A method for preparing a suspension gel of sucralfate and a method for detecting the protein affinity of the suspension gel

Abstract

The present application relates to a kind of preparation method of sucralfate suspension gel and the method for detecting its protein affinity, belong to chemical technical field.The method of the present application is simplified, with the characteristics such as intuitive and accurate, fast, the raw materials needed for detection are easy to obtain and low in cost.The process compared with prior art, using alkali solution method continuous reaction gel technology preparation sucralfate suspension gel, not only effectively avoid using concentrated hydrochloric acid, reduce the amount of sodium hydroxide, reduce production cost, reduce environmental pollution, and the sucralfate suspension gel prepared is compared with the sucralfate suspension gel currently produced on the market, the particle size is similar, and the average particle size is less than sucralfate suspension.

Description

Technical Field

[0001] This invention relates to a method for preparing sucralfate suspension gel and a method for detecting its affinity for proteins, belonging to the field of chemical technology. Background Technology

[0002] Gastric ulcers are a common digestive tract disease, mainly caused by the self-digestion of the gastric mucosa by gastric acid and pepsin. Sucralfate is a commonly used drug for treating gastric ulcers. Sucralfate preparations are mainly used for acute and chronic gastritis, gastric and duodenal ulcers, drug-induced and stress-induced gastric mucosal damage, reflux esophagitis, and esophageal ulcers. Sucralfate has a unique dioctahedral structure and is an aluminum sucrose ester salt containing eight sulfate ions. It is insoluble in water and most organic solvents, and only soluble in acidic or alkaline solutions. Under acidic gastric conditions, sucralfate dissociates into aluminum ions and octathose. Aluminum ions promote carbonate secretion, neutralize gastric acid, and regulate mucosal pH. Octathose aggregates to form a negatively charged, high-density, viscous colloid that binds to positively charged proteins exuded from damaged mucosa, forming a protective film on the gastric mucosa surface. This film protects the gastric mucosa from gastric acid, pepsin, and bile salts, and stimulates the gastric mucosa's defense mechanisms, promoting carbonate secretion and epithelial proliferation, thereby rapidly repairing damaged gastric ulcer tissue. Sucralfate suspension gel utilizes a novel process that alters the physical morphology of sucralfate. The gel particles form a three-dimensional network of bioctahedral structures, allowing water molecules to easily penetrate the interstices, creating a strong three-dimensional structure with excellent anti-precipitation and adhesion properties, resulting in a longer gastrointestinal retention time. Sucralfate suspension gel contains no suspending agents and maintains a stable gel state without relying on any suspending agents, making it more stable and allowing for excellent coating on mucosa, thus providing longer-lasting coverage and more reliable therapeutic effects. Sucralfate suspension gel was developed by the Italian pharmaceutical biochemistry laboratory (Lisapharma SpA). It is prepared by dissolving powdered sucralfate in concentrated hydrochloric acid solution, then adding sodium hydroxide solution to adjust the pH to 4-4.5 to induce precipitation, resulting in a stable sucralfate suspension without suspending agents. The sucralfate exists in a gel form with self-sustaining properties. Other publicly available information describes the preparation method of sucralfate suspension gel similarly: sucralfate powder is dissolved in concentrated hydrochloric acid, followed by acid-base neutralization with sodium hydroxide solution to adjust the pH to 4-4.5 and induce precipitation. Because the production process of sucralfate suspension gel is relatively complex, requiring large amounts of concentrated hydrochloric acid and sodium hydroxide solution, and because concentrated hydrochloric acid is volatile and generates waste gas that pollutes the environment, reducing the amount of concentrated hydrochloric acid used, adopting more environmentally friendly and milder preparation conditions, completing the dissolution and gelation reaction of sucralfate, shortening the process flow, and simplifying post-processing operations are of great significance for reducing environmental pollution, improving safety, lowering production costs, and increasing production efficiency. Due to the complex molecular structure of sucralfate, its affinity for proteins is a key determinant of its pharmacological effects and the efficacy of sucralfate preparations. However, an effective method for detecting the affinity between sucralfate and proteins is currently lacking. Therefore, developing a method for detecting the affinity between sucralfate and proteins is a pressing issue that needs to be addressed in order to accurately evaluate the efficacy of sucralfate preparations. Summary of the Invention

[0003] The purpose of this invention is to address the shortcomings of existing technologies by proposing a method for preparing sucralfate suspension gel and a method for detecting its affinity for proteins, thereby reducing the amount of concentrated hydrochloric acid used, making the preparation method mild, and lowering production costs.

