EA013570B1 - Pharmaceutical composition for treating tuberculosis and diseases mediated by helicobacter pylori based on solid lipid nanoparticles and a method for the treatment of tuberculosis - Google Patents

Abstract

The invention relates to a medicine, pharmacy and antitubercular preparations, namely, to the solid lipid nanoparticles (SLN)-based pharmaceutical composition for treating tuberculosis and diseases mediated by Helicobacter pilory, where this pharmaceutical composition contains a therapeutically effective amount of antitubercular antibiotics, selected from rifabutin and rifampicin, which are sorbed into the lipidic matrix, which consists of a combination of one or more long-chain fatty acids esters, cholesterol or its pharmaceutically acceptable derivative, and phospholipid. The invention also relates to the method of treating tuberculosis and diseases mediated by Helicobacter pilory, which method includes a peroral administration of solid lipid nanoparticles (SLN)-based pharmaceutical composition according with the invention in a therapeutically effective amount to a patient(s) needs thereof, in a certain therapeutic regime. The invention, versus a prototype, enables a higher drug-loading of solid lipid nanoparticles and a higher antitubercular activity.

Description

Technical field

The present invention relates to the field of medicine, pharmacy and nanotechnology, in particular, to a pharmaceutical composition for treating tuberculosis based on solid lipid nanoparticles containing a therapeutically effective amount of an anti-tuberculosis drug selected from rifabutin or rifampicin sorbed in a lipid matrix, to a method for producing such a composition and to a method for treating tuberculosis comprising administering such a pharmaceutical composition in a therapeutically effective amount to a needy person I'm in this patient.

BACKGROUND OF THE INVENTION (prior art)

In connection with the spread of tuberculosis in the world, caused by the emergence of drug-resistant strains and the spread of diseases of the immune system, including AIDS, it is necessary to create new anti-TB drugs. According to WHO estimates, by 2020, the number of newly infected with tuberculosis will reach 1 billion 200 million people will fall ill, and 35 million will die from tuberculosis if new, more effective treatments are not found.

In connection with the foregoing, along with the study of new antimycobacterial agents, especially new antibiotics, one of the most promising areas is the creation of new dosage forms of known antibiotics that allow targeted creation of a high concentration of active principle in the affected organs, without having significant systemic side effects on the whole body generally.

One of the most promising solutions to this problem is the creation of nanosomal forms of the antibiotics (rifampicin and rifabutin) most popular in the treatment of mycobacterial infection, based on a lipid-polymer composition, which is a composition of a biodegradable polymer and lipid.

Rifabutin (1 \ '4-didehydro-1-deoxy-1,4-dihydro-5 \' - (2-methylpropyl) -1-oxorifamycin XIV) is a semi-synthetic broad-spectrum antibiotic, first-line anti-tuberculosis drugs; is a highly lipophilic compound. Red-violet powder. Soluble in chloroform and methyl alcohol, sparingly soluble in ethyl alcohol, practically insoluble in water. The molecular weight of 847.02.

Rifabutin is effective against intracellular and extracellularly located microorganisms. Selectively inhibits DNA-dependent RNA polymerase of bacteria. It has a bactericidal effect. It is highly active against Musoacobacterium carr. (Muso-bac1etsti 1 and bc1ci1oz13. Muso-bac1etsti auscht, including those located intracellularly) and other atypical mycobacteria. Between 1/3 and 1/2 of the rhocampicin-resistant Muscoabacetinus bilobidae strains resistant to rifampicin are susceptible to rifabutin, which indicates incomplete cross-resistance between these antibiotics. It is also active against many gram-positive microorganisms.

Rifabutin is indicated for chronic multiresistant pulmonary tuberculosis caused by rifampicin-resistant strains of Musobacterium tuberculosis (as part of combination therapy), infections caused by Musobacterium tuberculosis bacillus tuberculosis muscularis tuberculosis.

In addition to the use of rifabutin in the treatment of mycobacterial infections, the efficacy of rifabutin in the eradication of bacteria of the genus Hepocabacet, especially of Hepocacipetcophen (for strains of this microorganism resistant to more traditional antibiotics such as clarithromycin), has also been shown. According to the information known to date, Hepocobactyl pylori plays an important role in the occurrence of diseases of the gastrointestinal tract, such as peptic ulcer and gastric ulcer, gastritis, colitis, proctitis, etc. According to WHO estimates, approximately 50% of the population of the planet .

