CN113321699A

CN113321699A - Preparation method and application of anserine cholate

Info

Publication number: CN113321699A
Application number: CN202110498664.5A
Authority: CN
Inventors: 吴伟伟; 李健雄; 冯议; 李海亮; 唐启明
Original assignee: Wuhan Jason Biotech Co ltd
Current assignee: Wuhan Jason Biotech Co ltd
Priority date: 2021-05-08
Filing date: 2021-05-08
Publication date: 2021-08-31
Anticipated expiration: 2041-05-08
Also published as: CN113321699B

Abstract

The invention discloses a preparation method and application of anserine cholate, namely application of the anserine cholate in medicines for resisting hyperuricemia and gout, and belongs to the technical field of new application of medicines. The invention proves that the anserine cholate can obviously improve the bioavailability of the anserine by SD rats for the first time. Meanwhile, the effect of the medicine on gout caused by oteracil potassium salt is found, the effect of the chenosine cholate on reducing uric acid and treating gout is found, the concentration of the chenosine cholate is as low as 1.1mg/kg, the effect is good, and the effect is enough to reduce the blood uric acid, the blood urea nitrogen and the blood creatinine level. The effect of anserine with high concentration and lower concentration is good, but the effect of anserine cholate with low concentration and the effect of anserine with low concentration and higher concentration are good.

Description

Preparation method and application of anserine cholate

Technical Field

The invention discloses a preparation method of anserine cholate and application of the anserine cholate in preparation of anti-hyperuricemia and anti-gout drugs, belonging to the technical field of new application of drugs.

Background

Hyperuricemia is a metabolic disease caused by the fact that a substance called purine is metabolized and disordered in human body, so that uric acid in blood is increased, the daily production amount and excretion amount of uric acid in the human body are approximately equal, in terms of production amount, one third is from food, two thirds is self-synthesized in the body, excretion route is one third is from intestinal tract, and two thirds is from kidney. Any of the above routes, if problematic, will cause uric acid elevation.

Gout is a crystal-related arthropathy caused by deposition of monosodium urate (MSU), is directly related to purine metabolic disorder and/or hyperuricemia caused by reduction of uric acid excretion, and belongs to the category of metabolic rheumatism. Gout can be complicated with kidney disease, and severe cases can cause joint destruction and renal function damage, often accompanied with hyperlipidemia, hypertension, diabetes, arteriosclerosis, coronary heart disease, etc.

As a chronic crystalline arthritis, gout, hyperuricemia is the basis for gout development. Genetic factors influence the whole process of occurrence and development of gout and hyperuricemia, monogenic genetic diseases may influence key enzymes on a uric acid metabolic pathway, and SNP may cause difference of uric acid transport proteins and degree of inflammatory reaction. Therefore, hyperuricemia is closely related to gout.

In the prior art, anserine can be used for reducing uric acid. The invention unexpectedly discovers that the anserine cholate has better effect.

Disclosure of Invention

The invention aims to provide a compound anserine cholate with high uric acid reducing activity, and a new application thereof in preparing anti-hyperuricemia and anti-gout drugs. The anserine cholate can obviously improve the bioavailability of the anserine; in addition, the applicant simultaneously proves that not all anserine salts have the effect of obviously improving the utilization rate of anserine, the acetate salt of anserine can only slightly improve the utilization rate, and the decanoate salt of anserine can obviously reduce the utilization rate of the anserine, but the cholate of anserine unexpectedly greatly improves the bioavailability, which is the core of the invention. The other salts of anserine do not have the effect of improving the utilization rate of anserine, and the anserine acetate and anserine caprate are taken as examples, and the description of the other salts is omitted.

The invention discloses a chenosine cholate which is prepared by the reaction of chenosine and cholic acid (salifying according to the molar ratio of 1: 1), and has better oral bioavailability than the chenosine.

In particular to application of anserine cholate in preparing medicines for resisting hyperuricemia and gout.

Furthermore, the anserine cholate is applied to medicines for reducing blood uric acid, blood urea nitrogen, blood creatinine and the like.

