Method for detecting the adsorption efficiency of drugs on nucleic acid metabolites and its application

Abstract

The present invention provides a method for detecting the adsorption efficiency of a drug on a nucleic acid metabolite. The method comprises: mixing a buffer containing a nucleic acid metabolite with a drug to obtain a test solution; performing high performance liquid chromatography on the test solution, and determining the adsorption efficiency of the drug on the nucleic acid metabolite based on the test result, wherein the nucleic acid metabolite comprises at least one selected from the following: uric acid, adenine, guanine, hypoxanthine, adenine nucleoside, guanine nucleoside, inosine and 5-adenosine monophosphate; the mobile phase used in the high performance liquid chromatography detection comprises a buffer and / or an organic solvent, and the pH value of the buffer is 3.6 to 4.0.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular, to a method for detecting the adsorption efficiency of a drug on a nucleic acid metabolite and an application thereof, and more particularly, to a method for detecting the adsorption efficiency of a drug on a nucleic acid metabolite and a method for screening drugs. Background Art

[0002] The 2019 Guidelines for the Diagnosis and Treatment of Hyperuricemia and Gout in China point out that hyperuricemia is a metabolic syndrome caused by purine metabolism disorders. Hyperuricemia (HUA) refers to the fasting blood uric acid level of two different days in men higher than 420μmol / L and women higher than 360μmol / L under normal purine diet, which is called hyperuricemia. When the blood uric acid exceeds its saturation in the blood or tissue fluid, uric acid crystals can form and deposit in the joints, inducing local inflammatory reactions and tissue destruction, namely gout; it can be deposited in the kidneys to cause acute kidney disease, chronic interstitial nephritis or kidney stones, which is called uric acid nephropathy. Many evidences show that hyperuricemia and gout are independent risk factors for diseases such as chronic kidney disease, hypertension, cardiovascular and cerebrovascular diseases and diabetes, and are independent predictors of premature death. At present, the prevalence of hyperuricemia in China is 13.3%, and the number of patients has exceeded 120 million; the prevalence of gout in China is 1.1%, and the number of patients has exceeded 17 million. Hyperuricemia and gout have many hazards. Among them, tophi are an important cause of death and disability caused by gout. In addition, joint deformities, gouty nephropathy, cardiovascular and cerebrovascular diseases, etc. cannot be ignored.

[0003] Uric acid is a decomposition metabolite of purine nucleotides in the human body. 80% of purine nucleotides are produced by cell metabolism, and 20% are derived from diet. Uric acid is mainly derived from self-synthesis, and very little comes from food. The synthesis of purine is mainly carried out in the liver and small intestine tissues containing xanthine oxidase. Nucleic acids in food mainly exist in the form of nucleoproteins, which are decomposed into nucleic acids and proteins by gastric acid in the stomach. Nucleic acids enter the intestine and are hydrolyzed by various enzymes into bases and pentoses. The nucleotides in human cells are hydrolyzed into nucleosides under the action of nucleotidases. Nucleosides are decomposed into free bases and 1-phosphoribose under the action of nucleoside phosphorylase. Purine bases can be further hydrolyzed. The decomposition of purines starts with hydrolysis and deamination. Adenine is hydrolyzed to hypoxanthine, and guanine is hydrolyzed to xanthine. Deamination reactions can be carried out at the base, nucleoside or nucleotide level. Hypoxanthine is oxidized to xanthine, which is then oxidized to uric acid. Purine bases in the human body are eventually hydrolyzed into uric acid.

[0004] Currently, there are drugs on the market such as allopurinol that inhibit xanthine oxidase to inhibit uric acid synthesis; benzbromarone, a drug that promotes uric acid excretion, promotes renal uric acid excretion by inhibiting the reabsorption of uric acid by the renal tubules. The market for polymer drugs that adsorb nucleotide metabolites is lacking, and methods for detecting the adsorption capacity of these drugs for nucleotide metabolites are also lacking, and the accuracy is not high.

[0005] It can be seen that it is necessary to develop a method that can efficiently, quickly and accurately detect the adsorption of drugs to various nucleotide metabolites, so as to provide a more convenient and effective method for the screening of polymer drugs that adsorb nucleotide metabolites. Summary of the invention

[0006] To this end, in a first aspect of the present invention, a method for detecting the adsorption efficiency of a drug on a nucleic acid metabolite is proposed. According to an embodiment of the present invention, the method comprises: mixing a buffer solution containing nucleic acid metabolites with a drug to obtain a test solution; performing high performance liquid chromatography on the test solution, and determining the adsorption efficiency of the drug on the nucleic acid metabolite based on the test result, wherein the nucleic acid metabolite comprises at least one selected from the following: uric acid, adenine, guanine, hypoxanthine, adenosine, guanosine, inosine and 5-adenosine monophosphate; the mobile phase used in the high performance liquid chromatography detection comprises a buffer solution and / or an organic solvent, and the pH value of the buffer solution is 3.6 to 4.0.

[0007] The method according to the embodiment of the present invention is based on HPLC, and can achieve baseline separation of up to 8 nucleotide metabolites in the test solution, providing a basis for accurately detecting the adsorption degree of uric acid, adenine, guanine, hypoxanthine, adenine nucleoside, guanine nucleoside, inosine and 5-adenosine monophosphate by polymer drugs that adsorb anions. For large-scale screening of the adsorption degree of polymer drugs that adsorb anions, it can improve detection efficiency, save detection time, and save detection costs.

