1991 (Antibiotics and Chemotherapy, 1991, v. 36, No. 6, pp. 28-31) by hydrolysis of eremomycin with a 0.2 M hydrochloric acid solution.

Thus, using a substance of eremomycin containing not more than 3% of the mass. deseremosaminyl-eremomycin, as well as compositions of eremomycin, which allow it to be stabilized against decay into deseremosaminyl-eremomycin, can effectively treat human diseases caused by gram-positive bacteria.

The authors also unexpectedly found that eremomycin more effectively suppresses human Clostridium difficile strains than vancomycin. In this regard, eremomycin is an excellent tool for the treatment of human clostridial infection. Moreover, therapy with eremomycin significantly reduces the number of relapses and adverse reactions, compared with vancomycin therapy. It was unexpected that eremomycin had a sweet taste, which eliminates the manifestation of nausea when a person takes the drug orally, which occurs in the case of vancomycin therapy.

Methods of treating a person using eremomycin are not disclosed in the prior art. There is also no information about the compositions of eremomycin, which can be used in these methods.

Disclosure of invention

In the present invention, the authors unexpectedly discovered the high efficiency of using eremomycin in the treatment of diseases caused by gram-positive bacteria in humans. The high efficiency of using eremomycin in the treatment of human diseases caused by gram-positive bacteria is manifested in a decrease in the number of side effects, relapse rates, and the suppression of resistant bacterial strains, which was not observed for other polycyclic glycopeptides, for example, vancomycin, teicoplanin, ristomycin, dalbavancin and others.

Moreover, the inventors unexpectedly found that the above effects from the use of eremomycin are possible only if the eremomycin used contains as an impurity no more than 3% of the mass. deseremosaminyl-eremomycin. Mentioned impurity in an amount of more than 3% of the mass. causes severe allergic reactions (up to anaphylactic shock), leading to the inability to conduct eremomycin therapy.

In this regard, the present invention is directed to a method of treating human diseases caused by gram-positive bacteria, comprising administering to a needy person a eremomycin or a pharmaceutically acceptable salt thereof, a solvate in an amount of from 20 to 2000 mg per day, wherein the administered eremomycin contains no more than an impurity 3% of the mass. deseremosaminyl-eremomycin.

In one embodiment of the invention, eremomycin or a pharmaceutically acceptable salt or solvate thereof is administered orally.

In a preferred embodiment of the invention, eremomycin or a pharmaceutically acceptable salt, solvate thereof is administered orally in the form of a capsule, tablet or oral solution.

In a preferred embodiment of the invention, the oral solution comprises eremomycin or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carbohydrate, and water.

In a preferred embodiment, the tablet contains eremomycin or a pharmaceutically acceptable salt, solvate and macrogol thereof.

In one embodiment, the capsule contains eremomycin or a pharmaceutically acceptable salt, solvate and pharmaceutically acceptable carbohydrate thereof.

In one embodiment of the invention, eremomycin or a pharmaceutically acceptable salt or solvate thereof is administered intravenously.

In a preferred embodiment, eremomycin or a pharmaceutically acceptable salt or solvate thereof is administered as an infusion solution.

In a preferred embodiment, the infusion solution comprises eremomycin or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carbohydrate, and water.

In one embodiment of the invention, eremomycin or a pharmaceutically acceptable salt or solvate thereof is administered in an amount of from 100 to 1500 mg per day.

In a preferred embodiment of the invention, eremomycin or a pharmaceutically acceptable salt or solvate thereof is administered in an amount of 250 to 1000 mg per day.

In one embodiment of the invention, eremomycin or a pharmaceutically acceptable salt or solvate thereof is administered in two divided doses.

In one embodiment of the invention, eremomycin or a pharmaceutically acceptable salt or solvate thereof is administered in four divided doses.

In a most preferred embodiment of the invention, eremomycin or a pharmaceutically acceptable salt or solvate thereof is administered in an amount of 1000 mg per day in four divided doses.

In one embodiment of the invention, gram-positive bacteria are selected from bacteria including bacteria of the genera Bacillus, Listeria, Staphylococcus, Streptococcus, Enterococcus and Clostridium.

In one embodiment of the invention, the human disease caused by gram-positive bacteria is selected from the group consisting of endocarditis, sepsis, pneumonia, lung abscess, meningitis, staphylococcal enterocolitis, pseudomembranous colitis associated with Clostridium difficile and other infections caused by Clostridium difficile, soft skin infections , bones and joints.

In addition, the present invention is directed to a method for treating clostridial infection in humans, comprising orally administering to a needy person eremomycin or a pharmaceutically acceptable salt thereof, a solvate in an amount of from 20 to 2000 mg per day.

In one embodiment of the invention, eremomycin or a pharmaceutically acceptable salt or solvate thereof is administered in an amount of 250 to 1000 mg per day.

In one embodiment of the invention, eremomycin or a pharmaceutically acceptable salt or solvate thereof is administered in two divided doses.

In a preferred embodiment, eremomycin or a pharmaceutically acceptable salt or solvate thereof is administered in four divided doses.

In a most preferred embodiment of the invention, eremomycin or a pharmaceutically acceptable salt or solvate thereof is administered in an amount of 1000 mg per day in four divided doses.

In one embodiment of the invention, eremomycin or a pharmaceutically acceptable salt, solvate thereof is administered orally in the form of a capsule, tablet or oral solution.

In a most preferred embodiment of the invention, the tablet contains eremomycin or a pharmaceutically acceptable salt, solvate and macrogol thereof.

In a most preferred embodiment, the oral solution comprises eremomycin or a pharmaceutically acceptable salt, solvate thereof, as well as a pharmaceutically acceptable carbohydrate and water.

In one embodiment of the invention, the administered eremomycin contains as an impurity not more than 3% of the mass. deseremosaminyl-eremomycin.

The present invention also provides a composition of eremomycin or a pharmaceutically acceptable salt thereof with a pharmaceutically acceptable excipient, the composition being characterized by a content of not more than 3% by weight. deseremosaminyl-eremomycin.

Eremomycin, for use in the claimed method of treatment, can be obtained in accordance with the methods of preparation known in SU 1475150, RU 2110578, RU 2352631, RU 2621866.

The pharmaceutically acceptable salt or solvate of eremomycin may be any suitable known salt or solvate thereof, for example, eremomycin sulfate, eremomycin hydrochloride, eremomycin trifluoroacetate, eremomycin mesylate and others.

The most preferred pharmaceutically acceptable salt of eremomycin is eremomycin sulfate.

