A process for the preparation of a dry powder of polymyxin B sulfate

Abstract

The application discloses a preparation method of a polymyxin B sulfate dry powder and belongs to the technical field of medicine preparation. The preparation of the polymyxin B sulfate dry powder is that the polymyxin B sulfate fermentation liquor is subjected to adsorption filtration by using a ZIF-8@polyurea / polyether sulfone composite membrane, and the filter cake is subjected to elution by using a sulfuric acid aqueous solution. The eluate is post-treated to obtain the polymyxin B sulfate dry powder. The application has the advantages of mild preparation condition, simple process, low reaction energy consumption and industrial application prospect.

Description

Technical Field

[0001] This invention relates to a method for preparing polymyxin B sulfate dry powder, belonging to the field of pharmaceutical preparation technology. Background Technology

[0002] Polymyxin B is a general term for a group of basic polypeptide antibiotics. Polymyxin B sulfate is also known as polymyxin B. Although polymyxin B has a strong bactericidal effect against Gram-negative bacteria, its clinical application was discontinued in the 1980s and 1990s due to its strong toxicity and neuromuscular blocking effect. However, with the increasing number of severe illnesses caused by drug-resistant Gram-negative bacterial strains with multiple antibodies in recent years, the clinical application of polymyxin B has regained attention, and clinical research is increasing. Polymyxin B sulfate is mostly produced by fermentation, using straight-rod-shaped Bacillus polymyxinus. The fermentation broth of polymyxin B sulfate contains impurities such as residual sugars, inorganic salts, pigments, proteins, and microbial cells from the culture medium, which can significantly affect the subsequent application effect. Therefore, the fermentation broth of polymyxin B needs to be separated and purified to ensure product quality.

[0003] There are many methods for the extraction and purification of polymyxin B sulfate. Currently, the main extraction and purification methods include precipitation, membrane filtration, adsorption, extraction, and ion exchange. Precipitation and extraction methods use organic solvents, which are not only costly but also harmful to operators and the environment. Adsorption methods require a large amount of adsorbent material, making them expensive. Ion exchange methods have disadvantages such as long production time and low adsorption yield.

[0004] Metal-organic frameworks (MOFs) are characterized by large specific surface area, high porosity, and tunable pore size. Among MOFs, ZIF-8 has significant potential for application in adsorption separation, wastewater treatment, and other technological fields due to its mild synthesis conditions and good physicochemical stability.

[0005] Diisocyanates are a class of compounds containing two highly reactive isocyanate groups (-N=C=O) in their molecules. They can react with polyamines to generate polyureas containing -NHCONH- functional groups. Polyureas have microporous, mesoporous, and macroporous structures and have good water resistance, aging resistance, and wear resistance. Summary of the Invention

[0006] The technical objective of this invention is to overcome the shortcomings of existing technologies and provide a method for preparing polymyxin B sulfate dry powder. Specifically, it involves adsorbing and filtering polymyxin B sulfate fermentation broth using a ZIF-8@polyurea / polyethersulfone composite membrane, eluting the filter cake with a sulfuric acid aqueous solution, and then post-treating the eluent to obtain polymyxin B sulfate dry powder. This method has a simple preparation process, low reaction energy consumption, and promising industrial application prospects.

[0007] The technical solution of the present invention is as follows:

[0008] A method for preparing polymyxin B sulfate dry powder involves adsorbing and filtering the polymyxin B sulfate fermentation broth using a ZIF-8@polyurea / polyethersulfone composite membrane, eluting the filter cake with a sulfuric acid aqueous solution, and then post-treating the eluent to obtain polymyxin B sulfate dry powder.

[0009] (1) The ZIF-8 is a metal-organic framework crystal prepared by reacting 2-methylimidazolium with zinc nitrate;

[0010] The polyurea is a porous network polymer generated by the cross-linking reaction of polymethylene polyphenyl polyisocyanate and triethylenetetramine.

[0011] The ZIF-8@polyurea / polyethersulfone composite film is a porous composite material in which polyurea with MOF nanocrystals embedded on the surface is supported on a polyethersulfone substrate.