[0008] According to an embodiment of the present invention, the above method may further include at least one of the following additional technical features:

[0009] According to an embodiment of the present invention, the pH value of the buffer solution is 4.0. After a large number of experiments, the inventors found that when the pH of the mobile phase buffer solution is 4.0, the separation between adjacent chromatographic peaks can be further improved, and the separation of uric acid, adenine, guanine, hypoxanthine, adenine nucleoside, guanine nucleoside, inosine and 5-adenosine monophosphate is good, the standard curve correlation coefficient r is greater than 0.999, and the repeatability of the determination is high.

[0010] According to an embodiment of the present invention, the buffer is selected from at least one of the following: phosphate buffer, citrate buffer, acetate buffer and formic acid buffer, preferably phosphate buffer. According to an embodiment of the present invention, the above four buffers as mobile phase A can achieve good separation effect of the above 8 substances. When the mobile phase A is phosphate buffer, especially potassium dihydrogen phosphate buffer, the separation and detection effect of the above 8 substances is the best.

[0011] According to an embodiment of the present invention, the organic solvent includes acetonitrile. According to an embodiment of the present invention, when the mobile phase B is acetonitrile, the amount of each substance in the test solution can be effectively determined, and then the adsorption degree of the drug to be detected on the above 8 substances can be obtained.

[0012] According to an embodiment of the present invention, the high performance liquid chromatography detection adopts a gradient elution method, and the conditions of the gradient elution are: at 0-15 minutes, the volume ratio of the buffer solution to the organic solvent is 100%:0%; at 15-20 minutes, the volume ratio of the buffer solution to the organic solvent is (95%-100%):(0%-5%); at 20-40 minutes, the volume ratio of the buffer solution to the organic solvent is 95%:5%; at 40-41 minutes, the volume ratio of the buffer solution to the organic solvent is (95%-100%):(0%-5%); at 41-60 minutes, the volume ratio of the buffer salt to the organic solvent is 100%:0%. According to an embodiment of the present invention, the peak shape obtained by the gradient elution method is the best.

[0013] According to an embodiment of the present invention, the conditions of the gradient elution are: 0-15 minutes, the volume ratio of the buffer to the organic solvent is 100%:0%; 15-20 minutes, the volume ratio of the buffer to the organic solvent is 100%:0%; 20-40 minutes, the volume ratio of the buffer to the organic solvent is 95%:5%; 40-41 minutes, the volume ratio of the buffer to the organic solvent is 95%:5%; 41-60 minutes, the volume ratio of the buffer to the organic solvent is 100%:0%. After a large number of experiments, the inventors found that the above-mentioned gradient elution method and mobile phase ratio can achieve the best separation effect and better peak shape for the above-mentioned 8 substances.

[0014] According to an embodiment of the present invention, the flow rate of the mobile phase is 0.2 mL / min-1.0 mL / min, preferably 0.6 mL / min. The method according to an embodiment of the present invention can improve the detection sensitivity and further improve the separation degree.

[0015] According to an embodiment of the present invention, the buffer containing nucleic acid metabolites contains a buffer reagent, and the buffer reagent includes at least one selected from the following: sodium hydroxide, phosphate, 2-(diethanolamino)ethanesulfonic acid (BES), tromethamine·hydrochloric acid, citrate and carbonate; preferably potassium dihydrogen phosphate. According to an embodiment of the present invention, the use of the above-mentioned buffer reagent can well simulate the in vivo environment, so that the prepared buffer containing nucleic acid metabolites can more accurately simulate the adsorption of nucleotide metabolites by the drug to be detected in the body, thereby making the detected adsorption closer to the adsorption of the drug to be detected in the body, making the detection more accurate.

[0016] According to an embodiment of the present invention, the pH of the buffer reagent is 6.0 to 8.0, preferably 6.5 to 7.0. According to an embodiment of the present invention, the buffer reagent is closer to the pH in the human body, and can more accurately simulate the environment in the human body, simulate the environment in which the drug to be detected adsorbs nucleotide metabolites in the human body, and make the detected adsorption degree closer to the adsorption degree of the drug in the human body.

[0017] According to an embodiment of the present invention, the mixing is carried out at 30°C to 40°C for 30min to 24h. According to an embodiment of the present invention, a buffer solution containing nucleic acid metabolites is prepared under the above mixing conditions, and the proportions of uric acid, adenine, guanine, hypoxanthine, adenine nucleoside, guanine nucleoside, inosine and 5-adenosine monophosphate simulate the proportions in vivo, so that the detected drug adsorption is closer to the actual value in vivo.

[0018] According to an embodiment of the present invention, the chromatographic column used in the high performance liquid chromatography detection is an octadecylsilane bonded silica gel column; the particle size of the chromatographic column is 3 μm to 5 μm, preferably 3 μm; the length of the chromatographic column is 100 mm to 300 mm, preferably 150 mm. According to an embodiment of the present invention, the separation degree of the detection of the above 8 nucleotide metabolites using the above chromatographic column is higher, and the standard curve correlation coefficient is high.

[0019] According to an embodiment of the present invention, the column temperature used in the high performance liquid chromatography detection is 20-40° C. According to an embodiment of the present invention, when the above column temperature is used, the separation degree is higher and the detection accuracy is better.

[0020] According to an embodiment of the present invention, the detection wavelength used in the HPLC detection is 220nm-300nm, preferably 260nm. According to an embodiment of the present invention, the detection sensitivity can be significantly improved by using the above detection wavelength.

[0021] According to an embodiment of the present invention, the adsorption efficiency is characterized in the form of the binding amount of each nucleic acid metabolite.

