CN115650917A

CN115650917A - Bulleyaconitine A polycrystalline type and preparation method and application thereof

Info

Publication number: CN115650917A
Application number: CN202211205169.1A
Authority: CN
Inventors: 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
Original assignee: Shanghai Pinshan Pharmaceutical Consulting Co ltd
Current assignee: Shanghai Pinshan Pharmaceutical Consulting Co ltd
Priority date: 2020-07-03
Filing date: 2020-07-03
Publication date: 2023-01-31
Also published as: CN111875541B; CN111875541A

Abstract

The invention discloses a bulleyaconitine A crystal form VI, a preparation method and application thereof, and the bulleyaconitine A crystal form VI is simple to prepare, and has good stability and better pharmacokinetic characteristic. 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.

Description

Bulleyaconitine A polycrystalline type and preparation method and application thereof

The application is divided into separate applications of patent application with application number 2020106364524, application date 7/3/2020 and named as bulleyaconitine A polycrystalline form and preparation method and application thereof.

Technical Field

The invention relates to the technical field of bulleyaconitine A preparation, in particular to a bulleyaconitine A crystal form VI, a preparation method and application thereof.

Background

Bulleyaconitine A (CAS-RN 79592-91-9) is a modern plant medicine, is separated from Aconitum plant Dianthi of Ranunculaceae, 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 central analgesia and topical analgesia. 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 increases local vascular permeability, further aggravates the inflammatory response, and thus produces a series of changes, such as: synovial cells and fibroblasts proliferate, angiogenesis, collagenase 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 of 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 at the posterior horn of the spinal cord, and simultaneously, the bulleyaconitine A is 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 medicine for treating primary erythromelalgia; CN 10724574A discloses the use of an amorphous bulleyaconitine A compound in preparing a medicament for treating pain caused by rheumatic or rheumatoid arthritis; bulleyaconitine A in CN106943402A is used for preparing medicine for preventing and treating osteoporosis or osteolysis.

The previous research (CN 110478350A) of the inventor finds that the bulleyaconitine A has the obvious effect of inhibiting spontaneous activity 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 few, 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. CN 10724574A discloses an amorphous bulleyaconitine A compound, the solubility and 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, a 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.

Another aspect according to the invention relates to form I of bulleyaconitine A, which has an XRPD pattern 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 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 the bulleyaconitine A crystal form VI is to crystallize the bulleyaconitine A crystal form VI by using 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 is also a pharmaceutical composition comprising an amorphous form of bulleyaconitine a, bulleyaconitine a form I or VI according to the invention; and pharmaceutically acceptable auxiliary materials.

Preferably, the pharmaceutical excipients are selected from at least one of disintegrants, diluents, lubricants, binders, wetting agents, flavoring agents, suspending agents, surfactants or preservatives; 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 aerosil, magnesium stearate, calcium stearate, stearic acid, talc or anhydrous silica gel; more preferably, the binder is selected from at least one of acacia, gelatin, dextrin, hydroxypropyl cellulose, methyl cellulose or polyvinylpyrrolidone; 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 glycerol 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 known use 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 bulleyaconitine A polymorphism has 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 polymorphic form of the bulleyaconitine A 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 is a schematic diagram: TGA and DSC of bulleyaconitine A amorphous form.

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

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

FIG. 5: an 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 are obtained.

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

FIG. 11: standard curve charts of bulleyaconitine A crystal forms I (1) and VI (2) in rat plasma are determined by an LC-MS/MS method.

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

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

FIG. 14 is a schematic view of: the blood concentration-time curve of the rats after single intragastric administration of the natural bulleyaconitine A is shown.

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

FIG. 16: mortality-log dose curve (a) and 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 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 D8 Advance (Bruker, GER). The 2 theta scan angle was from 3 deg. to 45 deg., the scan step size was 0.02 deg., and the exposure time was 0.2 seconds. The light tube voltage and current were 40kV and 40mA, respectively, for the test samples, and the sample disks were zero background sample disks.

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 and heated to the final temperature at a rate of 10 deg.C/min with a nitrogen purge rate in the furnace of 50mL/min.

