Carbonic anhydrase inhibitor composition as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a carbonic anhydrase inhibitor composition as well as a preparation method and application thereof, and the carbonic anhydrase inhibitor composition comprises salvianolic acid B and tanshinone IIA. The invention solves the technical problems of large systemic toxic and side effects and poor curative effect of carbonic anhydrase inhibitors represented by sulfonamides such as acetazolamide in the prior art, and is expected to develop drugs with better curative effect for treating or preventing glaucoma, ocular hypertension, cardiac edema and acute altitude sickness.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and in particular relates to a carbonic anhydrase inhibitor composition and a preparation method and application thereof. Background Art

[0002] Carbonic anhydrase (CA) is a family of zinc-containing metalloenzymes that catalyze the reversible hydration of carbon dioxide and bicarbonate. CA is widely distributed in human tissues and is involved in respiration, CO2 and HCO3 - It is an important link in transmembrane transport, acid-base balance, osmotic pressure regulation, calcification, signal transduction, tumorigenesis, etc.

[0003] At present, the traditional carbonic anhydrase inhibitors (CAI) used in clinical practice mainly include inorganic metal complexes and sulfonamide derivatives. CAI, led by sulfonamide drugs such as acetazolamide, can reduce H by inhibiting CA activity. + -Na + Exchange, HCO3 - Transport and other physiological processes, thereby achieving the effects of improving metabolic acidosis, lowering intraocular pressure, and diuresis. It is often used in the clinical treatment of glaucoma, cardiogenic edema, and acute mountain sickness. However, CAIs, led by sulfonamides such as acetazolamide, have large systemic toxic and side effects, including blood electrolyte disorders, numbness of the face and extremities, gastrointestinal discomfort, and easy induction of urinary tract stones. In addition, because it is a non-selective CAI, it has an inhibitory effect on almost all CAs, which leads to various adverse reactions of the drug and affects the efficacy. Therefore, the development of new CAIs is of great research significance.

[0004] Salvia miltiorrhiza is a traditional Chinese medicine with the effects of promoting blood circulation and removing blood stasis, relieving pain and cooling blood and eliminating carbuncle. Its main components include water-soluble phenolic acids and fat-soluble diterpenoid tanshinones. Among them, the phenolic acid components of salvia miltiorrhiza are mainly composed of phenolic acid components represented by salvianolic acid B; while the tanshinone components mainly include tanshinone IIA, cryptotanshinone, dihydrotanshinone I, tanshinone I, etc.

[0005] In the prior art, a Chinese patent with publication number CN105646418A discloses a method for preparing salvianolic acid B and its application in retinal diseases. Pharmacodynamic studies have found that salvianolic acid B can protect the retina of chronic high intraocular pressure by increasing HO-1, reducing the expression of cleaved Caspase3 protein, increasing the activity of free radical scavenger SOD, reducing the content of lipid peroxide MDA and inhibiting the apoptosis of retinal ganglion cells. A Chinese patent with publication number CN1836670A discloses a microsphere injection for treating eye diseases. The technical solution is to use tanshinone IIA as a traditional Chinese medicine vasodilator in combination with other drugs for treating eye diseases.

[0006] Salvianolic acid B is a phenolic acid component, while tanshinone IIA is a fat-soluble diterpenoid component. Both are the main components of Salvia miltiorrhiza. Salvianolic acid B has anti-tumor, cardiovascular protection, anti-inflammatory, and antioxidant effects; tanshinone IIA has anti-inflammatory, antioxidant, neuroprotective, and cardiovascular protective effects. Although the two have similar effects, their mechanisms of action are different. At present, there has been no report on the combined use of salvianolic acid B and tanshinone IIA to inhibit carbonic anhydrase. Summary of the invention

[0007] In order to solve the technical problems that the existing CAI represented by sulfonamide drugs such as acetazolamide has large systemic toxic side effects and poor therapeutic effects, the present invention provides a carbonic anhydrase inhibitor composition and a preparation method and application thereof, which provides a reference for the development of drugs based on salvianolic acid components and tanshinone components, and is expected to develop drugs with better efficacy for treating or preventing glaucoma, cardiogenic edema and acute mountain sickness.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0009] A first aspect of the present invention provides a carbonic anhydrase inhibitor composition, which comprises salvianolic acid B and tanshinone IIA.

[0010] Preferably, the mass ratio of salvianolic acid B to tanshinone IIA is 2-24:1, for example, it can be 2:1, 2.4:1, 5:1, 10:1, 12.2:1, 15:1, 20:1, 24:1, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0011] Preferably, the carbonic anhydrase inhibitor composition further comprises a pharmaceutically acceptable excipient.

