Sugar drug in vivo pharmacokinetic model, method for constructing same and application thereof

Abstract

The present application belongs to the technical field of pharmaceutical analysis, and particularly relates to a saccharide drug in-vivo pharmacokinetic model, a construction method thereof and application. The present application uses ion chromatography, high performance liquid chromatography and other detection technologies to realize in-vivo pharmacokinetic application analysis of saccharide drugs, quantitatively detects the content change of monosaccharide in tissue samples after administration, analyzes the monosaccharide change of serum and corresponding disease tissue, takes the monosaccharide with the maximum content change as an index to perform pharmacokinetic analysis, and constructs a corresponding pharmacokinetic model. The determination method used for constructing the model has good specificity, precision and high accuracy, a wide linear range and high sensitivity. The pharmacokinetic model constructed by the present application is suitable for non-preclinical pharmacokinetic research of saccharide drugs, provides theoretical and technical support for saccharide drug development, and solves the bottleneck problem of saccharide drug research.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical analysis, and particularly relates to an in-vivo pharmacokinetic model of a saccharide drug and a construction method and application thereof, and provides theoretical and technical support for non-clinical pharmacokinetic research of saccharide drugs and development of saccharide drugs. BACKGROUND

[0002] Saccharide drugs occupy an increasingly important position in the field of drugs due to their unique physiological activity. Saccharide drug research is the focus of biochemical drug development in this century. Fucoidan is a sulfated polysaccharide obtained by extraction and purification from marine brown algae. Fucoidan with a molecular weight of 6-13 KDa is obtained by degradation of fucoidan. Fucoidan can be used for treating chronic renal failure, diabetic nephropathy, and nephrotic syndrome, and has achieved good effects in treating chronic renal failure and dampness syndrome in clinical practice. Through non-clinical pharmacological research, it is revealed that low molecular weight fucoidan can significantly treat diabetic complications such as diabetic foot ulcer.

[0003] Pharmacokinetic research of drugs in the body is important in the process of innovative drug research. Fucoidan is a component with complexity, and there have been limitations in the study of its pharmacokinetics. The key technology is the determination and application of the drug detection method in plasma. Pharmacokinetics is the key to improving the success rate of innovative drug research and realizing safe and reasonable drug use in clinical practice. Some researchers use PMP pre-column derivatization high performance liquid chromatography to determine the fucose in serum. The sample needs to be treated in multiple steps, and there is a large human error in quantitative detection.

[0004] High performance liquid chromatography and ion chromatography have been well applied in sugar detection due to their excellent separation technology and high sensitivity detector. Through this detection technology, the content of sugar in plasma can be quantitatively detected, and the pharmacokinetic rules of drugs in the animal body can be better understood, and the pharmacological and pharmacodynamic effects of drugs can be better mastered. The use of this method can accurately quantitatively detect.

[0005] The monosaccharide composition of saccharide drugs is similar to that of the human body. Due to the limitations of in-vivo detection technology of saccharide drugs, the pharmacokinetics of saccharide drugs has always been a research difficulty. SUMMARY

[0006] The technical problem to be solved by the present application is to provide a saccharide drug in vivo pharmacokinetic model, a construction method and application thereof, to quantitatively detect the content change of each monosaccharide in a tissue sample after administration by using ion chromatography, high performance liquid chromatography and the like, to analyze the monosaccharide change of serum and corresponding disease tissue, to take the monosaccharide with the maximum content change as an index to perform pharmacokinetic analysis, to construct a corresponding pharmacokinetic model, to apply the pharmacokinetic model to nonclinical saccharide drug pharmacokinetic research, and to determine reasonable drug use.

[0007] The technical scheme adopted is as follows:

[0008] The construction method of the saccharide drug in vivo pharmacokinetic model quantitatively detects the content change of monosaccharide in a tissue sample after administration, analyzes the monosaccharide change of serum and corresponding disease tissue, takes the monosaccharide with the maximum content change as an index to perform pharmacokinetic analysis, and constructs a corresponding pharmacokinetic model.