[0012] The sulfuric acid aqueous solution is preferably 0.5 mol / L.

[0013] (2) The ZIF-8 nanocrystals are prepared by the following steps:

[0014] 2.5 g of 2-methylimidazole was dissolved in 30 mL of methanol, and 1.6 g of zinc nitrate hexahydrate was dissolved in 30 mL of ethanol. The two solutions were mixed and placed in a frequency conversion microwave synthesizer. The mixture was stirred at 40 °C for 10 min, cooled to room temperature, centrifuged, washed three times with ethanol, and dried at 80 °C to constant weight to obtain ZIF-8 nanocrystals.

[0015] The power of the frequency conversion microwave synthesizer is 0-1000W.

[0016] (3) The ZIF-8@polyurea / polyethersulfone composite membrane is prepared by the following steps:

[0017] 7.4 g of polymethylene polyphenyl polyisocyanate and 0.5 g of triethylenetetramine were dissolved in 35 mL of acetonitrile. 2-2.5 g of ZIF-8 nanocrystals were added, and the mixture was heated to 35-40 °C. The polyethersulfone porous membrane was then immersed in the solution and kept at this temperature for 10 min. After being removed, the membrane was air-dried at room temperature. This process was repeated 3-4 times to obtain the ZIF-8@polyurea / polyethersulfone composite membrane.

[0018] The polyethersulfone porous membrane is a commercially available membrane substrate with a pore size of 1.0 μm and a diameter of 50 mm.

[0019] The polymethylene polyphenyl polyisocyanate is produced by Yantai Wanhua Polyurethane Co., Ltd.

[0020] (4) The preparation steps of the polymyxin B sulfate fermentation broth are as follows:

[0021] 1) Preparation of seed culture medium

[0022] Each 1L of seed culture medium contains the following components: 40g soluble starch, 50g glucose, 8g ammonium sulfate, 7g potassium dihydrogen phosphate, 4g calcium carbonate, and the remainder is water; the seed culture medium is sterilized at 120℃ for 30min and cooled for later use.

[0023] 2) Preparation of polymyxin B sulfate fermentation broth

[0024] Freshly grown slant-cultured Paenibacillus polymyxa ATCC10401 was inoculated into seed culture medium and cultured on a shaker at 300 rpm and 30°C for 20 h to obtain polymyxin B sulfate fermentation broth.

[0025] 3) Preparation of polymyxin B sulfate fermentation broth

[0026] Each 1L of fermentation medium contains the following components: 80g whole wheat flour; 4g corn steep liquor powder; 9g ammonium sulfate; 0.3g potassium dihydrogen phosphate; 0.3g calcium carbonate; the remainder is water; the fermentation medium is sterilized at 120℃ for 20min and cooled for later use.

[0027] The inoculum was inoculated into the above fermentation medium at a volume fraction of 4%, stirred at 350 r / min, and cultured at 30°C for 38 h to obtain polymyxin B sulfate fermentation stock broth.

[0028] The pH of the fermentation broth was adjusted to 2 with oxalic acid, and 5.0% diatomaceous earth was added. After stirring for 15 minutes, the mixture was filtered to obtain the fermentation broth of polymyxin B sulfate.

[0029] (5) The post-treatment of the eluent is as follows:

[0030] The pH of the eluent was adjusted to 5.5-6.5 using a 10% sodium hydroxide solution, and then concentrated until solid precipitates to obtain a saturated polymyxin B sulfate solution.

[0031] At room temperature, ethanol was added dropwise to a saturated solution. When the amount of precipitated crystals no longer increased, the solution was filtered. The filter cake was washed three times with ethanol, heated to 40°C, and vacuum dried for 6 hours to obtain polymyxin B sulfate powder. The beneficial technical effects of this invention are:

[0032] (1) The preparation method of polymyxin B sulfate dry powder of the present invention is simple, has low reaction energy consumption, and has industrial application prospects.

