RU2520603C1 - Method for preparing high-bioavailability rifabutin composition, pharmaceutical composition and method of treating mycobacteriosis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: rifabutin is dissolved in a water-miscible solvent which dissolves rifabutin better than water does; a rifabutin solution is prepared; gelatin is dissolved in water to prepare a gelatin solution; the rifabutin solution is slowly added to the gelatin solution while stirring to prepare a semi-product. The semi-product is dried in a spray drier or lyophilised to prepare a product. The prepared product is used as a part of a pharmaceutical composition for treating mycobacteriosis and Helicobacter pylori infection.

EFFECT: higher bioavailability of rifabutin.

46 cl, 8 tbl, 5 ex, 7 dwg

Description

FIELD OF TECHNOLOGY

The present invention relates to the field of medicine, pharmacy and colloid chemistry, specifically to a pharmaceutical composition for treating tuberculosis based on rifabutin solubilized with gelatin.

BACKGROUND

By chemical structure, rifabutin is 4-deoxo-3,4 - [2-spiro [N-isobutyl-4-piperaidyl] 2,5-dihydro-1H-imidazole]. In terms of spectrum and mechanism of action, rifabutin is similar to rifampicin, however, it significantly surpasses it in pharmacodynamic and pharmacokinetic properties (Tsybanev A.A., Sokolova GB.The long-acting anti-tuberculosis antibiotic is rifabutin. The antimicrobial spectrum, pharmacodynamics and pharmacokinetics are especially important. , 1999; 44 (8), pp. 30-36).

Rifabutin is readily soluble in lipids, which leads to its penetration into tissues, a larger distribution volume, a smaller difference between the maximum (C _max ) and minimum (C _min ) concentrations in the blood serum, prolonged half-life from the body (Ungheri D., Delia Brunna C. , Sanfilippo A. Studies on the mechanism of action of spiropiperidyl-rifamycin on LM427 rifampicin-resistant M. tuberculosis. - Drugs under Experimental and Clinical Research 1984; 10: 681-689: 37: 7685-7694).

The antibacterial action of rifabutin, as well as other antibiotics from the rifamycin group (for example, rifampicin), is based on the suppression of DNA-dependent bacterial RNA polymerase (see the above article by A. Tsybanev). Rifabutin is also believed to have a direct inhibitory effect on bacterial cell DNA synthesis, resulting in its activity against rifampicin-resistant mycobacteria (see the aforementioned Ungheri D. article). Rifabutin has a wide spectrum of antibacterial action, is active against a wide range of gram-positive and gram-negative bacteria.

The most important property of rifabutin is its activity against Mycobacterium tuberculosis and Mycobacterium avium-intracellulare complex. The minimum inhibitory concentration (MIC) of rifabutin for rifampicin-sensitive strains of Mycobacterium tuberculosis is 0.03-0.06 μg / ml, i.e., within the range of concentrations that are maintained in the blood for 24 hours after a single oral administration of 300 mg of the drug. The minimum bactericidal concentration (MBC) is 2-4 times higher than the BMD for the same strains of mycobacteria. This level of antibiotic concentrations in the blood is detected within 6-12 hours after taking the above dose (see the above article by Tsybanev AA; Grosset JH New approaches in antimycobacterial chemotherapy. - Drugs Today 1988; 24: 291-301; Heifets LB et al. Rifabutin : minimal inhibitory concentration for Mycobacterium tuberculosis. - The American Review of Respiratory Diseases 1988; 137: 719-721).

An important property of rifabutin, which determines the possibility of its use in tuberculosis due to rifampicin-resistant mycobacteria, is the lack of complete cross-resistance of pathogens to these antibiotics. It has been shown that more than 35% of rifampicin-resistant mycobacteria remain susceptible to rifabutin. The effectiveness of rifabutin was confirmed in a model of experimental tuberculosis in mice caused by rifampicin-resistant strains, when the drug used in calculating the average daily doses for humans (300-400 mg) contributed to the rapid elimination of mycobacteria (Arme YA Antimycobacterial activity in vivo of LM 427 (Rifabutin ). - The American Review of Respiratory Diseases 1988, 138: 1254-267).

Rifabutin exceeds rifampicin activity in relation to clinical strains of atypical mycobacteria (Mycobacterium avium-intracellulare complex-MAC) (Brogden RN, Futton A. Rifabutin. A review of its antimicrobial activity, pharmacokinetic properties and therapeutic efficacy. - Drugs 1994; 47: 6: 983-1009). The values of IPC ₉₀ (the minimum concentration that suppresses the growth of 90% of mycobacteria) of rifabutin for most strains of these species ranges from 1.0-2.0 μg / ml. Rifabutin sensitivity of atypical mycobacteria (Mycobacterium kansasii, Mycobacterium fortuitum, Mycobacterium gordonae, Mycobacterium haemophilum) is significantly lower than that of MAC (MPC90, depending on the species, 2.5-8 μg / ml). Mycobacterium chelonae and Mycobacterium simiae are moderately sensitive or resistant (Yajko DM et al. Therapeutic implications of inhibition versus killing of Mycobacterium avium complex by antimicrobial agents. - Antimicrobial Agents and Chemotherapy 1987, 31: 117-20).

Most strains of Mycobacterium leprae also exhibit high sensitivity to rifabutin. There is a synergistic effect against Mycobacterium leprae in the combined use of rifabutin with fluoroquinolones (sparfloxacin, lomefloxacin, norfloxacin, ofloxacin, perfloksatsin, ciprofloxacin, levofloxacin or moxifloxacin) (Dhople AM et al. In vitro activity of three new fluoroquinolones and synergy with ansamycins against Mycobacterium leprae - The Journal of Antimicrobial Chemotherapy 1993; 32: 445-49).

One of the most important indications for rifabutin use today is tuberculosis in HIV-infected patients and in patients with AIDS (HIV / AIDS-associated tuberculosis) (Centers for Disease Control and Prevention (2000) Updated guidelines for the use of rifabutin or rifampin for the treatment and prevention of tuberculosis among HIV-infected patients taking protease inhibitors or nonnucleoside reverse transcriptase inhibitors. - MMWR Morb Mortal Wkly Rep 49 (9), 185 9. Available on-line: www.cdc.goy / nchstp / tb / ( at 09/29/2010)).

It is known that rifabutin is similar to rifampicin by the action on many opportunistic types of gram-positive and gram-negative microorganisms. Rifabutin is active against Staphylococcus aureus, coagulase-negative Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus haemolyticus, Streptococcus pyogenes and Streptococcus viridans (MPC <0.005-0.01 μg / ml) Infinitial Infinitum Infinitum Infinitum Infinitum infectious microbial fungus infectious microbial activity an official publication of the Infectious Diseases Society of America 1996; 22: Suppl 1: 3-14). Streptococcus pneumoniae and anaerobic cocci are sensitive to rifabutin; Enterococcus faecalis strains show only moderate sensitivity to rifabutin (MIC 25 μg / ml) (Kan N. et al. A comparative study of rifabutin and other antibiotics against a spectrum of nonmycobacterial microorganism. - 9th Meeting of the American Society of Microbiology, 1994, Abstr A-37, USA).

Rifabutin is active against methicillin-resistant strains of golden (MRSA) and coagulase-negative (MRCNS) staphylococci, however, staphylococci resistance to rifabutin in vitro and in vivo develops very quickly, almost after a single contact with the drug. Its development can be prevented by the combined use of rifabutin with fusidine, fluoroquinolones or erythromycin. Such combinations can be effectively used in the treatment of severe infections caused by methicillin-resistant Staphylococcus aureus (MRSA) and Coagulase-negative (MRCNS) (Wood C. A. Rifampicin-resistant Staphylococcus bacteriemia in patient with AIDS receiving rifabutin. - Lancet 1994; 343: 919-2 )

Rifabutin is characterized by the rapid development of resistance in pneumococci, especially in cases of its frequent use in the treatment of tuberculosis, MAC-mycobacteriosis (Mycobacterium avium-intracellulare complex). Among gram-positive bacteria, Clostridium spp., Including Clostridium difficile, are highly sensitive to rifabutin. Rifabutin is highly active against Neisseria (Neisseria meningitidis, Neisseria gonorrhoeae), although clinical data on the possibility of its use in these infections are practically absent (see the above article by Kan N.).

