Application of Buxusemine H and Buxusemine L, alkaloids from boxwood, in pharmaceutical manufacturing

Abstract

The invention discloses the application of Buxusemine H and Buxusemine L, a boxwood alkaloid, in pharmaceutical preparation, and belongs to the field of pharmaceutical technology. Specifically, the invention provides the application of Buxusemine H and Buxusemine L, a boxwood alkaloid, in preparing drugs for treating inflammatory pain, visceral pain and neuropathic pain, and in preparing Ca v 3.2 Application in selective inhibitors of low voltage-gated calcium channel subtypes. Buxusemine H and Buxusemine L inhibit Ca v 3.2 The activity of low voltage-gated calcium channels is prominent, and the response to Ca v 3.2 and Ca v 3.1 The selectivity of low voltage-gated calcium channels is 9 and 30 respectively, which is significantly better than the candidate molecule Z944 in clinical trials (in Ca v 3.1 and Ca v 3.2) and the boxwood alkaloid analog cyclovibrobuxin D (in Ca v 3.1 and Ca v Buxusemine L has no selectivity in Na v 1.7 Na v Buxusemine L has outstanding specificity in ion channels involved in pain conduction, such as TRPV1, TPRA1, TRPM8, ASIC3, P2X2 and P2X4. At the same time, Buxusemine L can significantly relieve L-cysteine-induced inflammatory pain, acetic acid-induced visceral pain and sciatic nerve ligation-induced neuropathic pain in mice, and its effect is comparable to that of the positive controls Mibefradil and Z944.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology and relates to the application of boxwood alkaloid compounds Buxusemine H and Buxusemine L in pharmaceutical preparation, and specifically relates to the application of boxwood alkaloids Buxusemine H and Buxusemine L and drug combinations thereof as active ingredients in the preparation of drugs for treating inflammatory pain, visceral pain, neuropathic pain, absence epilepsy, tremor and sleep disorders. Background Art

[0002] Ion channels are the second largest target for current drug development. v 3.2 Low Voltage-gated calcium channels (LVGCC, Ca v 3.1-3.3), is an important target for drug development for neurological diseases such as neuropathic pain, absence epilepsy, cerebellar ataxia, Parkinson's tremor and depression. In recent years, well-known foreign pharmaceutical companies such as Zalicus, MERK, Actelion, Idorsia and Cavion have begun to use it as a target for innovative drug development, and synthetic molecules represented by Z944, ABT-639, Suvecaltamide, MK-8998, CX-8998 and NBI-827104 have also entered clinical research. However, none of the above molecules has been approved for marketing so far. In addition, ABT-639 and MK-8998 terminated related clinical trials due to poor therapeutic effects and other reasons. At the same time, in the just-concluded Phase II clinical trial, the performance of NBI-827104 did not meet the expected goals. The above-mentioned Ca v 3.2 One of the main reasons for the slow progress of drug development is that the molecules entering clinical trials have no subtype selectivity in LVGCC, especially in Ca v 3.1 and Ca v 3.2 These two LVGCCs are the most widely distributed and expressed. Therefore, it is necessary to develop Ca2+ subtype-selective v 3.2 Active molecules are imminent.

[0003] The inventors disclosed in CN116139154A that cyclovirobuxine D (CVB-D), a Buxus alkaloid molecule, is clinically used to treat ischemic heart disease (coronary heart disease, angina pectoris), various arrhythmias and heart failure by inhibiting Ca v 3.2 Play a role in relieving inflammatory pain and neuropathic pain in vivo. However, it should be pointed out that CVB-D has a v 3.1 and Cav There is no selectivity in 3.2.

[0004] So far, there is no report in the prior art that Buxusemine H and Buxusemine L, the boxwood alkaloid compounds, have pharmacological effects against inflammatory pain, visceral pain, neuropathic pain, absence epilepsy, tremor and sleep disorders. v 3.2 Report on selective inhibitors of low voltage-gated calcium channel subtypes and their use as active molecules in the preparation of drugs for the treatment of inflammatory pain, visceral pain, neuropathic pain, absence epilepsy, tremor and sleep disorders. Summary of the invention

[0005] The present invention aims to v 3.2 In order to overcome the above shortcomings of inhibitors, the boxwood alkaloid compounds Buxusemine H and Buxusemine L are provided as novel Ca v 3.2 Low voltage-gated calcium channel subtype selective inhibitors, and their use in the preparation of drugs for the treatment of inflammatory pain, visceral pain and neuropathic pain.

