Aconitine polymorphs and preparation methods and applications thereof

Abstract

The invention discloses a crystalline form VI of aconitine A and a preparation method and application thereof, which is simple to prepare and has good stability and better pharmacokinetic properties. Compared with the raw material of aconitine A, the polymorph of aconitine A according to the invention has lower toxicity and better analgesic effect, further improving the application prospect of aconitine A.

Description

[0001] This case is a divisional application of the patent application with application number 2020106364524, application date July 3, 2020, and titled "Polymorphs of Aconitine A, Preparation Methods and Applications thereof". Technical Field

[0002] The invention relates to the technical field of aconitine preparation, and in particular to a conitine crystal form VI and a preparation method and application thereof. Background Art

[0003] Bulleyaconitine A (CAS-RN 79592-91-9) is a modern botanical medicine, isolated from the plant of the genus Aconitum of the Ranunculaceae family, and is a diterpene diester alkaloid, which is different from aconitine and mesaconitine. The drug has no tolerance and no addiction, and can be used as a potent analgesic and anti-inflammatory drug, with dual pharmacological effects of central analgesia and topical analgesia. At present, the China Food and Drug Administration (CFDA) has approved the use of bulleyaconitine injection and tablets for the treatment of chronic pain and rheumatoid arthritis. Clinically, bulleyaconitine A has also been widely used to treat rheumatoid arthritis, osteoarthritis, myofibromyitis, cervical spondylosis, cancer pain and chronic pain caused by various reasons.

[0004] Aconitine A has a significant inhibitory effect on both inflammation and pain. Prostaglandin PGE2 is produced under the action of inflammatory factors. It is an inflammatory mediator with inflammatory and pain-inducing effects produced and released by local tissues, which increases local vascular permeability, further aggravates the inflammatory response, and produces a series of changes, such as: synovial cell and fibroblast proliferation, angiogenesis, collagenase production, etc., which destroys bone and cartilage cells. Reducing serum PGE2 is one of the mechanisms of the anti-inflammatory effect of aconitine A. PGE2 can activate peripheral pain receptors and transmit pain signals. β-Endorphin is a neuropeptide with a strong analgesic effect. The analgesic effect of aconitine A may be related to its antagonism of 5-hydroxytryptamine (5-HT) in the brain, inhibiting the release of PGE2, and thus relieving the inhibition of β-endorphin.

[0005] At the same time, according to literature reports, aconitine A can directly induce the expression of dynorphin A in spinal microglia, thereby exhibiting analgesic effects, and shows an inhibitory effect on neuropathic pain at the C-fiber synaptic surface of the dorsal horn of the spinal cord. It is also proven that aconitine A can enhance the analgesic effect of morphine and inhibit the analgesic tolerance of morphine. In addition, aconitine A can also inhibit inflammatory chemokines.

[0006] At present, the research and application of aconitine A are still limited to the anti-inflammatory and analgesic aspects. For example, CN101468000 discloses the use of aconitine A as a drug for preparing a drug for treating primary erythromelalgia; CN107245054A discloses the use of an amorphous aconitine A compound in the preparation of a drug for treating pain diseases caused by rheumatism or rheumatoid arthritis; the aconitine A in CN106943402A is used to prepare a drug for preventing and treating osteoporosis or osteolysis.

[0007] The inventor's previous study (CN110478350A) found that aconitine A has a significant effect of inhibiting the symptoms of spontaneous activity caused by drugs, indicating that aconitine A and its derivatives have great application potential in inhibiting drug addiction.

[0008] However, there are relatively few studies on the polymorphic forms of aconitine A in the prior art. Most of the literature reports involve obtaining white, colorless and transparent aconitine A by crystallization and recrystallization. However, there are no XRPD reports on the polymorphic forms of aconitine A. CN107245054A discloses an amorphous aconitine A compound, which has slightly better solubility and analgesic effect than the reference substance. It is of great significance to further develop other polymorphic forms and amorphous aconitine A with better stability. Summary of the invention

[0009] One aspect of the present invention relates to a stable polymorphic form of aconitine A.

[0010] Another aspect of the present invention relates to polymorphs of aconitine A, which have better stability or pharmacokinetic properties than the known amorphous forms.

[0011] Specifically, the first aspect of the present invention relates to an amorphous form of aconitine A, whose XRPD pattern is substantially as shown in the attached Figure 1 Preferably, its TGA and DSC diagrams are substantially as shown Figure 2 shown.

[0012] According to another aspect of the present invention, the crystal form I of aconitine A is shown in the attached XRPD diagram. Figure 3 shown.

[0013] Preferably, the TGA and DSC diagrams of the crystalline form I are substantially as follows Figure 4 shown.