On the effectiveness of the use of rifabutin for the eradication of He11cobac1et ru1op, see, for example: Va n bet Roejep., Ka1e1apk pH. Thye eGGesbuepekk OG pGaNbp 1pr1e Shetaru Gog rabeShk iy b1Gysi11-1o-egab1sa1e He11sobas1et ru1op ίη s11shsa1 rtasbse. - Aitep! Ryattaso 1 Thiet. 2007 EU; 26 (11-12): 1537-42. Yer 2007 Ber 28; TotassYo B., Sarobyuaka B., Botha.) A ΌΒ, Sesch b., Magao b. KtGaibp Lakeb 1pr1e 1etaru Gog igabyubop og N. ru1op rtatu apb kesopbagu hekMaSh 1o Yshbakho1e apb s1ap1ygotus. - Ωίβ b1ueeg Όίδ, 2005 1ap; 37 (1): 33-8; Ret E., Eek1a V., C1etep1e V., 11 11ash MV, Oiyabato M., Sagiko Ν., Vegdoy M., Apstsch A. Kapbopi / к бу о Г 1 ио екс 1 ет ие 1 ет Г Г Н ей ей со со со еб еб ет ет ет ет ет ет ай OG to! appbag 1pr1e 1yetar1ek. - At 1 Oak1goep1ego1. 2001 1ap; 96 (1): 58-62.

Rifmapitsin is a semisynthetic broad-spectrum antibiotic, anti-TB drugs of the first row. At low concentrations, it has a bactericidal effect on Musoacobacterium bacillus, Brisella c. in high concentrations - on some gram-negative microorganisms. It is characterized by high activity against B1aryu1ossossik krr. (including penicillinase-forming and many methicillin-resistant strains), B1ter1ossossic cr., C1O1pbst cr., VasShik aptaak; gram-negative cocci: meningococci, gonococci.

Rifampicin acts on gram-positive bacteria in high concentrations. Active against intracellular and extracellularly located microorganisms. Suppresses DNA-dependent

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RNA polymerase of microorganisms. With monotherapy with the drug, selection of rifampicin-resistant bacteria is relatively fast. Cross-resistance with other antibiotics (with the exception of the rest of rifamycins) does not develop.

Rifampicin is indicated for all forms of tuberculosis (as part of combination therapy). When leprosy is used in combination with dapsone (multibacillary types of disease). Other indications: infectious diseases caused by susceptible microorganisms (in cases of resistance to other antibiotics and as part of combination antimicrobial therapy; after exclusion of the diagnosis of tuberculosis and leprosy); brucellosis - as part of combination therapy with an antibiotic of the tetracycline group (doxycycline); meningococcal meningitis (prophylaxis in persons who were in close contact with patients with meningococcal meningitis; in bacilli carriers No. 188epa ιηοηίη ^ ίΐίάίδ).

As well as rifabutin, rifampicin is effective in relation to Hsjeobaehlpuop (in particular, in relation to strains of this microorganism resistant to clarithromycin) (see, for example: Ayi) a V, Vyaya V, Bayadir! A 8, Ραίζαάα A, 8 yagta M.R. EGPsasu aib! O1ega1yu oG pGatrkt-bakeb hexie 1etaru Gog Ne11sobae1e rugop ebabeoyo Gabige sh rerbe u1seg b18ea8e. - B1§ B18 8εί. 2005 Arg; 50 (4): 630-3), however, rifampicin is significantly inferior to rifabutin in the degree of activity with respect to this microorganism (see: Ko881 K., B. aeb. EXCLUSIVELY ACCOUNT EXCEPT BITTER PROCESSING PROCESSING PROCEDURE the initial resistance of the strains M.! bibliso818 and N. ru1op to rifabutin is also much less common than to rifampicin.

However, clinical use of rifabutin and rifampicin has previously been limited due to their low bioavailability.

This problem can be overcome with the use of new drug delivery systems, in particular solid lipid nanoparticles.