[0022] The binding amount of each nucleic acid metabolite is determined by the following formula:

[0023]

[0024] Among them, a is the slope of the linear regression equation; b is the intercept of the linear regression equation; A 供 is the peak area of ​​the test solution; C 初 is the initial concentration of each nucleic acid metabolite in the buffer containing nucleic acid metabolites, in mg / L; V is the dilution volume of the test solution, in L; m is the sample weight of the test sample, in g; W is the drying loss of the test sample, in %. According to an embodiment of the present invention, based on the initial amounts and the amounts after adsorption of uric acid, adenine, guanine, hypoxanthine, adenine nucleoside, guanine nucleoside, inosine and 5-adenosine monophosphate, the adsorption degree of the drug to be tested on the above 8 substances, that is, the binding amount of each substance, can be obtained.

[0025] In the second aspect of the present invention, the present invention proposes a method for screening drugs. According to an embodiment of the present invention, the method comprises: providing a candidate drug; using the method proposed in the first aspect of the present invention to detect the adsorption efficiency of the candidate drug on nucleic acid metabolites, and determining whether the candidate drug is a target drug based on the obtained adsorption efficiency. According to an embodiment of the present invention, the method is simple to operate and has high accuracy. It can screen high-molecular drugs that efficiently adsorb anions in large quantities, improve detection efficiency, save detection time, and save detection costs.

[0026] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0028] Figure 1 is a high performance liquid chromatogram of the sample solution obtained under the conditions of Example 1;

[0029] Figure 2 is a high performance liquid chromatogram of the sample solution obtained under the conditions of Example 2;

[0030] Figure 3 is a high performance liquid chromatogram of the sample solution obtained according to the conditions of Example 3;

[0031] Figure 4 is a high performance liquid chromatogram of the sample solution obtained according to the conditions of Example 4;

[0032] Figure 5 The HPLC chromatogram of the sample solution obtained according to the conditions of Comparative Example 1;

[0033] Figure 6 The HPLC chromatogram of the sample solution obtained according to the conditions of Comparative Example 2;

[0034] Figure 7 The following is a high performance liquid chromatogram of the sample solution obtained under the conditions of Comparative Example 3. DETAILED DESCRIPTION

[0035] Embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0036] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0037] It should be noted that the nucleotide metabolites used in this article refer to the intermediates and final products produced during the catabolism of 5-adenosine monophosphate (AMP), guanine nucleotide (GMP) and inosine nucleotide (IMP) in the human body. Specifically, the nucleotide metabolites in this article refer to 5-adenosine monophosphate (AMP), guanine nucleotide (GMP) and inosine nucleotide (IMP), and at least one of their metabolic products uric acid, adenine, guanine, inosine, adenine nucleoside, guanosine and inosine.

[0038] Method for detecting the adsorption efficiency of drugs on nucleic acid metabolites

[0039] In one aspect of the present invention, the present invention proposes a method for detecting the adsorption efficiency of a drug on a nucleic acid metabolite. According to an embodiment of the present invention, the method comprises: mixing a buffer containing a nucleic acid metabolite with a drug to obtain a test solution; performing high performance liquid chromatography detection on the test solution, and determining the adsorption efficiency of the drug on the nucleic acid metabolite based on the detection result, wherein the nucleic acid metabolite comprises at least one selected from the following: uric acid, adenine, guanine, hypoxanthine, adenosine, guanosine, inosine and 5-adenosine monophosphate; the mobile phase used in the high performance liquid chromatography detection comprises a buffer and / or an organic solvent, and the pH value of the buffer is 3.6 to 4.0. According to an embodiment of the present invention, the detection method has high sensitivity, strong specificity, high precision, high accuracy, and convenient operation, and can effectively detect the amount of uric acid, adenine, guanine, hypoxanthine, adenosine, guanosine, inosine and 5-adenosine monophosphate adsorbed by a polymer drug that adsorbs anions.

[0040] According to some embodiments of the present invention, the buffer containing nucleic acid metabolites includes uric acid, adenine, guanine, hypoxanthine, adenine nucleoside, guanosine nucleoside, inosine and 5-adenosine monophosphate and a buffer reagent. The above eight nucleotide metabolites are formulated into a solution, and the pH and other conditions of the solution are close to the environment in the human body, thereby simulating the environment in which the drug adsorbs the nucleotide metabolites in the body.

[0041] According to some embodiments of the present invention, the drug to be tested is weighed and placed in a conical flask, a buffer containing nucleic acid metabolites is added, and the mixture is stirred with a magnetic stirrer for 30 min to 24 h in a water bath at 37°C ± 5°C for binding. The sample solution is then allowed to stand, and the supernatant is filtered to detect the amount of uric acid, adenine, guanine, hypoxanthine, adenosine, guanosine, inosine and 5-adenosine monophosphate adsorbed by the polymer drug that adsorbs anions.

[0042] According to some embodiments of the present invention, the environment in which the drug to be detected is combined with the nucleotide metabolites in the buffer containing nucleic acid metabolites is: water bath temperature 37°C ± 5°C, magnetic stirring for 30min to 24h, or air bath temperature 37°C ± 5°C, 100rpm to 300rpm for 30min to 24h, or water bath shaker 37°C ± 5°C, 100rpm to 300rpm. Preferably, the water bath temperature is 37°C ± 5°C, and magnetic stirring is 30min to 24h.

[0043] In some embodiments of the present invention, potassium dihydrogen phosphate buffer is used as mobile phase A, acetonitrile is used as mobile phase B, and the concentration of potassium dihydrogen phosphate buffer is 0.01 mol / L to 0.10 mol / L, preferably 0.05 mol / L.

