RU2420287C1 - Method of obtaining capsulated form of antituberculosis medications of rifampicin series - Google Patents

Abstract

FIELD: medicine, pharmaceutics. ^ SUBSTANCE: invention relates to field of pharmaceutics, namely to method of obtaining capsulated form of antibiotics of rifampicin series for treating tuberculosis. Claimed is method of obtaining liposomal form of antituberculosis medications of rifampicin series rifampicin (RM) or rifabutin (RB) which differs by the fact that antibiotic is dissolved in isotonic solution with medium pH value 2-3, after which on the surface of said solution applied in layers is powder of lipid mixture, which contains 50-70% of glycosphyngolipids, 30-50% phospholipids and 3% of cholesterol and triacetylglicerides with further dispersion with slight mixing at room temperature, pH of dispersion is brought to neutral value by base addition, dispersion is heated for 10-40 min at temperature 60-70C with further evaluation of degree of antibiotic inclusion by method of spectrophotometry after carrying out gel-filtration. ^ EFFECT: method ensures obtaining medication antibiotic of rifampicin series with high bioaccessibility. ^ 5 cl, 1 ex, 1 tbl, 2 dwg

Description

The invention relates to the field of pharmaceuticals, and in particular to a method for producing a liposomal form of anti-TB drugs for the treatment of tuberculosis.

For treatment of tuberculosis, a treatment regimen has been developed that includes at least several drugs. The semi-synthetic antibiotic rifampicin (RM) and its more modern analogue rifabutin (RB) are first-line drugs in the treatment of tuberculosis.

The mechanism of action of rifamycin-type antibiotics (PM and RB) is due to their ability to bind to the β-subunit of bacterial RNA polymerase and thereby inhibit the transcription process in Mycobacterium tuberculosis. A prerequisite for the successful implementation of such a mechanism is the penetration of an antibiotic into an infected macrophage.

However, both drugs (PM and RB) have serious drawbacks. They are slightly soluble in water (1 mg / ml and 0.01 mg / ml, respectively) and, with prolonged use, have a pronounced hepatotoxic effect. Therefore, such a dosage form for antibiotics is desirable that would increase their content in the aqueous phase. For this, antibiotics are enclosed in particles from natural phospholipids (liposomes), or in nanoparticles from biodegradable synthetic polymers. An important advantage of liposome preparations and polymer particles, which have a size from 0.2 to 0.5 microns, over traditional dosage forms is their ability to interact with macrophages by endocytosis. This feature is extremely important in the treatment of tuberculosis, because M. tuberculosis reproduce mainly in macrophages. Thus, using the encapsulated form of antibiotics, targeted delivery of the drug to the macrophage, to the breeding site of mycobacteria, can be carried out.

It should be noted that when an encapsulated form of the drug is introduced into the bloodstream, it is strongly diluted, which leads to the desorption of the drug from the surface of liposomes or nanoparticles and its exit from the inner water space of the liposomes outward along the concentration gradient. Therefore, for an anti-TB drug, it is important not so much the total content of the active substance in the capsule form as the amount (percentage) of its inclusion in the capsules themselves, be it liposomes or nanoparticles, which are then captured by macrophages.

Several compositions are known for PM and RB, each of which is embedded in polymer nanoparticles with a size of 0.2-0.4 microns.

A known method of obtaining a composition with RB, including separate mixing of rifabutin with target additives and a filler and another part of rifabutin with a lack of filler, their subsequent mixing, dusting with magnesium stearic acid and encapsulation (Patent RU 2320337, IPC A61K 31/395, A61K 9/48, published on 03/27/2008).

However, in this study there is no information on the solubility of this drug in the aqueous phase after dissolution of the capsule.

Known methods for producing compositions (Patent RU 2337711, IPC A61K 47/48, A61K 31/438, publ. 10/11/2008; Patent EA 200801388 A1, IPC A61K 31/438; A61K 9/16; publ. 28/08 / 2009, Patent WO 2009002227 A1, IPC A61 K9 / 395; A61K 31/10 publ. 31/12/2008), which are nanoparticles from polymers of lactic acid and copolymers of lactic and glycolic acids with RB included therein. Particles had a small size (0.2-0.4 microns) and a high percentage of antibiotic inclusion.

However, these studies did not study the kinetics of the release of RB from particles upon entry into the aquatic environment and the content of the antibiotic that remains in the particles after strong dilution.

There is also known a method for producing a PM preparation containing 1-22% rifampin (a synonym for rifampicin) in liposome form, consisting of 1-27% lecithin, 1-18% cholesterol and 70-100% distilled water (CN 1319395, IPC A61K 9 / 127; A61K 31/496, publ. 31/10/2001).

The inclusion of an antibiotic in liposomes was also not determined in this study.