[0012] Preferably, the auxiliary material comprises one or more of a solvent, a buffer, a filler, a binder, a wetting agent, an absorption enhancer and a surfactant.

[0013] Preferably, the carbonic anhydrase inhibitor composition is in liquid form or solid form.

[0014] Preferably, if the carbonic anhydrase inhibitor composition is in liquid form, the final concentration of the salvianolic acid B is 0.07-718.62 mg / L, for example, it can be 0.07 mg / L, 71.86 mg / L or 718.62 mg / L, but is not limited to the listed values, and other values ​​not listed in the numerical range are also applicable; the final concentration of the tanshinone IIA is 0.03-294.33 mg / L, for example, it can be 0.03 mg / L, 29.43 mg / L or 294.33 mg / L, but is not limited to the listed values, and other values ​​not listed in the numerical range are also applicable.

[0015] Further preferably, if the carbonic anhydrase inhibitor composition is in liquid form, the final concentration of the salvianolic acid B is 71.86-359.31 mg / L, for example, it can be 71.86 mg / L, 143.72 mg / L, 215.59 mg / L, 287.45 mg / L or 359.31 mg / L, but is not limited to the listed values, and other values ​​not listed in the numerical range are also applicable; the final concentration of the tanshinone IIA is 5.89-147.17 mg / L, for example, it can be 5.89 mg / L, 8.83 mg / L, 11.78 mg / L, 14.72 mg / L, 17.66 mg / L, 20.60 mg / L, 23.54 mg / L, 26.49 mg / L or 29.43 mg / L, but is not limited to the listed values, and other values ​​not listed in the numerical range are also applicable.

[0016] More preferably, if the carbonic anhydrase inhibitor composition is in liquid form, the final concentration of the salvianolic acid B is 71.86 mg / L, 143.72 mg / L or 287.45 mg / L, and the final concentration of tanshinone IIA is 11.77 mg / L or 29.43 mg / L.

[0017] Most preferably, if the carbonic anhydrase inhibitor composition is in liquid form, the final concentration of salvianolic acid B is 143.72 mg / L, and the final concentration of tanshinone IIA is 11.77 mg / L; or, the final concentration of salvianolic acid B is 71.86 mg / L, and the final concentration of tanshinone IIA is 29.43 mg / L.

[0018] The structural formulas of the salvianolic acid B and tanshinone IIA of the present invention are as follows: Figure 1 shown.

[0019] The second aspect of the present invention provides a method for preparing the carbonic anhydrase inhibitor composition, comprising the following steps: uniformly mixing according to the formula to obtain the carbonic anhydrase inhibitor composition.

[0020] Preferably, if the carbonic anhydrase inhibitor composition is in liquid form, the preparation method comprises the following steps:

[0021] S1, dissolving salvianolic acid B and tanshinone IIA in solvents to obtain salvianolic acid B mother solution and tanshinone IIA mother solution respectively;

[0022] S2, using a buffer solution to dilute the mother solution of salvianolic acid B and the mother solution of tanshinone IIA, respectively, to obtain a diluted salvianolic acid B solution and a diluted tanshinone IIA solution;

[0023] S3. Evenly mix the diluted tanshinone B solution and the diluted tanshinone IIA solution in a volume ratio of 1:1.

[0024] Preferably, the solvent is dimethyl sulfoxide (DMSO).

[0025] Preferably, the buffer is 3-morpholinepropanesulfonic acid (MOPS).

[0026] Preferably, if the carbonic anhydrase inhibitor composition is in solid form, the preparation method comprises the following steps: uniformly mixing salvianolic acid B, tanshinone IIA with one or more of a filler, a binder, a wetting agent, an absorption enhancer and a surfactant to obtain the composition.

[0027] The third aspect of the present invention provides use of the carbonic anhydrase inhibitor composition in the preparation of a medicament for inhibiting carbonic anhydrase.

[0028] The fourth aspect of the present invention provides the use of the carbonic anhydrase inhibitor composition in the preparation of a drug for treating or preventing glaucoma, ocular hypertension, cardiogenic edema or altitude sickness.

[0029] Preferably, the altitude sickness is acute altitude sickness.

[0030] Preferably, the acute mountain sickness is high altitude coma, high altitude cerebral edema or high altitude pulmonary edema.

