Preparation method of bicarbonate type colestyramine and application of bicarbonate type colestyramine in reducing renal uric acid

Abstract

The invention relates to the technical field of chemical drugs, and discloses a preparation method of bicarbonate type colestyramine and application of the bicarbonate type colestyramine in reducing kidney uric acid. The method comprises the following steps: replacing chlorine-type colestimine with bicarbonate-type colestimine, sulfate-type colestimine and acetate-type colestimine by using sodium bicarbonate, sodium sulfate and sodium acetate through an ion exchange column; it is found that compared with other salt type colestyramine, bicarbonate type colestyramine can reduce uric acid of kidney, liver and other tissues of KDY rats, play a role in reducing kidney uric acid and improve the kidney microstructure of the KDY rats, and has the potential of preventing and treating gout kidneys; meanwhile, other diseases caused by rising of kidney uric acid can be prevented or treated. Macromolecules of the medicine are not easily absorbed, the side effect risk of internal absorption of the medicine is reduced, and the medicine is suitable for popularization and application.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical drugs, and in particular to a preparation method of bicarbonate-type cholestyramine and application thereof in reducing renal uric acid. Background Art

[0002] The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.

[0003] Hyperuricemia is a chronic metabolic disease caused by a disorder of purine metabolism. Hyperuricemia is diagnosed when fasting blood uric acid levels exceed 70 μg / ml on two separate days while on a normal purine diet. Its etiology is primarily due to decreased uric acid excretion or increased uric acid production. Hyperuricemia not only contributes to gout but is also a risk factor for various cardiovascular diseases, metabolic syndrome, and chronic kidney disease. Because uric acid is most readily deposited in the kidneys, gouty kidney is the most common, difficult-to-detect, and most serious complication of hyperuricemia. Gouty kidney initially develops in the medulla, where damage to the medulla leads to interstitial hyperplasia and fibrosis. Early-stage gouty kidney has minimal impact on renal function. However, with persistent hyperuricemia, medullary lesions can worsen and spread to the cortical glomeruli, leading to severe renal dysfunction and potentially life-threatening consequences. Consequently, once gouty kidney develops, it is difficult to treat. Therefore, the key to treating gouty kidney is to reduce renal uric acid levels and thus reduce renal uric acid deposition.

[0004] To reduce uric acid in the body, current treatments for hyperuricemia primarily rely on drugs that promote uricosuria (such as probenecid and benzbromarone) or inhibit uric acid synthesis (such as allopurinol and febuxostat). These drugs can cause a range of serious adverse reactions after absorption, including immunotoxicity (such as the pathological immune response triggered by allopurinol), hepatotoxicity (febuxostat), and nephrotoxicity (benzbromarone). Since humans do not contain uricase, while marketed uricase preparations (such as rasburicase) are available, these drugs require injection and can only be used once, making them unsuitable for chronic conditions like gouty kidney disease.

[0005] Therefore, the development of new, safer drugs to lower renal uric acid and treat diseases such as nephrotic syndrome is urgent. Colestyramine is a bile acid-binding resin that inhibits intestinal bile acid absorption, leading to increased fecal bile acid excretion and, in turn, increased cholesterol bile acid synthesis. However, prior art does not report its uric acid-lowering effect. Summary of the Invention

[0006] The present invention aims to address the shortcomings of the current state of the art by providing a method for preparing a bicarbonate-type cholestyramine salt and its use in lowering renal uric acid. The present invention also provides a cationic polymer salt of cholestyramine for oral administration to reduce renal uric acid levels and prevent and treat gouty kidney. Because the cationic polymer cholestyramine salt is non-degradable in vivo and not absorbed by the human body, it is expected to reduce renal uric acid levels while avoiding safety issues associated with drug absorption.

