阅读说明：本技术 草乌甲素多晶型及其制备方法和应用 (Bulleyaconitine A polymorphism, preparation method and application thereof ) 是由 刘珉宇 连敏玲 魏武 刘全海 董玉琼 黄晓玲 邓轶方 张玉荣 汪蓉 梁晨 于 2020-07-03 设计创作，主要内容包括：本发明公开了草乌甲素多晶型及其制备方法和应用,其制备简单,具有良好的稳定性和更优的药代动力学特性。与草乌甲素原料相比,根据本发明的草乌甲素的多晶型具有更低的毒性和更好的镇痛效果,进一步提高了草乌甲素的应用前景。(The invention discloses a bulleyaconitine A polymorphism, a preparation method and application thereof, which have simple preparation, good stability and better pharmacokinetic property. Compared with the bulleyaconitine A raw material, the polymorphic form of the bulleyaconitine A has lower toxicity and better analgesic effect, and further improves the application prospect of the bulleyaconitine A.)

1. An amorphous form of bulleyaconitine A characterized by an XRPD pattern substantially as shown in figure 1; the TGA and DSC plots are substantially as shown in FIG. 2.

2. A bulleyaconitine A crystal form I is characterized in that the XRPD pattern is basically as shown in figure 3.

3. Form I according to claim 2, characterized by a TGA and DSC profile substantially as shown in figure 4.

4. A bulleyaconitine A crystal form VI is characterized in that the XRPD pattern is substantially as shown in figure 5.

5. The crystalline form VI according to claim 4 characterized by a TGA and DSC profile substantially as shown in figure 6.

6. The preparation method of bulleyaconitine A crystal form VI according to claim 4 or 5, which is characterized in that the bulleyaconitine A crystal form VI is obtained by crystallization with a mixed solvent of acetonitrile and water, preferably, the ratio of the acetonitrile to the water is 1: 2-4.

7. A pharmaceutical composition comprising the amorphous form of bulleyaconitine a of claim 1 or the form I or VI of bulleyaconitine a of any of claims 2 to 5; and pharmaceutically acceptable adjuvants.

8. Pharmaceutical composition according to claim 7, characterized in that it is a solid dosage form, preferably an oral dosage form.

9. Use of the amorphous form of bulleyaconitine A according to claim 1 or the bulleyaconitine A crystal form I or VI according to any of claims 2 to 5 in the preparation of anti-inflammatory and analgesic medicaments.

10. Use of the amorphous form of bulleyaconitine A according to claim 1 or the bulleyaconitine A crystalline form I or VI according to any of claims 2 to 5 in the preparation of a medicament for inhibiting drug addiction.

Technical Field

The invention relates to a polymorphic form, in particular to a polymorphic form of bulleyaconitine A and a preparation method and application thereof.

Background

Bulleyaconitine A (CAS-RN 79592-91-9) is a modern plant medicine, is separated from Ranunculaceae Umbelliferae plant Diancide Dulat, belongs to diterpene diester type alkaloid, and is an alkaloid different from aconitine and hypaconitine. The medicine has no tolerance and addiction, can be used as potent analgesic and anti-inflammatory, and has dual pharmacological effects of relieving pain in central nervous system and relieving pain by external application. Presently, the national Food and drug administration (CFDA) has approved the use of aconitine injections and tablets for the treatment of chronic pain and rheumatoid arthritis. Clinically, bulleyaconitine A has also been widely used for treating rheumatoid arthritis, osteoarthritis, fibromyositis, cervical spondylosis, cancer pain and chronic pain caused by various reasons.

The bulleyaconitine A has obvious inhibition effect on inflammation and pain. Prostaglandin PGE2 is an inflammatory mediator produced by inflammatory factors, produced and released locally in tissues, with inflammatory and nociceptive effects, which increase local vascular permeability, further accentuate the inflammatory response and thus produce a series of changes, such as: synovial cells and fibroblasts proliferate, angiogenesis, collagen production, etc., so that bone and cartilage cells are destroyed. The reduction of serum PGE2 is one of the mechanisms of the anti-inflammatory action of bulleyaconitine A, and PGE2 can activate peripheral pain receptors and transmit pain signals. The beta-endorphin is a neuropeptide with stronger analgesic effect, and the analgesic effect of the bulleyaconitine A is probably related to antagonizing intracerebral 5-hydroxytryptamine (5-HT) and inhibiting PGE2 release, thereby relieving the inhibition on the beta-endorphin.

Meanwhile, according to literature reports, bulleyaconitine A can directly induce the expression of spinal microglia dynorphin A, so that the analgesic effect is shown, the inhibitory effect on neuropathic pain is shown on the C-fiber synapse surface of the spinal cord posterior horn, the bulleyaconitine A is also proved to be capable of enhancing the analgesic effect of morphine and inhibiting the analgesic tolerance of morphine, and in addition, the bulleyaconitine A can also inhibit inflammatory chemotactic factors.

