RU2491288C1 - Method for preparing nanosized amphotericin b - Google Patents

Abstract

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

Description

The present invention relates to the field of medicine, pharmaceuticals and nanotechnology and, specifically, to a method for producing nanosized, deposited on aluminosilicate nanotubes, amphotericin B, a poorly soluble polyene macrocyclic antibiotic of the formula

,

,

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

Opened more than half a century ago, amphotericin B has until recently remained the ″ gold standard ″ of empirical antimycotic therapy, despite severe side effects. Amphotericin B is a polyene macrocyclic antibiotic produced by Streptomyces nodosus fungi isolated for the first time from a soil sample on the Orinoco River in Venezuela in 1955. The action of the drug is based largely on binding to the fungal membrane with ergosterol and a violation of its integrity.

Amphotericin B is a broad-spectrum drug that is active against most Candida species (excluding C. lusitaniae), as well as Blastomyces dermatitidis, Coccidioides immitis, Cryptococcus neoformans, Fusarium spp., Histoplasma capsulatum, Paracoccidioides brasiliensis, Rhodotorula s. Amphotericin B is also effective against pathogens of visceral and American cutaneous leishmaniasis. In addition, amphotericin B is in some cases active against Aspergillus spp. Trichosporon spp. and Pseudoallescheria boydii are resistant to AmB [(a) Mitrofanov V.S. Systemic antifungal drugs. Prob. honey. mycol. 2001; 3 (2): 6-14; (b) A. Lemke, A.F. Kiderlen, O. Kayser. Amphotericin B. Appl. Microbiol. 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: intravenously, inhaled, orally and topically (as an ointment). The main problem of the very limiting use of this substance is its low solubility in most organic solvents and water. Partially, the problem of low solubility of amphotericin B is solved by 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 B Lipid Complex Compared with Amphotericin B in the Treatment 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 of the listed dosage forms are not true solutions of amphotericin B.

A known method of producing nanosized suspensions of Amphotericin B for the treatment of amoebic brain diseases [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).]. In this method, a suspension of nanosized particles of Amphotericin B was obtained by high pressure homogenization using a pistol homogenizer. Amphotericin B was mixed with various surface-active substances in water, the amount of amphotericin B was 2%, and the surfactant was 1%. The resulting mass was repeatedly - for 20 cycles homogenized at a pressure of 1500 bar (approximately 1500 atmospheres) to obtain a nanosized suspension. In general, the method is very expensive and low-tech.

Known and accepted by us as a prototype is a method for producing nanosized amphotericin B on a gelatin carrier [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)]. A nanosized mixture of gelatin and amphotericin B was prepared as follows: 200 mg of gelatin was dissolved in 10 ml of distilled water at 40 degrees Celsius, then 10 ml of acetone was added to precipitate high molecular weight gelatin. The supernatant was removed, and the remaining high molecular weight gelatin was redissolved by adding 10 ml of distilled water with stirring at a speed of 600 rpm with constant heating. The pH of the gelatin solution was adjusted from 2 to 12. Amphotericin B, dissolved in 500 μl of dimethyl sulfoxide, was added to the water-polymer phase, then 30 ml of acetone was added, then a 25% aqueous solution of glutaraldehyde was added, which acted as a cross-linking reagent. The solution was mixed 12 hours at a speed of 600 rpm. Amphotericin B not bound to gelatin was removed by absorbing 10 ml of XAD 16 amberlite, followed by filtration (1-micron filter; Whatman Japan KK, Tokyo, Japan). Dimethyl sulfoxide was removed by repeated washing with distilled water. The obtained nanosized gelatin-amphotericin complex was lyophilized with trehalose disaccharide in order to obtain a mixture containing nanosized particles for subsequent studies.

The method is quite time-consuming and involves the production of nanosized amphotericin B exclusively for injection, since it is isolated in a lyophilized form.

The gelatin-amphotericin complex obtained by this method cannot be used for deposition on aluminosilicate nanotubes, since a significant amount of gelatin will be sorbed by the nanotubes and the complex will decompose. Nanotubes can aggregate into much larger formations when using gelatin. In addition, insoluble aluminosilicate nanotubes (inorganic in nature) cannot be lyophilized with trehalose. Also, in the case of purification by adsorption involving amberlite, a significant part of the nanotubes will be sorbed.

The objective of the present invention is to develop a convenient and easy-to-process method for producing nanosized amphotericin B deposited on a solid nanosized inactive carrier in order to use it in the form of another dosage form, for example, for oral administration in the form of tablets or for external use and thereby achieve a higher bioavailability of insoluble amphotericin B, in comparison with micro-sized forms of its use.

The problem is achieved by the proposed method for producing nanosized amphotericin B using a solution of amphotericin B in dimethyl sulfoxide and an inactive carrier, the distinguishing feature of which is that aluminosilicate nanotubes are used as an inactive carrier and the process is carried out by mixing a solution of amphotericin B in dimethyl sulfoxide at room temperature with aluminosiloxyl or at followed by precipitation of 1-20 wt.% amphotericin B on aluminosilicate nanotubes by processing the resulting mixture with water with vigorous stirring and a water flow rate of 10-20 ml / min.

