Preparation method of maltodextrin for synthesizing ferric carboxymaltose

Abstract

The invention discloses a preparation method of maltodextrin for synthesizing ferric carboxymaltose. The method comprises the following steps: adding pullulanase into commercially available maltodextrin in water to carry out branching reaction, and carrying out enzyme removal operation to obtain maltodextrin of which the branching degree (BD value) is 0-3.0% and the reducing sugar equivalent (DE value) is 8.0-16.0%. The maltodextrin prepared by the method is used for synthesizing ferric carboxymaltose, the particle size of the maltodextrin is 24.0-28.0 nm, the particle size distribution PDI is less than or equal to 0.14, the diameter of an iron core is 4.95-5.25 nm, and the maltodextrin is highly consistent with an original product.

Description

Technical Field

[0001] The invention belongs to the field of pharmacy and relates to a preparation method of maltodextrin for synthesizing carboxymaltose ferric. Background Art

[0002] Iron deficiency anemia is a common nutritional disease. It is caused by insufficient iron storage in the human body, which cannot meet the needs of normal red blood cell production. Iron supplementation is an effective treatment for iron deficiency patients. Oral iron supplementation is often used as the first choice for iron deficiency anemia because it is effective, cheap and safe. However, oral iron preparations often have obvious gastrointestinal discomfort symptoms. In addition, they are also affected by food ingredients and iron reserves in the body. Especially in many patients with chronic diseases and tumors, because inflammatory mediators in the body induce the liver to synthesize iron regulatory proteins, downregulate the expression of iron transport proteins on the surface of the gastrointestinal tract and iron storage cells in the body, thereby affecting the absorption and utilization of iron, oral iron preparations are often poor or even ineffective. Therefore, when iron reserves need to be restored quickly, and oral iron supplementation is not suitable or insufficient to quickly replenish the needs of the human body, intravenous iron supplementation is required.

[0003] Ferric carboxymaltose is a new generation of high-dose intravenous iron preparations. It has been clinically proven that intravenous injection of ferric carboxymaltose in patients with iron deficiency anemia can quickly supplement iron and increase the patient's hemoglobin level, thereby achieving significant therapeutic effects. Ferric carboxymaltose is composed of a unique ferric hydroxide core and a carboxymaltose shell, which can achieve a single sufficient iron supplement of 1000 mg. Similar to ferritin, ferric carboxymaltose has a stable molecular structure and a low free iron content. After entering the blood, it can be accurately taken up and stored by macrophages. The carboxymaltose shell can control the release of iron and supply it to target organs without iron overload. Ferric carboxymaltose is a non-dextrose derivative with a low risk of hypersensitivity reactions after injection, and no allergy test is required before use. In addition, ferric carboxymaltose is also commonly used in patients with chronic kidney disease and anemia.

[0004] Ferric carboxymaltose was developed by Weifu Pharmaceuticals and was first launched in Germany in 2007 and in the UK the following year. Its trade name is Ferinject, and it is used to treat iron-deficiency anemia for which oral iron is ineffective or cannot be used. As of 2014, ferric carboxymaltose has been launched in more than 50 countries. In November 2022, ferric carboxymaltose was approved for marketing in my country, which is expected to lead Chinese patients with iron deficiency and iron-deficiency anemia to quickly "get out of poverty" and usher in a new era of high-dose intravenous iron supplementation.

[0005] Patent CN1705682A applied by Weifu Pharmaceuticals provides the following method for synthesizing carboxymaltose ferric:

[0006] The method is to oxidize one or more maltodextrins with a hypochlorite aqueous solution under alkaline conditions, and then the obtained carboxymaltodextrin aqueous solution is complexed with an iron (III) salt to obtain carboxymaltose iron. When a single maltodextrin is used, the reducing sugar equivalent (hereinafter referred to as "DE value") of the maltodextrin is 5% to 20%, and when different types of mixed maltodextrins are used, the DE value of the mixture is also controlled to be 5% to 20%, and the DE value of each maltodextrin contained in the mixture is 2% to 40%.