In yet another embodiment of the invention, eremomycin, or a pharmaceutically acceptable salt or solvate thereof, can be used in the form of a composition containing conventional pharmaceutically acceptable excipients adopted in the preparation of medicines, such as binders, fillers, preservatives, flow regulators, emollients, wetting agents, dispersants, emulsifiers, solvents, antioxidants and / or propellants, prolonged action [Sucker et al .: Pharmazeutische Technologie, Thieme-Verlag, Stuttgard, 1991].

Unexpectedly, the substance of eremomycin has good solubility and acceptable technological properties. Thus, eremomycin to create a composition can be mixed with excipients in any suitable ratio. In one embodiment of the invention, the content of eremomycin in the composition is from 5 to 90% of the mass. In another embodiment of the invention, the content of eremomycin in the composition is from 20 to 85% of the mass.

The most preferred excipients are eremomycin stabilizing agents. Such excipients include pharmaceutically acceptable carbohydrates (mannitol, sucrose, lactose, cyclodextrins, cellulose and its derivatives, starch and its derivatives, sorbitol, fructose) and solid macrogols (macrogol 3350, macrogol 4000, macrogol 6000 and others). If necessary, suitable binders, fillers, preservatives, flow regulators, emollients, wetting agents, dispersants, emulsifiers, solvents, antioxidants and / or propellants, action prolongers to improve the physicochemical properties of the finished composition can be added to the above substances.

To obtain the finished eremomycin compositions, macrogol, stearic acid and its salts, talc, copovidone, silicon dioxide, polysorbates (e.g. polysorbate 80), poloxamers (e.g. poloxamer 188), magnesium oxide, cyclodextrins can be used as preferred pharmaceutically acceptable excipients (e.g. β-cyclodextrin), sodium carboxymethyl cellulose, microcrystalline cellulose, starch, calcium carbonate, basic magnesium carbonate, calcium phosphate, gelatin, lactose, pregelatinized starch, to Olin, mannitol, sorbitol.

More preferred adjuvants are mannitol, macrogol, lactose, sodium carboxymethyl cellulose, cyclodextrin, polysorbate, poloxamer and sorbitol.

The most preferred adjuvants are mannitol, macrogol, lactose and cyclodextrin. The inventors unexpectedly found that the use of macrogol in eremomycin compositions allows for more effective treatment of infected organs with eremomycin, as well as better stability of eremomycin.

In a most preferred embodiment of the invention, the eremomycin composition containing the usual pharmaceutically acceptable excipients may be in the form of an oral dosage form, for example, in the form of an optionally coated tablet, solution or suspension for oral administration. Eremomycin compositions can be performed by standard methods (Pharmaceutical Technology. Technology of Dosage Forms, 2nd ed., Moscow, 2006).

The pharmaceutical composition of the present invention can be coated with a suitable film coating, containing, for example, a suitable polymer (polyvinyl alcohol, hyprolose, ethyl cellulose and others), talc, titanium dioxide, polyethylene glycol (macrogol) and colorants. As a shell for the composition of the present invention, a finished shell may be used, for example, a shell of the Opadrai, Opasprey, and Opatint series. For example, you can use the finished casing Opadray II pink 85 F 14399 (polyvinyl alcohol - 40.00%, titanium dioxide - 24.85%, talc - 14.80%, macrogol 3350 - 20.20%, iron dye red oxide E172 - 0.15%).

The implementation of the invention

The following examples illustrate (without limiting the scope of claims) the most preferred embodiments of the invention, and also confirm the possibility of implementing the claimed method for the treatment of diseases caused by gram-positive bacteria and to achieve the indicated technical results.

Example 1

Determination of impurities in the substance of eremomycin by HPLC.

Below is a method for determining the content of eremomycin and impurities in it by HPLC.

0.6% solution of ammonium formate pH 8.0. In a volumetric flask with a capacity of 1000 ml, 6 g of ammonium formate are placed, dissolved in water, the solution volume is adjusted to the mark with the same solvent and mixed. The pH of the resulting solution was adjusted to 8.0 with 10% ammonia solution. The resulting solution is filtered and degassed in any convenient manner. The filter material is kapron or other material compatible with the resulting solution, with a pore size of not more than 0.45 microns.

Sample solvent. Mix 92 ml of the component of the mobile phase A and 8 ml of acetonitrile.

Test solution. About 25 mg of the substance (accurately weighed) is placed in a 50 ml volumetric flask, dissolved in a sample solvent, the solution volume is adjusted to the mark with the same solvent, mixed and filtered through a membrane filter (filter material is regenerated cellulose) with a pore size of not more than 0 , 45 microns, discarding the first portion of the filtrate.

The solution is used freshly prepared.

Solution for assessing the sensitivity of the chromatographic system. 2.0 ml of the test solution is transferred into a 100 ml volumetric flask, the volume of the solution is adjusted to the mark with a sample solvent and mixed thoroughly. 2.0 ml of the resulting solution was transferred into a 10 ml volumetric flask, the volume was adjusted to the mark with a sample solvent and mixed thoroughly. The concentration of eremomycin sulfate in the resulting solution is about 0.002 mg / ml.

Eremomycin sulfate amide solution. About 5 mg (accurately weighed) of eremomycin sulfate amide (standard sample of the company) is placed in a 50 ml volumetric flask, dissolved in a sample solvent, the solution volume is made up to the mark with the same solvent and mixed thoroughly.

The solution to verify the suitability of the chromatographic system. 5.0 ml of an eremomycin sulfate amide solution was transferred to a 10 ml volumetric flask, 2 ml of the test solution was added, the volume was adjusted to the mark with a sample solvent and mixed thoroughly.

Chromatographic conditions:

Kromasil C8; 250 × 4.6 mm; 5 μm (Akzo Nobel) Speaker - or similar; Mobile phase A - 0.6% solution of ammonium formate pH 8.0 Mobile phase B - acetonitrile Flow rate - 1.0 ml / min; Column temperature - 25 ° C; Detector - wavelength 280 nm; Sample Volume - 20 μl;

The solvent is chromatographed sequentially to obtain a blank chromatogram, a solution for evaluating the sensitivity of the chromatographic system, a solution for testing the suitability of the chromatographic system, and a test solution (at least 2 times). The retention time of eremomycin sulfate should be about 16.5 minutes.