[0031] The fifth aspect of the present invention provides a medicine comprising the above-mentioned carbonic anhydrase inhibitor composition.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The salvianolic acid B and tanshinone IIA in the carbonic anhydrase inhibitor composition provided by the present invention can inhibit carbonic anhydrase through a synergistic effect, which provides a reference for the development of drugs based on salvianolic acid components and tanshinone components, and is expected to develop drugs with better efficacy for treating or preventing glaucoma, ocular hypertension, cardiogenic edema and acute mountain sickness. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1Figure 1 is the structural formula of salvianolic acid B and tanshinone IIA, wherein Figure A is the structural formula of salvianolic acid B; Figure B is the structural formula of tanshinone IIA.

[0035] Figure 2 It is the CA inhibition rate of salvianolic acid B with a final concentration of 287.45 mg / L, tanshinone IIA with a final concentration of 11.77 mg / L, and the carbonic anhydrase inhibitor composition of Example 1, wherein A represents salvianolic acid B, B represents tanshinone IIA, and C represents the carbonic anhydrase inhibitor composition of Example 1.

[0036] Figure 3 It is the CA inhibition rate of salvianolic acid B with a final concentration of 143.72 mg / L, tanshinone IIA with a final concentration of 11.77 mg / L, and the carbonic anhydrase inhibitor composition of Example 1, wherein A represents salvianolic acid B, B represents tanshinone IIA, and C represents the carbonic anhydrase inhibitor composition of Example 2.

[0037] Figure 4 It is the CA inhibition rate of salvianolic acid B with a final concentration of 71.86 mg / L, tanshinone IIA with a final concentration of 29.43 mg / L, and the carbonic anhydrase inhibitor composition of Example 1, wherein A represents salvianolic acid B, B represents tanshinone IIA, and C represents the carbonic anhydrase inhibitor composition of Example 3. DETAILED DESCRIPTION

[0038] The present invention is further explained below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0039] In the following examples, unless otherwise specified, all raw materials used were commercially available or prepared by conventional methods in the art.

[0040] Tanshinone IIA and tanshinone B were purchased from Chengdu Dester Biotechnology Co., Ltd.

[0041] Example 1

[0042] A carbonic anhydrase inhibitor composition having a mass ratio of salvianolic acid B to tanshinone IIA of 24.4:1, and a preparation method comprising the following steps:

[0043] S1. Dissolve 8.98 mg of salvianolic acid B and 1.47 mg of tanshinone IIA in 100 μL of DMSO to obtain a mother solution of salvianolic acid B with a concentration of 89.80 g / L and a mother solution of tanshinone IIA with a concentration of 14.70 g / L, respectively;

[0044] S2, using MOPS to dilute the mother solution of salvianolic acid B and the mother solution of tanshinone IIA respectively, to obtain a diluted salvianolic acid B solution with a concentration of 1437.24 mg / L and a diluted tanshinone IIA solution with a concentration of 58.87 mg / L;

[0045] S3. Evenly mix the diluted tanshinone B solution and the diluted tanshinone IIA solution in a volume ratio of 1:1.

[0046] Example 2

[0047] The carbonic anhydrase inhibitor composition in which the mass ratio of salvianolic acid B to tanshinone IIA is 12.2:1 is different from Example 1 in that MOPS is used to dilute the salvianolic acid B mother solution and the tanshinone IIA mother solution respectively, so that the concentration of the diluted salvianolic acid B solution is 718.62 mg / L and the concentration of the diluted tanshinone IIA solution is 58.87 mg / L, and the rest are the same.

[0048] Example 3

[0049] The carbonic anhydrase inhibitor composition in which the mass ratio of salvianolic acid B to tanshinone IIA is 2.4:1 is different from Example 1 in that MOPS is used to dilute the salvianolic acid B mother solution and the tanshinone IIA mother solution respectively, so that the concentration of the diluted salvianolic acid B solution is 359.31 mg / L and the concentration of the diluted tanshinone IIA solution is 147.17 mg / L, and the rest are the same.

[0050] Test example:

[0051] 1. Determination of CA inhibitory activity:

[0052] 40 μL of the test solution (salvianolic acid B solution with concentrations of 179.66 mg / L, 359.31 mg / L, and 718.62 mg / L, respectively; tanshinone IIA solution with concentrations of 29.43 mg / L and 73.58 mg / L, respectively; the carbonic anhydrase inhibitor composition of Examples 1-3) was added to a 96-well plate and mixed with 50 μL of p-nitrophenyl acetate (PNPA, 6 mM) solution, and then 10 μL of carbonic anhydrase solution (100 nM) was added to start the reaction. The well plate was placed in a constant temperature mixer and incubated at 37°C and 600 r / min for 20 minutes, and then the test solution was detected at 405 nm using an ELISA instrument and the absorbance value of each well was recorded. The inhibition rate of the test solution was calculated according to the inhibition rate calculation formula (1), and the results are shown in Figure 2-4 :

[0053] Inhibition rate % = [1-(A1-A2) / (A3-A4)] × 100% (1)

[0054] Wherein, A1 represents the absorbance of the well containing the test solution and the carbonic anhydrase solution; A2 represents the absorbance of the well in which the carbonic anhydrase solution is replaced with MOPS buffer; A3 represents the absorbance of the well in which the test solution is replaced with MOPS buffer; A4 represents the absorbance of the well in which the test solution and the carbonic anhydrase solution are replaced with MOPS buffer.