U.S. Patent Application Publication No. 2007237826 (KoShraga Ko1e8 ^ ata Kao), published October 10, 2007, discloses a drug (protein or peptide) delivery system based on polymerized solid lipid nanoparticles containing solid lipids and long chain fatty acids, biologically active a substance (protein or peptide), adjuvant, lectin, at least one polymer and a pharmaceutically acceptable carrier. This system was proposed by the author only for the delivery of proteins or peptides and is not considered as a means for the treatment of tuberculosis.

US Patent Application No. 20060008531 (Ghetto Sogrohayots), published January 12, 2006, discloses a method for preparing a suspension / dispersion of solid lipid nanoparticles in a liquid phase, comprising: dissolving a lipid and a drug substance in a suitable organic solvent to form a solution; emulsification of the solution in a liquid medium, to obtain an emulsion consisting of a discontinuous phase containing an organic solvent, a drug and a lipid, and a continuous liquid phase; bringing the emulsion into contact with a supercritical fluid in order to extract an organic solvent from the resulting micelle; sedimentation of micelles as a precipitate in a liquid medium to obtain a suspension / dispersion of solid lipid nanoparticles containing the drug in a liquid medium. It should be obvious to a person skilled in the art that a similar method for producing solid lipid nanoparticles (in the form of a suspension / dispersion) is fraught with the risk of incomplete removal of the organic solvent, which limits the possibility of creating drugs in this way for clinical use.

In the publication of the international application PCT \ UO 00/06120 (Kogea J181yi1e OG 8s1ssiss apb Tssyio1o§u), publication date 02/10/2000, an oil-in-water emulsion and solid lipid nanoparticles used in accordance with this document are disclosed as drug delivery systems and DNA transfection systems. According to \ ¥ 0 00/06120, both the emulsion and the solid lipid nanoparticles obtained on its basis include 2-30% non-triglyceride oil (preferably squalane or squalene), 0.01-20% of one or more emulsifiers (preferably phospholipid or non-ionic surfactant), including cationic surfactants, and water (up to 100%); in a preferred embodiment, the emulsion and the nanoparticles obtained on its basis may additionally contain 0.01-10% hydrophilic polymer (preferably polyoxyethylene, polyethyloxazoline and polyethylene glycol).

In particular, examples 23-25 and FIG. 14 in the description of \ UO 00/06120 relate to the production of solid lipid nanoparticles with rifampicin. However, data related to the biological activity of such a dosage form of rifampicin are not provided in \ UO 00/06120, and therefore the development of new nanosomal formulations of anti-tuberculosis antibiotics (primarily rifabutin or rifampicin) based on solid lipid nanoparticles.

The closest analogue (prototype) of the claimed invention, in our opinion, are described in the publication Raibeu K. e1 a1. Og1 zoib Pr1b IAIORA ^ v ^ c1e-ba8eb aiΐybie ^ si1a ^ cietoIetaru - Tiebgi1o818 (Ebbb). 2005 8er-No; 85 (5-6): 415-20. EPI 2005 Oc1 26 solid lipid nanoparticles for the treatment of tuberculosis, containing simultaneously several antibiotics traditionally used as first-line anti-TB drugs - rifampicin, isoniazid and pyrazinamide.

In accordance with Raibeu K. e1 a1., 2005, solid lipid nanoparticles containing simultaneously rifampicin, isoniazid and pyrazinamide were prepared as follows: 10 mg of each drug (rifampicin, isoniazid and pyrazinamide) and 30 mg of stearic acid were placed in a mixture consisting of 12 ml

- 2013570 acetone and 12 ml of ethanol, then heated to a temperature of 60-70 ° C in a water bath. The lipid: drug ratio was maintained at 1: 1. The resulting solution was poured into a 1% (w / v) aqueous solution of polyvinyl alcohol at a temperature of 4-8 ° C and with mechanical stirring. Solid lipid nanoparticles were formed by centrifugation at 35,000 d for 30 min at a temperature of 48 ° C. The granules formed were washed three times with distilled water and vacuum dried.

The disadvantage of the solution proposed by Rapbeu Ya c1 a1., 2005 is that the loading of such solid lipid nanoparticles with the drug was only 41 ± 4% for pyrazinamide, 45 ± 4% for isoniazid and 51 ± 5% for rifampicin. Further, according to Rapbeu Ya. E1 a1., 2005, the content of residual polyvinyl alcohol in solid lipid nanoparticles obtained by the authors was at a rather high level - 10.5-12.5% v / v, which is extremely undesirable in light of the potential the use of such solid lipid nanoparticles in the clinical practice of TB doctors.