1.5 Dynamic moisture desorption analysis (DVS)

Dynamic water sorption and desorption analysis was determined using DVS Intrinsic (SMS, UK). The test adopts a gradient mode, the humidity change is 50% -95% -0% -50%, the humidity change of each gradient in the range of 0% -90% is 10%, and the gradient end point adopts d _m /d _t The mode is judged as d _m /d _t Less than 0.002% and maintained for 10 minutes as gradient endpoint. The sample after completion of the test was subjected to XRPD analysis.

Example 1

Dissolving 80mg of bulleyaconitine A in 1.6mL of methanol, quickly dripping the obtained clear solution into 80mL of water, carrying out suction filtration and separation on the solution, and drying the solid in vacuum to obtain white flocculent solid.

XRPD results (fig. 1) show that the resulting sample is amorphous. The TGA (fig. 2) results show that the sample has a small amount of adsorbed water; the DSC results show that the amorphous sample has an insignificant glass transition signal, recrystallization occurs at around 120 ℃, and the recrystallized solid then melts at around 160 ℃.

Example 2

1) Dissolving 97.3mg of raw material in 6mL of diethyl ether, filtering, and volatilizing at normal temperature; or,

2) Dissolving 200.4mg of raw material in 0.3mL of tetrahydrofuran, filtering, and volatilizing at normal temperature;

to obtain a crystal form I (FormI). XRPD (fig. 3), DSC and TGA (fig. 4) characterization was performed. The Form I is heated to 150 ℃ without obvious weight loss in the TGA characterization, and a melting endothermic peak exists at 162.8 ℃ in the DSC characterization, which indicates that the crystal Form is an anhydride.

Form I NMR results were consistent with anhydrous bulleyaconitine A. According to the DVS result, the weight loss of Form I in the humidity range of 0-95% is not more than 0.13%, which indicates that Form I has no obvious water loss or water absorption along with the humidity change, and the XRPD pattern does not change substantially before and after the test, indicating that the sample after the DVS test has no crystal Form change. In summary, form I is an anhydrous substance that is not prone to moisture absorption.

Example 3

Binary solvent cooling method

The binary solvent cooling crystallization experiments were performed using a combination of good solvents and 3 poor solvents, respectively, using 20mg of sample for each experiment, and the results are shown in table 1. Form V is obtained in a butyl formate/n-hexane system, form II is obtained in a dioxane/cyclohexane system, form III is obtained in a dichloromethane/cyclohexane system, form IV is obtained in a chloroform/cyclohexane system, and Form I is obtained in other experiments in which crystals are precipitated.

Form II lost 10.6% weight during TGA characterization heating to 150 ℃ and exhibited a significant weight loss step, indicating that the crystalline Form may be a hydrate or solvate. In DSC characterization, an endothermic peak exists at 81.7 ℃, which corresponds to a desolventizing weight loss process of TGA; an exothermic peak corresponding to recrystallization appeared at 99.7 ℃ and the crystal obtained by recrystallization exhibited a melting endothermic peak at 158.9 ℃ (this peak position is close to the melting endothermic peak of Form I). The Form II sample was heated to 120 ℃ before XRPD testing, which showed that Form II was transformed to Form I under these conditions. The Form II after being stored for several days at room temperature is subjected to XRPD test, and the result sample is a mixed crystal of the Form II and the Form I, which indicates that the Form II is unstable and spontaneously transforms into the Form I at room temperature. FormII NMR chart, 1.4ppm of cyclohexane appears as a characteristic peak. In view of the above information, form II should be a cyclohexane solvate.

Form III loses 5.1% weight during TGA characterization heating to 130 ℃ and exhibits a significant weight loss step, indicating that the crystalline Form may be a hydrate or solvate. In DSC characterization, an endothermic peak exists at 94.6 ℃, which corresponds to a desolventizing weight loss process of TGA; an exothermic peak corresponding to recrystallization appears at 109.0 ℃, and crystals obtained by recrystallization show a melting endothermic peak (the peak position is close to that of FormI) at 159.2 ℃; also exhibits a melting endotherm at 170.8 ℃ (this peak is located close to the melting endotherm of Form VI). The Form III sample was heated to 150 ℃ and then subjected to XRPD testing, resulting in Form III being transformed to Form I under these conditions. The XRPD test was performed on Form III after storage for several days at room temperature, and the result showed that the sample was completely converted to Form I, indicating that Form III is unstable and spontaneously crystallized to Form I at room temperature. In the NMR characterization chart of Form III, a characteristic peak of cyclohexane at 1.4ppm appears. In view of the above information, form III should be a cyclohexane solvate.