[0006] In order to achieve the above-mentioned object of the present invention, the present invention provides the following technical solutions:

[0007] Compounds Buxusemine H and Buxusemine L are used in the preparation of Ca v 3.2 Application in low voltage-gated calcium channel subtype selective inhibitors.

[0008] Buxusemine H and Buxusemine L were used in the preparation of Ca-inhibiting v 3.2 Application of drugs in diseases with low voltage-gated calcium channel activity.

[0009] According to the application, wherein the Ca v 3.2 Low voltage-gated calcium channel activity diseases are one or more of inflammatory pain, visceral pain, neuropathic pain, absence epilepsy, tremor and sleep disorders.

[0010] Application of the compounds Buxusemine H and Buxusemine L in preparing medicines for treating inflammatory pain, visceral pain and neuropathic pain.

[0011] Application of the compounds Buxusemine H and Buxusemine L in preparing medicines for treating pain, absence epilepsy, tremor, sleep disorder and depression.

[0012] According to the application, the dosage of the compounds Buxusemine H and Buxusemine L is 1 mg / kg to 30 mg / kg.

[0013] According to the application, it is characterized in that the compounds Buxusemine H and Buxusemine L exist in the form of racemates, mixtures of stereoisomers or stereoisomers.

[0014] According to the application, the compounds Buxusemine H and Buxusemine L exist in the form of salts.

[0015] According to the application, the compounds Buxusemine H and Buxusemine L exist in the form of solvates.

[0016] The present invention also provides Ca v 3.2 Low voltage-gated calcium channel subtype selective inhibitor, which has the compound Buxusemine H or Buxusemine L as the active ingredient.

[0017] Preferably, the dosage of Buxusemine H and Buxusemine L is 1 mg / kg to 30 mg / kg body weight.

[0018] Preferably, the specific dosage of Buxusemine H and Buxusemine L can be adjusted within the above dosage range according to the actual symptoms and administration methods.

[0019] Preferably, Buxusemine H and Buxusemine L are present in the form of a racemate, a mixture of stereoisomers or stereoisomers.

[0020] Preferably, the Buxusemine H and Buxusemine L are present in the form of salts.

[0021] Preferably, the Buxusemine H and Buxusemine L exist in the form of a solvate.

[0022] Preferably, the pharmaceutical composition is an oral preparation or an external preparation.

[0023] Preferably, the oral preparations include oral solid preparations and oral liquid preparations, the oral solid preparations are tablets, enteric-coated tablets, sustained-release tablets, granules or hard capsules, and the oral liquid preparations are oral solutions or syrups.

[0024] Preferably, the external preparation is an ointment, a transdermal patch, a suppository, an aerosol, a liniment, a gel or an injection.

[0025] The auxiliary materials and / or additives that can be used in the pharmaceutical preparations involved in the present invention can be selected from carrier materials, excipients, stent materials, glidants, fillers, solvents, cosolvents, diluents, colorants, flavoring agents, antioxidants, adhesives, transdermal enhancers, surfactants, propellants, pill bases, suppository bases, and / or ointment bases, and the selection of the types and amounts of these auxiliary materials and / or additives depends on the method of making and using the product. These oral or external preparations can be applied.

[0026] The pharmaceutically acceptable salts in the present invention refer to pharmaceutically acceptable salts, including salts formed with organic acids and inorganic acids. The organic acids are tartaric acid, citric acid, formic acid, acetic acid, oxalic acid, butyric acid, oxalic acid, maleic acid, succinic acid, oxalic acid, alginic acid, aspartic acid, benzenesulfonic acid, camphoric acid, camphorsulfonic acid, digluconic acid, cyclopentanepropionic acid, dodecylsulfonic acid, ethanesulfonic acid, glucoheptonic acid, glycerophosphoric acid, hemisulfuric acid, heptanoic acid, hexanoic acid, fumaric acid, 2-hydroxyethanesulfonic acid, lactic acid, maleic acid, methanesulfonic acid, nicotinic acid, 2-naphthalenesulfonic acid, pamoic acid, pectinic acid, 3-phenylpropionic acid, picric acid, pivalic acid, propionic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonate and undecanoate. The inorganic acid is hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid or phosphoric acid.