[0014] According to another aspect of the present invention, the crystal form VI of aconitine A is shown in the attached XRPD diagram. Figure 5 shown.

[0015] Preferably, the TGA and DSC graphs of the crystalline form VI are substantially as follows Figure 6 shown.

[0016] Another aspect of the present invention also relates to a method for preparing the above-mentioned polymorphic form of aconitine.

[0017] Specifically, the preparation method of amorphous aconitine A is as follows: aconitine A is dissolved in a lower alcohol (preferably methanol or ethanol), the resulting solution is quickly added dropwise to water, filtered and separated, and the solid is dried to obtain the amorphous aconitine A.

[0018] The preparation method of the crystal form I of aconitine A is characterized in that aconitine A is dissolved in a common organic solvent and volatilized at room temperature. Preferably, the common organic solvent includes at least one of ether, tetrahydrofuran, methanol, ethanol, n-propanol, isopropanol, acetone, ethyl acetate, ethyl formate, acetonitrile, toluene, dichloromethane, chloroform, tetrahydrofuran or dimethyl sulfoxide; preferably ether, tetrahydrofuran, ethanol, acetone, ethyl acetate, acetonitrile, dichloromethane, chloroform, tetrahydrofuran; most preferably at least one of ether, tetrahydrofuran, ethanol, acetone, ethyl acetate or dichloromethane.

[0019] The preparation method of the crystal form VI of aconitine A is to obtain it by crystallization with 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.

[0020] According to another aspect of the present invention, there is also a pharmaceutical composition comprising the amorphous form of aconitine A, aconitine A crystal form I or VI according to the present invention, and pharmaceutically acceptable excipients.

[0021] 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, cross-linked polyvinyl pyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, carboxymethyl cellulose, calcium carboxymethyl cellulose 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 micro powder silica gel, magnesium stearate, calcium stearate, stearic acid, talc or anhydrous silica gel; More preferably, the binder is selected from at least one of gum arabic, 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 gum arabic, gelatin, methyl cellulose, sodium carboxymethyl cellulose, hydroxymethyl cellulose 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.

[0022] According to another aspect of the present invention, the pharmaceutical composition is in a solid dosage form, preferably an oral dosage form.

[0023] Another aspect of the present invention also relates to the use of the polymorphic form of aconitine A according to the present invention.

[0024] Those skilled in the art will appreciate that the polymorphs of aconitine A according to the present invention can be applied to any known uses of aconitine A.

[0025] Specifically, the amorphous form of aconitine A or the crystal form I or VI of aconitine A according to the present invention can be used to prepare anti-inflammatory and analgesic drugs, and can also be used to prepare drugs that inhibit drug addiction.

[0026] The beneficial effects of the present invention are:

[0027] 1) The polymorphic form of aconitine A according to the present invention has a good stability form.

[0028] 2) Polymorphs of aconitine A, which have better pharmacokinetic properties than the known amorphous forms.

[0029] 3) Compared with the raw material of aconitine A, the polymorph of aconitine A according to the present invention has lower toxicity.

[0030] 4) Compared with the raw material of aconitine A, the polymorph of aconitine A according to the present invention has better analgesic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 : XRPD pattern of amorphous form of aconitine A.

[0032] Figure 2 : TGA and DSC diagrams of amorphous form of aconitine A.

[0033] Figure 3 : XRPD pattern of aconitine crystal form I.

[0034] Figure 4 : TGA and DSC diagrams of aconitine crystal form I.

[0035] Figure 5 : XRPD pattern of aconitine crystal form VI.

[0036] Figure 6 : TGA and DSC diagrams of aconitine crystal form VI.

[0037] Figure 7 : XRPD comparison diagram of aconitine crystal forms I-VI.

[0038] Figure 8 : Mass spectrum of blank plasma.

[0039] Fig. 9 : Measured sample spectra of aconitine crystal form Ⅰ (A) and Ⅵ (B) in the standard sample.

[0040] Fig.10 : Measured sample spectra of aconitine crystal form I (A) and VI (B) in plasma samples of rats after administration.

[0041] Fig.11 :Standard curve of aconitine crystalline forms Ⅰ(1) and VI(2) in rat plasma determined by LC-MS / MS.

[0042] Fig.12 : The blood concentration-time curve of aconitine A crystal form Ⅰ after single oral administration.

[0043] Fig.13 : The blood concentration-time curve of aconitine form VI after single oral administration.

[0044] Fig.14 :The blood concentration-time curve of natural aconitine A after single oral administration in rats.

[0045] Fig.15 :The blood concentration-time curve of amorphous aconitine A after single oral administration in rats

[0046] Fig.16 : Mortality-logarithmic dose curve (A) and probability unit-logarithmic dose curve (B) of crystal I.