[0007] The technical solutions of the present invention are as follows:

[0008] In one aspect, the present invention provides a method for preparing bicarbonate-type cholestyramine, comprising the following steps:

[0009] Step (1): Calculate the chloride ion content of the chlorocholestyramine to be replaced based on the amount of bicarbonate cholestyramine to be prepared; bicarbonate cholestyramine is prepared using chlorocholestyramine as a raw material. The chloride ion content of the chlorocholestyramine raw material is 15-17%. To more thoroughly elute the chloride ions, a maximum value of 17% is selected for calculation.

[0010] Step (2): Calculating the amount of bicarbonate required to replace the chloride ions based on the required chloride ion content and the charge number of the bicarbonate ions;

[0011] Step (3): Chloro-type cholestyramine is loaded onto a column, first washed with water, then washed with a sodium bicarbonate solution, wherein the total amount of bicarbonate ions used for washing the column is 3-5 times the total amount of chloride ions contained, and finally washed with distilled water to obtain bicarbonate-type cholestyramine.

[0012] Cholestyramine sulfate and cholestyramine acetate were prepared in a similar manner.

[0013] Another aspect of the present invention provides bicarbonate-type cholestyramine prepared by the above-described preparation method.

[0014] Another aspect of the present invention provides the use of bicarbonate-type cholestyramine in the preparation of a drug for lowering renal uric acid.

[0015] Another aspect of the present invention provides the use of bicarbonate-type cholestyramine in preparing a medicament for preventing, improving and treating gouty kidney.

[0016] Another aspect of the present invention provides use of bicarbonate-type cholestyramine in the preparation of a medicament for preventing, improving, and treating gout.

[0017] Another aspect of the present invention provides the use of bicarbonate-type cholestyramine in the preparation of a medicament for preventing, improving and treating uric acid stones.

[0018] Another aspect of the present invention provides use of bicarbonate-type cholestyramine in the preparation of a medicament for preventing, improving, and treating acute renal failure.

[0019] Another aspect of the present invention provides use of bicarbonate-type cholestyramine in the preparation of a medicament for preventing, improving, and treating chronic kidney disease (CKD).

[0020] According to a preferred embodiment, the bicarbonate-type cholestyramine is prepared as an oral preparation.

[0021] According to a preferred embodiment, the oral dosage of the bicarbonate-type cholestyramine is 2-12 g.

[0022] Compared with the existing technology, the beneficial effects of the present invention are:

[0023] 1. A method for preparing bicarbonate-type cholestyramine is provided. The method uses sodium bicarbonate to replace chlorocholestyramine with bicarbonate-type cholestyramine via an ion exchange column. Bicarbonate-type cholestyramine is successfully prepared. This preparation method uses chlorocholestyramine as a raw material, is relatively inexpensive, and the prepared drug does not contain chloride ions, thus avoiding the burden on the kidneys caused by chloride ion absorption.

[0024] 2. The present application also provides a new application of bicarbonate-type cholestyramine. It is found that bicarbonate-type cholestyramine can reduce uric acid in the kidneys, liver and other tissues of KDY rats compared with other salt forms of cholestyramine, thereby playing a role in lowering renal uric acid and improving the microstructure of the kidneys of KDY rats. It has the potential to treat gouty kidney; at the same time, it can prevent or treat other diseases caused by elevated renal uric acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 To study the effects of oral administration of different salts of cholestyramine (0.5 g / kg) on the body weight of uricase-deficient rats (KDY rats);

[0026] Note: Control group (equal volume of drinking water); Cl - , chloride salt type cholestyramine group; HCO 3- , carbonate type cholestyramine; SO4 2- , sulfate type cholestyramine; CH3COO - , cholestyramine acetate; the experimental data of each group were processed by independent sample t test, compared with the control group, *P < 0.05;

[0027] Figure 2 To study the effects of oral administration of different salts of cholestyramine (0.5 g / kg) on the serum uric acid level in uricase-deficient rats (KDY rats);