Of great interest are data on the high sensitivity to rifabutin of Helicobacter pylori (Rossi R. et al. In vitro activity in rifabutin, potential antibiotic in the therapy of Helicobacter pylori. - 6th International Congress of Infectious Diseases. Prague 1994; Abstr 48; Toracchio S. et al. Rifabutin based triple therapy for eradication of H. pylori primary and secondary resistant to tinidazole and clarithromycin. - Digestive and Liver Diseases 2005 Jan; 37 (1): 33-8; Canducci F. et al. Rifabutin-based Helicobacter pylori eradication 'resque therapy'. - Alimentary Pharmacology & Therapeutics 2000; 14: 311-16); the MIC value of ₉₀ rifabutin for a given microorganism is 0.007 μg / ml. Rifabutin is significantly superior to rifampicin in terms of activity against Helicobacter pylori (see the aforementioned work by Rossi R.). Such a property of rifabutin, as stability at wide fluctuations in pH values, gives reason to consider it as an active component in the complex therapy of peptic ulcer.

There is evidence of a high activity of rifabutin (IPC ₉₀ 0.008 μg / ml), superior to the action of rifampicin, in relation to Chlamydia trachomatis. Moreover, rifabutin is characterized by a slow formation of chlamydia resistance, while in vitro rifampicin resistance is formed in the presence of subinhibitory concentrations for several passages (Treharne JD et al. Chlamydia trachomatis susceptibility and resistance to rifampicin and rifabutin. - The Journal of Antimicrobial Chemotherapy, 1989; 33: 1393-1394).

An in vivo experiment found that rifabutin is a highly effective drug in the treatment of toxocoplasmosis (Aranjo F.G. et al. Rifabutin is active in murine model toxoplasmosis. - Antimicrobial Agents and Chemotherapy, 1994, 38: 570-75). This feature of the antibiotic is important because antibiotic therapy of toxocoplasmosis causes serious difficulties. A dose of rifabutin 100-200 mg for 10 days protects 100% of mice with experimental toxoplasmosis. This dose is significantly higher than that used in the treatment of tuberculosis or MAC infection (Mycobacterium avium-intracellulare complex), but it can be reduced when used in combination with sulfadiazine, pyrimethamine or clindamycin (ibid.). It is also known from this work that rifabutin (at a dose of 50 mg / kg) in combination with subtherapeutic doses of pyrimethamine or clindamycin prevented the death of 40% and 90% of animals, respectively.

Rifabutin is also active against many gram-negative bacteria, in particular against Pseudmonas aeruginosa (Pseudomonas aeruginosa), and its activity against this pathogen has been shown to increase when combined with polymyxin B (Vaara M. Comparative activity of rifabutin and rifampicin against gramnegative bacteria that have damaged or defected outer membranes. - The Journal of Antimicrobial Chemotherapy, 1993, 31: 799-801).

Rifabutin acts bactericidal on the microbial cell. For Mycobacterium avium and Mycobacterium intracellulare, which actively proliferate inside the cell, the bactericidal effect of the drug is more correlated with clinical efficacy than the BMD values. When studying the dynamics of the death of Mycobacterium avium, the MBC (minimum bactericidal concentration causing death of ≥ 99% of bacterial cells) for rifabutin was 8 μg / ml, while the BMD (minimum inhibitory concentration) was less than 0.03 μg / ml (Brogden RN, Futton A. Rifabutin. A review of its antimicrobial activity, pharmacokinetic properties and therapeutic efficacy. - Drugs 1994; 47: 6: 983-1009). To a greater extent, rifabutin activity characterizes the value of the MBK / MBC ratio, which for Mycobacterium avium ranges from 8 to 128. When determining the rifabutin MBC in vitro in a dynamic system that simulates the concentration of an antibiotic in the blood when taken orally, by counting the number of CFU (colony forming units) after exposure to a strain of Mycobacterium intracellulare with 5 μg / ml antibiotic showed a decrease in the number of viable individuals, starting from the 6th day of contact with complete death of the culture by the 8th day. The ratio of MBC to MIC of rifabutin in relation to the reference strain of Mycobacterium tuberculosis was 4, with a MBC value of 0.125 μg / ml (Heifets LB et al. Bactericidal activity in vitro of rifabutin against M.avium and M. tuberculosis. - The American Review of Respiratory Diseases 1990 141: 626-30).

The values of MBC and IPC of rifabutin and, consequently, the MBC / IPC ratios for other bacterial species (Legionella spp., Chlamidya trachomatis, Staphilococcus aureus) differed no more than 2 times (see the above article by A. Tsybanev).

Rifabutin is characterized by a prolonged post-antibiotic effect, manifested in the suppression of microbial growth after a short exposure period of the culture with the antibiotic and its subsequent removal from the nutrient medium. The duration of the post-antibiotic effect depended on the type of microorganism, the concentration of the drug, and the duration of exposure. When the exposure of the MAC culture with rifabutin for 1-2 hours at drug concentrations 2-4 higher than MBC, the duration of the post-antibiotic effect ranged from 21.5 to 47.5 hours (see the above article by Brogden R.N.).

The possibility of enhancing the antimicrobial activity of rifabutin when combined with other antibacterial drugs has been most thoroughly studied in relation to Mycobacterium avium-intracellulare complex (MAC), which is due to the difficulties in treating diseases caused by these pathogens. In vivo, rifabutin, used in monotherapy for generalized MAC infection, was characterized by moderate activity, manifested only in a decrease in the contamination of the lungs and spleen of mice, without protecting them from death. For prophylactic administration, the efficacy of rifabutin was comparable to that of clarithromycin or azithromycin (see the above article by A. Tsybanev).

The activity of rifabutin in combination with other anti-tuberculosis drugs was studied in various systems, using when processing the results in some cases, the determination of the index of fractional inhibitory concentration (FIC) (the sum of the IPC of the drugs in combination with the IPC of each drug separately), as well as the index of the fractional bactericidal concentration (FBK) (the sum of the MBK of each drug in combination, divided by the MBK of each drug separately). The synergistic effect of rifabutin with ethambutol was found for 43-100% MAC strains (FIC or FBC index <0.5) (Kent RJ et al. The in vitro bactericidal activities of combinations of antimicrobial agents against clinical isolates of Mycobacterium avium-intracellulare. - The Journal of Antimicrobial Chemotherapy 1992; 30: 643-50).

With the combination of rifabutin with clarithromycin, a significant increase in the sensitivity of MAC strains isolated in patients with HIV infection was observed (Mascellino M.T. et al. In vitro activity of clarithromycin alone or 10 combinations with other antimicrobial agents against Mycobacterium avium-intracellulare complex strains isolated from AIDS patients. - Journal of Chemotherapy 1991; 3: 357-62).

The most active against Mycobacterium avium-intracellulare complex in the mouse macrophage system were combinations of rifabutin with ethambutol and ciprofloxacin / amikacin (91% and 100% cell death, respectively). The combination of rifabutin with thiasetasone was also synergistic for MAC strains (Seydel J.K. et al. Development of effective drug combinations for the inhibition of multiple resistant Mycobacterium, especially of the Mycobacterium avium complex. - Chemotherapy 1992; 38: 159-168) .

The additive or synergistic effects of the combined use of rifabutin and ethambutol have been found in relation to Mycobacterium kansasii (Hjelm U. et al. Susceptibility of Mycobacterium kansasii to ethambutol with rifamycin, ciprofloxacin and isoniazid. - European Journal of Clinical Diseases 11; -54).

Combinations of rifabutin and ethambutol with clofazimine or cefazolin, as well as rifabutin with cefazolin and streptomycin, showed synergism against strains of Mycobacterium paratuberculosis (Chiodim RJ Bactericidal activities of various antimicrobial agents against human and animal isolates of Mycobacteric antigicrobial agents against Mycobactericidal anti-microbial agents of Mycobacteriobicides and Mycobacterias anti-microbial agents of Mycobacterium agratum 36. -67).

Rifabutin is one of the few antibacterial drugs that are highly effective in treating infections caused by intracellular localized pathogens, in particular in the treatment of mycobacteriosis. The basis of such a high activity of rifabutin is its rapid penetration into the cell, in particular into the macrophage, which occurs without a violation of phagocytic function. The ratios between intracellular and extracellular concentrations of rifabutin in mouse macrophages range from 9: 1 to 15: 1, compared with a similar 5: 1 for rifampicin (Van Der Awera T. et al. Intraphagocytic penetration of antibiotics. - Journal of Antimicrobial Chemotherapy 1988, 22: 185-92).