[0009] Preferably, the quantitatively detected tissue sample is serum and organ tissue, and the monosaccharide concentration at different times in the serum and organ tissue is quantitatively detected; wherein the quantitative detection includes sample preparation and detection.

[0010] Preferably, the monosaccharide includes fucose, glucose, galactose, mannose, rhamnose, glucosamine, galactosamine and acetylglucosamine; and the organ tissue includes heart, liver, spleen, lung and kidney.

[0011] Preferably, the sample preparation includes the following steps:

[0012] 1) Collect blood after administration, centrifuge (5000xg, 20min), take the upper serum in an appropriate amount (0.1-0.15ml), and place it in a hydrolysis bottle;

[0013] Add water in an appropriate amount (0.85-0.9ml), add acid, seal, hydrolyze at 105℃ for 4-6h, and hydrolyze saccharide substances into monosaccharides;

[0014] 2) Take organ tissue (heart, liver, spleen, lung, kidney, brain, gastrointestinal tissue, etc.) after administration, clean it, dry the surface water with a water absorption paper, weigh and take 100-150mg, and grind with water;

[0015] After grinding, add sodium hypochlorite and borax mixture for 2min, add formic acid solution, take about 2ml, add 2ml hydrolysis solution (i.e. acid) to hydrolyze, and hydrolyze saccharide substances into monosaccharides;

[0016] 3) After the hydrolysis in steps 1) and 2) is completed, filter the solution to a volumetric flask with filter paper, add an alkali solution to neutralize the filtrate, make up to the scale, and reserve.

[0017] Preferably, the hydrolysis solution in the steps 1) and 2) is any one of hydrochloric acid and trifluoroacetic acid, and 4 mol / L trifluoroacetic acid is preferred, and the amount of addition is 1 ml;

[0018] The alkali solution in the step 3) is any one of sodium hydroxide and ammonia water, and 2 mol / L sodium hydroxide solution is preferred.

[0019] Preferably, the detection method is any one of ion chromatography, high performance liquid chromatography and liquid chromatography-mass spectrometry.

[0020] Preferably, the content of each monosaccharide in serum is detected, a blood concentration-time curve is drawn, a serum drug-time curve of different monosaccharides is obtained, the peak time and peak concentration of different monosaccharides are obtained, the content of different monosaccharides in organ tissues is detected, a time-tissue concentration curve is drawn, the monosaccharide with the largest change amount in different organ tissues is obtained, and the peak time and peak concentration of different monosaccharides are obtained.

[0021] Preferably, for different disease types, the organ tissue where the disease occurs is analyzed, the monosaccharide with the largest content change in the organ tissue is obtained, and the change amount of the monosaccharide in serum is analyzed, so that the monosaccharide with the largest content change in serum and organ tissue is determined as an index for the in-vivo pharmacokinetic analysis of the saccharide drug.

[0022] The equation of the saccharide drug in-vivo pharmacokinetic model constructed by the above method is Y=0.0175*X-0.0637, where Y is the peak concentration, and the unit is μg / ml.

[0023] X is the drug dose, and the unit is mg / kg, and the range of X is 0-120 mg / kg.

[0024] When the drug dose of X is greater than 120 mg / kg, the peak concentration does not increase or changes little.

[0025] The saccharide drug in-vivo pharmacokinetic model provided by the application can be applied to the non-clinical pharmacokinetic research of saccharide drugs and the development of saccharide drugs.

[0026] The drug for administration is a saccharide drug preparation, including capsule, tablet, sugar-coated tablet, film-coated tablet, enteric-coated tablet, hard capsule, soft capsule, buccal tablet, granule, instant powder, pill, powder, paste, powder injection, injection, etc.

[0027] Compared with the prior art, the saccharide drug in-vivo pharmacokinetic model provided by the application has the following beneficial effects:

[0028] The present application quantitatively detects the content change of each monosaccharide in a tissue sample after administration, analyzes the monosaccharide change of serum and corresponding disease tissue, takes the monosaccharide with the largest content change as an index for pharmacokinetic analysis, constructs a corresponding pharmacokinetic model, and is applied to non-clinical pharmacokinetic study of saccharide drugs, thereby providing theoretical and technical support for saccharide drug development and scientific methods and basis for clinical rational drug use.