Aluminosilicate nanotubes are an inorganic material kaolinite Al ₂ O ₃ · 2SiO ₂ · 2H ₂ O corresponding in composition to the mineral. The outer diameter of the tubes is 90-140 nm, the inner diameter is 10-60 nm, the length is 300-2000 nm; the main composition is silicon oxide (43.13%) and aluminum oxide (34.37%). The proposed method is based on the preparation of a true solution of amphotericin B in the presence of nanotubes, to which, then, water is added with uniform speed with vigorous stirring. During the addition of water, a solvent system is formed in which amphotericin B is insoluble and precipitates onto the surface of aluminosilicate nanotubes; after precipitation, the solution is centrifuged, the mixture of water with dimethyl sulfoxyl is decanted. To remove residues of dimethyl sulfoxide, the resulting powder is mixed with water and centrifuged, the procedure is repeated two more times. After that, the powder of nanotubes coated with amphotericin B is dried in a vacuum desiccator to remove residual water.

In figure 1. initial aluminosilicate nanotubes are shown.

Figure 2. Aluminosilicate nanotubes with 10% by mass of Amphotericin B.

The technical result - the proposed method is simple and convenient in technological design and allows you to get a fundamentally new dosage form of amphotericin B, which is amphotericin B, applied to solid inorganic structures - alumosilicate nanotubes, which will subsequently develop new ointments, gels and lubes for the treatment of fungal diseases . The use of a nano-sized carrier promotes a uniform distribution of amphotericin B, and the aluminosilicate type of carrier, a good sorbent, ensures the prolonged action of amphotericin B, while the carrier (nanoscale tubes) does not break down, and also allows for a higher bioavailability of insoluble amphotericin B, in comparison with its micro-sized forms use.

The invention meets the criterion of "novelty", as in the well-known scientific, technical and patent literature there is no complete set of features characterizing the invention.

From the literature it is known that aluminosilicate nanotubes can be a carrier for organic substances, including biologically active ones [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", since up to now amphotericin B has not been applied to aluminosilicate nanotubes and no co-solvent has been used to precipitate amphotericin B on an inorganic carrier and, most importantly, it was not obvious that amphotericin B will settle on nanotubes, and not on the walls of the reaction vessel or precipitate in a form not associated with nanotubes.

The invention meets the condition of "industrial applicability", since amphotericin B is widely used to treat fungal diseases. The creation of carriers and dosage forms with its use is a solution to the key problem of using this sparingly soluble drug substance. The use of amphotericin B deposited on nanotubes obtained by the proposed method will allow the development of new tablets, ointments, gels and dabs for treating fungal diseases.

The invention is illustrated by the following examples, not limiting its scope.

Example 1

Alumosilicate nanotubes (5 g) were added to a solution of amphotericin B (0.6 g) in dimethyl sulfoxide (50 ml) with stirring at a temperature of 18–20 ° C and stirred for 15 minutes. To the resulting mixture, 100 ml of distilled water was added at a uniform rate with vigorous stirring for 10 minutes. Stirring was stopped, 120 ml of liquid was decanted from the precipitate, 100 ml of water was added, it was shaken, centrifuged, and 100 ml of liquid was decanted. The procedure was repeated two more times. The mixture was centrifuged, the entire liquid phase was decanted. The precipitate was dried in a vacuum (5-10 mm Hg) desiccator at room temperature for two days. Received 5.2 g of powder, which was analyzed using elemental analysis, the carbon content corresponded to a ten percent mass amount of amphotericin B in the mixture. A comparison of photographs taken on an electron microscope for the starting aluminosilicate nanotubes (Fig. 1) and for the final mixture (Fig. 2) showed that amphotericin B was sorbed by the nanotubes.

Example 2

Alumosilicate nanotubes (10 g) were added to a solution of amphotericin B (1.2 g) in dimethyl sulfoxide (100 ml) with stirring at a temperature of 25 ° C, and stirred for 15 minutes. To the resulting mixture was added at a uniform rate with vigorous stirring 200 ml of distilled water over 10 minutes. Stirring was stopped, 240 ml of liquid was decanted from the precipitate, 200 ml of water was added, it was shaken, centrifuged, 200 ml of liquid was decanted. The procedure was repeated two more times. The mixture was centrifuged, the entire liquid phase was decanted. The precipitate was dried in a vacuum (5-10 mm Hg) desiccator at room temperature for two days. Received 10.4 g of powder, which was analyzed using elemental analysis, the carbon content corresponded to ten percent by weight of amphotericin B in the mixture.

Example 3

Alumosilicate nanotubes (10 g) were added to a solution of amphotericin B (2.5 g) in dimethyl sulfoxide (100 ml) with stirring at a temperature of 25 ° C, and stirred for 15 minutes. To the resulting mixture was added at a uniform rate with vigorous stirring 200 ml of distilled water over 10 minutes. Stirring was stopped, 240 ml of liquid was decanted from the precipitate, 200 ml of water was added, it was shaken, centrifuged, 200 ml of liquid was decanted. The procedure was repeated two more times. The mixture was centrifuged, the entire liquid phase was decanted. The precipitate was dried in a vacuum (5-10 mm Hg) desiccator at room temperature for two days. Received 12 g of powder, which was analyzed using elemental analysis, the carbon content corresponded to twenty percent by weight of amphotericin B in the mixture.

Example 4

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

Claims (1)

A method of producing nanosized amphotericin B using a solution of amphotericin B in dimethyl sulfoxide and an inactive carrier, characterized in that aluminosilicate nanotubes are used as an inactive carrier and the process is carried out by mixing a solution of amphotericin B in dimethyl sulfoxide at room temperature with aluminosilicate nanotubes 1 to 20 followed by aluminosilicate nanotubes 1 to 20 followed by .% amphotericin B on aluminosilicate nanotubes by treating the resulting mixture with water with vigorous stirring and a feed rate of 10 ml / min

Images