[0028] Note: Control group (equal volume of drinking water); Cl -, chloride salt type cholestyramine group; HCO 3- , carbonate type cholestyramine; SO4 2- , sulfate type cholestyramine; CH3COO - , acetate type cholestyramine; experimental data of each group were processed using independent sample t test, compared with the control group, *P < 0.05. Preparation method of bicarbonate type cholestyramine and its application in lowering renal uric acid;

[0029] Figure 3 To study the effects of oral administration of different salts of cholestyramine (0.5 g / kg) on urinary uric acid (UUA) excretion in KDY rats;

[0030] Note: Control group (equal volume of drinking water); Cl - , chloride salt type cholestyramine group; HCO 3- , carbonate type cholestyramine; SO4 2- , sulfate type cholestyramine; CH3COO - , cholestyramine acetate; the experimental data of each group were processed by independent sample t test, compared with the control group, *P < 0.05; compared with W0 and W1, #P < 0.05;

[0031] Figure 4 To study the effects of oral administration of different salts of cholestyramine (0.5 g / kg) on tissue uric acid (Tissue UA) in KDY rats;

[0032] Note: Control group (equal volume of drinking water); Cl - , chloride salt type cholestyramine group; HCO 3- , carbonate type cholestyramine; SO4 2- , sulfate type cholestyramine; CH3COO - , cholestyramine acetate; the experimental data of each group were processed by independent sample t test, compared with the control group, *P < 0.05;

[0033] Figure 5 The effect of bicarbonate cholestyramine on the microstructure of the kidney in KDY rats (Masson staining);

[0034] Note: A, kidney of rats in the control group (low magnification); B, kidney of KDY rats in the bicarbonate-type cholestyramine group (low magnification); C, renal medulla of rats in the control group; D, renal medulla of KDY rats in the bicarbonate-type cholestyramine group. DETAILED DESCRIPTION

[0035] The specific embodiments listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of the present invention. Therefore, equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be included within the scope of the present invention. If specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without the manufacturer specified are conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are given in the specific embodiments below. Those skilled in the art should understand that the present invention can also be implemented without certain specific details. In other embodiments, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present invention.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the units used in this specification are International Standard Units, and the numerical values and numerical ranges appearing in this invention should be understood to include the inevitable systematic errors in industrial production.

[0037] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0038] Example 1 Preparation of bicarbonate-type cholestyramine

[0039] Prepare as follows:

[0040] Step (1): using cholestyramine chloride (chloride content of 15-17%) as a raw material, and calculating the chloride ion content to be replaced based on the amount of cholestyramine bicarbonate to be prepared (in order to elute the chloride ions more thoroughly, a maximum value of 17% is selected for calculation);

[0041] Step (2): Calculating the amount of bicarbonate required to replace the chloride ions based on the required chloride ion content and the charge number of the bicarbonate ions;

[0042] Step (3): Chlorocholestyramine is loaded onto a column and first washed with water. The column is then washed with a 0.5 mol / L sodium bicarbonate solution, where the total amount of bicarbonate ions used for washing the column is four times the total amount of chloride ions present. Finally, the column is washed with distilled water to obtain bicarbonate-type cholestyramine. Since chlorocholestyramine is a water-insoluble anion exchange resin, the theoretical yield of bicarbonate-type cholestyramine after sufficient exchange with sodium bicarbonate is 100% (calculated as cholestyramine).

[0043] Cholestyramine sulfate and cholestyramine acetate were prepared in a similar manner.

[0044] Cholestyramine sulfate:

[0045] Step (1): Calculate the chloride ion content to be replaced based on the amount of bicarbonate-type cholestyramine to be prepared (a maximum value of 17% is selected for calculation in order to elute the chloride ions more thoroughly);

[0046] Step (2): Calculating the amount of sulfate required to replace the chloride ions based on the required chloride ion content and the charge number of the sulfate ions;

[0047] Step (3): Chloro-cholestyramine is loaded onto a column, first washed with water, then washed with a 0.5 mol / L sodium sulfate solution, wherein the total amount of sulfate ions used for washing the column is 4 times the total amount of chloride ions contained, and finally washed with distilled water to obtain sulfate-type cholestyramine.