[0055] 2. Verification of synergistic effect: Calculate the expected additive effect and CI using formulas (2) and (3). The results are shown in Figure 2-4 .

[0056] Expected Additive Effect = E A +E B -E A ×E B (2)

[0057] CI=(E A +E B -E A ×E B ) / E AB (3)

[0058] Among them, E A represents the inhibition rate of salvianolic acid B, E B represents the inhibition rate of tanshinone IIA, E AB represents the inhibition rate of the carbonic anhydrase inhibitor composition; if CI is less than 1, and E AB This is greater than the expected additive effect, proving that salvianolic acid B and tanshinone IIA have a synergistic effect.

[0059] Depend on Figure 2 It can be seen that the expected additive effect of salvianolic acid B with a final concentration of 287.45 mg / L and tanshinone IIA with a final concentration of 11.77 mg / L is 50.4%, while the CA inhibition rate of the carbonic anhydrase inhibitor composition of Example 1 is 46.3%, and the CI is greater than 1. Therefore, there is no synergistic effect when the mass ratio of salvianolic acid B to tanshinone IIA is 24.4:1.

[0060] Depend on Figure 3 It can be seen that the expected additive effect of salvianolic acid B with a final concentration of 143.72 mg / L and tanshinone IIA with a final concentration of 11.77 mg / L is 46.5%, the CA inhibition rate of the carbonic anhydrase inhibitor composition of Example 2 is 60.0%, and the CI is less than 1. Therefore, when the mass ratio of salvianolic acid B to tanshinone IIA is 12.2:1, there is a synergistic effect.

[0061] Depend on Figure 4It can be seen that the expected additive effect of salvianolic acid B with a final concentration of 71.86 mg / L and tanshinone IIA with a final concentration of 29.43 mg / L is 44.3%, the CA inhibition rate of the carbonic anhydrase inhibitor composition of Example 3 is 47.7%, and the CI is less than 1. Therefore, when the mass ratio of salvianolic acid B to tanshinone IIA is 2.4:1, there is a synergistic effect.

[0062] Based on the results of Examples 1-3, it can be seen that the composition of salvianolic acid B and tanshinone IIA mixed in a mass ratio of 2-24:1 has good CA inhibitory activity, can exert the synergistic effect of the two compounds, and can be applied to drugs that inhibit carbonic anhydrase and develop drugs with better efficacy.

[0063] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A carbonic anhydrase inhibitor composition, characterized in that: The carbonic anhydrase inhibitor composition comprises salvianolic acid B and tanshinone IIA.

2. The carbonic anhydrase inhibitor composition according to claim 1, characterized in that: The mass ratio of the salvianolic acid B to tanshinone IIA is 2-24:

1.

3. The carbonic anhydrase inhibitor composition according to claim 1, characterized in that: The carbonic anhydrase inhibitor composition further comprises a pharmaceutically acceptable excipient.

4. The carbonic anhydrase inhibitor composition according to claim 1, characterized in that: The auxiliary materials include one or more of solvents, buffers, fillers, adhesives, wetting agents, absorption enhancers and surfactants.

5. A method for preparing the carbonic anhydrase inhibitor composition according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: mixing evenly according to the formula to obtain the product.

6. Use of the carbonic anhydrase inhibitor composition according to any one of claims 1 to 4 in the preparation of a medicament for inhibiting carbonic anhydrase.

7. Use of the carbonic anhydrase inhibitor composition according to any one of claims 1 to 4 in the preparation of a medicament for treating or preventing glaucoma, ocular hypertension, cardiogenic edema or altitude sickness.

8. The use according to claim 7, characterized in that: The altitude sickness is acute altitude sickness.

9. The use according to claim 8, characterized in that: The acute mountain sickness is high altitude coma, high altitude cerebral edema or high altitude pulmonary edema.

10. A drug, characterized in that The invention comprises the carbonic anhydrase inhibitor composition according to any one of claims 1 to 4.

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