In addition, even earlier in the literature it was shown that isoniazid, when used together with rifampicin, forms an inactive complex compound with it in the body.

Objects of the Present Invention

Thus, further development of pharmaceutical compositions for the treatment of tuberculosis, made on the basis of solid lipid nanoparticles and containing as an active principle an anti-tuberculosis drug selected from first-line anti-tuberculosis drugs, rifabutin or rifampicin, is necessary.

SUMMARY OF THE INVENTION

Within the framework of the present invention, the team of authors of the Nanosystem Scientific-Industrial Complex LLC established that to obtain solid lipid nanoparticles containing a therapeutically effective amount of an anti-TB drug selected from rifabutin or rifampicin, the previously described solid lipid nanoparticle production method can be successfully used Sauhash Ya. E1 a1., 1999 to obtain nanoparticles containing dihydrocortisone or progesterone as therapeutically active principle (see: Yaoyeya SauSh, E1epa P1ega, Oyo SarSho, Mala Yaoka Sacco.

Moreover, within the framework of the present invention, it was unexpectedly shown that the use of the method described by SaauSh Y. e1 a1., 1999 to obtain pharmaceutical compositions for the treatment of tuberculosis, including solid lipid nanoparticles containing rifabutin or rifampicin as a therapeutically active principle, allows to overcome the above disadvantages inherent in solid lipid nanoparticles in accordance with the prototype (Rapbeu Ya. e1 a1., 2005), and not only to obtain a dosage form based on solid lipid nanoparticles, distinguishing I have a higher “loading” of nanoparticles with a drug substance, but also increase the directed delivery of the therapeutically active principle to the target organs (lungs, spleen) and the bioavailability of the antibiotic (rifabutin or rifampicin).

It was also unexpectedly shown that the pharmaceutical composition in accordance with the invention provides, in comparison with the substance of the corresponding antibiotic, a significantly more pronounced antimycobacterial effect (by creating foci of the concentration of drugs in the target organs), which allows the use of a simplified treatment regimen (with lower doses Drugs with a lower frequency of administration and with increased intervals).

Thus, in accordance with the present invention, 2 objects are claimed:

1) A pharmaceutical composition for the treatment of tuberculosis and neurotransmitter-mediated diseases based on solid lipid nanoparticles dispersed in a pharmaceutically acceptable carrier containing a therapeutically effective amount of an anti-tuberculosis drug selected from rifabutin or rifampicin sorbed in a lipid matrix, characterized in that as a lipid matrix contains a combination of one or more esters of long chain fatty acids, cholesterol or a pharmaceutically acceptable derivative and phospholipid thereof; and

2) A method for treating tuberculosis and diseases mediated by Hepatobacter pylori, characterized in that they provide an oral administration to a patient in need of a therapeutically effective amount of the pharmaceutical composition of the invention three times a week for 4 weeks.

In accordance with the present invention, preferred long chain fatty acid esters can be selected from palmitic acid ester and monostearic acid ester, more preferably cetyl palmitate and glyceryl monostearate; a preferred pharmaceutically acceptable cholesterol derivative is potassium cholesterol sulfate; a preferred phospholipid is lecithin.

Pharmaceutical composition based on solid lipid nanoparticles, in accordance with the present

The invention also in a preferred embodiment also contains a surfactant and a cosurfactant. In a more preferred embodiment, the surfactant is a polyoxyethylated castor oil, most preferably Cheshorob Eb, and the cosurfactant is isopropyl alcohol.

A pharmaceutical composition based on solid lipid nanoparticles in accordance with the present invention may further comprise a cryoprotectant selected from 5-10% mannitol (ol) a, glucose or trehalose.

In the most preferred embodiment (but not limiting the scope of the claims to these quantitative values), the composition in accordance with the invention is made in the form for inhalation or oral administration and has the following preferred quantitative composition, wt.%:

cetyl palmitate - 20-25% glyceryl monostearate - 5-10% antibiotic selected from rifabutin or rifampicin - 20-25% lecithin - 10-13% potassium cholesterol sulfate - 7-10%

Cheshorog EG - 20-22% isopropyl alcohol - 3-5%.