Form IV lost 5.0% weight during TGA characterization heating to 120 ℃ and exhibited a significant weight loss step, indicating that the crystalline Form may be a hydrate or solvate. In DSC characterization, an endothermic peak exists at 84.6 ℃, which corresponds to a desolventizing weight loss process of TGA; an exothermic peak corresponding to recrystallization appears at 92.9 ℃, and the crystal obtained by recrystallization shows a melting endothermic peak (the peak position is close to the melting endothermic peak of FormI) at 158.1 ℃; a weak melting endotherm (peak position close to that of FormVI) is also present at 171.3 ℃. The XRPD test on Form IV after three days of storage at 0 ℃ shows the characteristic peak of Form I, which indicates that Form IV is unstable and spontaneously transforms into Form I even at 0 ℃. In the NMR characterization chart of Form IV, a peak at 1.4ppm is a characteristic peak of cyclohexane. In view of the above information, form IV should be a cyclohexane solvate.

Form V loses 5.8% weight during TGA characterization heating to 120 ℃ and exhibits a distinct weight loss step, indicating that the crystalline Form may be a hydrate or solvate. In DSC characterization, an endothermic peak exists at 112.0 ℃, which corresponds to a desolventizing weight loss process of TGA; then a wider endothermic signal was exhibited starting at 126 ℃. The Form V sample was heated to 115 ℃ and subjected to XRPD testing, which showed that Form V was transformed to Form I under these conditions.

TABLE 1

Example 4

Binary solvent suspension method

1) Weighing 20mg of a sample, suspending the sample in 0.8mL of a selected binary mixed solvent (acetonitrile/water (1;

2) 249.3mg of raw material and 007-23-15Form VI seed crystal are added into 1.2mL of acetonitrile/water (1.

The XRPD results for Form VI are shown in FIG. 5. The results of the TGA and DSC tests are shown in FIG. 6, form VI has no weight loss when heated to 150 ℃ in the TGA characterization, and has a melting endothermic peak only at 174.6 ℃ in the DSC characterization. The NMR results of Form VI are consistent with the structure of anhydrous bulleyaconitine A. In summary, form VI should be in the anhydrous crystalline Form.

The XRPD pattern for the forms I-VI is shown in FIG. 7, and the properties of the different forms are summarized in Table 2 below:

TABLE 2

The crystal Form research result shows that Form II, form III, form IV and Form V are unstable, can be transformed into Form I by heating, and can be transformed into Form I by spontaneous desolventizing even at room temperature.

Example 5 pharmacokinetic experiments

1 materials and methods

1.1 Instrument

A Waters ultra high performance liquid chromatography-mass spectrometry system (comprising an online degasser, an ultra high pressure gradient pump, a column incubator, an automatic sample injector, a Waters I-CLASS UPLC liquid phase system, a Xevo TQ-S mass spectrum and a UNIFI workstation); an Eppendorf 5810R full-automatic high-speed freezing centrifuge; eppendorf MixMate vortex instrument; METTLER TOLEDO XP105DR electronic balance.

1.1.1 reagents

Methanol was chromatographically pure (Merck, germany); acetonitrile in chromatographically pure form (Merck, germany); the water is commercially available Drech distilled water.

1.2 test animals

Strain: SD rat

Sex: male sex

Weight: about 250g

The source is as follows: shanghai Sphall-Bikai laboratory animals GmbH

Producing license number of experimental animal: SCXK 2018-0006

Feeding: 4 per cage are raised in an air-conditioning constant-temperature room with the room temperature of 20-24 ℃, the humidity of 40-70% and the illumination time of 12h. Can be taken freely and drunk freely. 1.3 methods of drug administration and sample Collection

1.3.1 dosage and route of administration

The administration mode comprises the following steps: intragastric administration;

administration dosage by intragastric administration: 2.7mg/kg (bulleyaconitine A crystal type I, VI, bulleyaconitine A raw material, amorphous bulleyaconitine A); 0.5mg/kg (amorphous bulleyaconitine A).

