Method for preparing nanosized amphotericin b

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to medicine, pharmaceutics and nanotechnologies, and more specifically to a method for preparing nanosized amphotericin B for coating aluminosilicate nanotubes, used as a poorly soluble polyene macrocyclic antibiotic widely used for treating fungal diseases. What is presented is the method for preparing nanosized amphotericin B by mixing a solution of amphotericin B in dimethyl sulphoxide at room temperature with the aluminosilicate tubes to make 1-20 wt % of amphotericine B precipitate on the aluminosilicate tubes by water treatment of the prepared mixture while stirring thoroughly at water feed rate 10 ml/min. The technical effect consists in the fact that the presented method is simple and easy to implement, and enables producing a novel dosage form of amphotericin B represented by amphotericin B coating the solid inorganic structures that are the aluminosilicate tubes; the above shall provide the further development of new ointments, gels and magmas for treating fungal diseases.

EFFECT: using the nanosized carrier promotes the uniform distribution of amphotericin B, while an aluminosilicate nature of the carrier is a good sorbent that provides prolonged action of amphotericin B with the carrier staying undisturbed that also allows for higher bioavailability of insoluble amphotericin B as compared to microforms thereof.

1 cl, 4 ex

 

The present invention relates to the field of medicine, pharmaceuticals, and nanotechnology, and specifically, to a method for producing nanoscale deposited on silica-alumina nanotubes, amphotericin b is poorly soluble macrocyclic polyene antibiotic of the formula

,

produced by Streptomyces nodosus, which has fungicidal or fungistatic activity against Candida spp., Cryptococcus neoformans, Aspergillus spp. and other fungi. The method may find application in the manufacture of drugs, pharmaceuticals and medicine.

Established over half a century ago amphotericin b until recently, remains the "gold standard" empirical antimycotic therapy, despite serious side effects. Amphotericin b is a macrocyclic polyene antibiotic produced by fungi Streptomyces nodosus, highlighted for the first time from a sample of soil on the river Orinoco (Orinoco in Venezuela in 1955. The drugs are based largely on the binding to ergosterol in the fungal membrane and a violation of its integrity.

Amphotericin b is the drug of broad-spectrum activity against most Candida species (excluding .lusitaniae), as well as Blastomyces dermatitidis, Coccidioides immitis, Cryptococcus neoformans, Fusarium spp., Histoplasma capsulatum, Paracoccidioides brasiliensis, Rhodotorula spp., Sporotrix spp. Amphotericin b is also effective protivoseborainey visceral and American cutaneous leishmaniasis. In addition, amphotericin b in some cases, active against Aspergillus spp.Trichosporon spp. and Pseudoallescheria boydii resistant to the CA [(a) Mitrofanov VS Systemic antifungal drugs. Probl. the honey. micol. 2001; 3(2): 6-14; (b) A. Lemke, A.F. Kiderlen, O. Kayser. Amphotericin B. Appl. Environ. Biotechnol. (2005) 68: 151-162; (C) V. Idemyor. Emerging opportunistic fungal infections: where are we heading? J. Natl. Med. Assoc. (2003) 95:1211-1215].