[0027] The invention provides a Ca-containing compound with Buxusemine H or Buxusemine L as an active ingredient. v 3.2 A pharmaceutical composition with selective inhibitory activity of low voltage-gated calcium channel subtypes, consisting of an active ingredient and a pharmaceutically acceptable excipient.

[0028] The present invention has no particular limitation on the pharmaceutically acceptable excipients. Specifically, the pharmaceutically acceptable excipients preferably include one or more of a drug carrier, a surfactant, a buffer substance, a disintegrant, a binder, a filler, a lubricant, an excipient, a solubilizer, a flavoring agent, and a coloring agent. The present invention has no particular limitation on the specific types of the above excipients, and they can be selected according to actual needs.

[0029] The present invention provides the use of the compound described in the above technical solution and its pharmaceutical composition in the preparation of a drug for treating inflammatory pain, visceral pain, neuropathic pain, absence epilepsy, tremor and sleep disorders.

[0030] The present invention has no particular limitation on the dosage form of the drug for treating inflammatory pain and neuropathic pain, and can be selected according to actual needs, and can specifically be tablets, capsules, granules, granules, pills, oral liquid preparations, ointments, transdermal patches and suppositories, etc. The present invention has no particular limitation on the preparation method of drugs in different dosage forms, and can be prepared by methods well known to those skilled in the art.

[0031] In the present invention, the dosage of the drug for treating inflammatory pain, visceral pain and neuropathic pain will vary with the compound used, the mode of administration, the desired treatment and the disorder indicated. For example, for oral administration, the daily dosage of Buxusemine H and Buxusemine L or a pharmaceutically acceptable salt thereof can be in the range of 1 mg / kg to 30 mg / kg of body weight.

[0032] The advantages of the present invention are as follows: the inventors found in the process of further in-depth research on boxwood alkaloids that 9 (10 / 19) abeo-artane-type boxwood alkaloid molecules Buxusemine H and Buxusemine L have a strong affinity for Ca v 3.2 has a significant inhibitory effect. Unlike CVB-D and other molecules with a 6 / 6 / 6 / 5 skeleton, Buxusemine H and Buxusemine L have a 6 / 7 / 6 / 5 molecular skeleton. At the same time, Buxusemine H and Buxusemine L have a significant inhibitory effect on Ca v 3.2 and Ca v 3.1 The selectivity of low voltage-gated calcium channels is 9 and 30 respectively, which is significantly better than the candidate molecule Z944 (in Ca v 3.1 and Ca v 3.2) and CVB-D (in Ca v 3.1 and Ca v Buxusemine L has no selectivity in Na v 1.7 Na v 1.8, TRPV1, TPRA1, TRPM8, ASIC3, P 2 X 2 and P 2 X 4 Buxusemine L has outstanding specificity in ion channels involved in pain transmission, such as acetic acid. In addition, Buxusemine L can significantly relieve L-cysteine-induced inflammatory pain, acetic acid-induced visceral pain, and sciatic nerve ligation-induced neuropathic pain in mice, and its effect is comparable to that of the positive control Mibefradil and Z944. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Buxusemine H and Buxusemine L of the present invention are Ca v 3.1 and Ca v AC in the figure represent the inhibitory effect of Buxusemine H on Ca v 3.1 Effect of different concentrations of Buxusemine H on Ca v 3.1 Inhibitory effect of peak current and Buxusemine H on Ca v 3.1 Dose-effect curve of the inhibitory effect on peak current. DF represents: Buxusemine H to Ca v 3.2 Effect of different concentrations of Buxusemine H on Ca v 3.2 Inhibitory effect of peak current and Buxusemine H on Ca v 3.2 Dose-effect curve of the inhibitory effect of peak current. GI represents: Buxusemine L on Ca v 3.1 Effect of different concentrations of Buxusemine L on Ca v 3.1 Inhibitory effect of peak current and Buxusemine L on Ca v 3.1 Dose-effect curve of the inhibitory effect on peak current. JL represents: Buxusemine L on Ca v 3.2 Effect of different concentrations of Buxusemine L on Ca v 3.2 Inhibitory effect of peak current and Buxusemine L on Ca v 3.2 Dose-effect curve of peak current inhibition.