[0047] Fig.17 : Mortality-logarithmic dose curve (A) and probability unit-logarithmic dose curve (B) of crystal VI. DETAILED DESCRIPTION

[0048] Raw material compound: aconitine A, molecular formula C35H49NO10, molecular weight 643.77, its structure is as follows:

[0049]

[0050] Purity ≥ 98%, purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0051] Batch number: HS0903XA13

[0052] Storage conditions: 4℃

[0053] Analytical methods

[0054] 1.1 Nuclear magnetic resonance spectroscopy (1H NMR)

[0055] Several milligrams of solid sample were dissolved in dimethyl sulfoxide-d6 solvent and subjected to NMR analysis on a Bruker AVANCE-III (Bruker, GER).

[0056] 1.2 X-ray powder diffractometer (XRPD)

[0057] The solid samples obtained in the experiment were analyzed by X-ray powder diffractometer Bruker D8 Advance (Bruker, GER). The 2θ scanning angle was from 3° to 45°, the scanning step was 0.02°, and the exposure time was 0.2 seconds. When testing the samples, the tube voltage and current were 40 kV and 40 mA, respectively, and the sample pan was a zero background sample pan.

[0058] 1.3 Thermogravimetric Analysis (TGA)

[0059] The model of the thermogravimetric analyzer is TA Discovery 55 (TA, US). 2-5 mg of sample was placed in a balanced open aluminum sample pan and automatically weighed in the TGA heating furnace. The sample was heated to the final temperature at a rate of 10 ° C / min, and the nitrogen purge rate at the sample was 60 mL / min, and the nitrogen purge rate at the balance was 40 mL / min.

[0060] 1.4 Differential scanning calorimetry (DSC)

[0061] The model of the differential scanning calorimeter was TA Discovery 2500 (TA, US). 1-2 mg of sample was accurately weighed and placed in a DSC Tzero sample pan with holes, and heated to the final temperature at a rate of 10°C / min, with a nitrogen purge rate of 50 mL / min in the furnace.

[0062] 1.5 Dynamic moisture desorption analysis (DVS)

[0063] Dynamic moisture adsorption and desorption analysis was performed using DVS Intrinsic (SMS, UK). The test used a gradient mode with humidity changes of 50%-95%-0%-50%. The humidity change of each gradient in the range of 0% to 90% was 10%. The gradient endpoint was d m / d t The method is to judge m / d t The gradient endpoint is less than 0.002% and maintained for 10 minutes. The samples after the test are subjected to XRPD analysis.

[0064] Example 1

[0065] 80 mg of aconitine A was dissolved in 1.6 mL of methanol, and the obtained clear solution was quickly added dropwise to 80 mL of water. The solution was filtered and separated, and the solid was vacuum dried to obtain a white flocculent solid.

[0066] XRPD results ( Figure 1 ) showed that the obtained sample was amorphous. TGA ( Figure 2 ) The results showed that the sample had a small amount of adsorbed water; the DSC results showed that the glass transition signal of the amorphous sample was not obvious, recrystallization occurred at around 120°C, and then the recrystallized solid melted at around 160°C.

[0067] Example 2

[0068] 1) Dissolve 97.3 mg of the raw material in 6 mL of ether, filter, and evaporate at room temperature; or,

[0069] 2) Dissolve 200.4 mg of the raw material in 0.3 mL of tetrahydrofuran, filter, and evaporate at room temperature;

[0070] The crystal form I (Form I) was obtained. XRPD ( Figure 3 )、DSC and TGA( Figure 4 In TGA characterization, Form I has no obvious weight loss when heated to 150°C, and in DSC characterization, there is a melting endothermic peak at 162.8°C, indicating that the crystal form is anhydrous.

[0071] The NMR results of Form I are consistent with those of anhydrous aconitine A. According to the DVS results, the weight gain and weight loss of Form I in the humidity range of 0-95% does not exceed 0.13%, indicating that Form I does not lose or absorb water significantly with humidity changes, and there is no substantial change in the XRPD pattern before and after the test, indicating that the sample after the DVS test has not undergone a change in crystal form. In summary, Form I is an anhydrous substance that is not easy to absorb moisture.

[0072] Example 3

[0073] Binary solvent cooling method

[0074] A variety of good solvents and three poor solvents were used in combination to conduct cooling crystallization experiments of binary solvents. 20 mg of sample was used in each experiment. The results are shown in Table 1. Form V was obtained in the butyl formate / n-hexane system, Form II was obtained in the dioxane / cyclohexane system, Form III was obtained in the dichloromethane / cyclohexane system, Form IV was obtained in the chloroform / cyclohexane system, and Form I was obtained in other experiments with crystal precipitation.