Depending on the concentrations used, rifabutin, as shown in the literature, showed inhibitory or bactericidal activity against murine macrophages propagating in culture Mycobacterium avium-intracellulare complex and Mycobacterium xenopi (Rastogi N. et al. Action of antituberculosis and β-lactam drugs against extra- and intracellularly growing Mycobacterium avium-intracellulare. - Ann Inst Pasteur (Microbiologic), 1988; 139: 225-32 and Rastogi N. et al. Drug action against intracellular growth of Mycobacterium xenopi. - Curr Microbiol, 1989, 19: 83-9 )

When the atypical mycobacteria isolated from AIDS patients were exposed for 2-5 days with therapeutic concentrations of rifabutin achieved in the blood plasma after its administration as an oral dosage form, a bacteriostatic effect was observed with a pronounced decrease in the number of CFU (colony forming units) (Fantonni L. et al. Activity of antimicrobial agents against Mycobacterium avium intracellulare complex (VFC) strains isolated in Italy from AIDS patients. - Zentralblatt Bacteriol, 1992, 276: 512-20).

With the developed infection of Mycobacterium avium-intracellulare complex in mice, the effect of rifabutin is manifested in a pronounced decrease in the number of viable mycobacteria in the lungs and spleen; for this indicator, the effect of rifabutin is superior to the similar effect of rifampicin, rifapentin or azithromycin (see the above article by A. Tsybanev).

In the prevention of mycobacteriosis in infected mice pre-vaccinated with the Calmette-Guerin vaccine, the protective (prophylactic) effect of rifabutin was manifested when this antibiotic was administered at a dose of 10 mg / day for 1 week, while rifampicin had a similar protective effect only after 12 weeks use in the same dose (see the aforementioned article by Mascellino M.T.).

With respect to strains of Helicobacter pylori resistant to clarithromycin and tinidazole, three-component therapy was found to be most effective, including the use of a proton pump inhibitor (pantoprazole), amoxicillin and rifabutin (Toracchio S. et al. Rifabutin based triple therapy for eradication of H. pylori primary resistant to tinidazole and clarithromycin. - Digestive and Liver Diseases. 2005.37 (1): 33-8).

Despite such a wide spectrum of antibacterial activity, the clinical use of rifabutin is limited by the fact that rifabutin, as noted above, is slightly soluble in water, which is why the preparation of rifabutin suitable for parenteral (intravenous and / or intramuscular) administration to a person in need this patient is a serious problem.

Traditionally available commercially available oral dosage forms of rifabutin (usually rifabutin is now available in the form of gelatin capsules) are characterized by low bioavailability, and therefore the doses of this antibiotic necessary for effective antibacterial therapy are quite high, which causes not only significant side effects effects on the digestive system (diarrhea, heartburn, indigestion, loss of appetite and nausea), but also such side effects, a fever, the appearance of skin itching and skin rashes, thrombocytopenia (according to the FDA); It has been shown that therapy using traditional oral forms of rifabutin may also be accompanied by the appearance of “flu-like” symptoms (Matteelli A, et al. Tolerability of twice-weekly rifabutin-isoniazid combinations versus daily isoniazid for latent tuberculosis in HIV-infected subjects: a pilot study. - International Journal of Tuberculosis and Lung Diseases, 2005, 3, 1043-6).

In quantitative terms, the bioavailability of rifabutin when taken orally is on average about 20% (Skinner M. N. et al. 1989. Pharmacokinetics of rifabutin. Antimicrob. Agents Chemother. 33: 1237-41). Being a sufficiently lipophilic substance (octanol / water distribution coefficient substantially depends on pH (Vostrikov VV et al. Distribution coefficient of rifabutin in liposome / water system as measured by different methods. European Journal of Pharmaceutics and Biopharmaceutics, 2007): in a neutral environment according to various authors, the log P value is 2.6–4.3 (3.2 according to Monograph MYCOBUTIN * (rifabutin capsules USP) Pfizer Canada Inc, 2008; 2.6 according to Narang PK 1995. Clinical pharmacology of rifabutin : a new antimycobacterial. Rev. Contemp. Pharmacother. 6: 129-15 and 4.3 according to Clinical Pharmacology)), online at Gold Standard Multimedia. Available at: http://www.gsm.com), rifabutin is easily but not completely absorbed from the digestive tract (Battaglia R. et al. 1990. Absorption, disposition and preliminary metabolic pathway of 14C-rifabutin in animals and man. J. Antimicrob. Chemother. 26: 813-22). The reason for such low bioavailability is probably the low solubility of rifabutin in water (about 190 μg / ml, according to the aforementioned article by Vostrikov VV) and its low dissolution rate, and therefore rifabutin, obviously, refers to the 2nd or 4th class BFK (compounds with low solubility and high or low permeability) (see articles Amidon GL et al. A theoretical basis for a biopharmaceutic drug classification: the correlation of in vitro drug product dissolution and in vivo bioavailability, Pharm. Res ., 1995, 12, 413-20 and Nighute A.V. et al. Enhancement of Dissolution rate of Rifabutin by preparation of Microcrystals using Solvent Change Method International Journal of PharmTech Research, 1 (2): 142-8, 2009). Medicinal substances are classified as low solubility with a dose / solubility ratio of not more than 250 ml in the entire pH range from 1.2 to 7.4 when absorbed by at least 85%. For rifabutin, these values exceed 2500 ml (at pH 7.0 and for a dose of 150 mg). With regard to permeability, there is no specific data in the literature, although, since the same source indicates that the substance is classified as highly permeable under the condition of absorption of 85% and higher, rifabutin seems to be more likely to belong to class 4 according to BFK.

The solubility of rifabutin is highly dependent on the pH of the medium and reaches maximum values at pH below 3.0, when most of the rifabutin molecules are in the ionized state (see the aforementioned article by Vostrikov V.V.). At the same time, it is known that ionized molecules penetrate worse through the cell membrane and are therefore more slowly absorbed (see http://howmed.net/pharmacology/absorption-of-drugs/ and Kaplan Pharmacology 2010, page 6, Absorption). Therefore, it can be assumed that the main limiting factor limiting bioavailability is the low solubility of rifabutin at a pH above pH 5.0, i.e. in the intestinal environment. Indeed, in the above article, Narang P.K. it was shown that the solubility of rifabutin decreases 16 times with increasing pH from 5 to pH 7.4. At the same time, another commercially available substance has a similar ratio of 8. This discrepancy, apparently, can be explained by the properties of the rifabutin substance obtained by various manufacturers. It is known, for example, that the solubility of rifampicin (a rifabutin analog similar in chemical properties) is largely determined by the structure of its amorphous form, and, depending on the preparation method, can vary in the range from 0.2 to 1.5 mg / ml (Becker C . et al. J Pharm Sci. 2009, 98 (7): 2252-67. Biowaiver monographs for immediate release solid oral dosage forms: rifampicin). However, in any case, the solubility of rifabutin is low. In this regard, it seemed appropriate to increase the solubility and dissolution rate of RB in the composition of various compositions in the intestine, that is, in accordance with the recommendations of the United States Pharmacopeia, at a pH of 6.8.

In this regard, the urgent task is to develop a soluble dosage form of rifabutin with high solubility and bioavailability.

Eurasian Patent EA 012121 B1 discloses a pharmaceutical composition for treating tuberculosis and Helicobacter pilory mediated diseases based on 100-800 nm polymer particles (nanoparticles) containing a rifabutin antibiotic in a therapeutically effective amount and adjuvants suitable for intravenous administration to a person in need thereof patient, characterized in that as auxiliary substances contains a polymer / polymers of lactic acid and / or a copolymer / copolymers of lactic and glycolic acids with glycol content acid in copolymers up to 50 mol% with or without an additional carboxyl group at the end of the molecule or copolymers of lactic acid polymers or copolymers of lactic or glycolic acid with polyethylene glycol; the molecular weight of the polymers and copolymers is from 2 to 200 kDa, and the composition is a lyophilisate, which, when added with water or physiological saline, forms a stable suspension with a particle size of 0.1-0.8 microns.

The specified composition additionally contains a water-soluble natural or synthetic polymer stabilizer with a molecular weight of not more than 70 kDa and, if necessary, a lipid plasticizer and fillers at a certain quantitative ratio of the components.