[0029] The sample pre-treatment of the present application is simple, and the index component of the drug is quantitatively detected, so that the detection result is accurate and the sensitivity is high. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Ion chromatograms of fucose in blank plasma samples and plasma after administration of fucoidan;

[0031] Figure 2 Standard curve graphs of each monosaccharide series;

[0032] Figure 3 Content change curve of each monosaccharide in serum after administration of fucoidan with time;

[0033] Figure 4 Content change curve of each monosaccharide in kidney tissue after administration of fucoidan with time;

[0034] Figure 5 Content change curve of each monosaccharide in lung tissue after administration of fucoidan with time;

[0035] Figure 6 Linear relationship graph of the administration dose and peak concentration in serum. DETAILED DESCRIPTION

[0036] The present application is further illustrated by the following examples, but is not limited to the present application.

[0037] Since the saccharide drug has complex components and the structure is difficult to analyze, the present application analyzes the in-vivo pharmacokinetic characteristics of the saccharide drug by taking the monosaccharide content as an index.

[0038] The saccharide medicine is different from the chemical medicine, and the main chemical component or the metabolite in the body is used as the index to study the pharmacokinetic characteristics. The fucose is the main component of the fucoidan, and the monosaccharide content in the serum and various tissues in the body is different, which shows that the administration of the saccharide medicine with high content of a certain monosaccharide can cause the content change of other monosaccharides in the tissues. For example, the fucose is the main component of the fucoidan, and the monosaccharide content in the serum is changed after administration. However, the fucose content in the tissues, such as kidney tissues, is greatly changed, which shows that the saccharide medicine has a greater impact on the kidney, and the fucose can be used as the index to analyze the pharmacokinetic characteristics of the saccharide medicine. For example, the galactose content in the lung tissue is greatly changed, which shows that the saccharide medicine has a greater impact on the lung tissue, and the galactose can be used as the index to analyze the pharmacokinetic characteristics of the saccharide medicine, which reflects the unique characteristics of the pharmacokinetic characteristics of the saccharide medicine in the body. On this basis, the pharmacokinetic model of the saccharide medicine is established, and is applied to the in-vivo pharmacokinetic study of different saccharide medicines.

[0039] The present application uses the fucoidan or the preparation with the fucoidan as the main medicine as the medicine, and is administered to animals by gavage. After the medicine is absorbed for a period of time, the blood is taken from the animal eyeball venous plexus (the serum is separated), and then the tissue samples are obtained. The serum and the tissue are measured by the instrument after the sample pre-treatment.

[0040] The screening method of the present application is as follows:

[0041] Example 1: The establishment of the analysis method of each monosaccharide in the serum sample

[0042] The high performance liquid chromatography is adopted:

[0043] The serum sample is pretreated

[0044] The blood after administration is centrifuged at 5000 r / min, and the serum 0.1 ml is sucked and added with 0.9 ml water, and then the hydrolysis liquid 4 mol / L trifluoroacetic acid 1 ml is added. The mouth is sealed, and the hydrolysis is carried out at 105℃ for 4-6h. After the hydrolysis is completed, the 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization reaction is carried out.

[0045] The verification of the analysis method

[0046] (1) The specificity of the method

[0047] The mouse blank serum 0.1 ml is mixed with the fucose control solution and the monosaccharide control solution with a certain concentration according to the method in the item of "the serum sample pretreatment", and the chromatogram of the blank serum sample and the fucose control and the monosaccharide control solution is obtained. The serum sample after oral gavage of the mouse is operated in the same way, and the chromatogram is obtained, which is shown in the attached Figure 1 and Table 1. Figure 1The results of Table 1 show that endogenous substances in serum do not interfere with the assay.