Amphotericin b is used in three ways: intravenous, inhalation, orally and topically (in the form of ointment). The main problem severely limit the use of this substance is its low solubility in most organic solvents and water. Part of the problem of the low solubility of amphotericin b is solved through the use of expensive dosage forms in which it is in the form of lipid complexes, liposomes and colloidal dispersions [(a) Walsh, Thomas J.; Finberg, Robert W.; Arndt, Carola; Hiemenz, John; Schwartz, Cindy; Bodensteiner, David; Pappas, Peter; Seibel, Nita; Greenberg, Richard N.; Dummer, Stephen; Schuster, Mindy; Dismukes, William E.; Holcenberg, John S.; Liposomal Amphotericin B for Empirical Therapy in Patients with Persistent Fever and Neutropenia. New England Journal of Medicine 1999; 340:764-771; (b) Janknegt, R.; de Marie, S.; Bakker-Woudenberg, I. A.; Crommelin, D. J. Liposomal and lipid formulations of amphotericin B. Clinical pharmacokinetics, 1992, 23(4):279-291; (c) Patricia K. Sharkey, John R. Graybill, Edward S. Johnson, Stephen G. Hausrath, Richard B. Pollard, Antonia Kolokathis, Donna Mildvan, Patty Fan-Havard, Robert H.K. Eng, Thomas F. Patterson, John C. Pottage, Jr., Michael S. Simberkoff, Judith Wolf, Richard D. Meyer, Renu Gupta, Lily W. Lee, and David S. Gordon. Amphotericin In Lipid Complex Compared with Amphotericin In the Treatent of Cryptococcal Meningitis in Patients with AIDS. din Infect Dis. 1996, 22: 308-314; (d) John W. Hiemenz and Thomas J. Walsh. Lipid Formulations of Amphotericin B: Recent Progress and Future Directions. Clin Infect Dis. 1996, 22 (Supplement 2): S133-S144.]; it is important to note that all these dosage forms are not true solutions of amphotericin b C.

A method of obtaining nano-suspensions of Amphotericin b for the treatment of amoebic diseases of the brain [Andreas Lemke, Albrecht F. Kiderlen, Boris Petri, Oliver Kayser. Delivery of amphotericin B nanosuspensions to the brain and determination of activity against Balamuthia mandrillaris amebas Nanomedicine: Nanotechnology, Biology, and Medicine, 6: 597-603 (2010).]. According to this method, a suspension of nano-sized particles of Amphotericin b was obtained by homogenization under high pressure, using a pistol homogenizer. Amphotericin b was mixed with different structure of surface-active substances in the water, the amount of amphotericin b was 2%, and surfactant 1%. The resulting mass is many times within 20 cycles homogenized at a pressure of 1500 bar (about 1500 atmospheres) to obtain nano-suspensions. In General, the method is very expensive and multihelical.

Known and accepted by us for the prototype method for producing nano-amphotericin b In the carrier - gelatin [Manoj Nahar, Dinesh Mishra, Vaibhav Dubey, Narendra Kumar Jain. Development, characterization, and toxicity evaluation of amphotericin B-loaded gelatin nanoparticles. Nanomedicine: Nanotechnology, Biology, and Medicine 4: 252-261 (2008)]. Nanoscale mixture of gelatin and amphotericin b is received as follows: 200 mg of gelatin was dissolved in 10 ml of distilled water at 40 degrees Celsius, then added 10 ml of acetone to precipitate high molecular weight gelatin. The supernatant was removed, and the remaining high molecular weight gelatin was re-dissolved by adding 10 ml of distilled water under stirring with a speed of 600 revolutions per minute at a constant heating. If the pH of the gelatin solution was regulated in the range from 2 to 12. Dissolved in 500 μl of dimethyl sulfoxide amphotericin b was added to the water-polymer phase was then added 30 ml of acetone, then pinned 25% aqueous solution of glutaraldehyde, acting as a crosslinking reagentes was stirred for 12 hours with a speed of 600 revolutions per minute. Not contacting gelatin amphotericin b was removed by absorbing 10 ml amberlite XAD 16, followed by filtration (filter with pore size of 1 μm; Whatman Japan KK, Tokyo, Japan). The dimethylsulfoxide was removed by repeated washing with distilled water. The obtained nano-gelatin-amphotericinb complex was subjected to lyophilization the disaccharide trehalose (trehalose) to obtain a mixture containing nano-sized particles for further research.

The method is quite laborious and requires receipt of a nano-amphotericin b In exclusively for injection, as emit it in liofilizirovannom.