Eurasian Patent EA 013569 B1 discloses a pharmaceutical composition for treating tuberculosis and Helicobacter pylori-mediated diseases, characterized in that it is a lyophilisate of rifabutin solubilized with human serum albumin, characterized by an average particle size of 4 to 10 nm, and when a pharmaceutically acceptable diluent is added or carrier - a stable solution suitable for intravenous administration to a patient in need of this, characterized by an average particle size of 4 to 10 nm, as well as a method for the preparation of such a pharmaceutical composition (rifabutin lyophilisate solubilized with human serum albumin) is characterized in that the aqueous mixture of rifabutin, albumin and an organic solvent is dispersed at a temperature of 0 to + 40 ° C, subjected to high pressure homogenization to obtain a nanoemulsion, and the organic nanoemulsion is removed from the obtained nanoemulsion the solvent is filtered, lyoprotectant is added, frozen and lyophilized, moreover, said aqueous mixture of rifabutin and albumin is obtained by dissolving 2-5% (m / o) albumin in demineralized water, the subsequent addition to the specified solution of albumin of 2-5% (v / v) organic solvent and rifabutin in an amount of from 0.2 to 20.0 wt.%, and a method for the treatment of tuberculosis and diseases mediated by Helicobacter pylori comprising the intravenous administration of a therapeutically effective amount of such a pharmaceutical composition to a patient in need thereof.

Although the composition in accordance with EA 013569 B1 does not contain potentially toxic biodegradable polymers, nevertheless, human serum albumin, necessary for obtaining such a composition, being one of the components of human blood plasma, can be infected with viral infections having a parenteral transmission route (HIV, hepatitis B and C, etc.); in addition, in some cases, intravenous administration to a patient of drugs based on albumin of human blood plasma can cause such side effects as urticaria, chills, fever, shortness of breath, tachycardia, lowering blood pressure, pain in the lumbar region.

All of the above limits the possibility of using the nanoparticles disclosed in EA 013569 B1 to create colloidal (nanosomal) forms of rifabutin.

OBJECT OF THE INVENTION

Since, as mentioned above, known oral forms have low bioavailability, forms for intravenous administration based on nanoparticles of biodegradable polymers have additional toxicity, while intravenous forms containing albumin pose an additional risk of infection with infectious diseases, the present invention is to create an oral drug forms of rifabutin with high bioavailability.

SUMMARY OF THE INVENTION

The technical result of the present invention is to increase the bioavailability of rifabutin in a pharmaceutical composition for oral administration.

The technical result is achieved due to the fact that the method of producing rifabutin with increased bioavailability, solubilized with gelatin, is as follows:

(a) rifabutin is dissolved in a pharmaceutically acceptable water-miscible solvent, the solubility of rifabutin in which is higher than in water, to obtain a rifabutin solution;

(b) the gelatin is dissolved in water to obtain a solution of gelatin in water;

(c) solution (a) is slowly added to solution (b) with stirring to obtain an intermediate;

(g) the intermediate obtained in stage (C) is dried in a vacuum spray dryer or frozen and lyophilized to obtain the product.

Dissolution or mixing of the components can be carried out at temperatures from 0 to 40 ° C.

The stirring addition in step (c) can be carried out gradually over at least 1 hour, preferably at least 2 hours, particularly preferably at least 3 hours.

Stirring after addition in step (c) can be carried out for at least 1 hour, preferably at least 2 hours, particularly preferably at least 3 hours.

The concentration of rifabutin in solution (a) can be 40 ÷ 80 mg / ml.

Solution (a), solution (b) and / or solution (c) can (although not necessary) be further filtered or centrifuged to obtain a filtrate or supernatant, respectively, and in the next step ((b), (c) and / or (g), respectively) used as a filtrate or supernatant, respectively.

Excess solvent from intermediate (c) can (although not necessary) be removed on a rotary evaporator, the resulting suspension in this case is filtered or centrifuged, and in step (d), the filtrate or supernatant is used as an intermediate, respectively.

In the present method, demineralized or deionized water may be used (although not necessarily).

Suitable types of gelatin may be pork gelatin, beef gelatin or goat gelatin. Succinylated liquid gelatin is preferred.

The concentration of gelatin in solution (b) in one of the preferred embodiments of the method is 10 ÷ 40 mg / ml.

The mass fraction of gelatin in solution (b) in one of the preferred embodiments of the method is at least 0.005, preferably at least 0.015, particularly preferably at least 0.03, even more preferably at least , 0.08.

The molecular weight of gelatin in one of the preferred embodiments of the method is from 15 to 40 kDa, preferably from 19 to 30 kDa, particularly preferably from 20 to 25 kDa.

Solution (b) in one preferred embodiment of the process further comprises polyvinylpyrrolidone. In one particularly preferred embodiment of the method, solution (b) further comprises

polyvinylpyrrolidone with a molecular weight of from 8 to 15 kDa, preferably from 9 to 12 kDa, particularly preferably about 10 kDa. In an even more preferred embodiment of the method, solution (b) further comprises polyvinylpyrrolidone at a concentration of up to 30 mg / ml.

In one embodiment of the method, the mass fraction of rifabutin in solution (a) is from 0.002 to 0.2.

Ethanol is used as a water miscible solvent in one preferred embodiment of the process. Preferably, the amount of water miscible solvent is at least sufficient to completely dissolve rifabutin.

N-methyl-2-pyrrolidone, methoxypolyethylene glycol, alkoxypolyethylene glycol, polyethylene glycol esters, glycofurol, glycerolformal, methyl acetate, ethyl acetate, methyl ethyl ketone, benzene methyl dimethyl benzene, dimethyl methyl sulfide, dimethyl benzene are also used as water miscible pharmaceutically acceptable solvent. ethyl benzoate, triacetin, diacetin, tributyrin, triethyl citrate, tributyl citrate, acetyl triethyl citrate, acetyl tributyl citrate, triethyl glyceride, triethyl phosphate , Diethyl phthalate, diethyl tartrate, ethyl lactate, propylene carbonate, ethylene carbonate, butyrolactone, 1-dodecylazacycloheptan-2-one and combinations thereof.

Freezing in accordance with the method is usually carried out to a temperature of -70 ° C or lower, preferably to a boiling point of liquid nitrogen.

Lyophilization is usually carried out at a temperature of from -60 to -70 ° C.

Preferably, a lyoprotectant is added before freezing, preferably dextrose, glucose, lactose, mannitol and / or trehalose, preferably to a concentration of from 0.1 to 10% by weight in solution.

Any pharmaceutically acceptable substance that, when incorporated into the lyophilized composition, can protect the ingredients of the composition from the adverse effects of freezing and vacuuming, such as those typically associated with lyophilization, such as damage, adsorption and loss from the vacuum used in lyophilization, can be used as a lyoprotectant. The present invention is not limited to the use of a particular lyoprotectant; examples of suitable lyoprotectants include, but are not limited to, hydrocarbons such as saccharides, mono-, di- or polysaccharides, for example glucose, galactose, fructose, sucrose, trehalose, maltose, lactose, amylose, amylopectin, cyclodextrins, dextran, inulin, soluble starch, hydroxyethyl starch (HES), sugar alcohols, for example mannitol, sorbitol, and polyglycols, such as polyethylene glycols. A solid list of substances with a lyoprotective effect is given in Acta Pharm. Technol. 34 (3), pp. 129-139 (1988). The lyoprotector can be added to the aqueous-organic mixture before emulsification and / or after emulsification.

In one preferred embodiment of the method, before adding the solution (a) to the solution (b), the pH of the solution (b) is adjusted to 5.4-6.8.

Mixing of the intermediate product (c) can (although not necessarily) be carried out by means of an immersion dispersant at a speed of 15,000 ÷ 20,000 rpm for 1 ÷ 2 minutes and / or mixing of the intermediate product (c) can be carried out on a high-pressure homogenizer, with a homogenization pressure from 15,000 to 45,000 psi and / or mixing of the intermediate (c) can be accomplished by exposure to ultrasound.

The product obtained in step (d) can be mixed with excipients and other excipients and use the resulting mixture for the manufacture of a solid oral dosage form.

In another aspect, the present invention relates to a pharmaceutical composition for the treatment of mycobacteriosis and Helicobacter pylori infection, containing an effective amount of rifabutin with increased bioavailability, solubilized with gelatin, and obtained as described above.