Currently, research and application of bulleyaconitine A are limited to anti-inflammatory and analgesic aspects, such as: CN101468000 discloses an application of bulleyaconitine A in preparing a medicament for treating primary erythromelalgia; CN107245054A discloses the use of an amorphous bulleyaconitine A compound in the preparation of medicaments for treating pain diseases caused by rheumatism or rheumatoid arthritis; the bulleyaconitine A in CN106943402A is used for preparing medicine for preventing and treating osteoporosis or osteolysis.

The prior research (CN110478350A) of the inventor finds that the bulleyaconitine A has the function of obviously inhibiting spontaneous activation symptoms caused by drugs, and shows that the bulleyaconitine A and the derivatives thereof have great application potential in the aspect of inhibiting drug addiction.

However, the research on the polymorphism of bulleyaconitine A in the prior art is relatively rare, and many reports in the literature relate to white, colorless and transparent bulleyaconitine A obtained by recrystallization such as crystallization. But does not report XRPD about bulleyaconitine A polymorphic form. CN107245054A discloses an amorphous bulleyaconitine A compound, the dissolubility and the analgesic effect of which are slightly better than those of a reference substance. Further developing other polymorphic forms and amorphous bulleyaconitine A with better stability has important significance.

Disclosure of Invention

One aspect of the present invention relates to a stable polymorphic form of bulleyaconitine A.

Another aspect of the invention relates to polymorphic forms of bulleyaconitine A having better stability or pharmacokinetic properties than the known amorphous forms.

In particular, the first aspect of the invention relates to an amorphous form of bulleyaconitine A, wherein the XRPD pattern is substantially as shown in figure 1; preferably, the TGA and DSC profiles are substantially as shown in figure 2.

According to another aspect of the invention, the XRPD pattern of form I of bulleyaconitine a is substantially as shown in figure 3.

Preferably, the TGA and DSC profile of said crystalline form I are substantially as shown in figure 4.

According to another aspect of the invention, the XRPD pattern of form VI of bulleyaconitine a is substantially as shown in figure 5.

Preferably, the TGA and DSC profile of said crystalline form VI is substantially as shown in figure 6.

According to another aspect of the invention, the invention also relates to a preparation method of the bulleyaconitine A polymorphism.

Specifically, the preparation method of the amorphous bulleyaconitine A comprises the following steps: dissolving bulleyaconitine A in lower alcohol (preferably methanol and ethanol), quickly dripping the obtained solution into water, filtering, separating, and drying the solid to obtain the final product.

The preparation method of the bulleyaconitine A crystal form I is characterized in that the bulleyaconitine A is dissolved in a common organic solvent and volatilized at normal temperature. Preferably, the common organic solvent includes at least one of diethyl ether, tetrahydrofuran, methanol, ethanol, n-propanol, isopropanol, acetone, ethyl acetate, ethyl formate, acetonitrile, toluene, dichloromethane, chloroform, tetrahydrofuran, or dimethyl sulfoxide; preferably diethyl ether, tetrahydrofuran, ethanol, acetone, ethyl acetate, acetonitrile, dichloromethane, chloroform, tetrahydrofuran; most preferably at least one of diethyl ether, tetrahydrofuran, ethanol, acetone, ethyl acetate or dichloromethane.

The preparation method of bulleyaconitine A crystal form VI is obtained by crystallizing a mixed solvent of acetonitrile and water; preferably, the ratio of acetonitrile to water is 1: 2-4; more preferably, the ratio of acetonitrile to water is 1: 3.

according to another aspect of the invention, a pharmaceutical composition is also related, comprising an amorphous form of bulleyaconitine a, bulleyaconitine a form I or VI according to the invention; and pharmaceutically acceptable adjuvants.

Preferably, the pharmaceutical excipient is selected from at least one of a disintegrant, a diluent, a lubricant, a binder, a wetting agent, a flavoring agent, a suspending agent, a surfactant, or a preservative; more preferably, the disintegrant is selected from at least one of corn starch, potato starch, crospovidone, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, croscarmellose sodium, carboxymethylcellulose calcium, or alginic acid; more preferably, the diluent is selected from at least one of lactose, sucrose, mannitol, corn starch, potato starch, calcium phosphate, calcium citrate or crystalline cellulose; more preferably, the lubricant is selected from at least one of micropowder silica gel, magnesium stearate, calcium stearate, stearic acid, talcum powder or anhydrous silica gel; more preferably, the binder is selected from at least one of acacia, gelatin, dextrin, hydroxypropyl cellulose, methyl cellulose or polyvinyl pyrrolidone; more preferably, the wetting agent is selected from sodium lauryl sulfate; more preferably, the flavoring agent is selected from at least one of aspartame, stevioside, sucrose, maltitol, or citric acid; more preferably, the suspending agent is selected from at least one of acacia, gelatin, methylcellulose, sodium carboxymethylcellulose, hydroxymethylcellulose, or aluminum stearate gel; more preferably, the surfactant is selected from at least one of lecithin, sorbitan monooleate, or glyceryl monostearate; more preferably, the preservative is selected from at least one of methyl paraben or propyl paraben.