[0048] Cholestyramine acetate:

[0049] Step (1): Calculate the chloride ion content to be replaced based on the amount of bicarbonate-type cholestyramine to be prepared (a maximum value of 17% is selected for calculation in order to elute the chloride ions more thoroughly);

[0050] Step (2): Calculating the amount of acetate required to replace the chloride ions based on the required chloride ion content and the charge number of the acetate ions;

[0051] Step (3): Chlorocholestyramine is loaded onto a column, first washed with water, then washed with a 0.5 mol / L sodium acetate solution, wherein the total amount of acetate ions used for washing the column is 4 times the total amount of chloride ions contained, and finally washed with distilled water to obtain acetate-type cholestyramine.

[0052] Example 2 Protective Effect of Bicarbonate-Type Cholestyramine on the Kidneys of Uricase-Deficient Rats with Gout

[0053] 1. Experimental Methods

[0054] Uricase-deficient rats (KDY rats) were divided into 10 groups of 6 rats each. These groups were divided into four control groups (equal volumes of water) and groups receiving cholestyramine chloride, cholestyramine bicarbonate, cholestyramine sulfate, and cholestyramine acetate. Each cholestyramine group received 0.5 g / kg (calculated as cholestyramine chloride) once daily for 14 days. On days 0, 7, and 14, the rats were placed in metabolic cages. Their food intake was recorded for 24 hours, and urine and feces were collected. Blood was then collected by tail amputation and centrifuged (5000 rpm for 5 minutes) to prepare serum. Following the experiment, the animals were anesthetized and the liver, kidney, duodenum, ileum, colon, and gastric antrum were collected to measure tissue uric acid levels.

[0055] Uric acid test: Serum is used directly for uric acid testing; organ tissues are added with 10 times the volume of 0.05 mol / L sodium bicarbonate solution and homogenized with an electric homogenizer. Centrifuge (5000 / min, 5 min) and the supernatant is collected for testing; urine is mixed and diluted 20 times with pure water for testing; feces are added with three times the weight of 0.1 mol / L Tris base solution, placed on a shaker for mixing (120 r / min, 4 h), and centrifuged to obtain the supernatant for testing.

[0056] Blood urea nitrogen (BUN) detection: Blood was collected from the tails of KDY rats and centrifuged at 5000 g for 5 min after blood coagulation to prepare serum. Blood urate nitrogen was detected using a BUN kit (diacetyloxime colorimetric method, Nanjing Jiancheng Bioengineering Institute).

[0057] Serum creatinine detection: Blood was collected from KDY rats by tail clipping. After blood coagulation, it was centrifuged at 5000 g for 5 min to prepare serum. Serum creatinine was detected using a serum creatinine kit (sarcosine oxidase method, Nanjing Jiancheng Bioengineering Institute).

[0058] 2. Experimental Data and Conclusions

[0059] 1. Bicarbonate-type cholestyramine has no significant negative effect on the body weight of KDY rats

[0060] There was no significant difference in the weight gain trend of KDY rats in each group before and after administration (P>0.05, Figure 1 A-1D). Since weight change is one of the important sensitive indicators of drug toxicity, it is shown that the above oral administration drugs (chloride, bicarbonate, sulfate and acetate forms of cholestyramine) have no obvious toxicity.

[0061] 2. Bicarbonate-type cholestyramine can reduce blood uric acid in KDY rats

[0062] Compared with the control group, the serum uric acid (SUA) level in the chloride-type cholestyramine group was significantly increased on day 14 (P<0.001, Figure 2 A). The bicarbonate-type cholestyramine group had the most significant effect in reducing SUA, and on the 14th day, SUA was significantly lower than that in the control group (P<0.01, Figure 2 B); SUA in the sulfated cholestyramine group showed an increasing trend compared with the control group on the 7th and 14th days ( Figure 2 C); the SUA levels at each time point in the cholestyramine acetate group were not significantly different from those in the control group (P>0.05, Figure 2 D).