Further, the application of the low-dose anserine cholate in the medicines for preventing and treating gout.

More specifically, low doses of anserine cholate, such as 1.1mg/kg, significantly reduced the levels of uric acid, blood urea nitrogen and blood creatinine in potassium oxonate-induced hyperuricemia mice.

Further, the medicament of this embodiment may include various pharmaceutically acceptable diluents or/and carriers.

Specifically, the drug in this embodiment is an oral agent.

The oral preparation in this embodiment includes a diluent, and the diluent is selected from physiological saline, a CMC-Na aqueous solution, and the like, and may also be other suitable excipients.

Specifically, the diluent in this embodiment is 5% Solutol HS15 physiological saline solution or 0.5% CMC-Na aqueous solution, etc., preferably 0.5% CMC-Na aqueous solution.

In addition, the embodiment of the invention also discloses a preparation method of the anserine cholate, which comprises the following steps: dissolving anserine and cholic acid in an organic solvent according to a molar ratio of 1:1.5-3.0, heating to 50-60 ℃, completely dissolving, evaporating to remove part of the solvent while hot, cooling and crystallizing when solid is separated out, filtering and drying to obtain the anserine cholate. Wherein, the organic solvent can be common alcohol solvent, such as methanol and ethanol. The method has simple process, and the prepared product meets the requirement of use.

The invention discloses a new application of anserine cholate, which is proved from two aspects, namely: the SD rat is used for providing anserine pharmacokinetic experiment to prove that the utilization rate of the anserine can be obviously improved by only anserine cholate (taking 0.5% CMC-Na aqueous solution for matching use as an example), and the highest utilization rate can reach more than 120% (the corresponding bioavailability of the anserine is only about 25%); other salts can not obviously improve the utilization rate, acetate is slightly improved, and decanoate reduces the utilization rate. The second aspect is that: by the effect of the drug on gout caused by oteracil potassium salt, we can obtain: the effect of low dose, such as 1.1mg/kg anserine cholate in reducing uric acid is obviously higher than that of a reference substance (0.4 mg/kg anserine), the effect of 3.2mg/kg anserine cholate in reducing blood urea nitrogen is higher than that of the corresponding reference substance (1.2 mg/kg anserine), the effect of 3.2mg/kg anserine cholate in reducing creatinine is higher than that of the reference substance (1.2 mg/kg anserine), and the effect of two groups of anserine cholates has no certain difference in three indexes. And the three indexes show that the effect of low dose, such as 1.1mg/kg anserine cholate, shows good effect, and at the concentration, the effect is enough to reduce the blood uric acid, blood urea nitrogen and blood creatinine level. The invention discovers the application of the anserine cholate in the aspects of reducing uric acid and the like for the first time. The effect of anserine with high concentration and lower concentration is good, but the effect of anserine with higher concentration is good even with low concentration. The effect of anserine cholate is not as good as the higher concentration, but is better even with lower concentration (and better with higher concentration).

Drawings

FIG. 1 is a bar graph comparing (umol/l) blood Uric Acid (UA) levels of groups;

FIG. 2 bar graph comparing UREA nitrogen (UREA) levels (mmol/l) for each group;

FIG. 3 bar graph comparing (ummol/l) blood Creatinine (CREA) levels for each group.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings.

Example 1: preparation of salts

Dissolving anserine (240.2 g, 1 mol) and cholic acid (2 mol) in ethanol (excessive), heating to completely dissolve, evaporating to remove part of solvent while hot, cooling to separate out solid when solid is separated out, filtering, drying, and measuring the salt formation ratio by an HPLC area normalization method to be 1:1.

example 2

Pharmacokinetic experiment of goose muscle peptide given to SD rat

1.1 materials

Gly-Gly-His, batch No. C1709032, available from Shanghai Allantin Biotechnology Ltd.

Anserine, anserine acetate, anserine cholate and anserine decanoate.

Blank substrate, cynomolgus monkey blank plasma, as blank substrate when processing standard curve.