According to another aspect of the invention, the pharmaceutical composition is in a solid dosage form, preferably an oral dosage form.

According to another aspect the invention also relates to the use of the bulleyaconitine A polymorph according to the invention.

It will be appreciated by those skilled in the art that the bulleyaconitine A polymorph according to the invention may be used in any of the known uses of bulleyaconitine A.

Specifically, the bulleyaconitine A amorphous form or the bulleyaconitine A crystal form I or VI can be used for preparing anti-inflammatory and analgesic drugs and also can be used for preparing drugs for inhibiting drug addiction.

The invention has the beneficial effects that:

1) the polymorphic form of bulleyaconitine A according to the invention has a good stable form.

2) Polymorphic forms of bulleyaconitine A, which have better pharmacokinetic properties than the known amorphous forms.

3) Compared with the bulleyaconitine A raw material, the bulleyaconitine A polymorphism has lower toxicity.

4) Compared with the bulleyaconitine A raw material, the polymorphic form of the bulleyaconitine A has better analgesic effect.

Drawings

FIG. 1: amorphous XRPD pattern of bulleyaconitine A

FIG. 2: amorphous TGA and DSC chart of bulleyaconitine A

FIG. 3: XRPD pattern of bulleyaconitine A crystal form I

FIG. 4: TGA and DSC of bulleyaconitine A crystal form I

FIG. 5: XRPD pattern of bulleyaconitine A crystal form VI

FIG. 6: TGA and DSC of bulleyaconitine A crystal form VI

FIG. 7: XRPD comparison chart of bulleyaconitine A crystal forms I-VI

FIG. 8: blank plasma mass spectrum

FIG. 9: actual measurement sample maps of bulleyaconitine A crystal forms I (A) and VI (B) in the labeled sample

FIG. 10: actual measurement sample maps of bulleyaconitine A crystal forms I (A) and VI (B) in plasma samples after administration to rats

FIG. 11: standard curve chart for determining bulleyaconitine A crystal forms I (1) and VI (2) in rat plasma by LC-MS/MS method

FIG. 12: a blood concentration-time curve after single gavage administration of bulleyaconitine A form I.

FIG. 13: a blood concentration-time curve after single gavage administration of bulleyaconitine A crystal form VI.

FIG. 14: the blood concentration-time curve of the rats after single intragastric administration of the natural bulleyaconitine A.

FIG. 15: blood concentration-time curve of single intragastric administration of amorphous bulleyaconitine A in rats

FIG. 16: a mortality-log dose curve (a) and a probability unit-log dose curve (B) for crystal I.

FIG. 17: mortality-log dose curve (a) and probability unit-log dose curve (B) for crystal VI.

Detailed Description

Starting compound (b): bulleyaconitine A has molecular formula of C35H49NO10, molecular weight of 643.77, and structure shown as follows:

the purity is more than or equal to 98 percent, and the product is purchased from Shanghai leaf Biotech limited company.

Batch number: HS0903XA13

Storage conditions are as follows: storing at 4 deg.C

Analytical method

1.1 Nuclear magnetic analysis (Nuclear magnetic resonance spectroscopy, 1H NMR)

Several milligrams of a solid sample were dissolved in dimethylsulfoxide-d 6 solvent and subjected to nuclear magnetic analysis on Bruker AVANCE-III (Bruker, GER).

1.2X-ray powder diffraction (X-ray powder diffractometer, XRPD)

The solid samples obtained from the experiments were analyzed by X-ray powder diffractometer Bruker D8Advance (Bruker, GER). The 2 theta scan angle was from 3 deg. to 45 deg., the scan step was 0.02 deg., and the exposure time was 0.2 seconds. The voltage and current of the light tube are respectively 40kV and 40mA when the sample is tested, and the sample disc is a zero background sample disc. 1.3 thermogravimetric analysis (TGA)

The thermogravimetric analyzer is model number TA Discovery 55(TA, US). 2-5mg of sample was placed in an equilibrated open aluminum sample pan and automatically weighed in a TGA oven. The sample was heated to the final temperature at a rate of 10 deg.C/min with a nitrogen purge rate of 60mL/min at the sample and 40mL/min at the balance. 1.4 Differential Scanning Calorimetry (DSC)

The model of the differential scanning calorimetry analyzer is TA Discovery 2500(TA, US). 1-2mg of sample was accurately weighed and placed in a perforated DSC Tzero sample pan, heated to the final temperature at a rate of 10 deg.C/min, and purged with nitrogen in the oven at a rate of 50 mL/min. 1.5 Dynamic moisture desorption analysis (DVS)

Dynamic water sorption desorption analysis was determined using DVS Intrinsic (SMS, UK). The test uses a gradient mode with a humidity change of 50% -95% -0% -50%, a humidity change of 10% per gradient in the range of 0% to 90%, and the end points of the gradients use d_m/d_tThe mode is judged as d_m/d_tLess than 0.002% and maintained for 10 minutes as gradient endpoint. The sample after completion of the test was subjected to XRPD analysis.