However, the composition may (although not necessary) additionally contain ascorbic acid.

The composition (in one of the possible options) can be obtained and used in the form of a lyophilisate or can be a powder obtained by vacuum spray drying.

The composition (in one of the possible options) may be a powder for the manufacture of solid oral dosage forms.

The composition (in one of the possible options) can be obtained and used in the form of a solid oral dosage form, for example, in the form of tablets, capsules, dragees, pills or granules.

Mycobacteriosis, for the treatment of which the composition may be used, may be caused by one or more pathogens selected from Mycobacterium tuberculosis, or Mycobacterium bovis, or Mycobacterium kansasii, or Mycobacterium scrofulaceum, or Mycobacterium avium-intracellulare complex, or Mycobacteriumium intiumllulare complex, or Mycobacteriumium intiumllulare complex, or Mycobacteriumium intiumllulare complex, or Mycobacteriumium intiumllulare complex, or Mycobacteriumium intiumllulare complex, or Mycobacteriumium intracellulare complex, or Mycobacteriumiumium Mycobacteriosis, for the treatment of which the composition can be used, can be HIV / AIDS-associated mycobacteriosis,

 in particular, HIV / AIDS-associated mycobacteriosis may be due to Mycobacterium avium-intracellulare complex or Mycobacterium tuberculosis.

In one preferred embodiment, the composition of the invention comprises succinylated gelatin and rifabutin in a weight ratio of 3: 1-5: 1.

In another embodiment, the invention relates to a method for treating mycobacteriosis and Helicobacter pylori infection, comprising orally administering a therapeutically effective amount of the above-described pharmaceutical composition to a patient in need thereof.

Mycobacteriosis, the treatment of which can be directed by the method, is caused by one or more pathogens selected from Mycobacterium tuberculosis, or Mycobacterium bovis, or Mycobacterium kansasii, or Mycobacterium scrofulaceum, or Mycobacterium avium-intracellulare complex, or Mycobacterium parium, lecd. Mycobacteriosis can be HIV / AIDS-associated mycobacteriosis, in particular HIV / AIDS-associated mycobacteriosis due to Mycobacterium tuberculosis, or HIV / AIDS-associated mycobacteriosis due to Mycobacterium avium-intracellulare complex.

The composition may be administered in the form of a solid oral dosage form to a patient in need thereof. Solid oral dosage forms can be tablets, capsules, dragees, pills or granules. In accordance with the proposed method, a therapeutically effective amount of one or more drugs selected from amoxicillin, pantoprazole, esomeprazole, rabeprazole, omeprazole, metronidazole, clarithromycin, bismuth citrate or tetracycline can be additionally administered to the patient.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the time during which 80% of rifabutin, which is part of various dosage forms, passes into an aqueous solution (pH 6.8, 37 ° C).

Figure 2 shows the effect of various excipients on the solubility of rifabutin, determined under the following conditions: pH 6.8; 37 ° C; excess rifabutin relative to the table value of solubility - 5 times; incubation time - 1 hour.

Figure 3 shows the amount of rifabutin transferred to an aqueous solution from its various forms in 20 minutes (37 ° C, pH 6.8).

Figure 4 shows the kinetics of dissolution of rifabutin, which is part of various compositions (pH 6.8; 37 ° C).

Figure 5 shows the effect of the method of drying the compositions on their dissolution rate: (spray micro- and nano-dryers, freeze dryer; pH 6.8; 37 ° C).

Figure 6 shows the dependence of the inoculation (from the lungs) of M. tuberculosis H37Rv mycobacteria on the dose and form of rifabutin used to treat tuberculosis in mice.

Figure 7 shows the dependence of the seeding (from the spleen) of M. tuberculosis H37Rv mycobacteria on the dose and form of rifabutin used to treat tuberculosis in mice.

DETAILED DESCRIPTION OF THE INVENTION

The following examples illustrating the preparation of rifabutin and albumin lyophilisate by the process claimed in accordance with the invention are provided only to illustrate the present invention and not to limit the scope of the claims.

EXAMPLE 1

Comparison of rifabutin-based preparations containing various auxiliary components and obtained in various ways

 For the manufacture of preparations according to the invention and comparative preparations used: rifabutin (purity 98.5%); rifabutin in capsules (as a part of excipients, 50% by weight - microcrystalline cellulose, colloidal silicon dioxide, sodium lauryl sulfate, magnesium stearate); succinyl gelatin (4% solution for infusion, average molecular weight 23.2 kDa); polyvinylpyrrolidone (PVP, average molecular weight 10 kDa); Poloxamer 407 (Pluronic F 127, S. N. Erbsloh).

The following equipment was used: Malvern Zetasizer Nano analyzer (Malvern Instruments Ltd, UK); spray dryer Nano-spray Dryer B-90 (Buchi, Switzerland); spray dryer Mini Spray Dryer B-290 (Buchi, Switzerland).

The solubility of the drugs is determined as follows. An assayed sample containing rifabutin calculated to be 26 mg (accurately weighed) is placed in 20 ml of a pH of 6.8 phosphate buffered saline (prepared according to the requirements of the United States Pharmacopeia). The vessel with the suspension is placed in a shaker incubator, observing the heating mode to 37 ± 0.5 ° C and stirred at 200 rpm. 3 samples are taken with a volume of 0.5 ml after 20, 40, 60, 80, 100, 130 minutes. Samples are centrifuged at 25 ° C, 13,200 rpm for 20 minutes. The Rb content in the supernatant is determined spectrophotometrically at λ = 513 nm. The equation of the calibration dependence: A = 0.0036 · c (Rb); R = 0.997992.

The dissolution rate of the drugs is determined as follows. An assayed sample containing 40 mg of rifabutin (accurately weighed) is placed in 200 ml of phosphate buffered saline pH 6.8 (prepared according to USP requirements). The vessel with the suspension is placed in a shaker incubator, observing the heating mode to 37 ± 0.5 ° C and stirred at 100 rpm. 3 samples are taken with a volume of 0.5 ml after 5, 10, 20, 30, 40, 50, 60, 80 minutes. Samples are centrifuged at 25 ° C, 13,200 rpm for 10 minutes. The Rb content in the supernatant is determined spectrophotometrically at λ = 513 nm. The equation of the calibration dependence: A = 0.0036 · c (Rb); R = 0.997992.

EXAMPLE 2

Obtaining a solid pharmaceutical composition using spray drying

A portion of the rifabutin substance is dissolved in ethanol and added dropwise with stirring to an aqueous solution of modified gelatin (Gelofusin). Stirring is continued in an open vessel on a magnetic stirrer for 3 hours; if necessary, remove the residual organic solvent on a rotary evaporator. Next, the suspension is filtered and dried on a spray dryer B-90.

Typically, the compositions of the invention have the following composition (wt.%):

Modified (Succinylated) Gelatin 40 ÷ 80 Rifabutin 3 ÷ 20 Polyvinylpyrrolidone (10 kDa) 0 ÷ 50

The following is an example of a composition 1 obtained in accordance with the above method (wt.%):

Modified (Succinylated) Gelatin 51.9 Rifabutin 9.0 Polyvinylpyrrolidone (10 kDa) 39.1

EXAMPLE 3

Obtaining a solid pharmaceutical composition using a spray micro-dryer B-290

A portion of the rifabutin substance is dissolved in ethanol and added dropwise with stirring to an aqueous solution of modified gelatin (Gelofusin). Stirring is continued in an open vessel on a magnetic stirrer for 3 hours; if necessary, remove the residual organic solvent on a rotary evaporator. Next, the suspension is filtered and dried on a spray dryer B-290.

The following is an example of a composition 2 obtained in accordance with the above method (wt.%):

Modified (Succinylated) Gelatin 50,4 Rifabutin 11.8

EXAMPLE 4

Obtaining a solid pharmaceutical composition using freeze drying

To obtain a gelatin-based composition using freeze-drying, a sample of rifabutin is dissolved in ethanol and added dropwise to an aqueous solution of modified gelatin (Gelofusin) with stirring. Stirring is continued in an open vessel on a magnetic stirrer for 3 hours; if necessary, remove the residual organic solvent on a rotary evaporator. The suspension is then filtered, frozen at -70 ° C and freeze-dried.