[0075] Form II lost 10.6% of its weight during heating to 150°C in TGA characterization, and showed an obvious weight loss step, indicating that the crystal form may be a hydrate or a solvate. In the DSC characterization, there is an endothermic peak at 81.7°C, corresponding to the desolvation weight loss process of TGA; an exothermic peak corresponding to recrystallization appears at 99.7°C, and the crystals obtained by recrystallization show a melting endothermic peak at 158.9°C (the peak position is close to the melting endothermic peak of Form I). The Form II sample was heated to 120°C and then subjected to XRPD testing. The results showed that Form II crystallized into Form I under this condition. XRPD testing was performed on Form II after it was stored at room temperature for several days. The result showed that the sample was a mixed crystal of Form II and Form I, indicating that Form II was unstable and spontaneously crystallized into Form I at room temperature. In the NMR graph of Form II, the characteristic peak of cyclohexane appeared at 1.4ppm. Based on the above information, Form II should be a cyclohexane solvate.

[0076] Form III loses 5.1% of its weight during heating to 130°C in TGA characterization, and shows an obvious weight loss step, indicating that the crystal form may be a hydrate or solvate. In DSC characterization, there is an endothermic peak at 94.6°C, corresponding to the desolvation weight loss process of TGA; an exothermic peak corresponding to recrystallization appears at 109.0°C, and the crystals obtained by recrystallization show a melting endothermic peak at 159.2°C (the peak position is close to the melting endothermic peak of Form I); a melting endothermic peak is also shown at 170.8°C (the peak position is close to the melting endothermic peak of Form VI). The Form III sample was heated to 150°C and then tested by XRPD. The results showed that Form III crystallized into Form I under this condition. XRPD test was performed on Form III after it was stored at room temperature for several days. The result showed that the sample was completely converted into Form I, indicating that Form III was unstable and spontaneously crystallized into Form I at room temperature. In the NMR characterization diagram of Form III, the characteristic peak of cyclohexane appeared at 1.4ppm. Based on the above information, Form III should be a cyclohexane solvate.

[0077] Form IV loses 5.0% of its weight during heating to 120°C in TGA characterization, and shows an obvious weight loss step, indicating that the crystal form may be a hydrate or a solvate. In the DSC characterization, there is an endothermic peak at 84.6°C, corresponding to the desolvation weight loss process of TGA; an exothermic peak corresponding to recrystallization appears at 92.9°C, and the crystals obtained by recrystallization show a melting endothermic peak at 158.1°C (the peak position is close to the melting endothermic peak of Form I); a weak melting endothermic peak is also shown at 171.3°C (the peak position is close to the melting endothermic peak of Form VI). XRPD testing was performed on Form IV after it was stored at 0°C for three days. The sample showed the characteristic peaks of Form I, indicating that Form IV is unstable and will spontaneously crystallize into Form I even at 0°C. In the NMR characterization diagram of Form IV, the peak of 1.4ppm is the characteristic peak of cyclohexane. Based on the above information, Form IV should be a cyclohexane solvate.

[0078] Form V lost 5.8% of its weight during heating to 120°C in TGA characterization, and showed an obvious weight loss step, indicating that the crystal form may be a hydrate or solvate. In DSC characterization, there was an endothermic peak at 112.0°C, corresponding to the desolvation weight loss process of TGA; then a broader endothermic signal began to appear from 126°C. The Form V sample was heated to 115°C and then tested by XRPD. The results showed that Form V transformed into Form I under this condition.

[0079] Table 1

[0080]

[0081]

[0082] Example 4

[0083] Binary solvent suspension method

[0084] 1) Weigh 20 mg of the sample and suspend it in 0.8 mL of a selected binary mixed solvent (acetonitrile / water (1:3)) at room temperature for 5 days to obtain a new crystal form Form VI, or;

[0085] 2) Add 249.3 mg of raw material and 007-23-15 Form VI seed crystals to 1.2 mL of acetonitrile / water (1:3) mixed solvent and suspend at room temperature for 5 days to obtain a new crystal form Form VI.

[0086] XRPD results of Form VI are shown in Figure 5 The TGA and DSC test results are as follows: Figure 6 As shown, Form VI has no weight loss when heated to 150°C in TGA characterization, and only has a melting endothermic peak at 174.6°C in DSC characterization. The NMR results of Form VI are consistent with the structure of anhydroaconitine A. In summary, Form VI should be anhydrous crystalline form.