[0034] Figure 2 . The effect of Z944 on Ca v 3.1 and Ca v 3.2. Figure A and B show the inhibitory effect of different concentrations of Z944 on Ca v 3.1 Inhibitory effect of peak current and Z944 on Ca v 3.1 Dose-effect curves of the inhibitory effect on peak current. C and D show the effects of different concentrations of Z944 on Ca v 3.2 Inhibitory effect of peak current and Z944 on Ca v 3.2 Dose-effect curve of peak current inhibition.

[0035] Figure 3. Buxusemine L (10 μM) of the present invention has an effect on TRPV1 (A), TRPM8 (B), TPRA1 (C), P 2 X 4 (D), P 2 X 2 (E), ASIC3 (F), Na v 1.7(G) and Na v 1.8(H) inhibitory effect.

[0036] Figure 4 . The relieving effect of Buxusemine L of the present invention in different mouse pain models. In the figure, A: Buxusemine L (intraperitoneal injection; 2.5, 5, 10 mg / kg) relieves mechanical allodynia in wild-type mice induced by L-cysteine ​​and compares it with the positive control Mibefradil (10 mg / kg); B: Buxusemine L (intraperitoneal injection; 2.5, 5, 10 mg / kg) relieves visceral pain in wild-type mice induced by acetic acid and compares it with the positive control Z944 (10 mg / kg); C: Buxusemine L (intraperitoneal injection; 2.5, 5, 10 mg / kg) relieves mechanical allodynia induced by sciatic nerve ligation and compares it with the positive control Z944 (10 mg / kg).

[0037] Figure 5 . The present invention shows the alleviating effect of Buxusemine H (intraperitoneal injection; 20 mg / kg) on ​​acetic acid-induced visceral pain in wild-type mice. DETAILED DESCRIPTION

[0038] The following will be combined with the accompanying drawings and the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] Example 1

[0040] Buxusemine H and Buxusemine L, the boxwood alkaloid compounds, can be prepared by the following method.

[0041] The preparation of compounds Buxusemine H and Buxusemine L comprises the following steps:

[0042] (1) Extracting the leaves and branches of Euonymus japonicus (15 kg) with alcohol, heating and refluxing for 3 times, each time for 3 hours, combining the alcohol extracts, recovering the solvent, dispersing the obtained crude alcohol extract with water, acidifying with 0.1 mol / L dilute hydrochloric acid to pH 5.0, extracting with ethyl acetate to remove non-alkaline fat-soluble components, and alkalizing the remaining acid aqueous solution to pH 10.0 with 0.1 mol / L sodium hydroxide, extracting with chloroform to obtain an extract of the total alkaloid-enriched part (120 g);

[0043] (2) subjecting the total alkaloid enrichment fraction extract to a first silica gel column chromatography separation (12 kg) to obtain component F1, component F2, component F3, component F4 and component F5, wherein the eluent for the first silica gel column chromatography separation comprises a petroleum ether-ethyl acetate-diethylamine solvent, wherein the volume ratio of petroleum ether:ethyl acetate:diethylamine in the petroleum ether-ethyl acetate-diethylamine solvent is (100:1:1 to 0:100:1), and the fractions are monitored by thin layer chromatography and then combined;

[0044] (3) The component F1 (6.0 g) was subjected to a second silica gel column separation (200 g) to obtain component F1a, component F1b, component F1c, component F1d, component F1e and component F1f, wherein the eluent for the second silica gel column chromatography separation comprises petroleum ether-acetone (containing 0.1% diethylamine in the total volume ratio), wherein the volume ratio of petroleum ether to acetone in the petroleum ether-acetone (containing 0.1% diethylamine in the total volume ratio) solvent is (100:1-2:1), and the fractions were monitored by thin layer chromatography and then combined; the component F1b (0.4 g) was subjected to a first LH20 gel column separation, wherein the eluent was methanol (containing 0.1% diethylamine), to obtain Buxusemine H (9.0 mg);