The following are examples of compositions 3-5, obtained in accordance with the above method.

Composition 3 (obtained on a spray mini-dryer B-290) (wt.%):

Modified (Succinylated) Gelatin 50,2 Rifabutin 12.0 Polyvinylpyrrolidone (10 kDa) 37.8 Composition 4 (wt.%): Modified (Succinylated) Gelatin 45.0 Rifabutin 5,0 Polyvinylpyrrolidone (10 kDa) 50,0 Composition 5 (wt.%): Modified (Succinylated) Gelatin 80 Rifabutin twenty Polyvinylpyrrolidone (10 kDa) 0

The results obtained in assessing the solubility and dissolution rate of the obtained compositions in comparison with the data obtained for a commercial preparation and for the substance of rifabutin are presented in table 1, in figures 1-3. All tests were performed in phosphate buffered saline at pH 6.8.

From the above results it follows that both compositions have an advantage in solubility and in dissolution rate both in comparison with the substance of rifabutin and in comparison with a commercial preparation. Moreover, in most parameters, the new compositions are approximately the same as commercial ones in which the latter surpasses the substance.

TABLE 1 Data on the dissolution of the obtained forms in comparison with the substance of rifabutin and rifabutin in capsules (pH 6.8) * The composition of the dosage form The dissolution time of 80% RB, min With _RB in solution for 20 min, μg / ml % RB dissolved in 20 minutes With _RB in solution for 40 min, mcg / ml Cs RB **, μg / ml RB > 80 min 59.4 29.7 79.8 258 RB capsules > 80 min 120.3 60.1 130.2 147 Gelatin Compositions RB + F + PVP (spray drying) 35 min 145.6 72.8 145.6 550 RB + F + PVP (lyophilisate) 10 min 159.2 79.6 159.7 550 * - Accepted abbreviations: W - gelatin, PVP - polyvinylpyrrolidone, Po407 - poloxamer 407, MCC - microcrystalline cellulose.
** - The solubility of RB in a buffer solution of pH 6.8, achieved during the hour of incubation of a five-fold excess of RB relative to its solubility (tabular value) at 37 ° C.

The diagram in figure 1 clearly shows that all of the proposed composition is superior in this parameter not only the substance of rifabutin, but also the traditionally used dosage form.

In addition, all new compositions make it possible to achieve higher solubilities of rifabutin (solubilities determined at pH 6.8, temperature 37 ° C and in excess of rifabutin, five times the tabulated value of its solubility; dissolution time - 1 hour). Moreover, the solubility of the substance of rifabutin, obviously, significantly exceeds the solubility of the substance used in capsules, even determined in the presence of excipients contained in the capsule. The results are presented in figure 2.

As follows from figure 2 and 3 in the parameter "Dissolution time of 20% of the active ingredient" new compositions also slightly exceed the traditionally used form (see figure 3).

Figure 4 clearly shows that, in general, the new ingredients increase the dissolution rate of rifabutin more efficiently than the known ones, and the advantages of the new compositions are especially pronounced after the first 30 minutes of dissolution.

Interestingly, the replacement of the type of spray dryer allowed to significantly improve the properties of the gelatin-based composition. Figure 5 clearly shows that the properties of the composition obtained using the spray micro-dryer B-290 are significantly superior to the properties of the composition obtained by drying a similar composition on the spray-dryer B-90. Structurally, these models differ in the design of the spray head - if for the B-290 model the jet is fed through the nozzle, then for the more modern B-90 model, the jet passes through the vibrating membrane. Obviously, for drying the gelatin-based rifabutin composition, the more well-known (and cheaper) modification of B-290 is optimal - the parameters of the composition obtained with its help approach the parameters of the lyophilisate.

Table 2 presents the compositions of the investigated compositions. Obviously, the relative amount of RB in the new compositions is less than in the contents of the capsules. Therefore, the choice of a new composition should be a comprehensive solution based on various factors, such as pharmacokinetic studies, safety, efficacy, economic feasibility, manufacturability, etc.

TABLE 2 The compositions of the used forms No. Composition RB, weight% Excipients, weight% one RB one hundred 0 2 RB capsules fifty BB - 50 (MCC, colloidal silicon dioxide, sodium lauryl sulfate, magnesium stearate) 3 RB + CD 36.7 GP-beta-CD-63.3 four RB + CD + Po407 36 GP-beta-CD-62.2 Po407-1.8 5 RB + CD + MCC 36 GP-beta-CD-62.2 MCC-1.8 6 RB-k + CD 26.8 BB-26.8 GP-beta-CD-46.4 7 RB + F + PVP atomizing mini-dryer V-90 9 Modified Gelatin - 51.9 PVP-39.1 8 RB + F + PVP lyophilisate 11.8 Modified Gelatin - 50.4 PVP-37.8 9 RB + F + PVP spray nano dryer V-290 12 Modified Gelatin - 50.3 PVP-37.7

EXAMPLE 5

Study of gelatin based compositions of the invention

Cells of the Mycobacterium tuberculosis H37Rv strain are used. Bacteria were cultured with aeration in atmospheric air (without CO ₂ ) in several 100-500 ml bottles in a modified Middelbrook 7H9 liquid medium (Himedia, India) supplemented with 10% OADC and 0.05% Tween 80 for 21 days at 37 ° C. Cultures are grown to an optical density corresponding to 5 × 10 ⁷ ÷ 10 ⁸ CFU, divided into aliquots, frozen and stored at minus 70 ° C. To confirm the concentration of living bacteria in frozen aliquots, control seeding was performed on Petri dishes with Middelbrook 7H11 medium (Himedia, India).

Dense medium Middelbrook 7H11 for the cultivation of mycobacteria is prepared as follows. A weighed portion of dry medium 10.25 g is placed in a volumetric dish, add 2.5 ml of glycerol and 0.5 g of L-asparagine, adjust the volume with distilled water pH (5.5 ± 0.1) to 450 ml and mix. The pH of the medium is adjusted to 6.6 ± 0.2, the medium is poured into flasks with a volume of 1000 ± 10 ml. The flasks are closed with cotton-gauze plugs and paper caps, sterilized at a temperature of (121 ± 1) ° C for 15 minutes. The finished medium is cooled to a temperature of (50 ± 1) ° C. Under aseptic conditions, sterile cattle serum is added at a rate of 50 ml per 450 ml of medium. The prepared medium is poured into sterile Petri dishes of 25 ml under aseptic conditions, dried for 24 hours at a temperature of (37 ± 1) ° C.

Suspensions of M. tuberculosis H37Rv cells for infection of animals are prepared as follows. A suspension of M. tuberculosis H37Rv cells frozen at a temperature of -70 ° C is thawed at room temperature and homogenized with an insulin syringe under aseptic conditions in compliance with safety regulations. Prepare ten-fold dilutions of the suspension in buffered saline (PBS) with 0.05% Tween 80. Part of the prepared suspension is used for control seeding on a solid Middelbrook 7H11 nutrient medium with 20% cattle serum.

Tuberculosis infection in mice is modeled as follows. Use female Balb / c mice (age 7-8 weeks, body weight 20-22 g). Animals are infected by intravenous administration of a suspension of M. tuberculosis H37Rv cells in the lateral tail vein at a dose of 5.9 × 10 ⁷ CFU / mouse, which led to the development of an acute tuberculosis process in animals.

The CFU content of M. tuberculosis H37Rv in the organs of infected animals is determined as follows. The animals are euthanized with carbon dioxide in a desiccator for 10 minutes under visual control, pathologically open to open parenchymal organs, observing aseptic rules. Target organs are homogenized in sterile mortars by adding 1 ml PBS per organ. To determine the concentration of colony forming units (CFU) of microorganisms, the homogenate is seeded by 10-fold dilution in PBS with 0.05% Tween-80 on a solid Middelbrook 7H11 nutrient medium (Himedia, India) containing 20% cattle serum. To suppress extraneous microflora (when plating from the initial dilution of the homogenate), hydrochloric acid is added to the suspension to a concentration of 3%. On the surface of the medium in a Petri dish, 0.1 ml of a suspension of the appropriate dilution is applied and thoroughly triturated with a spatula. Inoculated cups are placed in plastic bags and sealed. Incubation is carried out at a temperature of (37 ± 1) ° C for 25 days, then grown colonies are counted. Confirmation of the specificity of the grown colonies of mycobacteria is carried out using staining according to Ziehl-Nielson and light microscopy. The calculation results are processed by statistical methods indicating the average and relative deviations.