[0044] According to some embodiments of the present invention, the conditions for gradient elution are: at 0-15 minutes, the volume ratio of the potassium dihydrogen phosphate buffer to acetonitrile is 100%:0%; at 15-20 minutes, the volume ratio of the potassium dihydrogen phosphate buffer to acetonitrile is 100%:0%, 95%:5%; at 20-40 minutes, the volume ratio of the potassium dihydrogen phosphate buffer to acetonitrile is 95%:5%; at 40-41 minutes, the volume ratio of the potassium dihydrogen phosphate buffer to acetonitrile is 95%:5%, 100%:0%; at 41-60 minutes, the volume ratio of the potassium dihydrogen phosphate buffer to acetonitrile is 100%:0%.

[0045] According to a specific embodiment of the present invention, it can be implemented according to the following steps:

[0046] (1) Prepare a buffer solution containing nucleic acid metabolites (potassium dihydrogen phosphate buffer pH 6.8)

[0047] (2) Weigh the sample and place it in a conical flask. Add a buffer containing nucleic acid metabolites. Stir with a magnetic stirrer in a water bath at 37°C ± 5°C for 30 min to 24 h to allow the sample to bind. Then let the sample solution stand, filter the supernatant, and inject it into a high performance liquid chromatography instrument.

[0048] (2) The column temperature of the chromatographic column is set to 25° C.-35° C., preferably 30° C. The flow rate of the mobile phase is 0.5-0.7 mL / min, preferably 0.6 ml / min, and the detection wavelength is 254 nm-262 nm, preferably 260 nm.

[0049] (3) Take 2 μL-20 μL of the sample solution of (2), preferably 10 μL, and inject it into a high performance liquid chromatograph to complete the determination.

[0050] Based on the formula:

[0051]

[0052] Complete the adsorption efficiency of the drug to be tested on the nucleic acid metabolites,

[0053] Among them, a is the slope of the linear regression equation; b is the intercept of the linear regression equation; A 供 is the peak area of ​​the test solution; C 初 is the initial concentration of each nucleic acid metabolite in the buffer containing nucleic acid metabolites, in mg / L; V is the dilution volume of the test solution, in L; m is the sample weight of the test sample, in g; W is the drying loss of the test sample, in %.

[0054] The method of the invention can accurately measure the adsorption amount of uric acid, adenine, guanine, hypoxanthine, adenosine, guanosine, inosine and 5-adenosine monophosphate by the macromolecular drug, and can completely separate uric acid, adenine, guanine, hypoxanthine, adenosine, guanosine, inosine and 5-adenosine monophosphate, the separation degree between each peak is good, the operability is strong, the analysis method has strong specificity, high precision, high accuracy and convenient operation, and can accurately measure the adsorption amount of uric acid, adenine, guanine, hypoxanthine, adenosine, guanosine, inosine and 5-adenosine monophosphate by the macromolecular drug.

[0055] Methods for drug screening

[0056] In another aspect of the present invention, the present invention provides a method for screening drugs. According to the implementation of the present invention, it includes:

[0057] A candidate drug is provided; the adsorption efficiency of the candidate drug on nucleic acid metabolites is detected by the above method, and whether the candidate drug is a target drug is determined based on the obtained adsorption efficiency. The method of the present invention is simple to operate and has high accuracy. It can screen polymer drugs that efficiently adsorb anions in large quantities, improve detection efficiency, save detection time, and save detection costs.

[0058] The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

[0059] Instrument: Agilent 1260 high performance liquid chromatograph, 1260 Infinity diode array detector

[0060] Chromatographic column: YMC-Pack ODS-AQ C18 (150×4.6mm, 5μm)

[0061] Mobile phase A: 0.05 mol / L potassium dihydrogen phosphate buffer (adjust pH to 4.0 with phosphoric acid or sodium hydroxide solution)

[0062] Mobile phase B: acetonitrile

[0063] Gradient elution is shown in Table 1

[0064] Table 1:

[0065] Time (min) Phosphate buffer (%) Acetonitrile (%) 0 100 0 15 100 0 20 95 5 40 95 5 41 100 0 60 100 0

[0066] Flow rate: 0.6mL / min

[0067] Detection wavelength: 260nm

[0068] Column temperature: 30°C

[0069] Injection volume: 10 μL

[0070] Diluent: Potassium dihydrogen phosphate buffer (pH 6.8)

[0071] Running time: 60 minutes

[0072] Experimental steps:

[0073] Potassium dihydrogen phosphate buffer (pH 6.8): Weigh 13.658 g of potassium dihydrogen phosphate, place in a 1000 mL volumetric flask, add water to dissolve and dilute to the scale, adjust the pH to 6.8 with phosphoric acid or sodium hydroxide solution.

[0074] Mixed binding solution preparation:

[0075] Accurately weigh 450.34 mg of uric acid, 440.60 mg of adenine, 451.44 mg of guanine, 446.68 mg of hypoxanthine, 410.74 mg of adenosine, 455.40 mg of guanosine, 441.00 mg of inosine, and 458.64 mg of 5-adenosine monophosphate, respectively, place them in a 1000 mL volumetric flask, add diluent to dissolve and dilute to the scale, and shake well.

[0076] Mixed standard working solution: Accurately pipette the mixed binding solution into a 10 ml volumetric flask, dilute to the scale with water, shake well, and prepare 1 μg / ml, 5 μg / ml, 10 μg / ml, 15 μg / ml, 20 μg / ml respectively. The quantitative limit is the lowest point of linearity.