[0004] This invention achieves its objective through the following technical solution: First, it provides a method for preparing sucralfate suspension gel, comprising the following steps: Step 1: Add the prescribed amount of sucralfate powder to purified water, stir and disperse to form a suspension; Step 2: Transfer the sucralfate suspension to the first reactor with stirring at a flow rate of 10-60 mL / min, and then add 1 mol / L sodium hydroxide solution at the same flow rate until the sucralfate dissolves into a clear sucralfate solution. Step 3: Transfer the sucralfate solution obtained in Step 2 into a second reactor with a stirrer at a flow rate of 10-60 mL / min. Then add 1 mol / L dilute hydrochloric acid at the same flow rate to neutralize the solution and adjust the pH to 4.0-5.5 to form a colloid containing sucralfate gel with uniform particle size. Step 4: Centrifuge the colloid to collect the gel and discard the supernatant. Wash the gel with purified water and centrifuge again to obtain sucralfate gel. Step 5: Stir the sucralfate gel until homogeneous, add excipients, preservatives, and purified water, stir and homogenize to obtain sucralfate suspension gel.

[0005] The above method is a completely different sucralfate gelling process from existing publicly available technologies. It uses an alkaline solution to dissolve sucralfate powder and a continuous reaction device to prepare sucralfate suspension gel. This method effectively overcomes the technical problems currently faced in the industrial production of sucralfate suspension gel, and can effectively avoid the use of concentrated hydrochloric acid, reduce the amount of sodium hydroxide used, lower production costs, and reduce environmental pollution.

[0006] The present invention further achieves its objective through the following technical solutions: In step one, the ratio of sucralfate powder to purified water is 1:5-20.

[0007] In step three, the reaction temperature is controlled at 20-25℃.

[0008] The sucralfate gel has a particle size of less than 10 μm.

[0009] In step five, the weight and aluminum content of the sucralfate gel must first be tested. Then, the excipients and preservatives are added according to the following formulation range (by weight). The stirring time after adding excipients and preservatives to sucralfate gel is 30-60 minutes.

[0010] Secondly, to accurately evaluate the binding ability of sucralfate preparations to proteins, this invention establishes a method for detecting the affinity of sucralfate for proteins in an in vitro simulated gastric acid environment. Specifically, it is a method for detecting the affinity of sucralfate suspension gel for proteins, comprising the following steps: Step 1: Establish a standard curve for the absorbance of bovine serum albumin (BSA) content; Step 2: Take an appropriate amount of sucralfate suspension gel, place it in a centrifuge tube, add purified water to disperse it, then add BSA and mix it evenly. Shake it in a 37°C water bath for 30 minutes, then add hydrochloric acid-potassium chloride buffer (pH 1.0), mix it evenly, and filter it with a 0.22µm microporous filter to separate it from sucralfate and become insoluble BSA. Use the permeate as the test solution. Step 3: Add Coomassie Brilliant Blue G-250 reagent to the test solution, mix thoroughly, and measure the absorbance of the solution at a wavelength of 595 nm. Step 4: Based on the absorbance of the test solution, find the corresponding protein content on the standard curve and calculate the binding rate of sucralfate to protein.

[0011] Related studies (e.g., Huang Tianming, Xuan Xuejun, Ma Hailong. Observation on the efficacy of sucralfate suspension gel sprayed under emergency endoscopy in the treatment of peptic ulcer bleeding [J]. Chinese Medical Journal, 2013, 48(5):345-346) show that sucralfate suspension gel has stronger adhesion and longer adhesion time after entering the gastric mucosa, and can adhere to the ulcer surface, protecting the gastric mucosa from further damage. The sucralfate suspension gel prepared by this method has qualified quality control. Compared with the sucralfate suspension gel currently produced and marketed in China, its particle size is similar under a microscope, it does not easily separate after long-term storage, and it shows strong protein binding ability in in vitro detection models.