[0045] (4) The component F2 (1.5 g) was subjected to a first LH20 gel column separation, with methanol (containing 0.1% diethylamine) as the eluent, to obtain components F2a, F2b, F2c, F2d and F2e; the component F2d (0.1 g) was subjected to a first semi-preparative high performance liquid chromatography separation, with acetonitrile-water (containing 0.3% diethylamine) as the mobile phase in a volume ratio of 90:10, to obtain compound Buxusemine L (6.0 mg), with a peak elution time of 32.7 minutes;

[0046] (5) In the above separation process, the weight of the medicinal material can be increased to proportionally increase the amount of filler used in the subsequent separation to enrich a sufficient amount of compounds;

[0047] The structural formulas of the compounds Buxusemine H and Buxusemine L are as follows:

[0048]

[0049] Example 2

[0050] Buxusemine H and Buxusemine L of the present invention have an effect on Ca v 3.1 Ca v 3.2, TRPA1, TRPV1, TRPM8, P 2 X 2 , P 2 X 4 ,ASIC3,Na v 1.7 Na v 1.8 The experimental methods and results of inhibitory activity are as follows:

[0051] 1. Cell preparation and expression

[0052] Human embryonic kidney (HEK) 293 cells were cultured in DMEM (HyClone) supplemented with 10% fetal calf serum (Gibco) and penicillin (100 units / ml) and streptomycin (0.1 mg / ml) (Biological Industries). 293 cells in logarithmic growth phase were stained with LipoD293 TM (SignaGen Laboratories) transfection reagent was used to transform human pCDNA3.1-TRPA1, TRPV1, TRPM8, P 2 X 2 , P 2 X 4 ,ASIC3,Na v 1.7 Na v 1.8 Ca v 3.1 Ca v Transfected human embryonic kidney (HEK) 293 cells should be used within 48 hours.

[0053] 2. Electrophysiology Experiment

[0054] All experiments were performed at room temperature (22-25°C). Borosilicate glass pipettes (World Precision Instruments) were prepared as glass electrodes with an impedance of 2-4 MΩ using a microelectrode puller (P-1000, Sutter Instrument) and heated polishing and used for whole-cell current recording. The current was amplified by Axopatch 200B, and then the data was converted by Digidata 1440A (Molecular Devices). The current was passed through a low-energy filter at 2 kHz and then sampled at 10 kHz. pCLAMP 10 (Molecular Devices) was used for data acquisition and analysis.

[0055] Record Ca v 3.1 and Ca v 3.2 The extracellular solution contains (in mM) 142CsCl, 1MgCl 2 , 2CaCl 2 , 10Glucose and 10HEPES (pH = 7.4, adjusted with CsOH). The intracellular solution contains (in mM) 127Cs-methanesulphonate, 2MgCl 2 , 2Na 2 ATP, 10 HEPES and 11 EGTA (pH = 7.4, adjusted with CsOH). The current recording procedure is: clamp the cell membrane at -100 mV potential, stimulate the cell with a 150 ms depolarizing voltage of -30 mV to obtain the peak current, and the interval between each stimulation is 4 s; clamp the cell membrane at -100 mV potential, stimulate the cell with a 150 ms depolarizing voltage of -80 mV to +50 mV to obtain the current-voltage (IV) curve, and the interval between each stimulation is 4 s, with a voltage increment of 5 mV.

[0056] The extracellular solution for recording TRPA1, TRPV1, and TRPM8 currents contained (in mM) 150 NaCl, 1 MgCl 2 and 10 HEPES (pH = 7.4, adjusted with NaOH). The intracellular solution contained (in mM) 150 NaCl, 1 MgCl 2 , 1 EGTA and 10 HEPES (pH = 7.4, adjusted with NaOH). The current recording procedure is: the cell membrane is clamped at 0 mV potential, and the cell is stimulated with a ramp voltage from -100 mV to +100 mV for 500 ms, with each stimulation interval of 2 s.