[0063] 3. Bicarbonate-type cholestyramine reduces urinary uric acid excretion load in KDY rats

[0064] Compared with the control group, the urinary uric acid (UUA) in the chloride-type cholestyramine group did not show significant changes during the experimental period ( Figure 3A). It is noteworthy that in the bicarbonate-type cholestyramine group, UUA was significantly lower than that in the control group on day 7 (P<0.01) and also significantly lower than that in W0 (P<0.05); however, by day 14, UUA had returned to levels close to those in the control group, with no significant difference ( Figure 3 B), indicating that the body has entered a new equilibrium period in terms of uric acid excretion. In the sulfate-type cholestyramine group, UUA had no statistical difference with the control group ( Figure 3 C). The cholestyramine acetate group showed a sustained UUA-lowering effect, with UUA levels significantly reduced on day 7 (P<0.05 vs. W0) and day 14 (P<0.01 vs. W0), with no significant difference compared with the control group ( Figure 3 D).

[0065] Comparing the blood uric acid and urine uric acid levels at the same time points, it can be seen that after using bicarbonate cholestyramine, urine uric acid excretion increased and blood uric acid content decreased in the second week; thus, the bicarbonate cholestyramine of the present application increased urine uric acid excretion, thereby reducing blood uric acid content. During the two-week observation period, blood uric acid and urine uric acid levels fluctuated significantly. However, the direct cause of gouty kidney is the accumulation of uric acid in kidney tissue, which is directly related to renal uric acid content. However, changes in blood uric acid and urine uric acid levels are influenced by other factors and are provided for reference only.

[0066] 4. Bicarbonate-type cholestyramine reduces uric acid content in renal tissue of KDY rats

[0067] The uric acid in the intestinal tract of the chloride salt cholestyramine group showed a significant upward trend, and the uric acid in the duodenum, colon and gastric antrum was significantly higher than that in the control group (P < 0.05, Figure 4 Compared with the control group, the uric acid in the bicarbonate-type cholestyramine group showed a downward trend in all tissues except the duodenum, and the uric acid in the liver, kidney and gastric antrum decreased significantly (P < 0.001, Figure 4 B); compared with the control group, the intestinal uric acid in the sulfated cholestyramine group showed an upward trend, and the liver uric acid increased significantly (P < 0.05, Figure 4 C); In the cholestyramine acetate group, the uric acid in the kidney showed a downward trend, while the uric acid in other organs showed an upward trend, with the uric acid in the duodenum and gastric antrum significantly increased (P < 0.05, Figure 4 D).

[0068] 5. Bicarbonate-type cholestyramine improves the renal microstructure of KDY rats

[0069] KDY rats will spontaneously develop hyperuricemia, and the first part of the body to be damaged is the renal medulla, which is known as gouty kidney. This is manifested as renal medulla damage and fibrosis. The early effects on renal function are relatively small, but in the late stages, the renal cortex and glomeruli will be affected, leading to severe renal function decline. The kidneys of KDY rats gavaged for 14 days were fixed with conventional 4% neutral formaldehyde, paraffin sectioned, and Masson stained (to observe the fibrosis, blue indicates positive fiber staining). It was found that the control group ( Figure 5 A) The kidneys of rats were swollen and enlarged, while those of the bicarbonate-type cholestyramine group ( Figure 5 B) The kidneys of rats shrank, indicating that bicarbonate-type cholestyramine has a protective effect on the kidneys of KDY rats. Further observation showed that compared with the control group ( Figure 5 C) compared with the bicarbonate-type cholestyramine group ( Figure 5 D) The blue staining of the renal medulla became significantly lighter, interstitial proliferation was reduced, and the lumen area of the preserved renal tubules and collecting ducts was larger, indicating that bicarbonate-type cholestyramine can alleviate medullary fibrosis in KDY rats.