[0048] Table 1 results of specificity test

[0049]

[0050] (2) Standard curve and linear range

[0051] The standard solution of each monosaccharide was precisely measured as a control, and was diluted by several times, respectively. Six solutions with different concentrations were selected, and were injected into the column for detection according to the above chromatographic conditions. The peak areas of different concentrations were measured. The standard curves of each monosaccharide are shown in Table 1. Figure 2

[0052] Figure 2 In Table 1, the concentration C of the control was taken as the abscissa, and the peak area A was taken as the ordinate, to draw the standard curve.

[0053] The standard curve of mannose is y = 39.148x - 16.788, R2= 1. 2

[0054] The standard curve of glucosamine is y = 28.458x - 2.696, R2= 0.9999.

[0055] The standard curve of glucose is y = 29.172x - 15.186, R2= 1. 2

[0056] The standard curve of galactose is y = 43.136x - 15.239, R2= 0.9997. 2

[0057] The standard curve of glucuronic acid is y = 29.172x - 15.186, R2= 1. 2

[0058] The standard curve of galactosamine is y = 33.525x + 9.4439, R2= 0.9985. 2

[0059] The standard curve of xylose is y = 60.343x + 2.9043, R2= 0.9991. 2

[0060] The standard curve of fucose is y = 25.398x - 1.69, R2= 0.9999. 2

[0061] The standard curve of acetylglucosamine is y = 16.37x + 10.226, R2= 0.9999. 2

[0062] ​​​​​​​​​Conclusion: The correlation coefficient r of each monosaccharide in the concentration range was greater than 0.95, and the linear relationship was good.

[0063] (3) Precision

[0064] Take each parallel 5 portions of the limit of quantification concentration of fucose control solution, blank serum test sample, blank renal tissue test sample solution, according to the above chromatographic conditions, continuous sample, the data are shown in Table 2.

[0065] Table 2 Precision test results (n = 5)

[0066]

[0067] Among them, 1-1 is the limit of quantification concentration of fucose control; 1-2 is the blank serum test sample; 1-3 is the blank renal tissue solution test sample.

[0068] Conclusion: After continuous sampling of the limit of quantification concentration of fucose control solution, blank serum test sample, and blank renal tissue solution test sample, the relative standard deviation RSD was 5.10%, 1.13%, and 7.70%, respectively, all less than 15.0%, the method had good repeatability and good precision.

[0069] (4) Accuracy

[0070] Take the low, medium and high concentration serum treatment samples with 0.7, 1.75, and 2.8 μg of fucose control added in triplicate, from the test data, the intra-group recovery rate of low, medium and high concentrations in serum was between 75.61-78.90%, the inter-group recovery rate was 78.80%, and the relative standard deviation was 4.01%.

[0071] (5) Recovery rate of added sample in tissue sample

[0072] Take the low, medium and high concentration lung tissue treatment samples with 0.7, 1.75, and 2.8 μg of fucose control added in triplicate, from the test data, the intra-group recovery rate of low, medium and high concentrations in tissue sample was between 73.85-77.76%, the inter-group recovery rate was 72.98%, and the relative standard deviation was 5.07%.

[0073] Conclusion

[0074] A method for determining the pharmacokinetics of fucoidan in vivo was established, which was simple, specific, and not interfered by other components in serum.

[0075] Example 2: Pharmacokinetics after intragastric administration of fucoidan

[0076] Collection of serum samples

[0077] Take ICR mice 72, fasting 12h before the experiment, free drinking water. According to the dose of 60mg / kg, give brown algae polysaccharide sulfate solution by gavage, at 0min and 1, 2, 4, 6, 8, 14h, 0.5ml of blood is taken from the orbit, placed in the centrifuge tube, 5000r / min centrifugation, take serum, keep in-20℃ refrigerator for testing.

[0078] Pharmacokinetic determination results

[0079] After ICR mice were given brown algae polysaccharide sulfate by gavage, the average drug concentration-time curve of various monosaccharides was seen Figure 1 . Taking the kidney tissue as an example, the DAS software was used to process the blood concentration-time data by using the non-compartment model, and the pharmacokinetic parameters were obtained.