[0012] The beneficial effects of this invention are as follows: The method of this invention is simplified, and it features intuitive and accurate detection results, fast detection speed, readily available and low-cost raw materials. Compared with the prior art, this process uses an alkaline dissolution method for continuous reaction gelation to prepare sucralfate suspension gel, which not only effectively avoids the use of concentrated hydrochloric acid and reduces the amount of sodium hydroxide used, thus lowering production costs and reducing environmental pollution, but also, under a microscope, shows that the average particle size of the prepared sucralfate suspension gel is smaller than that of sucralfate suspension. Attached Figure Description

[0013] Figure 1 This is a schematic diagram of a continuous reactor device according to an embodiment of the present invention.

[0014] Figure 2 This is a process flow diagram of one embodiment of the present invention.

[0015] Figure 3 This is a photograph of the sucralfate suspension gel obtained in one embodiment of the present invention under a 400x microscope.

[0016] Figure 4 for Figure 3 Photographs showing the measured particle size.

[0017] Figure 5 This is a protein standard curve diagram according to an embodiment of the present invention. Detailed Implementation Example

[0018] This embodiment illustrates the continuous reactor apparatus. Figure 1 ,according to Figure 2 The specific preparation steps are as follows: 1. Prescription: (1) Sucralfate: 100g; (2) Preparation of 1N dilute hydrochloric acid: Take 90ml of concentrated hydrochloric acid and dilute to a final volume of 1000ml; (3) Preparation of 1N sodium hydroxide solution: Take 100g of sodium hydroxide and dissolve and dilute it to 2500ml; 2. Gel forming process: Add 1000ml of purified water to a 5000ml reaction flask. While stirring rapidly, add 100g of sucralfate in portions to disperse the solid powder into a suspension. Add 1000ml of 1mol / L sodium hydroxide solution dropwise. After dissolving, slowly add 1N dilute hydrochloric acid to adjust the pH to 4.0-5.5. Once the pH stabilizes, continue stirring for 30 minutes. After thoroughly cooling in an ice bath, place the reaction solution in a centrifuge and centrifuge at 3000 rpm for 15 minutes. Discard the supernatant and collect the precipitate. Add purified water to the precipitate, wash thoroughly, and centrifuge. Repeat the washing process several times until the conductivity of the supernatant is less than 2.0.

[0019] 3. Determination of aluminum content in intermediates: Accurately weigh an appropriate amount of this product (approximately equivalent to 0.5g of sucralfate), add 10ml of dilute hydrochloric acid, shake to dissolve the sucralfate, neutralize with ammonia solution until a precipitate just forms, then add dilute hydrochloric acid dropwise until the precipitate just dissolves. Add 20ml of acetate-ammonium acetate buffer (pH 6.0), then accurately add 25ml of disodium ethylenediaminetetraacetate titrant (0.05mol / L), boil for 3-5 minutes, cool to room temperature, add 1ml of xylenol orange indicator, and titrate with zinc titrant (0.05mol / L) until the solution changes from yellow to red. Correct the titration result with a blank test. Calculate the relative density based on the product. Each 1ml of disodium ethylenediaminetetraacetate titrant (0.05mol / L) is equivalent to 1.349mg of Al.

[0020] The content of sucralfate in the suspension gel was calculated based on the aluminum content determination results. Based on this, the amount of excipients, preservatives, and purified water added was calculated to control product quality and ensure that the aluminum content meets the requirements of the quality standard.

[0021] 4. Gel preparation process: Transfer the precipitate to a beaker and stir well. Based on the weight of the sucralfate suspension gel and the aluminum content, apply the proportions according to the content range in the table below: Add calculated amounts of sorbitol, flavorings and other excipients, sodium methylparaben and other preservatives, and purified water. Stir rapidly and vigorously for 30-60 minutes, then emulsify using a homogenizer to form a suspension gel (sucralfate content volume ratio of 5ml:1g).

[0022] 5. Testing: (1) Relative density: The relative density of this product should be 1.130 to 1.200.

[0023] (2) Acid-reducing power: Based on the relative density of this product, the amount of hydrochloric acid titrant (0.1mol / L) consumed per 1g of sucralfate shall not be less than 120ml.

[0024] (3) pH value: should be 4.0 to 5.5.