Grinding bulleyaconitine A crystal form I and VI powder and tween-80 with the volume of 1 percent of the total solution to be transparent, adding sodium carboxymethylcellulose to complement the volume, grinding while adding, and preparing into 0.27mg/mL liquid medicine for intragastric administration.

1.3.2 administration and blood drawing

SD rats were 12 animals, divided into three groups. One group was administered with bulleyaconitine form i, one group was administered with bulleyaconitine form vi, and one group was administered with amorphous bulleyaconitine form. After administration of bulleyaconitine form I, form VI and amorphous bulleyaconitine form A, 0.083,0.25,0.5,1,1.5,2,2.5,3,6,9,12,24h, 8000r/min after taking about 0.2mL of whole blood per time point, centrifuging for 10min, separating plasma, taking 50 μ L of one part, and backing up the rest plasma. All frozen at-80 deg.C for storage.

1.4 plasma sample processing method

50 μ L of plasma sample was mixed with 5 μ L of IS (500 ng/ml tolbutamide (JBHDN)), then 450 μ L of methanol was added to precipitate protein, the mixture was vortexed 30s, centrifuged 5min at 13000r/min, the supernatant was filtered and analyzed by LC/MS/MS.

1.5 establishing UPLC-MS/MS method to determine the contents of bulleyaconitine A crystal forms I, VI, bulleyaconitine A raw materials and amorphous bulleyaconitine A in SD rat plasma

1.5.1 solution preparation

1.5.1.1 preparation of bulleyaconitine A crystal form I and VI standard yeast working solution

Accurately weighing appropriate amount of bulleyaconitine A crystal form I and VI standard substance, dissolving with methanol, and diluting with 80% methanol water to obtain bulleyaconitine A crystal form I and VI stock solution with concentration of 1 mg/mL. Diluting the stock solutions of bulleyaconitine A crystal forms I and VI with 80% methanol water to obtain standard working solutions with bulleyaconitine A crystal forms I and VI concentrations of 10, 50, 100, 500, 1000, 2500 and 5000ng/mL respectively, and storing in a refrigerator at-20 deg.C.

The bulleyaconitine A raw material analysis takes bulleyaconitine A crystal form I working solution as a standard for analysis.

1.5.1.2 preparation of bulleyaconitine A crystal form I and VI quality control working solution

Accurately weighing appropriate amount of bulleyaconitine A crystal form I and VI standard substance, dissolving with methanol, and diluting with 80% methanol water to obtain quality control stock solution with concentration of 1 mg/mL. Diluting the quality control stock solution with 80% methanol water to obtain quality control working solution containing bulleyaconitine A crystal forms I and VI with concentrations of 30, 400 and 4000ng/ml, and storing in a refrigerator at-20 deg.C.

1.6 preparation of bulleyaconitine A crystal form I and VI labeled yeast samples and quality control samples

The 1.5.1.1 part of bulleyaconitine A crystal form I and VI labeled series solutions are prepared in blank plasma of SD rats according to the proportion of 1,5, 10, 50, 100, 250 and 500ng/mL respectively to obtain labeled series samples with the concentrations of 1,5, 10, 50, 100, 250 and 500 ng/mL. Diluting the quality control working solution under the item of '1.5.1.2' according to the same method to obtain plasma quality control samples with the concentrations of 3, 40 and 400ng/mL respectively.

1.7 sample measurement conditions

1.7.1 chromatographic conditions

The analytical column is ACQUITY UPLC BEH C18 (1.7 μm 2.1X 100 mm); mobile phase was water (0.1% fa): acetonitrile; gradient elution: the flow rate is 0.3mL/min; the sample injection amount is 1 mu L; the column temperature was 40 ℃; the elution gradient is shown in table 3 below:

TABLE 3 mobile phase gradient chart of bulleyaconitine A crystal form I and VI

1.7.2 Mass Spectrometry conditions

The ion source is an electrospray ionization (ESI); the source temperature is 150 ℃; the temperature of atomizing gas is 350 ℃; the capillary voltage was 3kv; the detection mode is positive ion detection; the scanning mode is Multiple Reaction Monitoring (MRM); the mass spectrometric detection parameters of the test substance and the internal standard are shown in table 4 below:

TABLE 4 Mass spectrometric detection parameter table of the substance to be measured

1.8 data processing method

Pharmacokinetic parameters of the SD rats after drug administration were calculated using a non-atrioventricular model of the DAS2.0 software. Including peak concentration C _max : adopting an actual measurement value; time to peak T _max : adopting an actual measurement value; area under the time curve AUC _(0-t) The value: calculating by adopting a trapezoidal method; AUC _(0-∞) ＝AUC _(0-t) +C _t /k _e ，C _t The blood concentration at the last measurable time point, k _e To eliminate the rate constant; elimination of half-life t _1/2 ＝0.693/k _e (ii) a Mean residence time MRT = AUMC/AUC; volume of distribution V _z ＝CL/k _e 。

And calculating the average value, standard deviation, precision and accuracy of the blood concentration of each sample by using Excel software.

2 results and analysis

2.1 sample analysis method

2.1.1 method specificity

In the experiment, bulleyaconitine A crystal form I and VI standard yeast working solution is added into blank rat plasma, the blank rat plasma is processed according to the method under the item '1.4', the LC-MS/MS analysis is respectively carried out under the chromatographic mass spectrum condition under the item '1.7', and the parameters in the test process are set as follows: channel name: integral formula, smoothing times are 2 times, the electrospray voltage is 30eV, the electrospray positive ion molecular weight is 644.29, and the daughter ion molecular weight is 583.99. The experimental result shows that the matrix does not contain components interfering the determination of the bulleyaconitine A crystal forms I and VI. The retention time of the bulleyaconitine A crystal forms I and VI is as follows: 1.92min. FIG. 8 is a blank plasma mass spectrum; FIG. 9 is a mass spectrum of an actually measured sample in a calibration sample; FIG. 10 is the mass spectrogram of the actual measurement sample of bulleyaconitine A crystal forms I and VI in the blood plasma of rats after drug administration.

2.1.2 accompanying standard curve and quality control

Weighting the corresponding concentration (C, X) by the peak area (As, Y) of the bulleyaconitine A crystal forms I and VI (1/X) ² ) Performing linear regression to obtain standard curves of bulleyaconitine A crystal forms I and VI in rat plasma, wherein the standard curve fitting equation of (1) in the graph 11 is Y =2.15e-002+ 3.19e-002X, the RSD is 6.372%, and R is ² Is 0.997827; FIG. 11 shows the standard curve fitting equation of (2) Y =2.01e-002+3.67e-002 x, RSD 5.112%, R ² And was 0.998776. The accuracy control results of each analysis batch are shown in table 5:

TABLE 5 calculated concentration of QC quality control samples for sample analysis batches

Acceptance criteria: each analysis batch should contain at least 2 sets of 3 horizontal quality control samples, and the total number of the quality control samples is not less than 5% of the number of the samples to be detected. At least 67% of the quality control samples per analysis batch should meet the criteria of accuracy within 15%, precision not exceeding 15%, and at least 50% of the quality control samples per concentration.

2.2 blood concentration and pharmacokinetic parameters of bulleyaconitine A in SD rat after administration of crystal forms I and VI and amorphous bulleyaconitine A

2.2.1SD rat bulleyaconitine A crystal forms I and VI and amorphous bulleyaconitine A single-time gavage administration blood concentration

The blood concentration of SD rat after single intragastric administration of bulleyaconitine A crystal form I is shown in Table 6-1, and the blood concentration-time curve is shown in FIG. 12; after single-time gavage administration of bulleyaconitine A crystal form VI, the blood concentration is shown in the table 6-2, and the blood concentration-time curve is shown in the table 13; the blood concentration of the rat after single intragastric administration of the natural bulleyaconitine A is shown in the table 6-3, and the blood concentration-time curve is shown in the figure 14; the blood concentration of the rats after single intragastric administration of the amorphous bulleyaconitine A is shown in tables 6-4 and 6-5, and the blood concentration-time curve is shown in figure 15;

TABLE 6-1 plasma drug concentrations (2.7 mg/kg) at different time points after single gavage administration of test agent to rats with bulleyaconitine A form I