[0080] As shown in Figure 3 , the time-blood concentration curve of each monosaccharide was analyzed by using fucose as an index, and the pharmacokinetic parameters were analyzed. After administration of low molecular weight brown algae polysaccharide sulfate (60mg / kg dose), the AUC (0-∞) was 3.72mg / L h, the t 1 / 2α , t 1 / 2β of low molecular weight brown algae polysaccharide sulfate were 0.5-0.8h, 6.5-6.9h, respectively. The mean residence time MRT of low molecular weight brown algae polysaccharide sulfate was 4.48h (60mg / kg), see Table 3 for details. Table 3 for pharmacokinetic parameters after oral administration of mice.

[0081] Table 3 Pharmacokinetic parameters after oral administration of mice

[0082]

[0083] Among them, C max : maximum blood concentration; T max : time to reach maximum blood concentration; AUC: area under the blood concentration-time curve; T 1 / 2α : distribution half-life; t 1 / 2β : elimination half-life; MRT: mean residence time.

[0084] Example 3 Tissue distribution of brown algae polysaccharide sulfate in mice

[0085] After the mice were given by gavage at a dose of 30mg / kg, they were killed at 0min before administration, 1, 1.5, 2, 3, 4, 6, 10h after administration, dissected, and heart, liver, spleen, lung, kidney tissues were taken.

[0086] As shown in Figure 4The content of each monosaccharide in the tissue was analyzed, taking the kidney as an example: through data analysis, it was found that the content of fucose in the kidney tissue changed the most, and through joint analysis of the blood drug concentration, for kidney diseases, fucose can be used as a pharmacokinetic monitoring index for fucoidan sulfate drugs, and the pharmacokinetic characteristics can be studied.

[0087] As shown in Figure 5 , the content of each monosaccharide in the tissue was detected after administration, taking the lung as an example: through the analysis of the content of each monosaccharide in the lung tissue, combined with the analysis of the blood drug concentration, for lung-related diseases, galactose can be used as an index for pharmacokinetic analysis of drugs, and a pharmacokinetic model of the drug can be constructed.

[0088] Example 4: Relationship between administration dose and peak concentration

[0089] Mice were administered at doses of 10, 30, 60, 120, 240, and 480 mg / kg, and serum samples were obtained at 1, 1.5, 2, 3, 4, 6, and 10 h, respectively, to determine the blood drug concentration. The peak concentration of fucose in the serum was 0, 0.53, 1.09, 1.98, 1.96, and 2.01 μg / ml, respectively, as shown in Figure 6 .

[0090] As can be seen from Figure 6 , the fucose content in the mouse body did not change at a dose of 10 mg / kg, it began to change at a dose of 30 mg / kg, and it changed with first-order kinetic characteristics up to a dose of 120 mg / kg. The peak concentration of the mouse did not increase again at a dose greater than 120 mg / kg. There was a certain relationship between the dose of 0-120 mg / kg and the peak concentration, and the equation was Y=0.0175*X-0.0637 (Y is the peak concentration, and X is the administration dose).

[0091] Example 5: Method for detecting fucose in plasma samples by ion chromatography

[0092] Analysis method: ion chromatography

[0093] Serum sample pretreatment:

[0094] Centrifuge the blood at 5000 r / min, take 0.1 ml of serum, add 0.9 ml of water, then add 1 ml of hydrolysis solution 4 mol / L trifluoroacetic acid, seal, and hydrolyze at 105°C for 4-6 h. After hydrolysis, adjust to neutral with sodium hydroxide solution, filter into a 10 ml volumetric flask, and dilute to volume. Prepare for use.

[0095] Precision

[0096] Take 5 parallel portions of the derivatized blank serum test sample, and continuously sample according to the above chromatographic conditions. The data are shown in Table 4.

[0097] Table 4 Precision test results (n = 5)

[0098]

[0099] Conclusion: After the blank serum test sample was continuously injected, the average sample amount was 69.71 mg / L, and the relative standard deviation RSD was 2.82%.