Reagent, Drech Drinking Water, lot number 20201031D; acetonitrile, Merck, batch No.: JA 097830; acetonitrile, Tedia, batch number: 20115148, respectively; ammonium acetate, ROE, batch No.: 5D 3094; perchloric acid, Shanghai test, batch number: 20170308, respectively; CMC-Na, Shanghai test, batch number: 20180412, respectively; solutol HS15, melphalan, batch number: D1223A.

1.2 Main instruments

Ultimate 3000 high performance liquid chromatography, Thermo fisher.

4500 mass spectrometer, Sciex.

Eppendorf Research plus pipettor, Eppendorf.

VXR BS25 was a circular shaker mixer, IKA.

Centrifuge 5424 small bench top high speed Centrifuge, Eppendorf.

1.3 conditions of analysis

Liquid phase conditions, as shown in table 1:

TABLE 1

Mass spectrometry conditions, as shown in table 2:

TABLE 2

1.4 method for diluting standard curve and internal standard working solution

The anserine stock solution was diluted to 10, 4, 1, 0.5, 0.25, 0.1. mu.g/ml with 1.6% perchloric acid water as a standard curve working solution.

Gly-Gly-His stock solution was diluted to 100. mu.g/ml with 1.6% perchloric acid water and 0.1. mu.g/ml with acetonitrile as an internal standard working solution.

1.5 plasma pretreatment method

5.1 blank sample

Sucking blank plasma 10 μ l, adding 1.6% perchloric acid water 10 μ l and acetonitrile 70 μ l, mixing by vortex for 2min, centrifuging at 12000rpm for 5min, transferring supernatant into a sample injection bottle, adding 1.6% perchloric acid water, sucking, and mixing.

5.2 zero concentration samples

Sucking blank plasma 10 μ l, adding 1.6% perchloric acid water 10 μ l and internal standard working solution 70 μ l, mixing for 2min by vortex, centrifuging at 12000rpm for 5min, collecting supernatant, transferring into sample injection bottle, adding 1.6% perchloric acid water, sucking, and mixing.

5.3 Standard Curve and quality control sample

Sucking blank plasma 10 μ l, adding standard curve working solution 10 μ l and internal standard working solution 70 μ l, vortex mixing for 2min, centrifuging at 12000rpm for 5min, transferring supernatant into a sample injection bottle, adding 1.6% perchloric acid water, sucking, and mixing.

5.4 plasma samples of unknown concentration

Sucking 10 μ l of animal plasma, adding 10 μ l of 1.6% perchloric acid water and 70 μ l of internal standard working solution, mixing for 2min by vortex, centrifuging at 12000rpm for 5min, taking the supernatant, transferring into a sample injection bottle, adding 1.6% perchloric acid water, sucking, beating, and mixing.

1.6 animal test methods

Test animals, male SD rats.

Blood sampling time points: before and after the medicine is taken, 5min, 15 min, 0.5h, 1h, 1.5h, 2h, 3h, 4h, 6h and 8 h.

The administration volume: 5ml/kg of vein and 10ml/kg of intragastric administration.

The blood sampling method comprises the following steps: taking 0.1ml of blood from the sinus jugular vein of the SD rat, placing the blood into a heparin anticoagulation tube, centrifuging the blood for 0.5min at 12000rpm, taking blood plasma, freezing the blood plasma at the temperature of minus 20 ℃ for detection, and testing the blood plasma by using the method, wherein the test process is shown in a table 3:

TABLE 3

Wherein: the administration dose of each salt was 50mg/kg calculated by molecular weight conversion based on anserine. Wherein, the solvent 1 is 5 percent Solutol HS15 normal saline solution. The solvent 2 is 0.5% CMC-Na aqueous solution.