A chromatographic system is considered suitable if the following conditions are met:

- on the chromatogram of the solution to check the suitability of the chromatographic system, the resolution between the peaks of eremomycin sulfate and the peak of eremomycin sulfate amide should be at least 3;

- on the chromatogram of the solution to check the sensitivity, the signal-to-noise ratio should be at least 10.

If necessary, the chromatographic conditions can be adjusted in order to fulfill the requirements of the test “Verification of suitability of the chromatographic system”.

Peaks of impurities present on the blank chromatogram, peaks whose area is less than the peak area in the chromatogram of the solution to assess the sensitivity of the chromatographic system (less than 0.4%), as well as peaks with a retention time of less than 4 minutes, are not taken into account when determining the content of impurities.

The content of a single impurity in tablets (X) in percent is calculated by the formula:

,

,

Where:

S _i is the peak area of a single impurity in the chromatogram of the test solution;

ΣS _i is the sum of the areas of all peaks in the chromatogram of the test solution.

The content of a single unidentified impurity is not more than 4.0%; the content of deseremosaminyl-eremomycin (relative retention time (relative to eremomycin 1.35) - not more than 3.0%; the amount of impurities - not more than 8.0%.

Example 2

Getting eremomycin.

Eremomycin is prepared in accordance with the following method previously disclosed in RU 2621866. The synthesis method involves the use of the Amycolatopsis orientalis VKM Ac-2717D strain and includes seed preparation, seed cultivation, eremomycin biosynthesis and isolation of eremomycin.

Sowing preparation.

Inoculum is prepared in Petri dishes using an agar medium (medium composition% wt.: Glucose - 2.0; soy peptone - 1.5; yeast extract - 0.1; agar - 2.0; pH = 7.0 before sterilization )

The agarized medium is inoculated with mycelium of the seed culture and incubated for 5 to 7 days at a temperature of 28 ° C. The culture on the cups should give a solid lawn, a folded surface, beige mycelium.

Growing seed.

The cultivation of seed in flasks is carried out on a vegetative medium (medium composition% wt: soy flour - 0.5, glycerol - 3.0, potassium nitrate - 1.0, sodium chloride - 1.0, magnesium sulfate - 0.01, water tap to 1 liter, pH 7.0 before sterilization).

The mycelium of the culture Amycolatopsis oriental is VKM Ac-2717D, after incubation for 5-7 days at 28 ° C on an agar medium, a vegetative nutrient medium is inoculated (100 ml in 1.0 L Erlenmeyer flasks) and grown at 28 ° C in for 24-48 hours on a thermostatic rocking unit at 230-250 rpm (eccentricity 5 cm). During microscopy, a thin basophilic network is observed, protoplasm in hyphae is differentiated.

Eremomycin biosynthesis.

A fermentation medium is prepared in the fermenter. For this, drinking water (7 liters) is poured into the fermenter and, with stirring, weighed portions of the following components: glycerin - 600 g; soy flour - 200 g; monosubstituted potassium phosphate - 2 g; magnesium chloride - 2 g; potassium nitrate - 60 g; calcium chloride - 30 g; yeast extract - 30 g; skimmed milk powder - 30 g. Bring the volume of the nutrient medium with drinking water to 10 liters.

Glycerin is used as an additional source of carbohydrates, in an amount of 5 g / l of medium per day. Glycerol supplementation is introduced on the 2nd, 3rd and 4th day of cultivation.

In a fermentation medium, create a pH (7.0 ± 0.2) with a 20% sodium hydroxide solution and sterilize for one hour. After sterilization, the fermenter with the medium is cooled to 29-30 ° C. The medium must be sterile and have a pH (7.6 ± 0.3).

Sowing of the fermenter with seed is carried out under aseptic conditions, while the amount of seed is 10% of the volume of medium in the fermenter. After inoculation of the fermenter, the temperature of the culture fluid is maintained at 28 ± 1 ° C.

In the fermenter is continuously fed sterile air in an amount of 300-600 l / h. Mixing at a speed of 100-500 rpm. The concentration of dissolved oxygen is maintained at 50%.

Fermentation time 144-168 hours.

Starting from 72 hours of growth, the eremomycin content is monitored by HPLC. With a maximum accumulation of eremomycin on the 6-7th day, the fermentation process is stopped.

By the end of biosynthesis (144 ± 24 hours), the culture fluid has a pH value of 8.3 ± 0.25.

The content of eremomycin 3300 mg / l (HPLC).

Microscopic description of mycelium: in a preparation stained with methylene blue, short, weakly branching, segmented, basophilic hyphae are observed.

Eremomycin Isolation

Eremomycin is isolated in the form of an acid addition salt by sorption on Purolite C-104 cation exchanger in the H + form.

To a culture fluid with a volume of 10 l (eremomycin content 3300 mg / l), 150 g of weakly acidic Purolite C-104 cation exchanger in the H + form are added in one go, the suspension is stirred for 1.5 hours and a sample is taken to determine the residual eremomycin content in the culture liquid (HPLC). In the absence of eremomycin in the selected sample, stirring is stopped and cation exchange resin is separated from the culture fluid. Cation exchange resin is thoroughly washed three times with water. Then, 600 ml of water is added to the cation exchange resin thus washed, and with vigorous stirring, ammonia is added dropwise to pH 9. The resin is filtered off and re-treated as described above. The filtrates obtained after the first and second treatment are combined and concentrated in vacuo at a temperature of 29-34 ° C to 2/3 of the initial volume, and then neutralized with a 40% sulfuric acid solution to a pH of 6.9 (if necessary, obtain another salt of eremomycin a suitable acid should be used at this stage).

From the obtained concentrate, eremomycin is crystallized in the form of sulfate with the addition of a double volume of isopropyl alcohol, cooled to 8-10 ° C and incubated for 2 hours. It is then filtered, washed on the filter with a small amount of isopropyl alcohol and dried in vacuum at 30 ° C for 6 hours. Obtain 28 g of eremomycin sulfate of the following structural formula:

The yield of the target product from the content in the culture fluid is at least 80% (the content of the main substance is at least 98%).

The resulting sulfate can be used in the preparation of suitable dosage forms.

Example 3

Comparison of the susceptibility of various PCR ribotypes of Clostridium difficile to eremomycin and vancomycin.

Currently, there are a significant number of resistant to existing therapy strains of Clostridium difficile. In this example, the task was to compare the activity of eremomycin and vancomycin in an in vitro experiment using various PCR ribotypes of Clostridium difficile as an example. To compare the activities of vancomycin and eremomycin, their minimum inhibitory concentrations (MICs) were determined against the group of the most common and human-aggressive European PCR ribotypes Clostridium difficile.