[0007] Maltodextrin is the key starting material of carboxymaltose iron. Maltodextrin of different sources and properties has a significant effect on the quality of carboxymaltose iron. Maltodextrin, also known as water-soluble dextrin or enzymatic dextrin, is a starch hydrolyzate obtained by acid or enzymatic treatment. The sources of starch include corn, potato, cassava, sweet potato, rice, wheat, etc. During acid hydrolysis, the glycosidic bonds in the starch are irregularly cut and the distribution of sugars is irregular. The enzymatic method generally uses α-amylase to catalyze hydrolysis. The main hydrolysis product is oligosaccharides, with relatively few monosaccharides, and the distribution of oligosaccharides often presents a more regular normal distribution. At present, the enzymatic process is used to produce maltodextrin at home and abroad, and the acid process has been basically eliminated. The DE value is usually used to indicate the degree of starch hydrolysis. Conventional maltodextrins are starch hydrolyzates with a DE value of less than 20%.

[0008] In addition to the DE value, the degree of branching is also one of the important properties of maltodextrin. Maltodextrin is a straight chain formed by D-glucose connected by α-1,4-glycosidic bonds. There is often a small amount of D-glucose connected to the straight chain by α-1,6-glycosidic bonds to form shorter branches. In the maltodextrin molecule, the ratio of the number of α-1,6-glycosidic bonds to the sum of the number of α-1,6-glycosidic bonds and α-1,4-glycosidic bonds is called the degree of branching of maltodextrin (hereinafter referred to as "BD value"). The BD value of commercially available maltodextrin is generally between 4.0% and 10.0%, which varies depending on the biological source of the starch.

[0009] Although the original research patent (CN1705682A) has a relatively detailed description of the synthesis method of carboxyl maltose iron, and the iron content and molecular weight of carboxyl maltose iron are also given by specific embodiments, there is no data on the molecular diameter and particle size distribution of relevant carboxyl maltose iron, nor the diameter data of iron core. In the existing literature method, such as journal papers (Zhao Wei et al., Research on Preparation Technology of Carboxyl Maltose Iron, Zhejiang Chemical Industry, 2018, 49 (11): 38-40), degree thesis (Lu Hua, Synthesis Research of Maltose Iron, Master's Degree Thesis, Southeast University, 2015) and Chinese patent (CN113004428A) etc., although the molecular weight of carboxyl maltose iron and the control strategy of molecular weight distribution are described to a certain extent, there is no molecular diameter and particle size distribution of carboxyl maltose iron and the relevant data of iron core diameter.

[0010] Carboxymaltose iron molecules are nanoscale iron complex particles. In order to ensure medical safety (embolism risk), the molecular size of nanoscale iron complexes needs to be evaluated. Carboxymaltose iron molecules are composed of an iron core composed of a β-FeOOH inorganic phase and a sugar shell composed of carboxymaltodextrin wrapped around the iron core. Therefore, the diameter of the entire particle (hydrodynamic diameter) and the diameter of the iron core (single nanocrystal size) can be used to evaluate.

[0011] After testing multiple batches of carboxymaltose iron injection produced by the original manufacturer Weifu Pharmaceutical, it was found that the particle size of the original carboxymaltose iron was 24.0-28.0nm, the particle size distribution coefficient PDI≤0.14, and the iron core diameter was about 4.95-5.25nm. Further research found that the particle size, particle size distribution and iron core diameter of carboxymaltose iron prepared using a variety of commercially available maltodextrins as raw materials were difficult to keep consistent with the original product.

[0012] In order to ensure the bioequivalence of generic drugs to the original drugs, it is necessary to develop a method to stably obtain carboxymaltose ferric whose particle size, particle size distribution and iron core diameter are highly consistent with those of the original product. Summary of the invention

[0013] As mentioned above, although maltodextrin is the key starting material of ferric carboxymaltose, there is not much description in many literatures about the impact of its properties on the quality attributes of ferric carboxymaltose, especially the impact of two important properties of maltodextrin, "DE value" and "BD value", on the particle size and particle size distribution of ferric carboxymaltose and the diameter of the iron core.

[0014] The present invention provides an improved method for preparing maltodextrin. The maltodextrin prepared by the method is used for synthesizing carboxymaltose iron, and the particle size and particle size distribution as well as the iron core diameter of the maltodextrin are highly consistent with those of the original research.