[0025] (4) Viscosity: should be 3.08-8.0 Pa·s (4) Particle fineness: Take this product, shake it thoroughly, add a few drops of water and stir well. Examine three fields of view under a 300-400x microscope. The diameter of more than 85% of the particles should not be greater than 10 μm.

[0026] A photograph of sucralfate suspension gel under a 400x microscope is shown below. Figure 3 As shown in the image, the particle size of the sucralfate suspension gel was measured using a microscope. Figure 4 As shown. Compared with domestically produced and marketed sucralfate suspension gels, its particle size is similar when observed under a microscope, and it does not easily separate into layers after prolonged storage.

[0027] 6. Protein binding ability: To evaluate the affinity of sucralfate suspension gel for proteins, which is one of the efficacy indicators of sucralfate, an in vitro model simulating the gastric acid environment and the binding ability of bovine serum albumin was established, and the following method was used for detection.

[0028] (1) Plotting the protein standard curve: Take 6 stoppered test tubes, number them, and add reagents according to Table 1.

[0029] Table 1

[0030] Replace the stopper and shake well. After standing for 5 minutes, measure the absorbance using spectrophotometry at a wavelength of 595 nm (the measurement should be completed within 1 hour). Plot a standard curve with bovine serum albumin content (μg) on ​​the x-axis and absorbance on the y-axis as shown below. Figure 5 As shown.

[0031] (2) Sample solution preparation: Take an appropriate amount of sucralfate suspension gel according to the aluminum content (6.0 mg aluminum content is approximately equivalent to 30 mg sucralfate), place it in a 100 ml centrifuge tube, add 5 ml of purified water to disperse it, then add 15 ml of BSA stock solution and 20 ml of hydrochloric acid-potassium chloride buffer (pH 1.0) and mix well. Shake in a 37℃ water bath for 30 min. Then filter with a 0.22 μm microporous membrane to separate the BSA that is bound to sucralfate and becomes insoluble. Use the permeate as the sample solution.

[0032] (3) Determination of absorbance of the test solution: Dilute the BSA stock solution to a standard solution of the same concentration as the sample solution (take 3 ml of BSA stock solution and add 1 ml of purified water and 4 ml of hydrochloric acid-potassium chloride buffer). Take 1 ml of the standard solution and dilute it with the sample solution to 20 ml to obtain the standard test solution and the sample test solution. Take two stoppered test tubes and accurately add 1 ml of the standard test solution and the sample test solution to each tube. Then add 5 ml of Coomassie Brilliant Blue G-250 reagent, mix thoroughly, and let stand for 5 min. Use test tube No. 1 of the standard curve as a reference and measure the absorbance at a wavelength of 595 nm using spectrophotometry (the measurement should be completed within 1 hour). Record the absorbance.

[0033] (4) Calculation of results: Based on the absorbance of the test solution, find the corresponding protein content P (μg) on ​​the standard curve, and calculate the protein binding capacity E of this sucralfate suspension gel as expressed as protein binding rate according to the following formula: In the formula: E represents the protein binding capacity of the sucralfate suspension gel, expressed as protein binding rate; P1 is the protein content (μg) measured in the permeate after binding with sucralfate suspension gel. P2 represents the protein content (μg) measured in a standard solution of the same concentration that was not bound to sucralfate suspension gel. Results are shown in Table 2. Table 2 Example

[0034] 1. Prescription: (1) Sucralfate powder: 50 kg; (2) Preparation of 1 mol / L dilute hydrochloric acid: Take 45 L of concentrated hydrochloric acid and dilute it to a final volume of 500 L; (3) Preparation of 1 mol / L sodium hydroxide solution: Take 50 kg of sodium hydroxide and dissolve and dilute it to 1250 L; 2. Gel forming process: Add 500L of purified water to reaction vessel A (mixing vessel). Add 50kg of sucralfate powder in portions while rapidly stirring to disperse the solid powder into a suspension. Use a peristaltic pump to transfer the sucralfate suspension to reaction vessel B (dissolving vessel) with a stirrer at a controlled flow rate. While rapidly stirring, add 1N sodium hydroxide solution at the same flow rate. Then, use a peristaltic pump to transfer the sucralfate solution to reaction vessel C (neutralizing vessel) with a stirrer at a controlled flow rate. While rapidly stirring, control the temperature of the neutralization reaction at 20-25℃. Add 1N dilute hydrochloric acid at the same flow rate to adjust the pH to 4.0-5.5. After the pH stabilizes, continue stirring for 30-60 minutes. After sufficient cooling, transfer the reaction solution to a centrifuge. After high-speed centrifugation, discard the supernatant and collect the precipitate. Add purified water to the precipitate and wash thoroughly. Centrifuge again and repeat the washing process with purified water multiple times until the conductivity of the supernatant is less than 2.0, indicating that the sodium chloride impurity has been largely removed.

