KR0137458B1 - Process and composition for the purification of amphotericin-b - Google Patents

Abstract

None.

Description

Method for purifying amphotericin B and composition therefor

The present invention relates to a process for the purification and crystallization of amphotericin B and compositions therefor.

A potent antifungal agent, amphotericin B, belongs to the group of compounds known as polyene macrolide antibiotics.

This compound is characterized by large lactone rings containing conjugated double bonds.

Methods of making them from amphotericin B and Streptomyces nodosus are described in US Pat. No. 2,908,611, issued October 13, 1959 to Dutcher et al.

Ampoterisin B is currently known to have the following structure.

The extraction and crystallization method of amphotericin described by Dutcher et al .:

a. Crude amphotericin in alcohol is slurried with acid and then neutralized with a strong base, or

b. Crude amphotericin is slurried with dimethylformamide and the slurry is treated with a water soluble alcohol or a water soluble ketone.

US Pat. No. 4,177,265 to Michael et al. Describes a process for purifying amphotericin B using a dissolution medium consisting of sodium iodide or sodium thiocyanide and acetone or methanol. The solubility of amphotericin B is drastically improved with either of these media, but with limited solubility in acetone or methanol alone.

It has been studied novel methods of purifying amphotericin B and compositions useful for this purification that allow higher crystalline products to be obtained in more consistent yields.

The present invention relates to a novel process for the purification of amphotericin B, in which a crystalline material can be obtained in a more consistent high yield, and a crystallization composition suitable therefor. The novel amphotericin B composition consists of amphotericin B with methanol, dimethylformamide, methylene chloride and water in solution. The novel purification process dissolves crude amphotericin B that is partially purified or contains impurities in methanol, dimethylformamide and acid solutions, the resulting extract mixture is separated from any insoluble components and the purified extract is chlorinated. Mixing with a solvent mixture of methylene and water, crystallizing amphotericin B, and recovering the crystalline product.

The inventors have found that the use of four solvent systems according to the method of the present invention can substantially improve the consistency of the amphotericin B production yield and the crystalline nature of the product. The four solvent systems consist of dimethylformamide in which amphotericin B is initially dissolved and methylene chloride and water added after filtration of methanol and methanol and the initial solution. The resulting crystallization composition of amphotericin B consists of an acid forming a salt with amphotericin B and amphotericin B in the four solvents. In addition, after purification, crystallization, filtration and washing, the product is dried to at least 2% by weight, preferably about 2 to 6% by weight, to obtain a more stable crystalline material.

The following table illustrates the advantages of the present invention. The amphotericin B products obtained by Dutcher et al. In US Pat. No. 2,908,611 and the amphotericin B products obtained by the method of the present invention were compared in the table below. Microbiological potency, yield, purity (measured as residue after product firing) and stability for each product were measured.

The exact mechanism of improvement as described above is unknown, but methylene chloride and water added to amphotericin B, methanol and dimethylformamide improve the crystalline structure, and thus the microbiological activity, purity and It is believed that stability is improved.

Ampoterisine B used as starting material in the purification process includes crude amphotericin B that has been partially purified or contains impurities. The term amphotericin B containing impurity refers to the inclusion of physical impurities such as dust particles, fibrous materials and other particulate elements which may render amphotericin B unavailable for pharmaceutical use.

The first step in the process of the present invention is to extract the amphotericin B by suspending the amphotericin B starting material in any order in a mixture of methanol and dimethylformamide and adding an acid to the mixture. The acid can be any acid that readily forms an acid with amphotericin B, for example citric acid, chloride, sulfuric acid and the like. Although the temperature is not critical, the extraction is preferably carried out by adjusting the temperature of the mixture thus formed to about 15-20 ° C. and stirring it until the amphotericin B starting material is dissolved.

Separation of the extraction mixture is carried out using methods known in the art. Preferably, filtration will be used to separate the amphotericin B extract from the insoluble material.