Methods

Clinical isolates of Clostridium difficile (n = 22) were provided by the Clostridium difficile Ribotyping Network (CDRN) laboratory in Leeds, UK. Isolates were selected to reflect the most common Clostridium difficile PCR ribotypes in Europe, according to the latest epidemiological reports (Lancet Infect Dis. 201414 (12): 1208-19; Clin Microbiol Infect. 2015 21 (3): 248.e9-248.e16 ) All isolates were transferred to the CDRN in 2015.

The susceptibility of the isolates to vancomycin and eremomycin was determined by the method of administration of Wilkins-Chalgren agar as described previously (J Antimicrob Chemother 2008; 62: 1046-1052). Briefly, the clinical isolates of Clostridium difficile were cultured in pre-reconstituted Shadler's anaerobic broths at 37 ° C anaerobically for 24 hours before dilution with isotonic solution. Diluted solutions were added to plates containing Wilkins-Chalgren agar and an antibiotic. The plates were incubated anaerobically at 37 ° C for 48 hours and examined for growth. MICs were defined as the lowest antimicrobial dilution in order to completely inhibit growth, or the growth was characterized only by individual insignificant colonies.

Results and discussion

Eremomycin turned out to be a more active agent against Clostridium difficile (arithmetic mean MIC = 0.18 mg / L, MIC ₅₀ = 0.125 mg / L, MIC ₉₀ = 0.25 mg / L). Eremomycin activity was approximately 4.6 times higher than vancomycin (the arithmetic mean value of the MIC was 0.83 mg / L, MIC ₅₀ = 1 mg / L, MIC ₉₀ = 1 mg / L) against the tested group of clinical isolates Clostridium difficile. MIC ranges were narrow and within two doubled dilutions for each agent.

Antimicrobials have been tested against the Clostridium difficile group, taken from the most common PCR ribotypes in Europe, including those that are known to support many antibiotic resistance - PCR ribotypes 027, 001 and 017 (Clin Microbiol Infect. 2015 21 (3) : 248.e9-248.e16). There were no ribotype-specific MIC differences for each of the tested agents, and the MIC range was narrow.

As can be seen from the above table, eremomycin was significantly more active against common PCR ribotypes of Clostridium difficile than vancomycin.

Example 4

Examples of eremomycin compositions.

The following are examples of preferred eremomycin compositions that can be used in the treatment method of the present invention. Eremomycin compositions are prepared by generally accepted standard methods (Pharmaceutical Technology. Dosage Form Technology, 2nd ed., Moscow, 2006).

Table number 2. Eremomycin formulations in tablet form.

The lyophilisates shown can be dissolved in suitable saline solutions (Ringer's solution, Acesol, isotonic solution) or water and administered intravenously at a concentration of not more than 5 mg / ml

The capsule shell may, for example, contain titanium dioxide, sodium lauryl sulfate and colorants.

Example 5

Comparison of the toxicity of eremomycin and vancomycin

The aim of this study was to study the acute toxicity of eremomycin and to identify toxic effects only after intravenous administration and to compare the data obtained with those for vancomycin (when administered orally, vancomycin and eremomycin are practically not absorbed into the blood and are considered absolutely safe compounds, their LD ₅₀ (oral)> 10000 mg / kg).

Since there was no information on the toxic doses of the test object of eremomycin, the choice of doses for the study was made on the basis of a preliminary pilot study and the known information on the safety of the antibiotic vancomycin from a similar pharmacotherapeutic group — tricyclic antibiotics of the glycopeptide group (Drugs, 2004, V. 64, P. 913 -936). According to the results of a preliminary study of mortality, doses for rats were - 375 mg / kg, 550 mg / kg, 820 mg / kg, 1200 mg / kg, 1750 mg / kg; for mice - 550 mg / kg, 730 mg / kg, 970 mg / kg, 1290 mg / kg, 1750 mg / kg.

According to toxicometry data, average lethal doses for the studied drugs (LD ₅₀ ) were established that characterize late delayed death of animals over a period of 14 days.

LD ₅₀ (iv, male rats) = 941 ± 179 mg / kg; LD ₁₀ (iv, male rats) = 308 mg / kg;

LD ₅₀ (iv, female rats) = 870 ± 157 mg / kg; LD ₁₀ (iv, male rats) = 311 mg / kg;

LD ₅₀ (iv, male mice) = 1380 ± 167 mg / kg; LD ₁₀ (iv, male mice) = 792 mg / kg;

LD ₅₀ (iv, female mice) = 1309 ± 159 mg / kg; LD ₁₀ (iv, female mice) = 748 mg / kg;

Thus, the maximum tolerated dose (MTD) for rats is <308 mg / kg (less than the calculated LD ₁₀ ), for mice - 550 mg / kg.

According to MSDS LD _50, vancomycin for intravenous administration to mice is 430 mg / kg; The LD ₅₀ for rats given intravenously is in the range of 251-319 mg / kg. Thus, in terms of the average lethal dose, eremomycin is less toxic than vancomycin from the glycopeptide group.

In experimental rats treated with eremomycin in the dose range from 550 mg / kg to 1750 mg / kg, the state was inhibited immediately after administration, which was manifested by a decrease in motor activity and a low response to external stimuli. At the same time, in animals treated with eremomycin at a dose of 550 mg / kg, intoxication symptoms were observed during the first days after administration, while in animals treated with the drug in the dose range from 820 mg / kg to 1750 mg / kg, intoxication symptoms were expressed throughout the study.

In all rats treated with eremomycin in the dose range from 820 mg / kg to 1750 mg / kg, hyperemia of the visible mucous membranes and skin was observed after administration. 24 hours after administration, the animals were ruffled.

On the 5-6th day after the introduction in groups of animals that received the test drug at doses of 820, 1200 and 1750 mg / kg, a general condition worsened. This was manifested by the appearance of ataxia, exhaustion, decreased muscle tone and reduced feed intake.

Throughout the study, the picture of intoxication was more pronounced in females compared with males who received the drug in the same doses.

In the experimental groups that received the test object at a dose of 375 mg / kg, visually, there were no signs of intoxication for the entire observation period in any animal.

The general condition of the surviving experimental rats receiving the study drug at doses of 820 and 1200 mg / kg was normalized by the 14th day of the study.

The death of rats occurred on 1-14 days after administration of the drug. The period of maximum animal mortality occurred on the 1st, 4th and 14th days of the study. The cause of death in all experimental rats was renal failure.