[0077] Sample processing:

[0078] Weigh 0.1g of polymer accurately, place in a conical flask, add mixed binding solution, stir in a water bath at 37℃±5℃, and allow to bind for 30min~24h. Then let the sample solution stand, filter the supernatant, and take the filtrate as the sample solution.

[0079] Take the sample solution and perform HPLC analysis under the above chromatographic conditions. Record the chromatogram. The results are shown in the attached Figure 1 .

[0080] Figure 1 The chromatographic peaks with retention times of 10.912min, 11.846min, 12.623min, 16.551min, 20.968min, 25.401min, 25.715min and 30.586min are the chromatographic peaks of uric acid, guanine, hypoxanthine, adenine, 5-adenosine monophosphate, inosine, adenosine and adenosine respectively. The minimum separation is between guanine and hypoxanthine, with a separation of 2.09, and the separation is greater than 1.5.

[0081] The adsorption efficiency is calculated as follows:

[0082]

[0083] Where:

[0084] a is the slope of the linear regression equation;

[0085] b is the intercept of the linear regression equation;

[0086] A 供 is the peak area of ​​the test solution;

[0087] C 初 is the initial concentration of each substance in the mixed binding solution in the test solution, mg / L;

[0088] V is the dilution volume of the test solution, L;

[0089] m is the sample weight of the test product, g;

[0090] W is the drying loss of the test sample, in %.

[0091] The linear range, regression equation, correlation coefficient, quantification limit and precision of various substances are shown in Table 2:

[0092] Table 2: Linear range, regression equation, correlation coefficient, quantification limit and precision of 8 mixed standards

[0093]

[0094] *Y: peak area; X: mass concentration, mg / ml

[0095] Repeatability test: Take the same anion-adsorbing polymer drug, prepare 6 sample solutions in parallel according to the sample processing method, inject 10 μl for chromatographic analysis, and calculate the RSD value based on the measured amount of uric acid, adenine, guanine, hypoxanthine, adenine nucleoside, guanine nucleoside, inosine and 5-adenosine monophosphate adsorbed by the anion-adsorbing polymer drug. The results show that the amount of the anion-adsorbing polymer drug binding to the seven components is 1.9% to 4.9%, and the determination repeatability is good. The results are shown in Table 3.

[0096] Table 3: Repeatability of the amount of uric acid, adenine, guanine, hypoxanthine, adenosine, guanosine, inosine and 5-adenosine monophosphate adsorbed by anionic polymer drugs

[0097]

[0098] Example 2 Column temperature 25°C

[0099] Instrument: Agilent 1260 high performance liquid chromatograph, 1260 Infinity diode array detector

[0100] Chromatographic column: YMC-Pack ODS-AQ C18 (150×4.6mm, 5μm)

[0101] Mobile phase A: 0.05 mol / L potassium dihydrogen phosphate buffer (adjust pH to 4.0 with phosphoric acid or sodium hydroxide solution)

[0102] Mobile phase B: acetonitrile

[0103] Gradient elution is the same as in Example 1

[0104] Flow rate: 0.6mL / min

[0105] Detection wavelength: 260nm

[0106] Column temperature: 25°C

[0107] Injection volume: 10 μL

[0108] Diluent: Potassium dihydrogen phosphate buffer (pH 6.8)

[0109] Running time: 60 minutes

[0110] Experimental steps:

[0111] Potassium dihydrogen phosphate buffer (pH 6.8): Weigh 13.658 g of potassium dihydrogen phosphate, place in a 1000 mL volumetric flask, add water to dissolve and dilute to the scale, adjust the pH to 6.8 with phosphoric acid or sodium hydroxide solution.

[0112] Preparation of mixed binding solution: accurately weigh 450.34 mg of uric acid, 440.60 mg of adenine, 451.44 mg of guanine, 446.68 mg of hypoxanthine, 410.74 mg of adenosine, 455.40 mg of guanosine, 441.00 mg of inosine, and 458.64 mg of 5-adenosine monophosphate, respectively, place in a 1000 mL volumetric flask, add diluent to dissolve and dilute to the scale, and shake well.

[0113] Take the mixed binding solution and perform HPLC analysis under the above chromatographic conditions. Record the chromatogram. The results are shown in the attached Figure 2 .

[0114] Figure 2The chromatographic peaks with retention times of 12.757min, 13.591min, 14.564min, 18.430min, 22.910min, 25.957min, 26.339min and 32.105min are the chromatographic peaks of uric acid, guanine, hypoxanthine, adenine, 5-adenosine monophosphate, inosine, adenosine and adenosine respectively. The minimum separation is between uric acid and hypoxanthine, with a separation of 2.04, and the separation is greater than 1.5.

[0115] The chromatograms showed that the eight substances could be well separated.

[0116] Example 3 Column temperature 35°C

[0117] Instrument: Agilent 1260 high performance liquid chromatograph, 1260 Infinity diode array detector

[0118] Chromatographic column: YMC-Pack ODS-AQ C18 (150×4.6mm, 5μm)

[0119] Mobile phase A: 0.05 mol / L potassium dihydrogen phosphate buffer (adjust pH to 4.0 with phosphoric acid or sodium hydroxide solution)

[0120] Mobile phase B: acetonitrile

[0121] Gradient elution is the same as in Example 1

[0122] Flow rate: 0.6mL / min

[0123] Detection wavelength: 260nm

[0124] Column temperature: 35°C

[0125] Injection volume: 10 μL

[0126] Diluent: Potassium dihydrogen phosphate buffer (pH 6.8)

[0127] Running time: 60 minutes

[0128] Experimental steps:

[0129] Potassium dihydrogen phosphate buffer (pH 6.8): Weigh 13.658 g of potassium dihydrogen phosphate, place in a 1000 mL volumetric flask, add water to dissolve and dilute to the scale, adjust the pH to 6.8 with phosphoric acid or sodium hydroxide solution.