[0033] (2) The ZIF-8@polyurea / polyethersulfone composite membrane prepared in this invention utilizes the highly active isocyanate groups (-N=C=O) in polyphenyl polymethylene polyisocyanate to undergo a cross-linking reaction with triethylenetetramine to generate a network polyurea. The polyurea is embedded with ZIF-8 nanocrystals on its surface through hydrogen bonding and other forces, and is loaded onto a polyethersulfone substrate. This composite membrane has many active sites, high specific surface area, large porosity, strong adsorption capacity for polymyxin B sulfate, and high yield. Detailed Implementation

[0034] The present invention will be further described below with reference to the embodiments. However, the scope of protection of the present invention is not limited to the embodiments. Any changes made to the technical solutions of the present invention by those skilled in the art should fall within the scope of protection of the present invention.

[0035] Example 1: A method for preparing ZIF-8 nanocrystals

[0036] 2.5 g of 2-methylimidazole was dissolved in 30 mL of methanol, and 1.6 g of zinc nitrate hexahydrate was dissolved in 30 mL of ethanol. The two solutions were mixed and placed in a frequency conversion microwave synthesizer. The mixture was stirred at 40 °C for 10 min, cooled to room temperature, centrifuged, washed three times with ethanol, and dried at 80 °C to constant weight to obtain ZIF-8 nanocrystals. The power of the frequency conversion microwave synthesizer was 0-1000 W.

[0037] Example 2: A method for preparing a ZIF-8@polyurea / polyethersulfone composite membrane

[0038] 7.4 g of polymethylene polyphenyl polyisocyanate and 0.5 g of triethylenetetramine were dissolved in 35 mL of acetonitrile, and 2 g of ZIF-8 nanocrystals were added. The mixture was heated to 35 °C, and a polyethersulfone porous membrane was immersed in the solution. The reaction was maintained at this temperature for 10 min. After being removed, the membrane was air-dried at room temperature. This process was repeated 3-4 times to obtain a ZIF-8@polyurea / polyethersulfone composite membrane. This ZIF-8@polyurea / polyethersulfone composite membrane is a porous composite material in which polyurea with MOF nanocrystals embedded on the surface is supported on a polyethersulfone substrate.

[0039] The polyethersulfone porous membrane is a commercially available membrane substrate with a pore size of 1.0 μm and a diameter of 50 mm.

[0040] The polymethylene polyphenyl polyisocyanate is produced by Yantai Polyurethane Co., Ltd.

[0041] Example 3: A method for preparing a ZIF-8@polyurea / polyethersulfone composite film

[0042] The steps and the product obtained are the same as in Example 2, except that 2.5g of ZIF-8 nanocrystals were added and the heating temperature was 40°C.

[0043] Example 4: A method for preparing polymyxin B sulfate dry powder

[0044] The polymyxin B sulfate fermentation broth was filtered through the ZIF-8@polyurea / polyethersulfone composite membrane prepared in Example 2. The filter cake was eluted with a 0.5 mol / L sulfuric acid aqueous solution to obtain polymyxin B sulfate eluent. The eluent was then post-treated to obtain polymyxin B sulfate dry powder.

[0045] The post-treatment of the eluent involves adjusting the pH of the eluent to 5.5-6.5 using a 10% sodium hydroxide solution, and then concentrating it until solid precipitates to obtain a saturated polymyxin B sulfate solution.

[0046] At room temperature, ethanol was added dropwise to a saturated solution. When the amount of precipitated crystals no longer increased, the solution was filtered. The filter cake was washed three times with ethanol, heated to 40°C, and vacuum dried for 6 hours to obtain polymyxin B sulfate powder.

[0047] The polymyxin B sulfate dry powder is off-white, with a drying loss of 4.4%, and the total mass of B1, B2, B3 and B1-1 is 83%, with an extraction yield of 65%.

[0048] Example 5: A method for preparing polymyxin B sulfate dry powder

[0049] The steps are the same as in Example 4, except that the polymyxin B sulfate fermentation broth is filtered using the ZIF-8@polyurea / polyethersulfone composite membrane prepared in Example 3; the obtained polymyxin B sulfate dry powder is off-white, with a drying loss of 4.3%, and the total mass of B1, B2, B3 and B1-1 is 89%, with an extraction yield of 69%.