In experimental mice that received eremomycin in the dose range from 730 mg / kg to 1750 mg / kg, inhibition was observed immediately after administration, which was manifested by a decrease in motor activity and a low response to external stimuli. Moreover, in mice that received eremomycin at a dose of 730 mg / kg, the general condition returned to normal on the 2nd day of the study. In most animals that received the drug in the dose range from 970 mg / kg to 1750 mg / kg, the general condition returned to normal on the 3rd day of the study. Only one male who received eremomycin at a dose of 970 mg / kg was inhibited until 7 days.

In the group of mice that received eremomycin at a dose of 550 mg / kg, visually, there were no signs of intoxication for the entire observational period.

The death of mice occurred 1-7 days after the administration of eremomycin, mainly at night. The period of maximum animal mortality occurred on the 1st day of the study. The cause of death in all experimental mice was acute heart failure.

Daily observation of the general condition of the animals, behavioral reactions in the arms and in the open area, as well as the study of individual behavior showed that a single injection of eremomycin did not have a delayed effect (on the 14th day of the study) on the general condition, orientational research activity, and emotional the status of intoxicated experimental rats in the dose range from 375 mg / kg to 1200 mg / kg, as in the intoxicated experimental mice in the dose range from 550 mg / kg to 1750 mg / kg.

At autopsy of rats that underwent elective euthanasia on the 15th day of the study, seven animals treated with eremomycin in the dose range from 550 mg / kg to 1200 mg / kg showed kidney dystrophy and, in one case, catarrhal hemorrhagic enteritis (in rats receiving eremomycin at a dose of 1200 mg / kg).

At autopsy of mice subjected to elective euthanasia on the 15th day of the study, in eight animals that received the studied eremomycin in the dose range from 970 mg / kg to 1750 mg / kg, kidney dystrophy was also detected.

Eremomycin in the dose range from 375 mg / kg to 1750 mg / kg in rats, and in the dose range from 550 mg / kg to 1750 mg / kg in mice with a single intravenous administration at a concentration of 50 mg / ml had no local irritant effect.

According to the results of the study, eremomycin was assigned to the 4th class of low toxic substances according to the classification of GOST 12.1.007-76 (LD ₅₀ iv / 700 mg / kg) and to the 5 hazard class according to the OECD classification (LD ₃₀ iv / 700 mg / kg )

Example 6

Study of the stability of eremomycin and its dosage forms in an accelerated aging experiment.

The stability study of the substance of eremomycin and eremomycin compositions in the accelerated aging experiment was carried out taking into account OFS 42-0075-07 “Shelf life of drugs”.

The studied compositions were stored at a temperature of 60 ° C and a humidity of 75%. The content of the resulting impurities was determined by HPLC after 7, 14 and 21 days. Total impurities (OD) as well as allergenic deeremosamininyl-eremomycin (DE) were determined.

The table below provides data on the stability of the substance of eremomycin sulfate and its compositions of the present invention. The stability of the composition was determined by the least amount of impurities formed.

As can be seen from the table, the substance of eremomycin turned out to be quite unstable, however, the compositions created from it exhibit very high stability, including to the formation of the most allergenic impurity of deseremosaminyl-eremomycin.

Example 7

Assessment of the effect of the deeremosaminyl-eremomycin (DE) impurity on the reaction of general anaphylaxis in guinea pigs

A comparative study of allergic reactions induced by eremomycin sulfate containing an admixture of deeremosaminyl-eremomycin (1.3-3.2% by weight) and vancomycin hydrochloride (“edicin”, manufactured by Lek dd .; registration certificate P No. 010233 dated 13.08. 2010) on males and females of guinea pigs (weighing 320-390 g, ZAO NPO HOUSE OF PHARMACY).

Sensitization. The positive control group was injected intraperitoneally with a 0.1% solution of ovalbumin at a dose of 0.3 mg / kg. The control group was injected with a solvent (saline solution) on the 1st day of the study - subcutaneously, on the 2nd and 3rd days of the study - intramuscularly. Animals from the groups of substances studied were injected with eremomycin sulfate (1.3–3.2% of the mass, deeremosaminyl-eremomycin) and vancomycin hydrochloride (“edicin”) at a dose of 33.3 mg / kg (highest therapeutic dose).

Stage of permission. On the 18th day of the study, animals from the positive control group were injected with 1% intracardiac ovalbumin solution in a volume of 0.5 ml. Animals of the groups of the studied substances were administered a permissive dose of the corresponding substance (167 mg / kg - 5-fold excess of the highest therapeutic dose) intracardially in a volume of 0.5 ml (with a concentration of 100 mg / ml) per animal, regardless of body weight.

Within 30 minutes after the introduction of the resolving dose, the intensity of anaphylactic shock was recorded on the Weigle scale (J. Immunol. 1960. V. 85: 469-77):

++++ - fatal shock +++ - severe shock (general cramps, asphyxia, the animal loses its ability to stay on its paws, falls on its side, does not die) ++ - moderate shock (small convulsions, severe bronchospasm) + - weak shock (some anxiety, rapid breathing, scratching of the muzzle, involuntary urination, bowel movements, hair tousled) 0 - shock did not develop, its signs are absent

Evaluation of the results was carried out by calculating the anaphylactic index Weigle (J. Immunol. 1960. V. 85: 469-77), reflecting the intensity of the reaction in the group according to the formula:

where N is the number of guinea pigs that have died;

N ₁ - the number of guinea pigs that developed severe shock;

N ₂ - the number of guinea pigs that developed moderate shock;

N ₃ - the number of guinea pigs that developed a mild shock;

N ₄ - the number of guinea pigs in which there was no shock.

When observing guinea pigs during the sensitization period, there were no abnormalities in the behavior and clinical condition of the animals. The results of the test "reaction of general anaphylaxis" are presented in table 7.

In animals sensitized with ovalbumin, the development of moderate anaphylactic shock, severe shock and fatal shock according to the Weigle index were recorded, which confirms the adequacy of the response. So, in the group of males, one animal showed signs of moderate shock with mild seizures and severe bronchospasm, in two males - severe shock with seizures, asphyxia, the animals fell to the side. The remaining 3 males died within 1 min after the administration of the resolving dose of ovalbumin. In the group of females, cases of the development of an anaphylactic reaction were also recorded: moderate, severe shock and two females died.

In animals sensitized with eremomycin sulfate (with 1.3% DE), cases of a positive reaction by the Weigle index were recorded at the resolution stage: 3 males and 2 females showed signs of weak anaphylactic shock: rapid breathing, anxiety, scratching of the muzzle.