[0130] Preparation of mixed binding solution: accurately weigh 450.34 mg of uric acid, 440.60 mg of adenine, 451.44 mg of guanine, 446.68 mg of hypoxanthine, 410.74 mg of adenosine, 455.40 mg of guanosine, 441.00 mg of inosine, and 458.64 mg of 5-adenosine monophosphate, respectively, place in a 1000 mL volumetric flask, add diluent to dissolve and dilute to the scale, and shake well.

[0131] Take the mixed binding solution and perform HPLC analysis under the above chromatographic conditions. Record the chromatogram. The results are shown in the attached Figure 3 .

[0132] Figure 3 The chromatographic peaks with retention times of 9.354min, 10.317min, 10.939min, 14.785min, 17.538min, 24.765min, 25.066min and 29.113min are the chromatographic peaks of uric acid, guanine, hypoxanthine, adenine, 5-adenosine monophosphate, inosine, adenosine and adenosine respectively. The minimum separation is between guanine and hypoxanthine, with a separation of 1.90, and the separation is greater than 1.5.

[0133] The chromatograms showed that the eight substances could be well separated.

[0134] Example 4 Buffer reagent is BES

[0135] Instrument: Agilent 1260 high performance liquid chromatograph, 1260 Infinity diode array detector

[0136] Chromatographic column: YMC-Pack ODS-AQ C18 (150×4.6mm, 5μm)

[0137] Mobile phase A: 0.05 mol / L potassium dihydrogen phosphate buffer (adjust pH to 4.0 with phosphoric acid or sodium hydroxide solution)

[0138] Mobile phase B: acetonitrile

[0139] Gradient elution is the same as in Example 1

[0140] Flow rate: 0.6mL / min

[0141] Detection wavelength: 260nm

[0142] Column temperature: 30°C

[0143] Injection volume: 10 μL

[0144] Diluent: BES buffer (pH 7.0)

[0145] Running time: 60 minutes

[0146] Experimental steps:

[0147] BES buffer (pH 7.0): Weigh 21.32 g of BES (N,N-(2-hydroxyethyl)-2-aminoethanesulfonic acid) and 4.67 g of sodium chloride, add to 1000 ml of purified water, dissolve, mix well, and adjust the pH to 7.0 with a saturated sodium hydroxide solution (about 3.5 ml).

[0148] Preparation of mixed binding solution: accurately weigh 225.78 mg of uric acid, 220.59 mg of adenine, 225.62 mg of guanine, 446.68 mg of hypoxanthine, 220.01 mg of adenosine, 225.17 mg of guanosine, 220.14 mg of inosine, and 240.39 mg of 5-adenosine monophosphate, place them in a 500 mL volumetric flask, add diluent to dissolve and dilute to the scale, and shake well.

[0149] Mixed standard working solution: accurately pipette the mixed binding solution into a 10 ml volumetric flask, dilute to the mark with water, shake well, and prepare 1 μg / ml, 5 μg / ml, 10 μg / ml, 15 μg / ml, and 20 μg / ml respectively.

[0150] Sample processing:

[0151] Weigh 0.1g of polymer accurately, place in a conical flask, add mixed binding solution, stir in a water bath at 37℃±5℃, and allow to bind for 30min~24h. Then let the sample solution stand, filter the supernatant, and take the filtrate as the sample solution.

[0152] Take the sample solution and perform HPLC analysis under the above chromatographic conditions. Record the chromatogram. The results are shown in the attached Figure 4 .

[0153] Figure 4 The chromatographic peaks with retention times of 10.912min, 11.846min, 12.623min, 16.553min, 20.969min, 25.402min, 25.715min, and 30.588min are uric acid, guanine, hypoxanthine, adenine, 5-adenosine monophosphate, inosine, adenosine, and adenosine, respectively. The separation degree is 2.61, the separation degree of guanine and hypoxanthine is 2.10, the separation degree of hypoxanthine and adenine is 7.72, the separation degree of adenine and 5-adenosine monophosphate is 7.00, the separation degree of inosine of 5-adenosine monophosphate is 11.45, the separation degree of inosine and guanosine is 2.24, and the separation degree of guanosine and adenine is 20.48. The separation degrees of the 8 substances are all greater than 1.5.

[0154] The amount of uric acid, adenine, guanine, hypoxanthine, adenosine, guanosine, inosine and 5-adenosine monophosphate adsorbed by the anion-adsorbing polymer drug is calculated as follows:

[0155]

[0156] Where:

[0157] a is the slope of the linear regression equation;

[0158] b is the intercept of the linear regression equation;

[0159] A 供 is the peak area of ​​the test solution;

[0160] C 初 is the initial concentration of each substance in the mixed binding solution in the test solution, mg / L;

[0161] V is the dilution volume of the test solution, L;

[0162] m is the sample weight of the test product, g;

[0163] W is the drying loss of the test sample, in %.