Obtained in this method, gelatin-amphotericinb to the complex cannot be used for drawing on aluminosilicate nanotubes, because a significant amount of gelatin to be adsorbed on the nanotubes and the complex will disintegrate. Nanotubes can be aggregated into much larger ones when using gelatin. In addition, insoluble aluminosilicate nanotubes (inorganic in nature) cannot be subjected to freeze-drying of trehalose. Also, in the case of purification by adsorption involving amberlite a significant portion of the nanotubes will be sorbed.

The present invention is to develop a simple and convenient process execution method of producing nano-amphotericin b deposited on solid nanosized inactive carrier for use in other dosage forms, for example, for oral administration in tablet form or for external use and thereby to achieve higher bioavailability of insoluble amphotericin b, in comparison with micro-size particles forms of its use.

This object is achieved by the proposed method of obtaining nano-amphotericin b using a solution of amphotericin b in dimethyl sulfoxide and inactive media, the distinguishing feature of which is that as inactive media use aluminosilicate nanotubes and the process is conducted by mixing the solution of amphotericin b In dimethylsulfoxide at room temperature aluminosilicate nanotubes with subsequent deposition of 1-20 wt.% of amphotericin b on aluminosilicate nanotubes by treating the mixture with water under vigorous stirring and speed of water flow 10-20 ml/min

Aluminosilicate nanotubes represent an inorganic material corresponding in composition to the mineral kaolinite Al2O3·2SiO2·2H2O. the External diameter of the tubes 90-140 nm, inner diameter of 10-60 nm, the length of 300-2000 nm; the main part of the silicon oxide (43,13%) and aluminum oxide (34,37%). The proposed method is based on the preparation of the true solution of amphotericin b in the presence of nanotubes, which, then, with uniform speed under intensive stirring, water. During the addition of water is the solvent system in which amphotericin b is not soluble and precipitates on the surface of the aluminosilicate nanotubes; after deposition, the solution is centrifuged, the mixture of water with dimethylsulfoxide decanted. To remove residual dimethyl sulfoxide, and the resulting powder is mixed with water and centrifuged, the procedure is repeated two more times. Then, the powder nanotubes coated with amphotericin b then dried in a vacuum desiccator to remove residual water.

Figure 1. shows the original aluminosilicate nanotubes.

Figure 2. shown aluminosilicate nanotubes coated with 10% by mass amount of amphotericin b C.

The technical result - the proposed method is simple and convenient in technological performance and allows you to get at is a brand new pharmaceutical form of amphotericin b, representing amphotericin b, deposited on a solid inorganic structures-aluminosilicate nanotubes, which will continue to develop new ointments, gels and mash for the treatment of fungal diseases. The use of nanosized carrier promotes a uniform distribution of amphotericin b, and aluminosilicate media type is a good sorbent provides prolonged action of amphotericin b, with the media (nano-tube) is not destroyed, and can achieve a higher bioavailability of insoluble amphotericin b, in comparison with micro-size particles forms of its use.

The invention meets the criterion of "novelty", as known in the scientific-technical and patent literature does not include a full set of features characterizing the invention.

From literature it is known that the aluminosilicate nanotubes can be a carrier for organic substances, including biologically active [D. Shchukin, R. Price, G. Sukhorukov, Y. Lvov, Halloysite Nanotubes as Biomimetic Nanoreactors. Small, 1, 510-513 (2005); M. Zhi, W. Jinye, G. Xiang, D. Tong, Q. Yongning. Application of Halloysite Nanotubes Progress in Chemistry, 24: 275-283 (2012)]. The present invention meets the criterion of "inventive step", because until now, amphotericin b is not inflicted on aluminosilicate nanotubes and not used zorastorian is for the deposition of amphotericin b on an inorganic carrier, and most importantly, it was not clear that amphotericin b will settle it on the nanotubes, and not on the walls of the reaction vessel or precipitate in not associated with the nanotubes form.

The invention meets the condition of "industrial applicability"because amphotericin b is widely used for the treatment of fungal diseases. Creating media and dosage forms with its use is the solution of key problems of the application of this poorly soluble drug substances. The use of amphotericin b deposited on the nanotubes obtained by the proposed method will allow the development of new tablets, ointments, gels and mash for the treatment of fungal diseases.