1.7 data statistics and processing

Because anserine is an animal endogenous substance, if the background of the pre-dose point is higher than the lower limit of quantification, the background is subtracted from the blood concentration of the subsequent points, otherwise, the background is not subtracted. The concentration of anserine in SD rat plasma was measured by LC-MS/MS, and the AUC was calculated by DAS 3.3_0-t。

1.8 results and analysis

The results of anserine exposure (μ g/ml) in plasma after 10mg/kg of anserine was intravenously administered to SD rats are shown in Table 4:

TABLE 4

The results of anserine exposure (μ g/ml) in plasma after oral administration of 50mg/kg anserine to SD rats are shown in Table 5:

TABLE 5

Time (h)	M4	M5	M6
					0	/	/	/
0.083	0.5	4.67	1.68
				0.25	4.1	12.79	6.97
0.5	3.46	4.6	3.4
				1	1.57	2.05	2.26
1.5	1.14	0.65	1.37
				2	0.64	0.2	0.8
3	0.17	/	/
				4	/	/	/
6	/	/	0.12
				8	/	0.14	0.31

The results of anserine exposure (μ g/ml) in plasma after intravenous administration of 50mg/kg anserine acetate to SD rats are shown in table 6:

TABLE 6

The results of anserine exposure in plasma (μ g/ml) after oral administration of 50mg/kg anserine cholate (dosed with 5% solutol HS15 physiological saline solution) in SD rats are shown in table 7:

TABLE 7

Time (h)	M10	M11	M12
					0	/	/	/
0.083	1.45	1.06	1.01
				0.25	3.17	2.75	3.78
0.5	2.99	4.1	4.44
				1	6.82	3.52	4.21
1.5	7.56	2.14	1.23
				2	6.5	1.53	0.49
3	1.53	0.96	1.12
				4	0.2	0.65	0.32
6	/	/	0.1
				8	/	0.14	/

The results of anserine exposure in plasma (μ g/ml) after oral administration of 50mg/kg anserine caprate (dosed in 5% solutol HS15 physiological saline solution) to SD rats are shown in table 8:

TABLE 8

Time (h)	M13	M14	M15
					0	/	/	/
0.083	0.25	0.24	0.67
				0.25	1.36	0.74	1.16
0.5	1.14	0.78	1.13
				1	0.88	0.44	0.57
1.5	0.26	0.12	0.34
				2	0.18	0.38	0.25
3	0.1	0.24	0.16
				4	0.18	/	0.2
6	/	/	/
				8	/	/	/

The results of anserine exposure (μ g/ml) in plasma after oral administration of 50mg/kg anserine cholate (dispensed in 0.5% CMC-Na aqueous solution) to SD rats are shown in Table 9:

TABLE 9

Time (h)	M16	M17	M18
					0	/	/	/
0.083	5.56	9.61	14.36
				0.25	28.2	40.3	30.31
0.5	31.23	37.52	38.64
				1	11.45	13.82	11.75
1.5	2.98	2.36	2.8
				2	0.61	0.37	0.46
3	0.23	/	0.14
				4	0.17	/	0.11
6	0.14	/	/

The results of anserine exposure (μ g/ml) in plasma after oral administration of 50mg/kg anserine caprate (dispensed in 0.5% CMC-Na aqueous solution) to SD rats are shown in Table 10:

watch 10

AUC of anserine and each salt_(0-t)(mg/L × h) and bioavailability are shown in table 11:

TABLE 11

As can be seen from tables 4, 5 and 11, the AUC (0-t) of anserine at the high dose is slightly improved relative to that at the low dose, and the bioavailability of anserine at 50mg/kg is 25.8%, and the bioavailability is lower. As can be seen from tables 5, 6 and 11, the bioavailability of acetate was slightly improved compared to anserine, and the effect was not significant. As can be seen from tables 5, 7, 9 and 11, the bioavailability of cholate is significantly improved relative to anserine, the AUC (0-t) of physiological saline is 10.70, the bioavailability is 44.2%, the AUC (0-t) of CMC-Na aqueous solution is 29.61, and the bioavailability is 122.3%. As can be seen from tables 5, 8, 10 and 11, the AUC (0-t) and the decrease in the availability of the decanoate salt was very large. In conclusion, the anserine cholate remarkably improves the AUC (0-t) value and bioavailability.