In animals sensitized with eremomycin sulfate (with 2.7% DE), a positive reaction was recorded at the Weigle index at the resolution stage: in 2 males and 1 females, signs of weak anaphylactic shock were noted - rapid breathing, anxiety, scratching of the muzzle; 2 males and 2 females showed signs of moderate shock with severe bronchospasm; 2 males and 1 females - severe shock with asphyxia, animals fell on their sides.

In animals sensitized with sulphate of eremomycin sulfate (with 3.2% DE), serious signs of anaphylactic reaction were noted. In 1 male and 1 female, signs of the development of anaphylactic shock of a weak type were recorded; 2 males and 1 females showed signs of moderate shock; in 1 male and 2 females, severe shock was observed.

In groups where males and females of guinea pigs were sensitized with a comparison drug of vancomycin hydrochloride, pronounced signs of an anaphylactic reaction were noted at the stage of resolving the corresponding substances at a concentration of 100 mg / ml. In 1 male and 1 female, signs of the development of anaphylactic shock of a weak type were recorded; 2 males and 2 females showed signs of moderate shock; in 1 male and 1 female, severe shock was observed.

The results of the test "reaction of general anaphylaxis" presented in table 7 demonstrate that eremomycin containing less than 3% (1.3-2.7%) DE as an impurity causes anaphylactic reactions of lower intensity compared to vancomycin. Eremomycin containing more than 3% (3.2%) DE as an impurity causes much more serious anaphylactic reactions compared to vancomycin.

Example 8

Evaluation of the effect of impurities of deseremosaminyl-eremomycin (DE) when testing a pseudo-allergic reaction

To study pseudo-allergic reactions, the method of non-immunological activation of histaminoliberization, the inflammatory response to concanavalin A, was used (Guidelines for preclinical studies of drugs, edited by A. Mironov, Moscow, 2012). The inflammation reaction to concanavalin A (a pseudo-allergic reaction) is based on the ability of concanavalin A non-specifically, i.e. without the involvement of reagins with allergens on the membranes of target cells, as a result of direct action on mast cell membranes and basophilic leukocyte receptors, release inflammatory mediators.

Studies were conducted on CBA mice, males weighing 26-28 g. The drugs were administered to mice at a dose of 167 mg / kg once intravenously. After 1 h, a solution of concanavalin A at a concentration of 5 mg / ml was injected into the right hind paw pad. The dose was 0.01 ml per 10 grams of mouse body weight. An adequate volume of isotonic sodium chloride solution was introduced into the pad of the opposite hind paw. After 1 h, the animals were sacrificed, the hind legs were amputated along the border of the hock joint and weighed using Sartorius scales.

The reaction index of inflammation (And _p ) was calculated by the formula:

And _p = (P _op -P _to ): P _to × 100%, where:

P _{op the} mass of the foot of the hind paw, in the pad of which was introduced concanavalin A,

P _to - the mass of the foot of the hind paw, in the pad of which was introduced an isotonic solution of sodium chloride.

Similarly, a study was conducted of the histamine-releasing activity of three samples of eremomycin sulfate containing an admixture of de-eremosaminyl-eremomycin (1.3-3.2% by weight) and vancomycin hydrochloride (“edicin”, manufactured by Lek dd; registration certificate P No. 010233 from 08.13.2010). The results are presented in table 8.

The results of testing pseudo-allergic reactions, presented in table 8, suggest that eremomycin with a low DE content (about 1.3%) does not significantly have histamine-releasing activity. An eremomycin preparation containing less than 3% (2.7%) DE has histamine-releasing activity, but causes a less allergic reaction than vancomycin. An eremomycin preparation containing more than 3% DE has a significantly greater allergic reaction than vancomycin.

Example 9

The use of eremomycin in the treatment of infections of the skin and soft tissues (causative agents of streptococcus and staphylococcus).

Treatment of diabetic foot ulcers.

Patient N., 56 years old, suffering from type 2 diabetes (sugar level 12.9 mmol / l; metformin therapy was unsuccessful), complained of deep bleeding ulcers and cracks in the feet (mainly the heels). Local hyperemia and hyperthermia were noted, purulent discharge was observed on the wound surface. Along with the local inflammatory process, the patient showed manifestations of a systemic inflammatory response syndrome (SIR) in the form of hectic fever (up to 39.1 ° C) with chills, leukocytosis (15.9 × 10 ⁹ / L) with a shift of the formula to the left to myelocytes, increase in C-reactive protein to 126 mg / l. Dressings with the antibacterial ointment levomecol and dioxicol, as well as systemic antibiotic therapy with cefazolin 6 g per day for 7 days were not accompanied by a clinical effect. Staphylococcus aureus resistant to oxacillin and cephalosin (MRSA) and sensitive to glycopeptides was isolated from the wound.

The patient was prescribed two-week therapy with eremomycin at a dose of 1 g per day (250 mg at intervals of 6 hours) intravenously (composition No. 17 from example No. 3, the content of deeremosaminyl-eremomycin, according to HPLC, 0.41% by weight). Also, the patient was ligated three times a day. Additionally, sitagliptin was prescribed instead of metformin as an effective hypoglycemic drug.

After 2 weeks of antibiotic therapy, the ulcers stopped bleeding, the purulent discharge stopped, and almost all ulcers healed. There was a decrease in the severity of SSVR: normalization of leukocytes in the blood, the absence of a left shift in the formula, a decrease in C-reactive protein to 12 mg / L. Adverse events were not observed during the administration of eremomycin, and renal function, assessed by creatinine clearance, did not change.

Boil treatment

A 19-year-old patient with immunodeficiency (hepatitis C without exacerbation) was admitted with a rapidly progressing boil in the right nostril. The inflammatory process encompassed the entire nasolabial triangle with the phenomena of SSVR (temperature rose to 38.4 ° C, leukocytosis - 12.8 × 10 ⁹ / L). Visual inspection of the boil showed a large accumulation of pus.

An incision was made on the outer surface of the right nostril, sanitation of the purulent focus was made, and eremomycin was prescribed at a dose of 250 mg intravenously every 6 hours (composition No. 13 from Example No. 3, the content of deeremosaminyl-eremomycin, according to HPLC, 0.91% by weight).