[0035] 3. Determination of aluminum content in intermediates: Accurately weigh an appropriate amount of this product (approximately equivalent to 0.5g of sucralfate), add 10ml of dilute hydrochloric acid, shake to dissolve the sucralfate, neutralize with ammonia solution until a precipitate just forms, then add dilute hydrochloric acid dropwise until the precipitate just dissolves. Add 20ml of acetate-ammonium acetate buffer (pH 6.0), then accurately add 25ml of disodium ethylenediaminetetraacetate titrant (0.05mol / L), boil for 3-5 minutes, cool to room temperature, add 1ml of xylenol orange indicator, and titrate with zinc titrant (0.05mol / L) until the solution changes from yellow to red. Correct the titration result with a blank test. Calculate the relative density based on the product. Each 1ml of disodium ethylenediaminetetraacetate titrant (0.05mol / L) is equivalent to 1.349mg of Al.

[0036] 4. Gel preparation process: Transfer the precipitate to a preparation tank and stir well. Based on the weight and aluminum content of the sucralfate suspension gel, add the calculated amounts of excipients, preservatives, and purified water. Stir rapidly and vigorously for 30-60 minutes, then emulsify with a homogenizer to form a suspension gel (sucralfate content volume ratio of 5ml:1g).

[0037] 5. Testing: (1) Relative density: The relative density of this product should be 1.130 to 1.200.

[0038] (2) Acid-reducing power: Based on the relative density of this product, the amount of hydrochloric acid titrant (0.1mol / L) consumed per 1g of sucralfate shall not be less than 120ml.

[0039] (3) pH value: should be 4.0 to 5.5.

[0040] (4) Viscosity: should be 3.08-8.0 Pa·s (4) Particle fineness: Take this product, shake it thoroughly, add a few drops of water and stir well. Examine three fields of view under a 300-400x microscope. The diameter of more than 85% of the particles should not be greater than 10μm.

[0041] 6. Protein binding ability: To evaluate the binding ability of sucralfate, one of its therapeutic effects, to serum proteins, an in vitro model simulating the gastric acid environment was established, and the following methods were used for testing.

[0042] Protein standard curve plotting: Take 6 stoppered test tubes, number them, and add reagents according to Table 3.

[0043] Table 3

[0044] Replace the stopper and shake well. After standing for 5 minutes, measure the absorbance at 595 nm using spectrophotometry (the measurement should be completed within 1 hour). Plot a standard curve with bovine serum albumin content (μg) on ​​the x-axis and absorbance on the y-axis.

[0045] (2) Sample solution preparation: Take an appropriate amount of sucralfate suspension gel according to the aluminum content (6.0 mg aluminum content is approximately equivalent to 30 mg sucralfate), place it in a 100 ml centrifuge tube, add 5 ml of purified water to disperse it, then add 15 ml of BSA stock solution and 20 ml of hydrochloric acid-potassium chloride buffer (pH 1.0) and mix well. Shake in a 37℃ water bath for 30 min. Then filter with a 0.22 μm microporous membrane to separate the BSA that is bound to sucralfate and becomes insoluble. Use the permeate as the sample solution.

[0046] (3) Determination of absorbance of the test solution: Dilute the BSA stock solution to a standard solution of the same concentration as the sample solution (take 3 ml of BSA stock solution and add 1 ml of purified water and 4 ml of hydrochloric acid-potassium chloride buffer). Take 1 ml of the standard solution and dilute it with the sample solution to 20 ml to obtain the standard test solution and the sample test solution. Take two stoppered test tubes and accurately add 1 ml of the standard test solution and the sample test solution to each tube. Then add 5 ml of Coomassie Brilliant Blue G-250 reagent, mix thoroughly, and let stand for 5 min. Use test tube No. 1 of the standard curve as a reference and measure the absorbance at a wavelength of 595 nm using spectrophotometry (the measurement should be completed within 1 hour). Record the absorbance.