[0015] According to the method provided by the original research patent (CN1705682A) and other documents, the synthesis of carboxymaltose iron generally uses maltodextrin with a DE value of 5% to 20% as a raw material, oxidizes it with a sodium hypochlorite solution under alkaline conditions to obtain carboxymaltodextrin, and then complexes it with ferric chloride to form a complex with ferric hydroxide as the core and carboxymaltodextrin as the shell, and obtains carboxymaltose iron after alcohol precipitation.

[0016] The inventors have creatively discovered through a large number of experiments and summarizing the rules: 1) When the ratio of maltodextrin and ferric chloride, the concentrations of ferric chloride solution and sodium carbonate solution, and the dropping speed of sodium carbonate solution are determined, the DE value of maltodextrin is negatively correlated with the particle size (D) of carboxymaltose iron, which can be predicted by the formula: D≈(0.64 / DE)+20. (Unit: nm); 2) When the ratio of maltodextrin and ferric chloride, the concentrations of ferric chloride solution and sodium carbonate solution, and the dropping speed of sodium carbonate solution are determined, the DE value and BD value of maltodextrin synergistically affect the particle size distribution (PDI) of carboxymaltose iron, which can be predicted by the formula: PDI≈0.14(1+2*BD) / (1+2*DE); 3) The diameter of the iron core of carboxymaltose iron has nothing to do with the DE value of maltodextrin, the ratio of maltodextrin and ferric chloride, the concentration of ferric chloride solution and sodium carbonate solution, the dripping speed of sodium carbonate solution, the batch size of the reaction, etc. The diameter of the iron core (Φ) is only positively correlated with the BD value of maltodextrin, which can be predicted by the formula: Φ≈4.95*(1+BD) 2 .(Unit: nm).

[0017] Based on the above findings, the inventors further clarified that when maltodextrin with a BD value of 0-3.0% and a DE value of 8.0%-16.0% is used to synthesize carboxymaltose iron according to certain process conditions, the particle size of the resulting product can be precisely controlled between 24.0 and 28.0 nm, the particle size distribution coefficient PDI≤0.14, and the iron core diameter is between 4.95 and 5.25 nm, which is highly consistent with the original product.

[0018] Since it is impossible to buy maltodextrin that satisfies both the BD value of 0-3.0% and the DE value of 8.0%-16.0% on the market, the inventors, based on a full understanding of the existing maltodextrin preparation technology, transformed the BD value of maltodextrin through a debranching reaction, thereby completing the present invention:

[0019] Commercially available maltodextrin (BD value is generally 3.0% to 10.0%, DE value is 8.0% to 16.0%) is dissolved in water, pullulanase (CAS No.: 9075-68-7) is added, pH is adjusted to 6.0 to 8.0, and debranching reaction is carried out at 0 to 80°C for 2 to 12 hours; pullulanase can be conveniently purchased directly from reagent companies such as Sigma-Aldrich or other enzyme preparation companies; the amount of pullulanase added is 100 to 500 U / g maltodextrin; after the debranching reaction, the enzyme can be inactivated by high temperature, or filtered with a filter with a pore size of 0.2 um to remove the enzyme; the maltodextrin aqueous solution after enzyme removal can be precipitated and crystallized by adding organic solvents such as ethanol and acetone, or by spray drying or freeze drying to obtain solid maltodextrin with a BD value of 0 to 3.0%; the maltodextrin aqueous solution after enzyme removal can also be directly used to prepare carboxymaltose iron. Maltodextrin with a BD value of 0 to 3.0% and a DE value of 8.0% to 16.0% can also be obtained by adding pullulanase and α-amylase to hydrolyze starch.

[0020] The maltodextrin prepared according to the method provided by the present invention can be used to synthesize carboxymaltose iron, and the synthesis method can refer to the original research patent or literature. Because the synthesis process of carboxymaltose has been reported in many literatures and is not the focus of the present invention, the present invention does not elaborate too much on the synthesis process of carboxymaltose iron.

[0021] The beneficial effects achieved by the present invention are as follows:

[0022] The present invention provides a method for preparing maltodextrin with a BD value of 0 to 3.0% and a DE value of 8.0% to 16.0%, based on fully revealing how the DE value and BD value of maltodextrin synergistically affect the particle size and particle size distribution of carboxymaltose iron and the diameter of the iron core. The maltodextrin obtained by this method is used to synthesize carboxymaltose iron, and the particle size of the obtained product can be accurately controlled between 24.0 and 28.0 nm, the particle size distribution coefficient PDI ≤ 0.14, and the iron core diameter is between 4.95 and 5.25 nm, which is highly consistent with the original research product and is easy to pass the bioequivalence evaluation of generic drugs.