[0087] XRPD comparison of Form I-VI is shown below: Figure 7 As shown, the properties of different crystal forms are summarized in Table 2:

[0088] Table 2

[0089]

[0090]

[0091] The results of crystal form research show that Form II, Form III, Form IV and Form V are unstable and will transform into Form I when heated. Even at room temperature, they will spontaneously desolvate and transform into Form I.

[0092] Example 5 Pharmacokinetic Experiment

[0093] 1 Materials and methods

[0094] 1.1 Instrument

[0095] Waters ultra-high performance liquid chromatography-mass spectrometry system (including online degasser, ultra-high pressure gradient pump, column oven, autosampler, Waters I-CLASS UPLC liquid phase system, Xevo TQ-S mass spectrometer, UNIFI workstation); Eppendorf5810R fully automatic high-speed refrigerated centrifuge; Eppendorf MixMate vortex instrument; METTLER TOLEDO XP105DR electronic balance.

[0096] 1.1.1 Reagents

[0097] Methanol was of chromatographic grade (Merck, Germany); acetonitrile was of chromatographic grade (Merck, Germany); and water was commercially available distilled water from Watsons.

[0098] 1.2 Experimental animals

[0099] Strain: SD rat

[0100] Gender: Male

[0101] Weight: about 250g

[0102] Source: Shanghai Xipu-Bikai Laboratory Animal Co., Ltd.

[0103] Laboratory Animal Production License Number: SCXK(Shanghai)2018-0006

[0104] Feeding: 4 mice per cage, kept in an air-conditioned constant temperature room, room temperature 20-24℃, humidity 40-70%, light for 12h. Free access to food and water. 1.3 Dosing method and sample collection

[0105] 1.3.1 Dosage and route of administration

[0106] Administration method: intragastric administration;

[0107] Dosage for oral administration: 2.7 mg / kg (aconitine A crystal forms I and VI, aconitine A raw material, amorphous aconitine A); 0.5 mg / kg (amorphous aconitine A).

[0108] The aconitine crystal form I and VI powders were added with 1% Tween-80 of the total solution volume and ground to make it transparent, and then sodium carboxymethyl cellulose was added to make up the volume while grinding to prepare a 0.27 mg / mL drug solution for oral administration.

[0109] 1.3.2 Medication and blood collection

[0110] Twelve SD rats were divided into three groups. One group was given crystalline form I of aconitine A, one group was given crystalline form VI of aconitine A, and one group was given amorphous aconitine A. After the administration of crystalline form I, VI, and amorphous aconitine A, about 0.2 mL of whole blood was taken at each time point at 0.083, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 6, 9, 12, and 24 h, and then centrifuged at 8000 r / min for 10 min to separate the plasma, with 50 μL aliquots and the remaining plasma as backup. All were frozen and stored at -80°C.

[0111] 1.4 Plasma sample processing method

[0112] 50 μL of plasma sample was added with 5 μL IS (500 ng / ml tolbutamide (JBHDN)) and mixed, and then 450 μL methanol was added to precipitate protein. The mixture was vortexed for 30 s and centrifuged at 13000 r / min for 5 min. The supernatant was filtered and analyzed by LC / MS / MS.

[0113] 1.5 Establishment of UPLC-MS / MS method for determination of aconitine A crystal forms I, VI, aconitine A raw material and amorphous aconitine A in SD rat plasma

[0114] 1.5.1 Solution preparation

[0115] 1.5.1.1 Preparation of the working solution of the standard curve of aconitine A crystal form I and VI

[0116] Accurately weigh an appropriate amount of aconitine crystal form I and VI standard, dissolve them in methanol, and then dilute them with 80% methanol water to obtain aconitine crystal form I and VI stock solution with a concentration of 1 mg / mL. Dilute the aconitine crystal form I and VI stock solution with 80% methanol water to obtain aconitine crystal form I and VI with a concentration of 10, 50, 100, 500, 1000, 2500, 5000 ng / mL standard curve working solution, and store them in a -20°C refrigerator.

[0117] The analysis of aconitine A raw material was carried out using aconitine A crystal form I working solution as the standard.

[0118] 1.5.1.2 Preparation of quality control working solution of aconitine A crystal form I and VI

[0119] Accurately weigh an appropriate amount of aconitine crystal form I and VI standard, dissolve them in methanol, and then dilute them with 80% methanol water to a quality control stock solution with a concentration of 1 mg / mL. Dilute the quality control stock solution with 80% methanol water to obtain aconitine crystal form I and VI with a concentration of 30, 400, and 4000 ng / ml quality control working solution, and store them in a -20°C refrigerator.