TABLE 6-2 plasma drug concentrations (2.7 mg/kg) at various time points following gavage administration of the test agent bulleyaconitine A form VI to rats

TABLE 6-3 plasma drug concentrations (2.7 mg/kg) at different time points after gavage administration of the test drug bulleyaconitine A raw material in rats

TABLE 6-4 blood concentration of rats dosed with 0.5mg/kg of amorphous bulleyaconitine A administered by oral gavage

Remarking: * Representative of being below the limit of detection

TABLE 6-5 blood concentration of rats dosed with 2.7mg/kg of amorphous bulleyaconitine A administered by oral gavage

Remarking: representing death of rat

2.2.2SD rat post-dose pharmacokinetic parameters

The corresponding pharmacokinetic parameters of single administration of bulleyaconitine A crystal forms I, VI, bulleyaconitine A raw materials and amorphous bulleyaconitine A to SD rats are shown in tables 7-1,7-2,7-3 and 7-4:

TABLE 7-1 pharmacokinetic parameters (2.7 mg/kg) following gavage administration of the test drug bulleyaconitine A form I in rats

TABLE 7-2 pharmacokinetic parameters (2.7 mg/kg) following gavage administration of the test drug bulleyaconitine A form VI to rats

TABLE 7-3 pharmacokinetic parameters after gavage administration of the test agent Natural bulleyaconitine A in rats (2.7 mg/kg)

TABLE 7-4 pharmacokinetic parameters (0.5 mg/kg) following gavage administration of test agent amorphous bulleyaconitine A to rats

The test results show that the average peak reaching time of the bulleyaconitine A blood concentration in vivo after the bulleyaconitine A raw materials and the amorphous bulleyaconitine A are respectively (0.38 +/-0.18) h, (1.063 +/-0.591) h, (0.875 +/-0.8) h and (0.5 +/-0.354) h after the rats are fed with single gavage, the average peak reaching concentration is respectively (10.38 +/-1.85) ng/mL, (20.575 +/-8.32) ng/mL, (21.098 +/-4.40) ng/mL and (11.809 +/-4.153) ng/mL, and the area AUC under the curve when the drug is averaged _0-t Respectively (73.2 +/-18.6) ng/mL h, (115.544 +/-29.598) ng/mL h (124.25 ± 32.85) and (34.363 ± 6.582) ng/mL _ h; after the gastric lavage administration of rats, the half-life period of elimination in vivo is averagely (21.48 +/-2.12) h, (12.547 +/-14.311) h, (4.700 +/-2.489) h and (2.085 +/-0.539) h respectively.

EXAMPLE 6 comparative analgesia test

1. Animals: SPF level mouse (Shanghai Spiral-BiKai laboratory animals Co., ltd., animal quality certificate number: SCXK 2018-0006, weight about 18-22 g)

2. The method comprises the following steps: 110 male mice were taken and randomly divided into 11 groups: a vehicle control group; positive control (aspirin 200 mg/kg) group; 0.2, 0.4 and 0.8mg/kg of bulleyaconitine A raw material group; 0.1, 0.2 and 0.4mg/kg of bulleyaconitine A crystal form I; amorphous bulleyaconitine A group 0.04, 0.08, 0.16 mg/kg; each group had 10. The animals of each group were each administered once by gavage with a dose, the vehicle control group was administered 1% of CMC-Na by 20mL/kg 30min after the administration, and was intraperitoneally injected with 0.1mL/10g of 0.6% glacial acetic acid solution, the number of times of body writhing of each mouse was observed and recorded within 15min, and the results of comparing the inhibition rates of the groups to the acetic acid-induced writhing were as shown in the following Table 8:

inhibition (%) = (mean number of twists in vehicle group-mean number of twists in drug group)/mean number of twists in vehicle group:. 100%

Table 8 effect of bulleyaconitine A crystal form on acetic acid induced pain in mice

From the above table 8, it can be seen that the effects of the bulleyaconitine A crystal form I and the bulleyaconitine A amorphous form are both better than those of the bulleyaconitine A raw material, and especially the bulleyaconitine A amorphous form is obviously better than that of the bulleyaconitine A raw material.