The purified amphotericin B extract is then mixed with a solvent mixture of methylene chloride and water. Although the temperature and order of addition of methylene chloride and water are not critical, it will be desirable to cool the water to about 5-10 ° C. The pH of this mixture is adjusted to about 5 to 7, preferably about 6, using a water soluble base (eg, water soluble triethanolamine) or any other suitable base. This mixture precipitates the amorphous product and the resulting slurry (slurry of amorphous product and solvent) provides a suitable environment to promote the conversion of amphotericin B to crystalline material.

The slurry of amorphous amphotericin B is converted to crystalline material by heating at a temperature of about 40 ° C. to about 60 ° C., preferably about 45 ° C. to about 55 ° C. Optionally, small amounts of nuclei crystals can be added to the slurry to accelerate crystallization.

Recovery of crystalline amphotericin B can be carried out using conventional methods such as centrifugation or filtration. The isolated material may be washed with a suitable solvent before drying if necessary. Preferably, the isolated crystalline material should be dried to contain at least 2% by weight and optionally 2-6% by weight of water. Examples of solvents for the washing step include mixtures of acetone, acetone and water, mixtures of methanol, methanol and water, water, or mixtures thereof.

The operating conditions for carrying out the method can be selected from among the methods for obtaining sterile products.

The present invention will be illustrated by the following examples, but is not meant to be limited to these descriptions.

Example 1

Amphotericin B about 10 BK (base kilograms, ie kilograms of pure active substance) and 225 liters of dimethylformamide were added to about 565 liters of methanol. About 18.5 KG citric acid monohydrate (or equivalent if acid anhydride) was added. The mixture was kept at 15-20 ° C. and stirred until the input dissolved completely. The thick mixture was then filtered to clear.

Next, about 140 L of methylene chloride was added to the filtrate. 225 L of water cooled to about 5-10 ° C was added. The pH was then adjusted to about 6 to precipitate amphotericin B. It was adjusted using 40% v / v of water-soluble triethylamine. The resulting slurry was then heated to about 44-46 ° C. for about 30 minutes. When the product crystallized under the microscope, the mixture was cooled to about 10 ° C. over 2-3 hours. The purified product was then isolated by filtration and washed with about 85 L of cooled 40% v / v of aqueous methanol. The wet cake was then slurried in about 140 L of acetone. After filtration, the cake was washed with about 28 liters of acetone and then dried to contain 2% to 6% moisture. Ampoterisin B recovered about 9.7 BK.

Claims (10)

An amphotericin B composition consisting of an acid capable of forming a salt with amphotericin B and amphotericin B in a solution consisting of methanol, dimethylformamide, methylene chloride and water. The composition of claim 1 further comprising a reagent suitable for adjusting the pH of the composition to about 5-7. (a) forming an amphotericin B solution in an acid capable of forming a salt with methanol, dimethylformamide and amphotericin B; (b) separating the amphotericin B extract from the insoluble material; (c) mixing the isolated amphotericin B extract with a solvent mixture consisting of methylene chloride and water; (d) adjusting the pH of the mixture thus formed to about 5-7; (e) heating the formed slurry to promote formation of crystalline material; (f) A method for purifying and crystallizing amphotericin B, comprising recovering crystalline amphotericin B. The method of claim 3 wherein the recovery of the crystalline material (Iii) separating the crystalline material formed in step (e) from the residual solvent; (Ii) washing the separated crystals and drying the crystals to contain about 2 to 6% by weight of water. The method of claim 3, wherein step (a) is performed at a temperature of about 15 to 20 ° C. 5. 4. A process according to claim 3, characterized in that in step (a) amphotericin B, methanol and dimethylformamide are mixed together, followed by addition of citric acid. The method of claim 3 wherein said separation is performed by filtration. 4. The method of claim 3, wherein in step (c) the methylene chloride is added to the extract, followed by addition of about 5-10 ° C. water to the methylene chloride and the extract. 4. The method of claim 3, wherein in step (d) the pH of the mixture is adjusted to about 6. The method of claim 3, wherein the amorphous amphotericin B is heated to about 40-60 ° C. to convert to crystalline amphotericin B. 5.