[0120] 1.6 Preparation of standard samples and quality control samples of aconitine crystal forms I and VI

[0121] The standard series solutions of aconitine crystal form I and VI under "1.5.1.1" were prepared in the blank plasma of SD rats at a ratio of 1:10 to obtain standard series samples with concentrations of 1, 5, 10, 50, 100, 250, and 500 ng / mL, respectively. The quality control working solution under "1.5.1.2" was diluted in the same way to obtain plasma quality control samples with concentrations of 3, 40, and 400 ng / mL, respectively.

[0122] The analysis of aconitine A raw material was carried out using aconitine A crystal form I working solution as the standard.

[0123] 1.7 Sample measurement conditions

[0124] 1.7.1 Chromatographic conditions

[0125] The analytical column was ACQUITY UPLC BEH C18 (1.7 μm 2.1×100 mm); the mobile phase was water (0.1% FA): acetonitrile; gradient elution: flow rate was 0.3 mL / min; injection volume was 1 μL; column temperature was 40° C.; the elution gradient was shown in Table 3 below:

[0126] Table 3 Mobile phase gradients for aconitine A crystal forms Ⅰ and Ⅵ

[0127]

[0128] 1.7.2 Mass spectrometry conditions

[0129] The ion source is an electrospray ionization source (ESI); the source temperature is 150°C; the nebulizing gas temperature is 350°C; the capillary voltage is 3kV; the detection method is positive ion detection; the scanning method is multiple reaction monitoring (MRM); the mass spectrometry detection parameters of the analyte and the internal standard are shown in Table 4 below:

[0130] Table 4 Mass spectrometry detection parameters of the analytes

[0131]

[0132] 1.8 Data processing methods

[0133] The non-compartmental model of DAS2.0 software was used to calculate the pharmacokinetic parameters of SD rats after administration. max : Use the measured value; peak time T max : Use the measured value; Area under the drug-time curve AUC (0-t) Value: Calculated using the trapezoidal method; AUC (0-∞) =AUC (0-t) +C t / k e, C t is the blood drug concentration at the last measurable time point, k e is the elimination rate constant; elimination half-life t 1 / 2 =0.693 / k e ; Mean residence time MRT = AUMC / AUC; Distribution volume V z =CL / k e .

[0134] Excel software was used to calculate the mean, standard deviation, precision and accuracy of the blood drug concentration of each sample.

[0135] 2 Results and analysis

[0136] 2.1 Sample analysis method

[0137] 2.1.1 Specificity of the assay method

[0138] In this experiment, the standard working solution of aconitine crystal form I and VI was added to the blank rat plasma, and the method under "1.4" was used for processing. The LC-MS / MS analysis was performed using the chromatographic mass spectrometry conditions under "1.7". The parameters during the test were set as follows: channel name: integral, smoothing times 2 times, triple quadrupole MRM mode, electrospray voltage of 30eV, electrospray positive ion mode, parent ion molecular weight of 644.29, and daughter ion molecular weight of 583.99. The experimental results show that there are no components in the matrix that interfere with the determination of aconitine crystal form I and VI. The retention time of aconitine crystal form I and VI is: 1.92min. Figure 8 is the mass spectrum of blank plasma; Fig. 9 is the mass spectrum of the sample measured in the calibration curve sample; Fig.10 This is the mass spectrum of the measured samples of aconitine crystalline forms I and VI in the plasma of rats after administration.

[0139] 2.1.2 Standard curve and quality control

[0140] The peak areas (As, Y) of aconitine crystalline forms I and VI were weighted (1 / X 2 ) linear regression, and the standard curve of aconitine crystal form I and VI in rat plasma was obtained. Fig.11 ,in, Fig.11 The standard curve fitting equation of (1) is Y = 2.15e-002 + 3.19e-002 * X, RSD is 6.372%, R 2 is 0.997827; Fig.11 The standard curve fitting equation in (2) is Y = 2.01e-002 + 3.67e-002 * X, RSD is 5.112%, R 2 The quality control accuracy results of each analysis batch are shown in Table 5:

[0141] Table 5 Calculated concentrations of QC samples in sample analysis batches

[0142]

[0143] Acceptance criteria: Each analytical batch should contain at least 2 sets of 3 levels of quality control samples, and the total number of quality control samples should not be less than 5% of the number of samples to be tested. At least 67% of the quality control samples in each analytical batch should meet the standards of accuracy within ±15% and precision not exceeding 15%, and at least 50% of the quality control samples at each concentration meet the above standards.