[0164] Table 4 shows the amount of adenine, guanine, hypoxanthine, adenosine, guanosine, inosine and 5-adenosine monophosphate adsorbed by the anionic polymer drug

[0165] Table 4:

[0166] Serial number Compound Binding amount (mg / g) 1 Uric acid 71.611 2 Guanine 18.891 3 Hypoxanthine 28.855 4 Adenine 17.837 5 5-Adenosine monophosphate 319.585 6 Inosine 14.973 7 Guanine Adenosine 10.763 8 Adenosine 6.147

[0167] Comparative Example 1 The pH of mobile phase A buffer is 3.5

[0168] Instrument: Agilent 1260 high performance liquid chromatograph, 1260 Infinity diode array detector

[0169] Chromatographic column: YMC-Pack ODS-AQ C18 (150×4.6mm, 5μm)

[0170] Mobile phase A: 0.05 mol / L potassium dihydrogen phosphate buffer (adjust pH to 3.5 with phosphoric acid or sodium hydroxide solution)

[0171] Mobile phase B: acetonitrile

[0172] Gradient elution is the same as in Example 1

[0173] Flow rate: 0.6mL / min

[0174] Detection wavelength: 260nm

[0175] Column temperature: 30°C

[0176] Injection volume: 10 μL

[0177] Diluent: Potassium dihydrogen phosphate buffer (pH 6.8)

[0178] Running time: 60 minutes

[0179] Experimental steps:

[0180] Potassium dihydrogen phosphate buffer (pH 6.8): Weigh 13.658 g of potassium dihydrogen phosphate, place in a 1000 mL volumetric flask, add water to dissolve and dilute to the scale, adjust the pH to 6.8 with phosphoric acid or sodium hydroxide solution.

[0181] Preparation of mixed binding solution: accurately weigh 450.34 mg of uric acid, 440.60 mg of adenine, 451.44 mg of guanine, 446.68 mg of hypoxanthine, 410.74 mg of adenosine, 455.40 mg of guanosine, 441.00 mg of inosine, and 458.64 mg of 5-adenosine monophosphate, respectively, place in a 1000 mL volumetric flask, add diluent to dissolve and dilute to the scale, and shake well.

[0182] Take the mixed binding solution and perform HPLC analysis under the above chromatographic conditions. Record the chromatogram. The results are shown in the attached Figure 5 .

[0183] Figure 5 The chromatographic peaks with retention times of 10.367min, 10.687min, 11.194min, 12.027min, 15.686min, 25.179min, 25.474min and 28.239min are the chromatographic peaks of uric acid, guanine, hypoxanthine, adenine, 5-adenosine monophosphate, inosine, adenosine and adenosine respectively. The separation degree between uric acid and guanine is 0.93, the separation degree between guanine and hypoxanthine is 1.33, and the separation degree between other substances is greater than 1.5.

[0184] The chromatograms showed that the eight substances could not be well separated.

[0185] Comparative Example 2 The pH of mobile phase A buffer is 4.2

[0186] Instrument: Agilent 1260 high performance liquid chromatograph, 1260 Infinity diode array detector

[0187] Chromatographic column: YMC-Pack ODS-AQ C18 (150×4.6mm, 5μm)

[0188] Mobile phase A: 0.05 mol / L potassium dihydrogen phosphate buffer (adjust the pH to 4.2 with phosphoric acid or sodium hydroxide solution)

[0189] Mobile phase B: acetonitrile

[0190] Gradient elution is the same as in Example 1

[0191] Flow rate: 0.6mL / min

[0192] Detection wavelength: 260nm

[0193] Column temperature: 30°C

[0194] Injection volume: 10 μL

[0195] Diluent: Potassium dihydrogen phosphate buffer (pH 6.8)

[0196] Running time: 60 minutes

[0197] Experimental steps:

[0198] Potassium dihydrogen phosphate buffer (pH 6.8): Weigh 13.658 g of potassium dihydrogen phosphate, place in a 1000 mL volumetric flask, add water to dissolve and dilute to the scale, adjust the pH to 6.8 with phosphoric acid or sodium hydroxide solution.

[0199] Preparation of mixed binding solution: accurately weigh 23.17 mg of uric acid, 22.48 mg of adenine, 22.80 mg of guanine, 20.64 mg of hypoxanthine, 23.00 mg of guanosine, 22.50 mg of inosine, and 23.40 mg of 5-adenosine monophosphate, place them in a 1000 mL volumetric flask, add diluent to dissolve and dilute to the scale, and shake well.

[0200] Take the mixed binding solution and perform HPLC analysis under the above chromatographic conditions. Record the chromatogram. The results are shown in the attached Figure 6 .

[0201] Figure 6 The chromatographic peaks with retention times of 10.371min, 11.746min, 12.182min, 18.779min, 22.055min, 25.146min, 25.465min and 30.564min are the chromatographic peaks of uric acid, guanine, hypoxanthine, adenine, 5-adenosine monophosphate, inosine, adenosine and adenosine nucleoside, respectively. The separation between guanine and hypoxanthine is 1.18, the separation between 5-adenosine monophosphate and inosine is 0.49, and the separation between other substances is greater than 1.5.

[0202] The chromatograms showed that the eight substances could not be well separated.

[0203] Comparative Example 3: The organic solvent of mobile phase B is methanol

[0204] Instrument: Agilent 1260 high performance liquid chromatograph, 1260 Infinity diode array detector

[0205] Chromatographic column: YMC-Pack ODS-AQ C18 (150×4.6mm, 5μm)

[0206] Mobile phase A: 0.05 mol / L potassium dihydrogen phosphate buffer (adjust pH to 4.0 with phosphoric acid or sodium hydroxide solution)

[0207] Mobile phase B: methanol

[0208] Gradient elution is the same as in Example 1

[0209] Flow rate: 0.6mL / min

[0210] Detection wavelength: 260nm

[0211] Column temperature: 30°C

[0212] Injection volume: 10 μL

[0213] Diluent: Potassium dihydrogen phosphate buffer (pH 6.8)

[0214] Running time: 60 minutes

[0215] Experimental steps:

[0216] Potassium dihydrogen phosphate buffer (pH 6.8): Weigh 13.658 g of potassium dihydrogen phosphate, place in a 1000 mL volumetric flask, add water to dissolve and dilute to the scale, adjust the pH to 6.8 with phosphoric acid or sodium hydroxide solution.