The preparation of working solutions of the preparation according to the invention and control substances is as follows. A solution of the preparation according to the invention is prepared by dissolving 5 mg of a sample in 1 ml of sterile water. A control solution is prepared by dissolving 30 mg of rifabutin substance in 0.3 ml of alcohol and adjusting the volume to 5 ml with distilled water. All solutions are used for 24 hours, then new portions are prepared.

The therapeutic effect of the preparation according to the invention is studied using three therapeutic doses per pure rifabutin: 100, 200 and 400 μg / mouse. Rifabutin solutions were administered to animals intragastrically (through a thin metal probe with an “olive” soldered at the end) at 0.1 ml / mouse. Treatment of mice infected with M. tuberculosis H37Rv TB pathogen was started on the seventh day after infection and continued for 8 weeks according to the schedule — three injections per week. On 63-66 days after infection, mice were euthanized and organ homogenates were plated on a solid nutrient medium.

Significance of differences was determined by Student's parametric criterion for p <0.05.

The therapeutic efficacy of the rifabutin composition according to the invention is studied in comparison with the rifabutin substance according to the following scheme.

Infection 1 day Start treatment 8th day Autopsy 1 day Strain, name Mycobacterium tuberculosis H37RV Infection dose per mouse (calculated) 5 × 10 ⁷ (0.1 ml each); Duration of treatment / General 9 weeks / 10 weeks Treatment regimen 3 times a week (Mon, Wed, Fri), 9 weeks

Formed 7 groups of animals. For each group, the average rate of dissemination of the lungs and spleen was determined. The individual values of the indicators determined the average value of CFU for this group (table 3).

TABLE 3 Groups of animals in an experiment to study the therapeutic effect of the composition according to the invention Group (9 mice each) Rifabutin form Dose, mcg / mouse Route of administration one The composition according to the invention one hundred orally 2 200 3 400 four Rifabutin substance one hundred orally 5 200 6 400 7 control (without treatment) - -

Comparison of the average indicators of contamination of the lungs and spleen in animals in each experimental group showed that for both drugs a dose-dependent therapeutic effect is observed. Moreover, the composition according to the invention has a much more pronounced therapeutic effect than the control rifabutin substance. The differences were most pronounced when using doses of drugs equal to 200 μg / mouse. Treatment of animals with the composition of the invention at this dose leads to eradication of the pathogen from the lungs and spleen, while treatment with rifabutin-substance reduces the rate of contamination of organs to 10 ³ CFU / mouse. The use of higher doses of drugs (400 μg / mouse) led to the eradication of the pathogen from the lungs and from the spleen both in the case of using the composition according to the invention and the control rifabutin substance (FIGS. 6 and 7).

Table 4 presents data on the values of CFU M. tuberculosis H37R V, determined in the homogenates of the organs of each experimental animal after treatment with rifabutin (sample No. 1532):

TABLE 4 The results of seeding homogenates after treatment with the composition according to the invention The dose of the composition according to the invention μg / mouse, the introduction of intragastric Mouse number / CFU in the lungs Mouse / CFU number in the spleen No. 1 - 1.6 × 10 No. 1 - 1.0 × 10 ² No. 2 - 2.1 × 10 ³ No. 2 -5.6 × 10 ² No. 3 - 1.0 × 10 ³ No. 3 - 0 No. 4 - 6.3 × 10 ² No. 4 - 1.5 × 10 ² No. 5 - 2.6 × 10 ² No. 5 - 1.1 × 10 ² No. 6 - 1.6 × 10 ² No. 6-1.5 × 10 No. 7 - 0 No. 7 - 4.3 × 10 No. 8 - 3.6 × 10 No. 8 - 8.0x10 Average 5.2 × 10 ² Average 1.3 × 10 ² 200 No. 1-0 No. 1-0 one hundred No. 2 - 0 No. 2 - 0 No. 3-4-4.3 × 10 No. 3 - 0 No. 4 - 0 No. 4 - 0 No. 5 - 0 No. 5 - 0 No. 6 - 0 No. 6 - 0 No. 7 - 0 No. 7 - 4.3 × 10 No. 8 - 0 No. 8-0 No. 9 - 0 No. 9 - 0 Average 0.47 Average 0.14 400 No. 1-0 No. 1-0 No. 2 - 0 No. 2 - 0 No. 3 - 0 No. 3 - 0 No. 4 - 0 No. 4 - 0 No. 5 - 0 No. 5 - 0 No. 6 - 0 No. 6 - 0 No. 7 - 0 No. 7 - 0 No. 8 - 0 No. 8 - 0 No. 9 - 0 No. 9 - 0 Average 0 Average 0

Table 5 presents data on the values of CFU of M. tuberculosis H37RV, determined in the homogenates of the organs of each experimental animal after treatment with rifabutin-substance:

TABLE 5 The results of seeding homogenates after treatment with rifabutin-substance The dose of the composition according to the invention μg / mouse, the introduction of intragastric Mouse number / CFU in the lungs Mouse / CFU number in the spleen one hundred No. 1 -5.0 × 10 ³ No. 1 -8.8 × 10 ² No. 2 - 9.7 × 10 ³ No. 2 -9.9 × 10 ³ No. 3 -3.1 × 10 ³ No. 3 - 5.1 × 10 ² No. 4 -9.9 × 10 ³ No. 4 -3.5 × 10 ³ No. 5 - 1.5 × 10 ³ No. 5 - 1.6 × 10 ³ No. 6 - 1.2 × 10 ⁴ No. 6 -3.2 × 10 ³ No. 7 - 1.2 × 10 ⁴ No. 7 -2.8 × 10 ³ No. 8 -6.5 × 10 ³ No. 8 -2.9 × 10 ³ No. 9 -6.6 × 10 ³ No. 9 - 1.7 × 10 ³ Average 7.4 × 10 ³ Average 2.9 × 10 ³ 200 No. 1 - 1.6 × 10 No. 1 -2.0 × 10 No. 2 - 0 No. 2 -3.3 × 10 ² No. 3 -3.3 × 10 ³ No. 3 - 5.8 × 10 ² No. 4 - 1.3 × 10 ² No. 4 -3.0 × 10 ³ No. 5 - 1.0 × 10 ² No. 5-4.0 × 10 No. 6 -2.3 × 10 ² No. 6 - 0 No. 7 - 0 No. 7 -5.0 × 10 ² No. 8 - 1.3 × 10 ³ No. 8 -6.7 × 10 ³ Average 6.3 × 10 ² Average 1.4 × 10 ³ 400 No. 1-0 No. 1-0 No. 2 - 0 No. 2 - 0 No. 3 - 0 No. 3 - 0 No. 4 - 0 No. 4 - 0 No. 5 - 0 No. 5 - 0 No. 6 - 0 No. 6 - 0 No. 7 - 0 No. 7 - 0 No. 8 - 0 No. 8 - 0 No. 9 - 0 No. 9 - 0 Average 0 Average 0

Table 6 presents data on the values of CFU of M. tuberculosis H37RV, determined in the homogenates of the organs of each experimental animal without treatment.

TABLE 6 The results of seeding homogenates without treatment The dose of the composition according to the invention μg / mouse, the introduction of intragastric Mouse number / CFU in the lungs Mouse / CFU number in the spleen the control No. 1 -6.7 × 10 ⁶ No. 1-5.8 × 10 ⁶ No. 2- 1.1 × 10 ⁷ No. 2 - 8.1 × 10 ⁶ No. 3 - 8.6 × 10 ⁶ No. 3 - 6.3 × 10 ⁶ No. 4 - 1.2 × 10 ⁷ No. 4 -4.9 × 10 ⁶ No. 5 - 8.1 × 10 ⁶ No. 5 -3.9 × 10 ⁶ No. 6 -9.5 × 10 ⁶ No. 6 -3.2 × 10 ⁶ No. 7- 1.1 × 10 ⁷ No. 7 -4.3 × 10 ⁶ No. 8 - 9.1 × 10 ⁶ No. 8 -2.2 × 10 ⁶ No. 9 - 1.0 × 10 ⁷ No. 9 -5.2 × 10 ⁶ Average: 9.5 × 10 ⁶ Average: 4.9 × 10 ⁶

Thus, treatment of a tuberculosis infection with the composition of the invention in a model of acute tuberculosis in mice is more effective than treatment with a rifabutin-substance preparation (table 7).