Example 3 Effect of the drug on gout caused by Potassium Oxonate

2.1 experimental mice:

kunming male mouse (KM) 46 mice, 5w, 26 + -2 g, the size of the batch is kept consistent.

2.2 Experimental reagents and materials:

preparing 1.5g/100ml suspension with sterilized hot distilled water, and performing intraperitoneal injection at a dose of 250mg/kg body weight; materials: syringes, gavage needles, centrifuge tubes, centrifuges, kits, and the like.

2.3 Experimental groups:

grouping: mice were randomly divided into 8 groups: normal control (n = 6), model (n = 8), experimental one 1.2mg/kg control (n = 8), experimental two 0.4mg/kg control (n = 8), experimental three 3.2mg/kg salts (n = 8), experimental four 1.1mg/kg salts (n = 8); and performing normal saline intragastric perfusion on the control group and the model group, performing intragastric perfusion on the experimental group I and the experimental group II according to the anserine with the specified concentration, and performing intragastric perfusion on the experimental group III and the experimental group IV according to the anserine cholate with the specified concentration.

2.4 Experimental methods:

after the mice are adapted for 3 days, formal experiments are started, drug intragastric administration is carried out, the control group and the model group are subjected to normal saline intragastric administration, and the experimental groups are respectively subjected to intragastric administration according to required concentration, 1 time a day and 7 days continuously. Carrying out intraperitoneal injection on the mice in each group except the control group according to 250mg/kg of oteracil potassium salt every day; and (3) performing intragastric administration on each group 1h after 7d intraperitoneal injection, taking blood from an eyeball 1h after intragastric administration, standing for 1h, centrifuging at 2500r/min for 5min, and measuring blood Uric Acid (UA), blood UREA nitrogen (UREA) and blood creatinine value (CREA). All values are expressed as mean + standard deviation, SPSS12.0 software is used for data statistics, and t-test is used for group comparisons.

2.5 test results

The levels of blood Uric Acid (UA), blood UREA nitrogen (UREA) and blood Creatinine (CREA) in the mice of each group are shown in table 12:

TABLE 12

Group of	UA（umol/l）	UREA（mmol/l）	CREA（umol/l）
				Blank group	62.65±7.69****	13.09±0.76****	17.51±0.51****
Model set	149.75±26.03	21.46±0.97	23.75±1.26
				1.2mg/kg control	78.42±7.13****	19.07±0.58****	20.99±0.35**
0.4mg/kg control	103.35±5.67****	16.39±1.32****	20.05±0.48****
				3.2mg/kg salt	76.58±11.15****	15.79±1.11****	18.46±1.50****
1.1mg/kg salt	61.08±12.82****	14.65±1.90****	18.18±0.82****

2.6 analysis of results

As can be seen from the results summarized in table 12 and fig. 1 to 3, blood Uric Acid (UA), blood urea nitrogen (UAEA) and blood creatinine level (CREA) were significantly increased (P <0.0001) in the model group compared to the control group, while the respective drug groups were significantly decreased in comparison to the model group.

As can be seen from table 12 and fig. 1, by comparison of the blood Uric Acid (UA) results, the model group >0.4mg/kg control >1.2mg/kg control >3.2mg/kg salt > blank >1.1mg/kg salt; through statistical analysis, it can be concluded that both the control drug and the corresponding salt significantly reduced the level of hematuria acid (P < 0.0001); the 1.2mg/kg reference substance has a certain difference with the 0.4mg/kg reference substance, and the 1.2mg/kg reference substance has a stronger uric acid reducing effect than the 0.4mg/kg reference substance; the two groups of salts have no significant difference; the results of the comparison product of 1.2mg/kg and the comparison product of 3.2mg/kg have no significant difference, and the comparison product of 0.4mg/kg and the comparison product of 1.1mg/kg have significant difference (P <0.01), show that the effect of 1.1mg/kg anserine cholate in reducing uric acid is significantly higher than that of the comparison product, and the effect of 1.1mg/kg anserine cholate in reducing uric acid is significantly lower than that of the comparison product of 1.2mg/kg (P <0.0001), and the effect of reducing uric acid of low-concentration anserine cholate is stronger than that of high-concentration anserine.