[0057] Record ASIC3, P 2 X 2 and P 2 X4 The extracellular solution contained (in mM) 150 NaCl, 1 MgCl 2 and 10HEPES (pH=7.4, adjusted with NaOH). The intracellular solution contained (in mM) 140KCl, 5EGTA and 10HEPES (pH=7.4, adjusted with NaOH). The current recording procedure was: the cell membrane was clamped at -60 mV potential, and the current was recorded in gap-free mode.

[0058] Record Na v 1.7 and Na v The extracellular solution at 1.8 current contains (in mM) 135K-gluconate, 5KCl, 5Mg-ATP, 0.5Na 2 GTP, 5HEPES, 2MgCl 2 , 5EGTA and 0.5CaCl 2 (pH = 7.4, adjusted with KOH) The intracellular solution contains (in mM) 140 NaCl, 5 KCl, 2 CaCl 2 , 2MgCl 2 , 10 HEPES and 10 glucose (pH=7.4, adjusted with NaOH). The current recording procedure was as follows: the cell membrane was clamped at a potential of -110 mV, and the cell was stimulated with a depolarizing voltage of -30 mV for 10 ms, with an interval of 5 s between each stimulation.

[0059] Buxusemine H and Buxusemine L were prepared into a stock solution with a concentration of 100 mM and diluted to the required concentration in the extracellular fluid for testing.

[0060] 3. Data analysis and statistics

[0061] Data collection and statistical analysis were performed using GraphPad 8.0.1 (GraphPad Software, San Diego, CA USA). 50 The Hill coefficient is calculated by the Hill equation Y = I Min +(I Max -IC Min ) / [1+10(LogEC 50 -C)×Hillslope] is calculated using the collected data. Here IC 50 is the concentration at which the channel current is inhibited by half, C is the concentration of the compound, and I Min is the minimum inhibition rate, I Max is the maximum inhibition rate, Hillslope is the Hill coefficient. All data are mean ± standard deviation.

[0062] 4. Effects of Buxusemine H and Buxusemine L on Ca v 3.1 and Ca v 3.2 Calcium channel inhibitory activity experiments, such as Figure 1 and 2 shown.

[0063] The experimental results show that under the above experimental conditions, Buxusemine H has a strong v 3.1 and Ca v 3.2 IC of peak current inhibition 50 30.71μM and 3.50μM, respectively, for Ca v 3.2 and Ca v The selectivity of Buxusemine L for Ca v 3.1 and Ca v 3.2 IC of peak current inhibition 50 14.91μM and 0.50μM, respectively, for Ca v 3.2 and Ca v The selectivity of 3.1 is 30. The positive control Z944 is for Ca v 3.1 and Ca v 3.2 IC of peak current inhibition 50 0.29μM and 0.65μM respectively. v 3.1 and Ca v 3.2 No significant selectivity (about 2). Buxusemine H has no significant selectivity for Ca v The inhibitory effect of 3.2 was not as good as that of Z944, but its selectivity was significantly better than that of Z944. v The inhibitory activity of 3.2 is comparable to that of Z944, and its selectivity is significantly better than that of Z944 and Buxusemine H.

[0064] 5. Effects of Buxusemine L on different ion channels involved in pain transmission in peripheral nociceptors

[0065] like Figure 3 As shown, high concentration of Buxusemine L (10 μM) had an effect on TRPV1, TRPM8, TPRA1, P 2 X 4 , P 2 X 2 ,ASIC3,Na v 1.7 and Na v 1.8 There is no obvious effect on a series of ion channels involved in pain transmission in peripheral nociceptors, and the selectivity is outstanding.

[0066] In order to illustrate the effect of the drug combination with Buxusemine H and Buxusemine L as active ingredients in the treatment of inflammatory pain, visceral pain and neuropathic pain, the present invention carried out the following pharmacological experiments.

[0067] Example 3

[0068] Buxusemine L relieves L-cysteine-induced inflammatory pain.