The following are examples related to the production of solid lipid nanoparticles of rifabutin and rifampicin and compositions based on them, in accordance with the invention, and to confirm their unexpectedly high anti-tuberculosis activity, do not limit the scope of claims, but are given only to illustrate the present invention.

Example 1. Obtaining solid lipid nanoparticles with rifabutin (TL-33) and rifampicin (TL-34)

To obtain solid lipid nanoparticles containing rifabutin or rifampicin as therapeutically active principle, the dispersion method of hot microemulsions in cold water was used, which was first described in the aforementioned publication SauSh K e1 a1., 1999 for nanoparticles with dihydrocortisone or progesterone. A solution of a surfactant (Cheshorog Eb) and a cosurfactant (isopropyl alcohol) in 2 ml of a hot aqueous phase (vigorous stirring, Voglax, 70 ° C) was mixed with a transparent melt of lipids with an added drug (70 ° C) under mechanical stirring. The resulting hot microemulsion was introduced into 30 ml of cold (4 ° C) water with stirring on a magnetic stirrer. The resulting suspension of solid lipid nanoparticles was filtered through a No. 1 glass filter, cryoprotectant (mannitol (ol) 10% w / v, or glucose 5-10%, or trehalose 5-10%) was added, frozen in vials (1 ml / vial) and freeze-dried (Syp81A1bba2-4, Magyp Syp81 OsHN, Oegshapu) for 36 hours. The composition of the lipid and hot aqueous phases is presented in table. one.

Table 1. The composition of the components of the microemulsion to obtain solid lipid nanoparticles with rifabutin and rifampicin

No. Comp, lipid phase Comp. Hot water phase Turning on, % Soda rye, mg / m l Cryo prote Dimensions, nm Resusp. 33 Cetyl palmitate (0.27 g), Glyceryl monostearate (0.07 g), Rifabutin | (300.6 mg) Lecithin (0.144 g), Potassium Cholesterol Sulfate (0.10 g), Heterogas Eb (0.238 g), Isopropyl alcohol (0.051 g) 94.8 4.24 Mann IT 458 ± 110 + 34 Cetyl palmitate (0.27 g), Glyceryl monostearate (0.073 g), Rifampicin | (307 mg) Lecithin (0.145 g), Potassium Cholesterol Sulfate (0.10 g), Hetethorog Eb (0.248 g), Isopropyl alcohol (0.041 g) 79 4.14 Mann IT 29Η73 +

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Example 2. The study of the effectiveness of nanosomal dosage forms of rifabutin according to the invention (composition TL-33) on a model of experimental tuberculosis (mouse model)

To study the effectiveness of various drug rifabutin nanoforms in an acute tuberculosis infection model in mice, an accelerated or fast screening method was used. The method is an alternative way to test new anti-TB agents. The duration of therapy is 3 days, in contrast to the classical model for testing anti-TB drugs, where the duration of therapy is 4 weeks. The fast method is, in fact, an adaptation of the method of testing new antibiotics, first used on the model of Hepatobacter pylori. To assess the adequacy of the fast method, several studies of anti-TB drugs were previously conducted according to the classical 4-week regimen, followed by comparison with the 2-day treatment regimen with the same antibiotics. The rapid testing method was developed to accelerate the screening process of new anti-TB agents in mouse models.

Materials and research methods

Experimental animals: Ba1b / c linear mice, females, age 7-8 weeks, weight 20-22 g. There are 10 animals in each experimental group.

Samples of organs for seeding: Lungs (L) and spleen (C) of mice.

Infection: intravenous infusion into the lateral caudal vein, dose 5x10 ⁶ CFU Musoacanthillus 1begi1o818 Η37Κν. The strains Musobacillus 1bbci818 Η37Κν are taken from the stock kit, which is stored at -70 ° C.

Rifabutin (antibiotic control) and solid lipid nanoparticles containing rifabutin were prepared no more than an hour before the start of therapy daily, in accordance with passport recommendations. The rifabutin substance (antibiotic control) was also administered orally.

Antibiotic treatment:

Three times daily oral administration, at a dose of 1 mg / mouse, respectively, according to the active component.