Due to the deep spread of pus after 24 hours, the second stage of the operation was performed - an incision from the upper gum and a drainage was inserted into the incision for the outflow of pus and exudate. Eremomycin therapy continued for another 7 days (composition No. 14 from example No. 3, the content of deseremosaminyl-eremomycin, according to HPLC, 1.19% of the mass.).

The therapy led to a complete recovery of the patient without the need for additional surgical interventions. No adverse events were observed during treatment.

Carbuncle treatment

Patient I., aged 18, complained of a neglected carbuncle in the lower abdomen on the right side. The duration of the disease was 1 week. At the time of admission, the patient had fever (38.1 ° C) and leukocytosis (12.2 × 10 ⁹ / L).

Surgical sanitation of the purulent focus was performed, and, given the large volume of the lesion, drainage was left. In the first three days after the rehabilitation of the carbuncle, the cavity was washed with isotonic solution and hydrogen peroxide. Along with local wound treatment, given the extent of the lesion and the presence of SSVR, systemic antibiotic therapy with eremomycin was administered intravenously at a dose of 250 mg with an interval of 12 hours (composition No. 14 from example No. 3, the content of deeremosaminyl-eremomycin, according to HPLC, 1.19% of the mass .).

After 2 days of treatment, purulent discharge completely stopped, the temperature returned to normal, the wound healed by primary intention. The duration of eremomycin administration was 4 days. No adverse events were noted during treatment.

Erysipelas treatment

A 29-year-old patient M. complained of pain, itching, and redness of the skin in the region of the right lower leg and thigh, observed for 5 days. Local erythematous manifestations on the skin were accompanied by intoxication, headaches, nausea and high fever (up to 38.8 ° C) with chills. Other signs of CVD were also noted (leukocytosis 14.8 × 10 ⁹ / L, C-reactive protein 86 mg / L, tachycardia up to 126 per min. Previously 2 times treated in a hospital for erysipelas streptococcal etiology of a protracted course.

The picture of the disease was fully consistent with streptococcal erysipelas. At home, she took a macrolide antibiotic clarithromycin at a dose of 1 g per day for 2 days without effect. Given the history, examination data, previous ineffective macrolide treatment, the patient was prescribed intravenous antibiotic therapy with eremomycin in a dose of 125 mg with an interval of 6 hours (composition No. 16 from example No. 3, the content of deseremosaminyl-eremomycin, according to HPLC, 1.68% by weight) . Also, the patient was prescribed antihistamine therapy (cetirzine 10 mg 1 time per day). On the second day of treatment with erythromycin, a decrease in intoxication and pain was noted, on the 3rd day, erythema significantly decreased and the temperature returned to normal. The duration of treatment with eremomycin was 7 days. At the end of treatment, signs of SSVR were not observed, leukocytes in the blood normalized and the level of C-reactive protein, erythematous rashes on the skin disappeared.

During treatment with eremomycin, no adverse events or laboratory signs of renal damage were noted.

Treatment of postoperative wound infection

Patient S., 58 years old, was hospitalized with signs of acute destructive cholecystitis, where he underwent emergency cholecystectomy in an open way by laparotomy. The patient suffered from type II diabetes mellitus and chronic obstructive pulmonary disease, suffered acute myocardial infarction two years ago. Preoperative prophylaxis was carried out by cefazolin 2 g once.

On the third postoperative day, the patient noted pain in the area of the postoperative wound and sacral discharge from the wound. On the 4th day, purulent discharge appeared, the appearance of SSVR was noted: fever up to 38.8 ° C, leukocytosis 24.2 × 10 ⁹ / L with a shift to young forms - 20%, C-reactive protein was 188 mg / L. A postoperative wound infection was diagnosed, the edges of the wound were diluted, local treatment of the wound was started, and systemic antibiotic therapy with ceftriaxone 2 g / day intravenously for 2 days was prescribed, and then, due to the lack of positive dynamics, levofloxacin was added at a dose of 1 g per day intravenously.

After another 3 days, no positive dynamics were obtained in the area of the postoperative wound, and SSVR was preserved. The result of a bacteriological study of the wound: staphylococcus aureus in the amount of 10 ⁸ CFU / ml (resistant to beta-lactams - MRSA) and pyogenic streptococcus - 10 ⁶ CFU / ml were isolated. Previous antibiotics were canceled due to the ineffectiveness and resistance of the pathogen, and an antibiotic eremomycin was prescribed intravenously at a dose of 250 mg with an interval of 6 hours. On the third day of treatment, positive clinical dynamics were noted, purulent discharge from the wound ceased, SSRP decreased and the temperature returned to normal. On the 5th day, the wound cleared, on the 7th day, white blood cells in the blood returned to normal. Repeated bacteriological studies of the wound discharge did not reveal the growth of pathogenic microflora. The duration of eremomycin therapy was 8 days. The wound healed by secondary intention, the patient was discharged in satisfactory condition. During treatment with eremomycin, no adverse events were noted; renal function did not change.

The above examples show the high efficacy of eremomycin in the treatment of infections of the skin and soft tissues caused by gram-positive bacteria.

Example 10

The use of eremomycin in the treatment of respiratory tract infections (pneumonia)

Patient N., 39 years old, male, was admitted with suspected pneumonia. The disease is 10 days old. On admission, a fever of 39.3 ° C was observed with chills, a cough with copious discharge of mucopurulent sputum, breathing was difficult due to pleural pain. Marked shortness of breath 10 per min, auscultation recorded small bubbly moist rales, area of bronchial respiration. Anamnesis is burdened: chronic alcohol intoxication, chronic obstructive pulmonary disease. At home, he took two courses of antibiotic therapy with ciprofloxacin and azithromycin without effect.

An X-ray examination revealed bilateral pleuropneumonia in the lower lobes of both lungs. In the study of sputum, Streptococcus pneumoniae sensitive to cefotaxime, levofloxanin, and vancomycin was isolated. Ceftriaxone prescribed for 2 days at a dose of 1 g per day did not lead to a positive effect.

Due to the ineffectiveness of treatment and the presence of aggravating factors, the patient was prescribed therapy with an intravenous antibiotic eremomycin and a mucolytic agent. The dose of eremomycin was 250 mg with an interval of 6 hours (composition No. 14 from example No. 3, the content of deseremosaminyl-eremomycin, according to HPLC, 1.19% by weight), and acetylcysteine at a dose of 800 mg per day was prescribed as a mucolytic agent.

The therapy led to the complete recovery of the patient without the manifestation of any significant adverse events (nevertheless, slight redness was observed at the injection site). The duration of eremomycin therapy was 10 days.