[0069] like Figure 4 As shown in the left figure of A, the sensitivity of mice to mechanical stimulation after intraplantar injection of L-cysteine ​​was greatly increased, which was manifested by a significant decrease in the threshold of the paw withdrawal reaction stimulated by von Frey fibers. The steady state of the lower threshold after 1 hour of L-cysteine ​​injection gradually recovered from 2 hours after injection, but the threshold was still far lower than that of the vehicle control group 4 hours after injection. Buxusemine L (2.5, 5, 10 mg / kg) intraperitoneal injection showed a dose-dependent analgesic effect ( Figure 4 In particular, the 5 and 10 mg / kg dose groups could substantially reverse the mechanical allodynia induced by plantar injection of L-cysteine ​​in mice starting from 3 hours after administration ( Figure 4 The positive control Mibefradil (10 mg / kg) also showed significant pain relief effect ( Figure 4 However, the pain relief effect produced by the drug 30 minutes after administration was weaker than that of 10 mg / kg Buxusemine L, indicating that Buxusemine L takes effect more quickly. Figure 4 As shown in the right figure of A, by calculating the area under the analgesic curve of each dosing group from 30 minutes to 4 hours after administration, it can be seen that the overall analgesic effect of 5 mg / kg Buxusemine L is equivalent to that of 10 mg / kg Mibefradil.

[0070] Experimental methods:

[0071] SPF grade C57BL / 6 experimental mice aged 8-10 weeks were adapted to the SPF barrier system environment for 3 days before the experiment.

[0072] On the day of the experiment, the 50% mechanical paw withdrawal threshold (PWT) of the right hind limb of the mouse was measured in advance by Von frey, and the 50% mechanical paw withdrawal threshold (PWT) of the right hind limb of the mouse was recorded before the experiment. Then, the drug was administered with a 1mL syringe, and 0.5 hours after the administration, 25μL of L-cysteine ​​(10nM) was injected subcutaneously into the sole of the right hind limb of the mouse with a microsyringe. After the injection of L-cysteine, the PWT was recorded by Von frey, and the pain response of the mouse was observed 0.5, 1, 2, 3, and 4 hours after the injection, and the PWT of the mice in each experimental group was statistically analyzed and compared.

[0073] Experimental groups:

[0074] 1. Vehicle control group (n=12): The vehicle was injected subcutaneously into the plantar surface of the right hind limb of mice, with a volume of 25 μl.

[0075] 2. Model group (n=12): L-cysteine ​​(10 nM) was injected subcutaneously into the plantar surface of the right hind limb of mice in a volume of 25 μl.

[0076] 3. Test drug + inflammatory pain model group (n=12): intraperitoneal injection 30 minutes before L-cysteine ​​injection.

[0077] Note: When the number of animals is even, half are male and half are female.

[0078] Drug dosage:

[0079] Mibefradil: 10 mg / kg, intraperitoneal injection

[0080] Buxusemine L: 2.5, 5, 10 mg / kg, intraperitoneal injection.

[0081] Drug vehicle: physiological saline solution containing 20% ​​(v / v) β-cyclodextrin and 0.4% DMSO

[0082] Animal mechanical stimulation pain sensitivity test: The mice were placed in a plastic box with a circular grid (aperture 6mm) on the bottom (length: 45cm, width: 29cm). The paws were stimulated with 0.008-4g von Frey fibers. The appropriate fibers were determined according to the current threshold of the mice. If there was a reaction, a fiber with a smaller intensity was selected. If there was no reaction, a fiber with a larger intensity was selected. The paw retraction reaction of the mice under stimulation of different intensities was recorded. Each fiber of each intensity was stimulated 3 times, and the thresholds of 6 fibers with different intensities were recorded. The recorded data were then substituted into the Vonfrey threshold table to find the corresponding threshold.

[0083] NS: Normal saline; L-Cys: L-cysteine; Mib: Mibefradil; 10: Buxusemine L

[0084] The method for significant difference analysis between groups was Brown-Forsythe and Welch ANOVA test with TamhaneT2multiple comparisons test (MCT).***P<0.001,****P<0.0001 (vs.Vehicle+NSgroup); ns indicates no significant difference. All data are mean ± standard deviation.

[0085] Example 4

[0086] Buxusemine L relieves acetic acid-induced visceral pain.