[0144] 2.2 Blood concentration and pharmacokinetic parameters of aconitine A crystal forms Ⅰ, Ⅵ and amorphous aconitine A after administration in SD rats

[0145] 2.2.1 Blood drug concentrations of aconitine A crystal forms I, VI and amorphous aconitine A in SD rats after single oral administration

[0146] The blood drug concentration of SD rats after single oral administration of aconitine crystal form I is shown in Table 6-1, and the blood drug concentration-time curve is shown in Fig.12 ; The blood drug concentration after single oral administration of aconitine crystal form VI is shown in Table 6-2, and the blood drug concentration-time curve is shown in Fig.13 The blood drug concentration of natural aconitine A in rats after single oral administration is shown in Table 6-3, and the blood drug concentration-time curve is shown in Fig.14 ; The blood drug concentration of amorphous aconitine A in rats after single oral administration is shown in Tables 6-4 and 6-5, and the blood drug concentration-time curve is shown in Fig.15 ;

[0147] Table 6-1 Plasma drug concentrations at different time points after a single oral administration of the test drug to rats (2.7 mg / kg)

[0148]

[0149]

[0150] Table 6-2 Plasma drug concentrations at different time points after rats were intragastrically administered with the test drug aconitine crystalline form VI (2.7 mg / kg)

[0151]

[0152] Table 6-3 Plasma drug concentrations at different time points after rats were gavaged with the test drug aconitine A raw material (2.7 mg / kg)

[0153]

[0154] Table 6-4 Blood drug concentration of amorphous aconitine A in rats after oral administration of 0.5 mg / kg

[0155]

[0156]

[0157] Note: * indicates below the detection limit

[0158] Table 6-5 Blood drug concentration of amorphous aconitine A in rats after oral administration of 2.7 mg / kg

[0159]

[0160] Note: / indicates rat death

[0161] 2.2.2 Pharmacokinetic parameters after administration in SD rats

[0162] The corresponding pharmacokinetic parameters of SD rats after single administration of aconitine A crystal forms I, VI, aconitine A raw material and amorphous aconitine A are shown in Tables 7-1, 7-2, 7-3 and 7-4:

[0163] Table 7-1 Pharmacokinetic parameters of rats after oral administration of the test drug aconitine crystalline form I (2.7 mg / kg)

[0164]

[0165] Table 7-2 Pharmacokinetic parameters of rats after oral administration of the test drug aconitine crystalline form VI (2.7 mg / kg)

[0166]

[0167]

[0168] Table 7-3 Pharmacokinetic parameters of rats after oral administration of the test drug natural aconitine (2.7 mg / kg)

[0169]

[0170] Table 7-4 Pharmacokinetic parameters of amorphous aconitine A in rats after oral administration (0.5 mg / kg)

[0171]

[0172] The above test results show that after a single oral administration of aconitine A crystal form I, VI, aconitine A raw material and amorphous aconitine A to rats, the average peak time of aconitine A blood concentration in vivo was (0.38±0.18) h, (1.063±0.591) h, (0.875±0.8) h and (0.5±0.354) h, respectively, and the average peak concentration was (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, respectively. The average area under the concentration-time curve (AUC) 0-t They were (73.2±18.6) ng / mL*h, (115.544±29.598) ng / mL*h, (124.25±32.85) ng / mL*h and (34.363±6.582) ng / mL*h respectively; the average elimination half-lives in rats after oral administration were (21.48±2.12) h, (12.547±14.311) h, (4.700±2.489) h and (2.085±0.539) h respectively.

[0173] Example 6 Analgesia Comparative Test

[0174] 1. Animals: SPF mice (Shanghai Xipu-Bikai Laboratory Animal Co., Ltd., animal certificate number: SCXK (Shanghai) 2018-0006, weighing about 18-22g)

[0175] 2. Methods: 110 male mice were randomly divided into 11 groups: vehicle control group; positive control (aspirin 200 mg / kg) group; aconitine raw material group, 0.2, 0.4, 0.8 mg / kg; aconitine crystal form I group 0.1, 0.2, 0.4 mg / kg; amorphous aconitine group 0.04, 0.08, 0.16 mg / kg group; 10 mice in each group. Each group of animals was gavaged once according to the dose, and the vehicle control group was given 1% CMC-Na at 20 mL / kg. 30 minutes after the administration, 0.6% glacial acetic acid solution 0.1 mL / 10 g was intraperitoneally injected, and the number of painful twists of each mouse within 15 minutes was observed and recorded. The inhibition rate of acetic acid-induced painful twists of each group was compared. The results are shown in Table 8 below:

[0176] Inhibition rate (%) = (average number of twists in the vehicle group - average number of twists in the drug group) / average number of twists in the vehicle group * 100%

[0177] Table 8 Effect of aconitine A crystal form on acetic acid-induced pain in mice

[0178]

[0179] It can be seen from Table 8 above that the effects of aconitine crystalline form I and aconitine amorphous form are better than the aconitine raw material, especially aconitine amorphous form is significantly better than the aconitine raw material.