[0023] The method of the invention is simple, has low requirements on equipment and technology, and is particularly suitable for industrial operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the 1H NMR spectrum of commercially available maltodextrin (BD value is: 1.34 / (20.38+1.38+1.34)*100%=5.80%, and the position at 4.978 ppm is the proton signal peak of α-1,6-linked C1 glycoside).

[0025] Figure 2This is the 1H NMR spectrum of debranched maltodextrin (BD value is 0; the α-1,6-linked C1 glycoside proton signal peak near 4.9 to 5.0 ppm completely disappears). DETAILED DESCRIPTION

[0026] The present invention can be more specifically understood through the following examples, which are intended to illustrate rather than limit the scope of the present invention.

[0027] Example 1: Effects of different enzymes on the DE and BD values ​​of maltodextrin

[0028] 100g of commercially available maltodextrin, add 300ml of water and stir to dissolve, adjust pH = 6.5-7.5, add different types of enzymes (the amount added is 300U / g maltodextrin), carry out debranching reaction at 50±5℃ for 6h, heat to 95±2℃ to inactivate enzyme for 0.5 hours, then cool to room temperature, and filter with 0.22um water membrane. 900ml of anhydrous ethanol is added to the filtrate, stirred and crystallized at 0-10℃ for 8h, filtered, and the obtained carboxyl maltodextrin solid is vacuum dried at 40℃ to constant weight to obtain about 95-98g of debranched maltodextrin, and its DE value and BD value are detected. The relevant results are shown in Table 1. The maltodextrin obtained by pullulanase can simultaneously meet the DE value control of 8.0%-16.0% and BD value of 0-3.0%. Table 1. Effects of different enzymes on the DE and BD values ​​of maltodextrin Note: Enzyme 1 : The amount of enzyme added in this group of experiments was 300U / g maltodextrin; Pullulanase 2 :Purchased from reagent company Sigma-Aldrich; starch branching enzyme 3 : Plant origin.

[0029] Example 2: Effect of the amount of pullulanase added on the DE value and BD value of maltodextrin

[0030] 100g of commercially available maltodextrin, add 300ml of water and stir to dissolve, adjust pH=6.5-7.5, add different amounts of pullulanase (50-600U / g maltodextrin), carry out debranching reaction at 50±5℃ for 6h, heat to 95±2℃ to inactivate enzyme for 0.5 hours, then cool to room temperature, and filter with 0.22um water membrane. 900ml of anhydrous ethanol is added to the filtrate, stirred and crystallized at 0-10℃ for 8h, filtered, and the obtained carboxyl maltodextrin solid is vacuum dried at 40℃ to dryness, and about 95-98g of debranched maltodextrin is obtained, and the DE value and BD value are detected. The relevant results are shown in Table 2. When the amount of pullulanase is above 100U / g, the BD value of the obtained maltodextrin is below 3.0%. Table 2. Effect of the amount of pullulanase added on the DE value and BD value of maltodextrin Note: Pullulanase 1 : Purchased from reagent company Sigma-Aldrich.

[0031] Example 3: Effect of temperature, time and pH value of debranching reaction on DE value and BD value of maltodextrin

[0032] 100g of commercially available maltodextrin was added with 300ml of water and stirred to dissolve, pH was adjusted to 6.0-8.0, and pullulanase (300U / g maltodextrin) was added. The debranching reaction was carried out at different temperatures for 2-10h, and the temperature was raised to 95±2℃ to inactivate the enzyme for 0.5h, then cooled to room temperature and filtered through a 0.22um water membrane. 900ml of anhydrous ethanol was added to the filtrate, and the mixture was stirred and crystallized at 0-10℃ for 8h, and then filtered. The obtained carboxyl maltodextrin solid was vacuum dried at 40℃ to dryness, and about 95-99g of debranched maltodextrin was obtained. The DE value and BD value were tested. The relevant results are shown in Table 3: Table 3. Effects of debranching reaction temperature, time and pH on DE and BD values ​​of maltodextrin Note: The pullulanase in this group of experiments was purchased from the reagent company Sigma-Aldrich, and the added amount was 300U / g maltodextrin.