[0217] Preparation of mixed binding solution: accurately weigh 450.34 mg of uric acid, 440.60 mg of adenine, 451.44 mg of guanine, 446.68 mg of hypoxanthine, 410.74 mg of adenosine, 455.40 mg of guanosine, 441.00 mg of inosine, and 458.64 mg of 5-adenosine monophosphate, respectively, place in a 1000 mL volumetric flask, add diluent to dissolve and dilute to the scale, and shake well.

[0218] Take the mixed binding solution and perform HPLC analysis under the above chromatographic conditions. Record the chromatogram. The results are shown in the attached Figure 7 .

[0219] Figure 7The chromatographic peaks with retention times of 10.645min, 11.604min, 12.333min, 16.377min, 20.543min, 29.536min and 31.254min are the chromatographic peaks of uric acid, guanine, hypoxanthine, adenine, 5-adenosine monophosphate, inosine and guanosine, and adenine nucleoside, respectively. Inosine and guanine completely overlap.

[0220] The chromatograms showed that the eight substances could not be well separated.

[0221] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0222] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for detecting the adsorption efficiency of a drug on a nucleic acid metabolite, characterized in that: include: The buffer containing nucleic acid metabolites is mixed with the drug to obtain a test solution, wherein the buffer containing nucleic acid metabolites contains a buffer reagent, the buffer reagent is potassium dihydrogen phosphate, the pH of the buffer reagent is 6.0-8.0, and the mixing is performed at 30° C.-40° C. for 30 min-24 h; The test solution is subjected to high performance liquid chromatography detection, and the adsorption efficiency of the drug on nucleic acid metabolites is determined based on the detection results, Wherein, the nucleic acid metabolites include: uric acid, adenine, guanine, hypoxanthine, adenine nucleoside, guanine nucleoside, inosine and 5-adenosine monophosphate; The mobile phase used in the high performance liquid chromatography detection includes a buffer solution and an organic solvent, and the pH value of the buffer solution is 3.6-4.0; The buffer is a phosphate buffer, and the organic solvent includes acetonitrile; The high performance liquid chromatography detection adopts a gradient elution mode, and the conditions of the gradient elution are: At 0-15 minutes, the volume ratio of the buffer solution to the organic solvent is 100%:0%; At 15-20 minutes, the volume ratio of the buffer solution to the organic solvent is (95%-100%): (0%-5%); At 20-40 minutes, the volume ratio of the buffer solution to the organic solvent is 95%:5%; At 40-41 minutes, the volume ratio of the buffer solution to the organic solvent is (95%-100%): (0%-5%); At 41-60 minutes, the volume ratio of the buffer solution to the organic solvent is 100%:0%; The chromatographic column used in the high performance liquid chromatography detection is an octadecylsilane bonded silica gel column; The particle size of the chromatographic column is 3µm~5µm; The length of the chromatographic column is 100 mm to 300 mm; The column temperature used in the high performance liquid chromatography detection is 20°C-40°C; The detection wavelength adopted by the high performance liquid chromatography detection is 220nm-300nm.

2. The method according to claim 1, characterized in that The pH value of the buffer solution is 4.

0.

3. The method according to claim 1, characterized in that The high performance liquid chromatography detection adopts a gradient elution mode, and the conditions of the gradient elution are: At 0-15 minutes, the volume ratio of the buffer solution to the organic solvent is 100%:0%; At 15-20 minutes, the volume ratio of the buffer solution to the organic solvent is 100%:0%; At 20-40 minutes, the volume ratio of the buffer solution to the organic solvent is 95%:5%; At 40-41 minutes, the volume ratio of the buffer solution to the organic solvent is 95%:5%; At 41-60 minutes, the volume ratio of the buffer solution to the organic solvent is 100%:0%.

4. The method according to claim 3, characterized in that The flow rate of the mobile phase is 0.2 mL / min-1.0 mL / min.

5. The method according to claim 3, characterized in that: The flow rate of the mobile phase was 0.6 mL / min.

6. The method according to claim 1, characterized in that The pH of the buffer reagent is 6.5-7.

0.

7. The method according to claim 1, characterized in that The particle size of the chromatographic column is 3 μm.

8. The method according to claim 1, characterized in that The length of the chromatographic column is 150 mm.

9. The method according to claim 1, characterized in that: The detection wavelength adopted by the high performance liquid chromatography detection is 260nm.

10. The method according to claim 1, characterized in that The adsorption efficiency is characterized by the binding amount of each nucleic acid metabolite. The binding amount of each nucleic acid metabolite is determined by the following formula: Where a is the slope of the linear regression equation; b is the intercept of the linear regression equation; A 供 is the peak area of ​​the test solution; C 初 is the initial concentration of each nucleic acid metabolite in the buffer containing nucleic acid metabolites, in mg / L; V is the dilution volume of the test solution, in L; m is the sample weight of the test product, in g; W is the drying loss of the test sample, in %.

11. A method for screening drugs, characterized in that: include: Providing drug candidates; The method according to any one of claims 1 to 10 is used to detect the adsorption efficiency of the candidate drug on the nucleic acid metabolite, and whether the candidate drug is a target drug is determined based on the obtained adsorption efficiency.

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