[0180] Example 7 Study on the acute toxicity of aconitine A to KM mice

[0181] 1. Experimental Animals

[0182] Species: KM mice

[0183] Grade: SPF grade

[0184] Number and gender of animals purchased: 30, male

[0185] Source: Shanghai Xipu-Bikai Experimental Animal Co., Ltd.

[0186] Production license number: SCXK (Shanghai) 2018-0006

[0187] Experimental Animal Use License: SYXK(Shanghai)2014-0018

[0188] 2. Experimental methods

[0189] 2.1 Grouping

[0190] The mice were adaptively raised for 1-2 days, and 15 mice were fed with each of the aconitine crystal form I and crystal form VI. The aconitine crystal form I was randomly divided into 5 groups, with 3 mice in each group: blank group, crystal form I 2.8 mg / kg, crystal form I 3.79 mg / kg, crystal form I 5.06 mg / kg, crystal form I 6.75 mg / kg, and crystal form I 9.0 mg / kg; the crystal form VI was grouped in the same way as the crystal form I.

[0191] 2.2 Experimental methods

[0192] Before administration, fasting was performed and the physiological state of the animals was observed. Except for the blank group, mice were given various doses of aconitine crystal form I or crystal form VI by gavage in the remaining groups, and the doses of each group were 2.8, 3.79, 5.06, 6.75, and 9.0 mg / kg (the two crystal forms were consistent). The poisoning symptoms of the animals were observed, and the number of deaths was recorded.

[0193] 2.3 Calculation method

[0194] The median lethal dose of mice was calculated using Bliss software. The data were obtained and the mortality-logarithmic dose curve and the probability unit-logarithmic dose curve were drawn.

[0195] 3. Experimental results

[0196] LD of Form I and Form VI 50The calculation reports are shown in Tables 9 and 10, respectively. The results are shown in Table 9. The mortality-logarithmic dose curves and the probability unit-logarithmic dose curves of Form I and Form VI are shown in Table 9 and 10, respectively. Fig.16 and 17 As shown in Tables 9 to 10 and Fig.16 and 17 It can be seen that after administration of aconitine A, the data showed that the LD 50 is 4.4 mg / kg, with a 95% confidence limit of 3.6-5.3 mg / kg; the LD of Form VI 50 5.9 mg / kg, 95% confidence limit 4.8 to 7.3 mg / kg

[0197] Table 9 LD of Form I 50 Calculation Report

[0198]

[0199] Table 10 LD of Form VI 50 Calculation Report

[0200]

[0201] 4. Conclusion

[0202] Both forms I and VI of aconitine A are toxic to a certain extent.

[0203] LD of Form I 50 The 95% confidence interval was 3.6-5.3 mg / kg, and the LD after Feiller correction was 4.4 mg / kg. 50 is 4.3 mg / kg; the LD of Form VI 50 The LD after Feiller correction was 5.9 mg / kg, with a 95% confidence limit of 4.8 to 7.3 mg / kg. 50 It is 6.1mg / kg.

[0204] The acute toxicity of the two crystalline forms I and VI of the non-standard aconitine A was measured by dissolving them in the corresponding solvents and adjusting them with Tween 80 to make an oral solution.

[0205] After exploring the dosage, the inventors tested the pharmacokinetics of the crystalline drug. It was proved that the crystalline forms I and VI were more effective than amorphous aconitine C. max It is significantly increased, indicating that the toxicity of crystal form I and crystal form VI is significantly lower than that of amorphous aconitine A.

Claims

1. A crystal form VI of aconitine A, characterized in that: Its XRPD diagram is basically as shown in Figure 5. The crystal form VI of aconitine A is obtained by crystallization using a mixed solvent of acetonitrile and water using a binary solvent suspension method, and the ratio of acetonitrile to water is 1:2-4.

2. The crystal form VI according to claim 1, characterized in that Its TGA and DSC graphs are basically shown in Figure 6.

3. The method for preparing the crystal form VI of aconitine A according to claim 1, characterized in that: The ratio of acetonitrile to water is 1:

3.

4. A pharmaceutical composition, characterized in that The invention comprises the crystal form VI of aconitine A according to claim 1 or 2; and pharmaceutically acceptable excipients.

5. The pharmaceutical composition according to claim 4, characterized in that It is a solid dosage form.

6. The pharmaceutical composition according to claim 5, characterized in that It is an oral preparation.

7. Use of the aconitine crystal form VI according to claim 1 or 2 in the preparation of anti-inflammatory and analgesic drugs.

8. Use of the aconitine crystal form VI according to claim 1 or 2 in the preparation of a drug for inhibiting drug addiction.

Images