As can be seen from table 12 and fig. 2, by comparing the results of blood urea nitrogen (UAEA), the model group >1.2mg/kg control >0.4mg/kg control >3.2mg/kg salt >1.1mg/kg salt > blank group, by statistical analysis, it can be found that the salt of 3.2mg/kg has significant difference (P <0.001) with the control of 1.2mg/kg, the salt of 1.2mg/kg also has significant difference (P <0.01) with the control of 0.4mg/kg, the results show that the effect of the salt on reducing blood urea nitrogen is obviously higher than that of the reference substance, the effect of 1.1mg/kg of the salt on reducing urea nitrogen is obviously higher than that of the 1.2mg/kg of the reference substance (P <0.0001), and the two concentrations of the salt (3.2 mg/kg and 1.1 mg/kg) are equivalent in effect without obvious difference, the results show that 1.1mg/kg anserine cholate is the most effective in lowering blood urea nitrogen.

As can be seen from table 12 and fig. 3, by comparison of blood Creatinine (CREA) results, the model group >1.2mg/kg control >0.4mg/kg control >3.2mg/kg salt >1.1mg/kg salt > blank; through statistical analysis, it can be concluded that both the control drug and the corresponding salt significantly reduced the blood creatinine level (P < 0.0001); the results of the 1.2mg/kg control and the 3.2mg/kg salt were significantly different (P <0.001), and the 0.4mg/kg control and the 1.1mg/kg salt were not significantly different, indicating that the 3.2mg/kg salt had significantly higher effect in reducing creatinine than the control, and the 1.1mg/kg salt had significantly different effect in reducing creatinine level from the 1.2mg/kg control (P <0.0001), while the two groups of salts had no significant difference in reducing creatinine level, and it can be seen that the 1.1mg/kg anserine cholate had the best effect in reducing creatinine level.

The effect of the low-dose 1.1mg/kg anserine cholate on reducing uric acid is obviously higher than that of a reference substance, the effect of free anserine (0.4 mg/kg of the reference substance and the same molar quantity) on reducing blood urea nitrogen is higher than that of the reference substance (1.2 mg/kg of the reference substance), the effect of 3.2mg/kg of salt on reducing creatinine is higher than that of the reference substance (1.2 mg/kg of the reference substance), and the effects of two groups of salts are not different in three indexes. And the three indexes show that the low dose, such as 1.1mg/kg of anserine cholate has the best effect, and at the concentration, the low dose is enough to reduce the blood uric acid, the blood urea nitrogen and the blood creatinine level.

The above description is only exemplary of the present invention and should not be taken as limiting the invention, as any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The anserine cholate is characterized by being prepared by reacting anserine with cholic acid, and has better oral bioavailability than the anserine.

2. Use of anserine cholate as claimed in claim 1 for preparing anti-hyperuricemia and anti-gout drugs.

3. The use according to claim 2, wherein anserine cholate is used in a medicament for reducing blood uric acid, blood urea nitrogen and blood creatinine.

4. The use of claim 2, wherein the low dose of anserine cholate is used in the preparation of drugs for preventing and treating gout.

5. Use according to claim 2, wherein the medicament comprises a pharmaceutically acceptable diluent or/and carrier.

6. The use of claim 5, wherein the medicament is an oral agent.

7. The use according to claim 5, wherein the oral formulation comprises a diluent selected from the group consisting of physiological saline or an aqueous CMC-Na solution.

8. The method for preparing anserine cholate according to claim 1, wherein the method comprises: dissolving anserine and cholic acid in an organic solvent according to a molar ratio of 1:1.5-3.0, heating to 50-60 ℃, completely dissolving, evaporating to remove part of the solvent while hot, cooling and crystallizing when solid is separated out, filtering and drying to obtain the anserine cholate.