[0070] The key cause of gouty kidney is the deposition of uric acid in renal tissue. Only by reducing the deposition of uric acid in the kidney can gouty kidney be fundamentally alleviated; pathological changes in renal tissue can more reliably reflect renal damage. The uric acid metabolism of uricase-deficient rats is more similar to that of humans. By comparing the uric acid-lowering effects of different salts of cholestyramine on uricase-deficient rats, it was found that bicarbonate cholestyramine can lower serum uric acid levels, significantly reduce renal uric acid content, and improve renal microstructure. This suggests that bicarbonate cholestyramine has an anti-gouty kidney effect, which is also not possessed or not significantly possessed by other salts of cholestyramine.

[0071] Although blood creatinine and blood urea nitrogen are commonly used indicators of renal function, with blood creatinine reflecting the glomerular filtration rate and blood urea nitrogen reflecting the excretion of waste products in the body, the kidneys have a strong compensatory capacity. Significant changes in blood creatinine and blood urea nitrogen will only occur when the condition is particularly severe, causing the two parameters to exceed the reference value range. Since the animal model in this example has not yet developed severe renal damage, the change trends of blood creatinine and blood urea nitrogen within the reference range have a low reference value. Therefore, this example focuses on observing more sensitive indicators: renal uric acid deposition and renal tissue pathological changes. This is consistent with the principle of gouty kidney and the concept of early intervention for the prevention and treatment of gouty kidney.

[0072] The above-described embodiments merely represent specific implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of protection of the present application. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the technical concept of the present application, and all such variations and improvements fall within the scope of protection of the present application.

Claims

1. A method for preparing bicarbonate-type cholestyramine, characterized in that: The steps include: Step (1): using cholestyramine chloride as a raw material, and calculating the chloride ion content to be replaced based on the amount of cholestyramine bicarbonate to be prepared; Step (2): Calculating the amount of bicarbonate required to replace the chloride ions based on the required chloride ion content and the charge number of the bicarbonate ions; Step (3): Chloro-type cholestyramine is loaded onto a column, first washed with water, then washed with a sodium bicarbonate solution, wherein the total amount of bicarbonate ions used for washing the column is 3-5 times the total amount of chloride ions contained, and finally washed with distilled water to obtain bicarbonate-type cholestyramine.

2. A bicarbonate-type cholestyramine, characterized in that The preparation method according to claim 1 is used for preparation.

3. Use of bicarbonate-type cholestyramine in the preparation of a drug for lowering renal uric acid, characterized in that the bicarbonate-type cholestyramine is as described in claim 2.

4. Use of bicarbonate-type cholestyramine in the preparation of a medicament for preventing, improving, and treating gouty kidney, wherein the bicarbonate-type cholestyramine is as described in claim 2.

5. Use of bicarbonate-type cholestyramine in the preparation of a medicament for preventing, improving, and treating gout, wherein the bicarbonate-type cholestyramine is as described in claim 2.

6. Use of bicarbonate-type cholestyramine in the preparation of a medicament for preventing, improving, and treating uric acid stones, wherein the bicarbonate-type cholestyramine is as described in claim 2.

7. Use of bicarbonate-type cholestyramine in the preparation of a medicament for preventing, improving, or treating acute renal failure, wherein the bicarbonate-type cholestyramine is as described in claim 2.

8. Use of bicarbonate-type cholestyramine in the preparation of a medicament for preventing, improving, or treating chronic kidney disease, wherein the bicarbonate-type cholestyramine is as described in claim 2.

9. The use of bicarbonate-type cholestyramine according to claim 8, characterized in that: The bicarbonate-type cholestyramine is prepared into an oral preparation.

10. The use of bicarbonate-type cholestyramine according to claim 9, characterized in that: The oral dosage of the bicarbonate-type cholestyramine is 2-12 g.

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