The day after the end of treatment, animals were euthanized and organs were sown to calculate the number of CFU in homogenates of the lungs and spleen. Dilutions of organ homogenates were plated on Middlebrook 7H11 agar. For seeding, -1 and -2 dilutions were used in groups with antibiotics and control lungs and -2 and -3 for spleens of the control group of mice. CFU counting was performed 21 days after sowing.

Oral administration of rifabutin.

A dose of rifabutin 1 mg / mouse was used when administered orally.

The results (decimal logarithms of the number of CFU in each of the groups, in the lungs and in the spleen) are presented in FIG. 1, and the values of decimal logarithms (b§) for each value of CFU in the table. 2.

Thus, the composition of TL-33 (based on solid lipid nanoparticles containing rifabutin, in accordance with the present invention) had the most pronounced antimycobacterial activity.

Example 3. The study of the effectiveness of nanosomal dosage forms of rifampicin according to the invention (composition TL-34) on a model of experimental tuberculosis (mouse model).

Materials and research methods - similar to example 2.

Control, rifampicin (a substance) and solid lipid nanoparticles containing rifampicin were prepared no more than an hour before the start of therapy daily, in accordance with passport recommendations. The rifampicin substance (antibiotic control) was also administered orally.

Table 2. The number of CFU in the lungs and spleen of animals with the introduction of various dosage forms of rifabutin

The control Rifabutin subst. Rifabutin (TL-33) Lungs 4.1 3.02 2.48 Spleen 5.10 4.31 3.83

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Table 3. The number of CFU in the lungs and spleen of animals with the introduction of various dosage forms of rifampicin

The control Rifampicin Subst. Rifampicin 8ΕΝ (TL-34) Lungs 4.43 4.16 3.95 Spleen 6.14 5.28 4.95

The results are presented in table. 3 and in FIG. 2.

Claims (15)

CLAIM 1. A pharmaceutical composition for the treatment of tuberculosis and diseases mediated by Hepocobacter pylori, made on the basis of solid lipid nanoparticles dispersed in a pharmaceutically acceptable carrier containing a therapeutically effective amount of an anti-tuberculosis drug selected from rifabutin or rifampicin sorbed in a lipid matrix, characterized in that as a lipid matrix contains a combination of one or more esters of long chain fatty acids, cholesterol or a pharmaceutically acceptable derivative thereof and a phospholipid. 2. The pharmaceutical composition according to claim 1, characterized in that the esters of long chain fatty acids are selected from an ester of palmitic acid and an ester of monostearic acid. 3. The pharmaceutical composition according to claim 2, characterized in that the palmitic acid ester is cetyl palmitate. 4. The pharmaceutical composition according to claim 2, characterized in that the ester of monostearic acid is glyceryl monostearate. 5. The pharmaceutical composition according to claim 1, characterized in that the pharmaceutically acceptable derivative of cholesterol is potassium cholesterol sulfate. 6. The pharmaceutical composition according to claim 1, characterized in that the phospholipid is a lecithin. 7. The pharmaceutical composition according to claim 1, characterized in that it further comprises a surfactant and cosurfactant. 8. The pharmaceutical composition according to claim 7, characterized in that the surfactant is a polyoxyethylene castor oil. 9. The pharmaceutical composition of claim 8, wherein the polyoxyethylated castor oil is Cheshoryog Eb. 10. The pharmaceutical composition according to claim 7, characterized in that the cosurfactant is isopropyl alcohol. 11. The pharmaceutical composition according to claim 1, characterized in that it further comprises a cryoprotectant. 12. The pharmaceutical composition according to claim 11, characterized in that the cryoprotectant is selected from mannitol (ol) a, glucose and trehalose. 13. The pharmaceutical composition according to any one of claims 1 to 12, characterized in that it has the following composition, wt.%: Cetyl palmitate - 20-25%, glyceryl monostearate - 5-10%, antibiotic selected from rifabutin or rifampicin - 20 -25%, lecithin - 10-13%, cholesterol potassium sulfate - 7-10%, Sheshoryog EB - 20-22%, isopropyl alcohol - 3-5%. 14. The pharmaceutical composition according to claim 1, characterized in that it is made in a form suitable for oral or inhalation administration. 15. A method for the treatment of tuberculosis and diseases mediated by Neusobacillus pylori, characterized in that they provide an oral administration to a patient in need of a therapeutically effective amount of the pharmaceutical composition according to any one of claims 1-14 three times a week for 4 weeks.