This example demonstrates the good efficacy of eremomycin in the treatment of respiratory tract infections caused by gram-positive bacteria.

Example 11

The use of eremomycin in the treatment of antibiotic-associated clostridial diarrhea and pseudomembranous colitis in patients in a hospital

This study presents the combined practice of using eremomycin and vancomycin in the treatment of antibiotic-associated clostridial infections, and analyzes the effectiveness of treatment with eremomycin and vancomycin.

Eremomycin or vancomycin were prescribed to 17 patients. In 16 cases, clostridial infection was caused by previous antibiotic therapy (3rd generation cephalosporins or fluoroquinolones). In 1 case, clostridial infection was caused by previous anticancer chemotherapy. The diagnosis of clostridial infection was confirmed by the detection of toxin A and B by ELISA, colonoscopy was used to diagnose pseudomembranous colitis. In all patients, the development of clostridial infection was characterized as an infection of "moderate severity".

After diagnosis of clostridial infection, patients were randomized into 2 groups. In the table below, a detailed description of groups of patients with clostridial infection who received eremomycin or vancomycin therapy.

All patients received antibiotics for 10 days. Eremomycin was used at a dose of 250 mg every 6 hours orally in the form of a solution (composition No. 8 from Example No. 3 diluted to 30 ml; the content of deseremosaminyl-eremomycin, according to HPLC, 1.43% by weight). Vancomycin was administered orally at a dose of 250 mg every 6 hours in the form of a solution with a volume of 30 ml (the drug “Editsin” manufactured by Lek dd .; registration certificate P No. 010233 of 08/13/2010.

During therapy in the vancomycin group, 3 adverse reactions were detected in the form of nausea and vomiting. The researchers associated these adverse reactions with the unpleasant bitter taste of vancomycin. In the eremomycin group, no adverse reactions associated with taking the antibiotic were found. Patients in the eremomycin group noted a sweetish taste of the antibiotic, which contributed to adherence to therapy.

After 10 days of treatment, all patients had no symptoms of clostridial infection, but patients continued to be monitored for another 10 weeks for relapses. No relapse was found in the eremomycin group. In the vancomycin group, two patients (on the 9th and 14th day after the end of therapy) recurred clostridial infection in a mild form, and they resumed vancomycin therapy (10 days of therapy), but already in an increased dose (500 mg every 6 hours). Thus, the total effectiveness of eremomycin was observed in all 8 patients and amounted to 100%. The total effectiveness of vancomycin was observed in 7 of 9 patients and amounted to 77.8%.

As can be seen from this example, eremomycin has been shown to be effective in the treatment of antibiotic-associated clostridial diarrhea and pseudomembranous colitis in humans. In addition, eremomycin was more effective (no side effects and relapses) than its related antibiotic, vancomycin.

Claims (25)

1. A method of treating human diseases caused by gram-positive bacteria, comprising administering to a needy person a eremomycin or a pharmaceutically acceptable salt thereof, a solvate in an amount of from 20 to 2000 mg per day, wherein the administered eremomycin contains not more than 3% by weight as an impurity. deseremosaminyl-eremomycin. 2. The method according to claim 1, wherein the eremomycin or a pharmaceutically acceptable salt or solvate thereof is administered orally. 3. The method according to p. 2, in which eremomycin or its pharmaceutically acceptable salt, MES is administered in the form of a capsule, tablet or oral solution. 4. The method of claim 3, wherein the oral solution comprises eremomycin or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carbohydrate, and water. 5. The method of claim 3, wherein the tablet comprises eremomycin or a pharmaceutically acceptable salt, solvate and macrogol thereof. 6. The method of claim 3, wherein the capsule contains eremomycin or a pharmaceutically acceptable salt, solvate and pharmaceutically acceptable carbohydrate thereof. 7. The method according to p. 1, in which eremomycin or its pharmaceutically acceptable salt, MES is administered intravenously. 8. The method of claim 7, wherein the eremomycin or a pharmaceutically acceptable salt thereof, the solvate is administered as an infusion solution. 9. The method of claim 8, wherein the infusion solution comprises eremomycin or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carbohydrate, and water. 10. The method according to p. 1, in which eremomycin or its pharmaceutically acceptable salt, MES is administered in an amount of from 100 to 1500 mg per day. 11. The method according to p. 10, in which eremomycin or its pharmaceutically acceptable salt, MES is administered in an amount of from 250 to 1000 mg per day. 12. The method according to p. 1, in which eremomycin or its pharmaceutically acceptable salt, MES is administered in two doses. 13. The method according to p. 1, in which eremomycin or its pharmaceutically acceptable salt, MES is administered in four doses. 14. The method according to p. 1, in which eremomycin or its pharmaceutically acceptable salt, MES is administered in an amount of 1000 mg per day in four doses. 15. The method according to claim 1, wherein the gram-positive bacteria are selected from bacteria including bacteria of the genera Bacillus, Listeria, Staphylococcus, Streptococcus, Enterococcus and Clostridium. 16. The method according to claim 1, in which the human disease caused by gram-positive bacteria is selected from the group comprising endocarditis, sepsis, pneumonia, lung abscess, meningitis, staphylococcal enterocolitis, pseudomembranous colitis associated with Clostridium difficile, infections of the skin, soft tissues, bones and joints. 17. A method of treating a clostridial infection in humans, comprising orally administering to a needy person eremomycin or a pharmaceutically acceptable salt thereof, a solvate in an amount of from 20 to 2000 mg per day. 18. The method according to p. 17, in which eremomycin or its pharmaceutically acceptable salt, MES is administered in an amount of from 250 to 1000 mg per day. 19. The method according to p. 17, in which eremomycin or a pharmaceutically acceptable salt or solvate thereof is administered in two doses. 20. The method according to p. 17, in which eremomycin or its pharmaceutically acceptable salt, MES is administered in four doses. 21. The method according to p. 17, in which eremomycin or its pharmaceutically acceptable salt, MES is administered in an amount of 1000 mg per day in four doses. 22. The method according to p. 17, in which eremomycin or its pharmaceutically acceptable salt, MES is administered in the form of a capsule, tablet or oral solution. 23. The method according to p. 22, in which the tablet contains eremomycin or its pharmaceutically acceptable salt, MES and macrogol. 24. The method of claim 22, wherein the oral solution comprises eremomycin or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carbohydrate, and water. 25. The method according to any one of paragraphs. 17-24, in which the administered eremomycin contains as an impurity not more than 3 wt.% Deseremosaminyl-eremomycin.