The closest technical solution to the proposed method for producing an encapsulated form of PM is a method of obtaining a liposomal form of PM, which includes preparing a solution of PM together with phospholipids in an organic solvent, removing the solvent to obtain a thin film that is dispersed in an aqueous medium to obtain a suspension of multilamellar vesicles enclosed in them RM The resulting suspension is homogenized at high pressure or forced through a filter until liposomes of 0.1-0.5 microns are obtained. As phospholipids take a mixture containing, wt.%: Phosphatidylcholine 70-80, the rest is a mixture of any other lipids of egg yolk, and / or soy, and / or cattle. A solution of RM together with phospholipids is prepared at a ratio of rifampicin: phospholipids equal to 1: 3-15 (RF Patent 2223764, IPC A61K 31/496, A61K 9/127, publ. 20/02/2004).

The disadvantage of this method is that the content of PM in the composition of liposomes was not determined, and the method of producing liposomes is very time-consuming and contains energy-intensive operations such as evaporation of an organic solvent and the use of special equipment for homogenizing and forcing MLV.

The closest technical solution to the proposed method for producing an encapsulated form of RB is a method for producing a RB composition in liposomes from an individual soybean phosphatidylcholine and its mixture with other lipids. The method consists in obtaining a lipid film containing an antibiotic by evaporating an organic solvent from a lipid solution with an antibiotic, dispersing the lipid film in a small volume of the aqueous phase, then the obtained MLV was diluted in a large volume of the aqueous phase and centrifuged. The supernatant was pressed through a filter with a defined pore size and received unamellar vesicles, in which the inclusion of RB was determined by its content in the liposome fraction, which were separated from unbound RB by gel filtration. RB inclusion was calculated as the ratio of the liposomes included in the liposomes of the liposomes in which it was located (Gaspar M.M., Neves S., Portaels F., Pedrosa J., Silva M.T., Cruz ME Therapeutic efficacy of liposomal rifabutin in a Mycobacterium avium model of infection // Antimicrob. Agents Chemother. 2000, Vol.44, No.9, pp.2424-2430).

The disadvantage of this method is that the proposed calculation of the inclusion rate of the antibiotic does not imply the ability to reliably estimate the ratio of liposomes containing RB and empty liposomes, and the method of producing liposomes is very time-consuming and contains energy-intensive operations such as evaporation of the organic solvent, centrifugation and forcing MLV.

The technical result of the invention is the development of a technologically advanced and cost-effective way to obtain liposomal forms of anti-TB drugs RM and RB, without the use of toxic organic solvents, as well as energy-intensive stages of their removal.

The specified technical result is achieved due to the fact that in our proposed method receive liposomes of small size (not more than 0.5 microns) containing PM or RB included in liposomes in high concentration.

The antibiotic is dissolved in an isotonic solution with a pH value of 2-3, and then a powder of a mixture of lipids containing 50-70% glycosphingolipids, 30-50% phospholipids and 3% cholesterol and triacylglycerides is layered on the surface of this solution, then dispersed with gentle stirring at room temperature, adjust the pH of the dispersion to a neutral value by adding a base, the dispersion is heated for 10-40 minutes at a temperature of 60-70 ° C, followed by assessment of the degree of inclusion of the antibiotic by spectrophotometry after gel-fil tration.

To obtain liposomes, a mixture of lipids of the following composition is used:

- glycosphingolipids 60%;

- phospholipids - 37%;

- cholesterol and triacylglycerides - 3%.

As glycolipids, a mixture of cerebrosides and cerebroside sulfates of natural or synthetic origin is taken in any ratio.

Densitometric analysis by TLC determined the qualitative and quantitative composition of the phospholipids of the drug. It was established that phosphatidylserine is present in the initial preparation of phospholipids - 28%; phosphatidylethanolamine - 8% and trace amounts of phosphatidylcholine and sphingomyelin (SM). The glycosphingolipid fraction consists mainly of cerebrosides (TS - 46%) and cerebroside sulfates (CS - 14%).

Particle morphology was investigated by electron microscopy. Particle size estimates were also obtained from turbidimetric analysis data.

The conditions for obtaining liposomes with PM or RB were optimized by varying the temperature of liposome production from long half-nanotubes from 40 to 80 ° C. At temperatures below 55 ° C, the initial half-nanotubes were observed, as shown in FIG. Above 80 ° C, spherical particles are formed from the semi-nanotubes (Fig. 2), but under these conditions the lipid oxidation index rises from 0.2 to 1.0, which indicates that lipid oxidation and degradation are ongoing. Thus, the temperature range is from 55 to 70 ° C.

To obtain liposomes with PM or with RB, different ratios of GSL and PL are used at a constant concentration of PM or RB equal to 5 mg / ml. To separate the antibiotic included in the liposomes from the antibiotic in solution, a microcolumn with a Sephadex G-25 sorbent was used. An increase in the FL content in the GSL / PL mixture reduces the inclusion of antibiotics in liposomes, as does an increase in the total concentration of the lipid mixture above 15 mg / ml.

Upon receipt of liposomes, the content of PM or RB in an isotonic solution of 1 to 5 mg / ml is used. An increase in the initial concentration of the antibiotic leads to the fact that their content in liposomes increases. But with an increase in antibiotic concentration to 10 mg / ml, a precipitate forms which is not dispersed by a mixture of lipids.