[0100] The relative standard deviations of the precision of ion chromatography and pre-column derivatization high performance liquid chromatography were 0.92% and 2.82%, respectively. According to the data, it can be seen that the ion chromatography and pre-column derivatization high performance liquid chromatography for detecting monosaccharide content can be used for in vivo pharmacokinetic analysis of drugs; from the sample processing operation, the ion chromatography for serum and tissue sample determination before processing is simple, which can reduce the loss of sample amount and reduce human operation errors.

[0101] Fuc-fucose;

[0102] Rha-rhamnose;

[0103] Ara-arabinose;

[0104] Gal-galactose;

[0105] Man-mannose;

[0106] Glc-glucose;

[0107] PMP-1-phenyl-3-methyl-5-pyrazolone.

[0108] Of course, the above description is not a limitation of the present application, and the present application is not limited to the above examples. Changes, modifications, additions or substitutions made by those skilled in the art within the scope of the present application should also be within the protection scope of the present application.

Claims

1. A method for constructing an in vivo pharmacokinetic model of carbohydrate drugs, characterized in that, The changes in monosaccharide content in tissue samples after drug administration were quantitatively detected, and the changes in monosaccharide content in serum and corresponding disease tissues were analyzed. The monosaccharide with the largest content change was used as an indicator for pharmacokinetic analysis, and a corresponding pharmacokinetic model was constructed. The tissue samples for quantitative detection are serum and organ tissues, and the concentration of monosaccharides in serum and organ tissues at different time points is quantitatively detected; among them, quantitative detection includes sample preparation and detection; Sample preparation includes the following steps: 1) Collect blood after drug administration, centrifuge and take the upper serum layer, place it in a hydrolysis bottle, add hydrolysis solution to hydrolyze and degrade it into monosaccharides; 2) Take the organs and tissues after administration, clean them, and grind them with water; after grinding, add sodium hypochlorite and borax and mix them, then add formic acid solution for oxidation treatment to obtain a clear liquid. Add hydrolysis solution to the clear liquid to hydrolyze it into monosaccharides. 3) After hydrolysis in steps 1) and 2), filter the solution through filter paper into a volumetric flask, add an alkaline solution to the filtrate to neutralize it, and bring the volume to the mark for later use; The detection method is any one of ion chromatography, high performance liquid chromatography, or liquid chromatography-mass spectrometry. The content of various monosaccharides in serum was detected, and a blood drug concentration-time curve was plotted to obtain the drug-time curves of different monosaccharides in serum, and the peak time and peak concentration of different monosaccharides were obtained; the content of different monosaccharides in organs and tissues was detected, and a tissue drug concentration-time curve was plotted to obtain the monosaccharides with the largest changes in different organs and tissues, and the peak time and peak concentration of different monosaccharides were obtained. For different disease types, the organs and tissues where the disease occurs are analyzed to obtain the monosaccharides with the greatest changes in content in the organs and tissues. Corresponding analysis with the changes in monosaccharides in serum is performed to determine the monosaccharides with the greatest changes in content in serum and organs and tissues as indicators for in vivo pharmacokinetic analysis of this type of sugar drug. The in vivo pharmacokinetic model of carbohydrate drugs was constructed, and the equation for the relationship between the administered dose and peak concentration was: Y = 0.0175 * X - 0.0637; Where Y is the peak concentration, in μg / ml; X represents the dosage, expressed in mg / kg, and ranges from 0 to 120 mg / kg.

2. The method for constructing an in vivo pharmacokinetic model of carbohydrate drugs according to claim 1, characterized in that, The monosaccharides include fucose, glucose, galactose, mannose, rhamnose, glucosamine, galactosamine, and acetylglucosamine; the organs and tissues include the heart, liver, spleen, lungs, and kidneys.

3. The method for constructing an in vivo pharmacokinetic model of carbohydrate drugs according to claim 1, characterized in that, The hydrolysate in steps 1) and 2) is either hydrochloric acid or trifluoroacetic acid; the alkaline solution in step 3) is either sodium hydroxide or ammonia.

4. The method for constructing an in vivo pharmacokinetic model of carbohydrate drugs as described in any one of claims 1-3, and its application in non-clinical pharmacokinetic studies of carbohydrate drugs and in the development of carbohydrate drugs.

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