The invention is illustrated by the following examples without limiting its scope.

Example 1.

To a solution of amphotericin b (0.6 g) in dimethyl sulfoxide (50 ml) under stirring and the temperature of 18-20°C was added aluminosilicate nanotubes (5 g), was stirred for 15 minutes. To the mixture was added at a uniform speed under vigorous stirring 100 ml of distilled water for 10 minuteperiod stopped, with sludge decantation 120 ml of liquid was added to 100 ml of water, which, centrifuged, decantation 100 ml of liquid. The procedure was repeated two more times. The mixture was centrifuged, decantation all liquid phases is. The precipitate was dried in vacuum (5-10 mm Hg) desiccator at room temperature for two days. Got to 5.2 g of powder, which was analyzed using elemental analysis, carbon content corresponded to ten percent of the mass amount of amphotericin b in the mixture. Comparison of photographs taken with an electron microscope, for the original aluminosilicate nanotubes (figure 1) and for a finite mixture (figure 2)showed that amphotericin b adsorbed on the nanotubes.

Example 2.

To a solution of amphotericin b (1.2 g) in dimethyl sulfoxide (100 ml) under stirring and a temperature of 25°C was added aluminosilicate nanotubes (10 g), was stirred for 15 minutes. To the mixture was added at a uniform speed under vigorous stirring to 200 ml of distilled water for 10 minutes. The stirring was stopped, with sludge decantation 240 ml of liquid was added 200 ml of water, which, centrifuged, decantation 200 ml of liquid. The procedure was repeated two more times. The mixture was centrifuged, decantation entire liquid phase. The precipitate was dried in vacuum (5-10 mm Hg) desiccator at room temperature for two days. Received 10.4 g of powder, which was analyzed using elemental analysis, carbon content corresponded to ten percent of the mass number of the amp is terizin In the mixture.

Example 3.

To a solution of amphotericin b (2.5 g) in dimethyl sulfoxide (100 ml) under stirring and a temperature of 25°C was added aluminosilicate nanotubes (10 g), was stirred for 15 minutes. To the mixture was added at a uniform speed under vigorous stirring to 200 ml of distilled water for 10 minutes. The stirring was stopped, with sludge decantation 240 ml of liquid was added 200 ml of water, which, centrifuged, decantation 200 ml of liquid. The procedure was repeated two more times. The mixture was centrifuged, decantation entire liquid phase. The precipitate was dried in vacuum (5-10 mm Hg) desiccator at room temperature for two days. Received 12 g of powder, which was analyzed using elemental analysis, carbon content corresponded to twenty percent mass amount of amphotericin b in the mixture.

Example 4.

To a solution of amphotericin b (0.2 g) in dimethyl sulfoxide (100 ml) under stirring and a temperature of 25°C was added aluminosilicate nanotubes (10 g), was stirred for 15 minutes. To the mixture was added at a uniform speed under vigorous stirring to 200 ml of distilled water for 10 minutes. The stirring was stopped, with sludge decantation 240 ml of liquid was added 200 ml of water, which, centrifuged, decantation 200 m of the liquid. The procedure was repeated two more times. The mixture was centrifuged, decantation entire liquid phase. The precipitate was dried in vacuum (5-10 mm Hg) desiccator at room temperature for two days. Received 9.6 g of powder, which was analyzed using elemental analysis, carbon content corresponded to one percent of the mass amount of amphotericin b in the mixture.

The method of obtaining nano-amphotericin b using a solution of amphotericin b in dimethyl sulfoxide and inactive carrier, characterized in that as inactive media use aluminosilicate nanotubes and the process is conducted by mixing the solution of amphotericin b in dimethyl sulfoxide at room temperature aluminosilicate nanotubes with subsequent deposition of 1-20 wt.% of amphotericin b on aluminosilicate nanotubes by treating the mixture with water under vigorous stirring and the speed of water flow 10 ml/min



 

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