[0047] (4) Calculation of results: Based on the absorbance of the test solution, find the corresponding protein content P (μg) on ​​the standard curve, and calculate the protein binding capacity E of this sucralfate suspension gel as expressed as protein binding rate according to the following formula: In the formula: E represents the protein binding capacity of the sucralfate suspension gel, expressed as protein binding rate; P1 is the protein content (μg) measured in the permeate after binding with sucralfate suspension gel. P2 represents the protein content (μg) measured in a standard solution of the same concentration that has not been bound to sucralfate suspension gel.

[0048] The test results are shown in Table 4: Table 4

[0049] In addition to the above-described embodiments, the present invention may have other implementations. All technical solutions formed by equivalent substitution or equivalent transformation fall within the protection scope claimed by the present invention.

Claims

1. A method for preparing a sucralfate suspension gel, comprising the following steps: Step 1: Add the prescribed amount of sucralfate powder to purified water, stir and disperse to form a suspension; Step 2: Transfer the sucralfate suspension into the first reactor equipped with a stirrer at a flow rate of 10-60 mL / min and stir. At the same time, add 1 mol / L sodium hydroxide solution at the same flow rate and react until the sucralfate dissolves into a clear sucralfate solution. Step 3: Transfer the sucralfate solution obtained in Step 2 into a second reactor with a stirrer at a flow rate of 10-60 mL / min and stir. Then add 1 mol / L dilute hydrochloric acid at the same flow rate to neutralize the solution and adjust the pH to 4.0-5.

5. After the pH stabilizes, continue stirring for 30 min and cool thoroughly in an ice bath to form a colloid containing sucralfate gel with uniform particle size. Step 4: Centrifuge the colloid to collect the gel and discard the supernatant. Wash the gel with purified water and centrifuge again to obtain sucralfate gel. Step 5: Stir the sucralfate gel until homogeneous, add excipients, preservatives, and purified water, stir and homogenize to obtain sucralfate suspension gel.

2. The method for preparing sucralfate suspension gel according to claim 1, characterized in that... In step one, the ratio of sucralfate powder to purified water is 1:5-20.

3. The method for preparing sucralfate suspension gel according to claim 1, characterized in that: In step three, the reaction temperature is controlled at 20-25℃.

4. The method for preparing sucralfate suspension gel according to claim 3, characterized in that: The sucralfate gel has a particle size of less than 10 μm.

5. The method for preparing sucralfate suspension gel according to claim 1, characterized in that: In step five, the weight and aluminum content of sucralfate gel are measured before stirring, and the following excipients and preservatives are added in the following weight ratios: sorbitol 1-5%, flavoring 0.5-2%, aspartame 0.1-0.5%, sodium p-hydroxybenzoate 0.1-0.3%, and sodium benzoate 0.1-0.3%.

6. The method for preparing sucralfate suspension gel according to claim 5, characterized in that: The stirring time is 30-60 minutes.

7. A method for detecting the affinity of sucralfate suspension gel obtained by the preparation method of sucralfate suspension gel according to claim 1 for protein, comprising the following steps: Step 1: Establish a standard curve for the absorbance of bovine serum albumin (BSA) content; Step 2: Take an appropriate amount of sucralfate suspension gel, place it in a centrifuge tube, add purified water to disperse it, then add BSA and mix it evenly. Shake in a 37°C water bath for 30 minutes, then add bovine serum albumin solution prepared with pH 1.0 hydrochloric acid-potassium chloride buffer, mix evenly, and filter it with a 0.22µm microporous filter to separate it from sucralfate and become insoluble BSA. Use the permeate as the test solution. Step 3: Add Coomassie Brilliant Blue G-250 reagent to the test solution, mix thoroughly, and measure the absorbance of the solution at a wavelength of 595 nm using the Coomassie Brilliant Blue G-250 staining method. Step 4: Based on the absorbance of the test solution, find the corresponding protein content on the standard curve and calculate the binding rate of sucralfate to protein.

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