TABLE 7 Inoculation of M. tuberculosis H37Rv mycobacteria from organs of mice treated with various forms of rifabutin Organ Rifabutin form LgKOE / Dose of the drug, mcg / mouse LgKOE in control one hundred 200 400 lungs composition according to the invention 2.74 ± 0.34 0 0 7.08 ± 0.24 rifabutin substance 3.70 ± 0.22 2,50 ± 0,58 0 spleen composition according to the invention 2.07 ± 0.26 0 0 6.66 ± 0.09

Organ Rifabutin form LgKOE / Dose of the drug, mcg / mouse LgKOE in control one hundred 200 400 rifabutin substance 3.30 ± 0.15 3.06 ± 0.44 0

Thus, the oral form of rifabutin showed high efficacy in the treatment of experimental tuberculosis in mice, which is higher compared to the control drug - the substance of rifabutin.

The dosage form of rifabutin according to the invention in doses of ≥ 200 μg / ml leads to complete eradication of the pathogen from the organs of an animal infected with tuberculosis.

Claims (46)

1. A method of producing rifabutin with increased bioavailability, solubilized with gelatin, in which
(a) rifabutin is dissolved in a pharmaceutically acceptable water-miscible solvent, the solubility of rifabutin in which is higher than in water, to obtain a rifabutin solution;
(b) the gelatin is dissolved in water to obtain a gelatin solution;
(c) solution (a) is slowly added to solution (b) with stirring to obtain an intermediate;
(g) the intermediate obtained in stage (C) is dried in a spray dryer or frozen and lyophilized to obtain the product. 2. The method according to claim 1, in which the dissolution or mixing of the components is carried out at a temperature of from 0 to 40 ° C. 3. The method according to claim 1, in which the addition with stirring in stage (C) is carried out gradually over at least 1 hour, preferably at least 2 hours, particularly preferably at least 3 hours. 4. The method according to claim 1, in which, after adding at the stage (C) carry out stirring for at least 1 hour, preferably at least 2 hours, particularly preferably at least 3 hours. 5. The method according to claim 1, in which the concentration of rifabutin in solution (a) is 40 ÷ 80 mg / ml 6. The method according to claim 1, in which the solution (a), solution (b) and / or solution (c) is additionally filtered or centrifuged to obtain a filtrate or supernatant, respectively, and in the next step ((b), (c) and / or (g), respectively) using a filtrate or supernatant, respectively. 7. The method according to claim 1, in which the excess solvent from the intermediate (c) is removed on a rotary evaporator, the resulting suspension is filtered or centrifuged, and in step (d), the filtrate or supernatant is used as an intermediate, respectively. 8. The method according to claim 1, in which demineralized water is used. 9. The method according to claim 1, in which pork, beef or goat gelatin is used. 10. The method according to claim 1, in which succinylated liquid gelatin is used. 11. The method according to claim 1, in which the concentration of gelatin in solution (b) is 10 ÷ 40 mg / ml 12. The method according to claim 1, in which the mass fraction of gelatin in solution (b) is at least 0.005, preferably at least 0.015, particularly preferably at least 0.03, even more preferably at least 0.08. 13. The method according to claim 1, in which the molecular weight of the gelatin is from 15 to 40 kDa, preferably from 19 to 30 kDa, particularly preferably from 20 to 25 kDa. 14. The method according to claim 1, in which the solution (b) further comprises polyvinylpyrrolidone. 15. The method according to claim 1, in which the solution (b) further comprises polyvinylpyrrolidone with a molecular weight of from 8 to 15 kDa, preferably from 9 to 12 kDa, particularly preferably about 10 kDa. 16. The method according to claim 1, in which the solution (b) further comprises polyvinylpyrrolidone at a concentration of up to 30 mg / ml. 17. The method according to claim 1, in which the mass fraction of rifabutin in solution (a) is from 0.002 to 0.2. 18. The method according to claim 1, in which ethanol is used as a water miscible solvent. 19. The method according to claim 1, in which use the amount of a miscible with water solvent, at least sufficient to completely dissolve rifabutin. 20. The method according to claim 1, in which freezing is carried out to a temperature of -70 ° C or lower, preferably to a boiling point of liquid nitrogen. 21. The method according to claim 1, in which the lyophilization is carried out at a temperature of from -60 to -70 ° C. 22. The method according to claim 1, in which, before freezing, a lyoprotector, preferably dextrose, glucose, lactose, mannitol and / or trehalose, is added, preferably to a concentration of from 0.1 to 10 wt.% In solution. 23. The method according to claim 1, in which before adding the solution (a) to the solution (b), the pH of the solution (b) is adjusted to 5.4 ÷ 6.8. 24. The method according to claim 1, in which the intermediate product (C) is mixed by means of a submersible dispersant at a speed of 15000 ÷ 20,000 rpm./min, for 1 ÷ 2 minutes 25. The method according to claim 1, in which the intermediate (b) is mixed on a high pressure homogenizer, at a homogenization pressure of from 15,000 to 45,000 psi. 26. The method according to claim 1, in which the intermediate product (C) is stirred by ultrasound. 27. The method according to claim 1, in which the product obtained in step (d) is mixed with excipients and other excipients and the resulting mixture is used to make a solid oral dosage form. 28. A pharmaceutical composition for the treatment of mycobacteriosis and Helicobacter pylori infection, containing an effective amount of rifabutin with increased bioavailability, solubilized with gelatin, obtained by the method according to any one of claims. 1-27. 29. The composition according to p. 28, characterized in that it further comprises ascorbic acid. 30. The composition according to p. 28, characterized in that it is a lyophilisate. 31. The composition according to p. 28, characterized in that it is a powder obtained by spray drying. 32. The composition according to p. 28, characterized in that it is a powder for the manufacture of solid oral dosage forms. 33. The composition according to p, characterized in that the solid oral dosage forms are tablets, capsules, dragees, pills or granules. 34. The composition according to p. 28, characterized in that the mycobacteriosis is caused by one or more pathogens selected from Mycobacterium tuberculosis, or Mycobacterium bovis, or Mycobacterium kansasii, or Mycobacterium scrofulaceum, or Mycobacterium avium-intracellulobereru complex, or . 35. The composition according to p. 28, characterized in that the mycobacteriosis is HIV / AIDS-associated mycobacteriosis. 36. The composition according to p. 35, characterized in that the HIV / AIDS-associated mycobacteriosis is caused by Mycobacterium avium-intracellulare complex. 37. The composition according to clause 35, characterized in that the HIV / AIDS-associated mycobacteriosis is caused by Mycobacterium tuberculosis. 38. The composition according to p. 35, characterized in that it contains succinylated gelatin and rifabutin in a mass ratio of 3: 1-5: 1 39. A method of treating mycobacteriosis and Helicobacter pylori infection, comprising orally administering a therapeutically effective amount of a pharmaceutical composition according to any one of paragraphs 28-38 to a patient in need thereof. 40. The method according to § 39, characterized in that the mycobacteriosis is caused by one or more pathogens selected from Mycobacterium tuberculosis, or Mycobacterium bovis, or Mycobacterium kansasii, or Mycobacterium scrofulaceum, or Mycobacterium avium-intracellulareererobuberum, or Mycobacterium avium-intracellulareer complex, or Mycobacterium lecidobacterium, or Mycobacterium lecidium or Mycobacterium tuberculosis, . 41. The method according to § 39, characterized in that the mycobacteriosis is HIV / AIDS-associated mycobacteriosis. 42. The method according to § 39, characterized in that the HIV / AIDS-associated mycobacteriosis is caused by Mycobacterium tuberculosis. 43. The method according to § 39, characterized in that the HIV / AIDS-associated mycobacteriosis is caused by Mycobacterium avium-intracellulare complex. 44. The method according to § 39, characterized in that said composition is administered in the form of a solid oral dosage form to a patient in need thereof. 45. The method according to item 44, wherein the solid oral dosage forms are tablets, capsules, dragees, pills or granules. 46. The method according to § 39, characterized in that the patient is additionally administered a therapeutically effective amount of one or more drugs selected from amoxicillin, pantoprazole, esomeprazole, rabeprazole, omeprazole, metronidazole, clarithromycin, bismuth citrate or tetracycline.

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