[0033] Example 4: Effect of maltodextrin DE value and BD value on particle size and particle size distribution of carboxymaltose iron and iron core diameter

[0034] 50 g of maltodextrin with different DE values ​​and BD values ​​after debranching reaction is selected, 150 ml of water is added and stirred to dissolve, 0.5 g of sodium bromide is added, 20-30% sodium hydroxide solution is dropped, the pH value of the maltodextrin solution is adjusted to 9.0-11.0, the temperature is controlled at 15-40° C., 50-100 g of 10-16% sodium hypochlorite solution (1.5-3.0 eq of available chlorine) is added dropwise under stirring to obtain a carboxymaltodextrin solution.

[0035] The carboxymaltodextrin solution and the ferric chloride solution (ferric chloride concentration 75% w / w, prepared by adding water to 101.8 g of ferric chloride hexahydrate) were first mixed, stirred, and the temperature was controlled at 10-50° C. Then, a sodium carbonate solution (concentration 15% w / w, prepared by adding water to 40 g of sodium carbonate) was added dropwise at a uniform rate. The sodium carbonate solution was added in about 3 hours. After the reaction is completed, add 20-30% sodium hydroxide solution, adjust the pH value of the solution to 10.0-12.0, control the temperature at 50-70°C, and stir for about 0.5 hour (alkalization); add 20% hydrochloric acid solution dropwise, adjust the pH value of the solution to 5.0-6.0, control the temperature at 50-70°C, and stir for about 0.5 hour (acidification); raise the temperature to 90-100°C, continue stirring for about 0.5 hour (high temperature curing) to obtain a stable carboxymaltose iron complex solution; after cooling to room temperature, add 500 ml of anhydrous ethanol to precipitate carboxymaltose iron, filter, and vacuum dry at 50-70°C for 12 hours to obtain about 70 g of carboxymaltose iron.

[0036] The samples were sent for testing of the particle size, particle size distribution PDI, iron core diameter, molecular weight, etc. of carboxymaltose iron. The relevant results are shown in Table 4: Table 4. Effect of maltodextrin DE and BD values ​​on particle size and particle size distribution of carboxymaltose iron and iron core diameter Note: The pullulanase in this group of experiments was purchased from the reagent company Sigma-Aldrich, and the added amount was 300U / g maltodextrin.

Claims

1. A method for preparing maltodextrin for synthesizing ferric carboxymaltose, characterized in that: Pullulanase (CAS No.: 9075-68-7) is added to commercially available maltodextrin in water to perform a debranching reaction, and maltodextrin with a branching degree (BD value) of 0 to 3.0% and a reducing sugar equivalent (DE value) of 8.0% to 16.0% is obtained after the enzyme removal operation.

2. The method according to claim 1, wherein the commercially available maltodextrin used has a BD value of 3.0% to 10.0% and a DE value of 8.0% to 16.0%.

3. The method according to claim 1, wherein the amount of pullulanase added is 100-500 U / g maltodextrin.

4. The method according to claim 1, wherein the debranching reaction is carried out in an aqueous solution, wherein: The temperature of the debranching reaction is 10 to 70°C; The time for the debranching reaction is 2 to 12 hours; The pH of the debranching reaction is 6.0 to 8.

0.

5. The method according to claim 1, wherein after the debranching reaction is completed, the enzyme can be inactivated by high temperature or filtered through a filter with a pore size of 0.2 μm.

6. The method according to claim 1, wherein the maltodextrin aqueous solution after enzyme removal can be precipitated and crystallized by adding an organic solvent such as ethanol or acetone, or can be spray-dried or freeze-dried to obtain solid maltodextrin with a BD value of 0-3.0%.

7. The method according to claim 1, wherein the maltodextrin aqueous solution after enzyme removal can also be directly used to prepare carboxymaltose iron.

8. The method according to claim 1, wherein the prepared maltodextrin is used to synthesize carboxymaltose iron, and the particle size is 24.0 to 28.0 nm, the particle size distribution is PDI ≤ 0.14, and the iron core diameter is 4.95 nm to 5.25 nm.

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