Upon receipt of liposomes, the concentration of the GSL / PL mixture (3: 2) in the initial dispersion is varied from 15 to 40 mg / ml. An increase in lipid concentration above 40 mg / ml led to a decrease in the inclusion of antibiotics. Perhaps this was because under these conditions there is no complete transition of the semi-nanotubes into spherical particles, which follows from the data on the particle size.

The best result of obtaining the encapsulated form of the antibiotics PM and RB according to the proposed method is described in example 1.

Example 1

First, 0.01 M HCl is added to the 0.9% NaCl solution so that the pH of the solution becomes 3.0 or lower. PM or RB is added to this solution so as to reach a final antibiotic concentration of 5 mg / ml. Next, a lipid mixture powder is layered on the surface of the solution and dispersed with gentle stirring with a magnetic stirrer at room temperature for 30 minutes. The mixture of glycosphingolipids and phospholipids (GSL / FL) used at a ratio of 3: 2 with this dispersion method forms extended structures in aqueous solutions with a diameter of 200 nm and a length of more than a micron. They have the form of a gutter and they are called semi-nanotubes (figure 1). The resulting dispersion is adjusted with a base solution to a pH of 6.0-7.0 and heated for 10 minutes at a temperature of 60 ° C. When this happens, the transformation of the semiconductors into spherical nanoparticles - liposomes (figure 2). A homogeneous emulsion of liposomes having a size of not more than 0.5 μm and containing a high percentage of antibiotics PM or RB is obtained after a strong dilution of the drug during the gel filtration process.

The degree of inclusion of an antibiotic in liposomes is estimated spectrophotometrically based on the dependence of light absorption at certain wavelengths on the concentration, which obeys the Lambert-Bouger-Behr law in the concentration range of interest. In particular, the content of PM can be determined from the value of light absorption at a wavelength of 475 nm, taking the molecular absorption coefficient equal to 15300 l · mol ^-1 · cm ^-1 . The content of RB is calculated by the light absorption of the solution at a wavelength of 500 nm (the molecular absorption index is taken to be 3100 l · mol ^-1 · cm ^-1 ).

To determine the amount (percent) of the inclusion of PM or RB in liposomes, liposomes containing anti-TB drugs are separated from their aqueous forms by gel filtration. For this, liposomal antibiotic preparations are applied to a column filled with Sephadex G-25 and eluted with saline or Tris buffer.

The first fractions collected from the column contained drug-containing liposomes and drug-free liposomes ("empty" liposomes), and then fractions containing free drug not included in the liposomes were followed. The content of both components (liposomes and antibiotic) was determined by spectrophotometry. The presence of liposomes was confirmed by fixing the turbidity of the incubation medium at 780 nm. To determine the number of antibiotics associated with liposomes, they were destroyed with a detergent, for which 0.2 ml of a 5.4% sodium deoxycholate solution was added to 3 ml of liposomes.

Examples 2-6

Homogeneous emulsions of liposomes, including PM and RB, were obtained analogously to example 1, their characteristics, such as the ratio of antibiotic: liposomes and evaluation of the effectiveness of the inclusion of antibiotics in liposomes, are shown in the table.

Table Characteristics of liposomes containing PM or RB from a GSL / PL mixture obtained under different conditions Lipid component The initial end of the antibiotic mg / ml Mg / ml lipids The effectiveness of inclusion. RM,% The effectiveness of inclusion. RB,% Particle size, nm GSL / FL (1: 2) 5 fifteen 17 10 325 GSL / FL (1: 1) 5 fifteen 25 twenty 370 GSL / FL (3: 2) one fifteen fifteen 23 250 GSL / FL (3: 2) 5 fifteen thirty 33 250 GSL / FL (3: 2) 10
sediment fifteen - - - GSL / FL (3: 2) 5 40 25 28 Above 1 micron

Claims (5)

1. A method of obtaining a capsule form of rifamycin rifampicin (PM) or rifabutin (RB) antibiotics in the form of liposomes, characterized in that the antibiotic is dissolved in an isotonic solution with a pH value of 2-3, and then a lipid mixture powder is layered on the surface of this solution, containing 50-70% glycosphingolipids, 30-50% phospholipids and 3% cholesterol and triacylglycerides, and dispersed with gentle stirring at room temperature, the pH of the dispersion is neutralized by adding base, the dispersion is heated in for 10-40 minutes at a temperature of 60-70 ° C, followed by assessment of the degree of inclusion of the antibiotic by spectrophotometry after gel filtration. 2. The method according to claim 1, characterized in that all these actions are carried out under sterile conditions, using an isotonic sterile NaCl solution. 3. The method according to any one of claims 1 and 2, characterized in that cerebrosides and cerebroside sulfates of natural or synthetic origin in any proportions are taken as glycolipids. 4. The method according to claim 3, characterized in that the antibiotic solution together with the lipid mixture is prepared at an antibiotic: lipid ratio of from 1: 3 to 1:15 (wt.). 5. The method according to claim 4, characterized in that the determination of PM or RB included in the liposome is carried out in the liposome fraction separated from free PM or RB using the gel filtration method, and the content of PM or RB in the obtained liposome fraction is determined spectrophotometrically.

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