[0050] Example 6: A method for preparing polymyxin B sulfate fermentation broth

[0051] The preparation steps of the polymyxin B sulfate fermentation broth described in Examples 4 and 5 are as follows:

[0052] (1) Preparation of seed culture medium

[0053] Each 1L of seed culture medium contains the following components: 40g soluble starch, 50g glucose, 8g ammonium sulfate, 7g potassium dihydrogen phosphate, 4g calcium carbonate, and the remainder is water; the seed culture medium is sterilized at 120℃ for 30min.

[0054] (2) Preparation of polymyxin B sulfate fermentation broth

[0055] Freshly grown slant-cultured Paenibacillus polymyxa ATCC10401 was inoculated into seed culture medium at a volume fraction of 4%. The temperature of the shaker was 30℃, the shaking speed was 300 rpm, and the culture time was 20 h to obtain the polymyxin B sulfate fermentation broth.

[0056] (3) Preparation of polymyxin B sulfate fermentation broth

[0057] Each 1L of fermentation medium contains the following components: whole wheat flour 80; corn steep liquor powder 4; soybean flour 4.5; ammonium sulfate 9; potassium dihydrogen phosphate 0.3; calcium carbonate 0.3; and the remainder is water. The fermentation medium is sterilized at 120℃ for 30min. A 4% (v / v) inoculum is added to the fermentation medium, stirred at 260r / min, and cultured at 30℃ for 38h to obtain polymyxin B sulfate fermentation stock solution.

[0058] The pH of the fermentation broth was adjusted to 2 with oxalic acid, and 5.0% diatomaceous earth was added. After stirring for 15 minutes, the mixture was filtered to obtain the fermentation broth of polymyxin B sulfate.

Claims

1. A method for preparing polymyxin B sulfate dry powder, characterized in that, The polymyxin B sulfate fermentation broth was adsorbed and filtered using a ZIF-8@polyurea / polyethersulfone composite membrane, and the filter cake was eluted with sulfuric acid aqueous solution; the eluent was then post-treated to obtain polymyxin B sulfate dry powder. The ZIF-8@polyurea / polyethersulfone composite film was prepared by the following method: 7.4 g of polymethylene polyphenyl polyisocyanate and 0.5 g of triethylenetetramine were dissolved in 35 mL of acetonitrile, and 2-2.5 g of ZIF-8 nanocrystals were added. The mixture was heated to 35-40 °C, and a polyethersulfone porous membrane was immersed in the solution. The reaction was maintained at this temperature for 10 min, and then the membrane was removed and air-dried at room temperature. This process was repeated 3-4 times to obtain a ZIF-8@polyurea / polyethersulfone composite membrane. The polyethersulfone porous membrane was a polyethersulfone membrane substrate with a pore size of 1.0 μm. The ZIF-8 nanocrystals were prepared by the following method: 2.5 g of 2-methylimidazole was dissolved in 30 mL of methanol, and 1.6 g of zinc nitrate hexahydrate was dissolved in 30 mL of ethanol. The two solutions were mixed and placed in a frequency conversion microwave synthesizer with a power of 0-1000 W. The mixture was stirred at 40 °C for 10 min, cooled to room temperature, centrifuged, washed three times with ethanol, and dried at 80 °C to constant weight to obtain ZIF-8 nanocrystals. The sulfuric acid aqueous solution has a concentration of 0.5 mol / L; The post-treatment of the eluent includes the following steps: The eluent was adjusted to pH 5.5-6.5 with a 10% sodium hydroxide solution, and then concentrated until solid precipitated to obtain a saturated polymyxin B sulfate solution. Ethanol was added dropwise to the saturated solution at room temperature. When the precipitated crystals no longer increased, the solution was filtered. The filter cake was washed three times with ethanol, heated to 40°C, and vacuum dried for 6 hours to obtain polymyxin B sulfate powder.