Example 7 investigation of bulleyaconitine A on KM mouse acute toxicity test

1. Laboratory animal

The species are as follows: KM mouse

Grade: SPF stage

Number and sex of purchased animals: 30, male

The source is as follows: shanghai Sphall-Bikai laboratory animals Co., ltd

Producing license numbers: SCXK 2018-0006 (Shanghai)

License for use of experimental animal: SYXK 2014-0018

2. Experimental method

2.1 grouping

The mice are bred adaptively for 1-2 days, 15 mice are respectively bred in a bulleyaconitine A crystal form I and a bulleyaconitine A crystal form VI, the bulleyaconitine A crystal form I is randomly divided into 5 groups, and each group comprises 3 mice: blank group, crystal form I2.8 mg/kg, crystal form I3.79 mg/kg, crystal form I5.06 mg/kg, crystal form I6.75 mg/kg, crystal form I9.0 mg/kg; the crystal form VI is consistent with the crystal form I in groups.

2.2 Experimental methods

Prior to dosing, fasting was performed and the animal was observed for physiological status. Except for the blank group, the other groups adopt mice to administrate various doses of bulleyaconitine A crystal form I or crystal form VI, and the doses of the various groups are respectively 2.8, 3.79, 5.06, 6.75 and 9.0mg/kg (the two crystal forms are consistent). Animals were observed for symptoms of intoxication and the number of deaths was recorded.

2.3 calculation method

The median lethal dose of the mice was calculated by using the Bliss method software. Data were obtained and mortality-log dose curves and probability unit-log dose curves were plotted.

3. Results of the experiment

LD of crystal I and crystal VI ₅₀ The results are shown in tables 9 and 10, respectively, as mortality-log dose curves for form I and form VI and probability unit-log dose curves, respectively, in fig. 16 and 17. As can be seen from tables 9-10 and FIGS. 16 and 17, the data after administration of bulleyaconitine A shows that the LD of form I ₅₀ 4.4mg/kg, with a 95% confidence limit of 3.6-5.3 mg/kg; LD of crystal form VI ₅₀ 5.9mg/kg,95% confidence limit of 4.8-7.3 mg/kg

TABLE 9 LD of Crystal form I ₅₀ Computation reporting

LD of form VI of Table 10 ₅₀ Computation reporting

4. Conclusion

The bulleyaconitine A crystal forms I and VI have certain toxicity.

LD of crystal form I ₅₀ 4.4mg/kg,95% confidence limit of 3.6-5.3 mg/kg, and Feiller corrected LD ₅₀ 4.3mg/kg; LD of crystal form VI ₅₀ 5.9mg/kg, with a 95% confidence limit of 4.8-7.3 mg/kg; feiller corrected LD ₅₀ It was 6.1mg/kg.

Dissolving two crystal forms I and VI of the non-normal bulleyaconitine A in corresponding solvents, and adjusting to the acute toxicity measured by oral solution by using Tween 80.

After the inventor grops the dosage, the pharmacokinetics of the crystal form drug is tested.Proves that the crystal form I and the crystal form VI are compared with the amorphous bulleyaconitine A C _max Obviously improved, which shows that the toxicity of the crystal form I and the crystal form VI is obviously lower than that of the amorphous bulleyaconitine A.

Claims

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

2. The crystalline form VI according to claim 1 characterized by a TGA and DSC profile substantially as shown in figure 6.

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

4. The method for preparing bulleyaconitine A crystal form VI according to claim 3, wherein the proportion of the acetonitrile and the water is 1:2-4.

5. The method for preparing bulleyaconitine A crystal form VI according to claim 3, wherein the ratio of the acetonitrile to the water is 1:3.

6. a pharmaceutical composition comprising the bulleyaconitine A form VI of claim 1 or 2; and pharmaceutically acceptable adjuvants.

7. Pharmaceutical composition according to claim 6, characterized in that it is a solid dosage form.

8. Pharmaceutical composition according to claim 7, characterized in that it is an oral agent.

9. The use of the bulleyaconitine form VI of claim 1 or 2 in the preparation of anti-inflammatory and analgesic medicaments.

10. The use of the bulleyaconitine form VI of claim 1 or 2 in the preparation of medicaments for inhibiting drug addiction.