[0087] like Figure 4 As shown in the left figure of B, mice showed visceral pain response after intraperitoneal injection of acetic acid, showing writhing reactions with abdomen concave, anterior abdominal wall close to the cage bottom, hip twisting and hind limb extension. Intraperitoneal injection of Buxusemine L, Z9944 and solvent alone did not induce writhing reactions in mice ( Figure 4 Buxusemine L (2.5, 5, 10 mg / kg) intraperitoneal injection can reduce the acetic acid-induced writhing reaction in mice in a dose-dependent manner ( Figure 4 The pain relief effect of Buxusemine L (10 mg / kg) was comparable to that of the positive control Z944 (10 mg / kg) ( Figure 4 In addition, Buxusemine L (2.5, 5, 10 mg / kg) can significantly prolong the time before the first writhing reaction occurs in mice. There is no significant difference in the effects of each dose group, and the effect is also equivalent to Z944 (10 mg / kg) ( Figure 4 (as shown in the left picture of B).

[0088] Experimental methods:

[0089] SPF grade C57BL / 6 experimental mice aged 8-10 weeks were adapted to the SPF barrier system environment for 3 days before the experiment.

[0090] The acetic acid writhing model was selected as the visceral pain model. 0.5 h after intraperitoneal injection, the mice were intraperitoneally injected with 0.6% acetic acid (0.1 mL / 10 g) to establish the model, and the number of writhing times of the mice within 20 min after acetic acid administration and the time of the first writhing were observed.

[0091] Experimental groups:

[0092] 1. Vehicle control group (n=8): Mice were intraperitoneally injected with the vehicle in a volume of 200 μl.

[0093] 2. Visceral pain model group (n=8): Mice were intraperitoneally injected with 200 μl of 0.6% acetic acid solution.

[0094] 3. Test drug group (n=8): mice were intraperitoneally injected with 200 μl of the drug.

[0095] 4. Test drug + visceral pain model group (n=10-12): 200 μl of the test drug was injected intraperitoneally 30 minutes before modeling.

[0096] Note: When the number of animals is even, half are male and half are female.

[0097] Drug dosage:

[0098] Z944: 10 mg / kg, intraperitoneal injection

[0099] Buxusemine L: 2.5, 5, 10 mg / kg, intraperitoneal injection.

[0100] Drug vehicle: physiological saline solution containing 20% ​​(v / v) β-cyclodextrin and 0.4% DMSO

[0101] NS: Normal saline; AA: Acetic acid; 10: Buxusemine L

[0102] The method of significant difference analysis between groups was: One-way ANOVA test with Tukey MCT. ****P<0.0001, ## P<0.01, ### P<0.001, and #### P<0.0001 (vs.0.6% AA group); ns indicates no significant difference. All data are mean ± SD.

[0103] Example 5

[0104] Buxusemine L relieves neuropathic pain induced by sciatic nerve ligation.

[0105] like Figure 4 As shown in the left figure of C, the sensitivity of mice to mechanical stimulation after sciatic nerve ligation was greatly improved compared with the sham operation group, as shown by a significant decrease in the threshold of paw withdrawal response to von Frey fiber stimulation. Intraperitoneal injection of Buxusemine L (2.5, 5, 10 mg / kg) showed a dose-dependent analgesic effect ( Figure 4In particular, the 5 and 10 mg / kg dose groups showed a significant pain relief effect that lasted from 1 hour after administration to 6 hours ( Figure 4 In contrast, the 2.5 mg / kg dose group had a slow onset and short duration of action—lasting from 3 hours to 6 hours after administration. The positive control Z944 (10 mg / kg) also showed a significant pain relief effect ( Figure 4 (as shown in the left figure of C in the figure). Z944 has a faster onset of action than Buxusemine L, starting 30 minutes after administration and lasting up to 4 hours. Compared with Z944, Buxusemine L has a relatively mild pain relief effect and lasts longer. In addition, Figure 4 As shown in the right figure of C, by calculating the area under the pain relief curve after administration of each medication group, it can be seen that the overall pain relief effect of 5 mg / kg Buxusemine L is equivalent to that of 10 mg / kg Z944.

[0106] Experimental methods:

[0107] The mice were anesthetized with 5% isoflurane and maintained with 2.5% isoflurane. The legs of the mice were shaved, the skin of the mice was wiped with alcohol, the skin at the sciatic nerve was cut with ophthalmic scissors, the muscles were bluntly separated with two toothpicks, the sciatic nerve was visible, the nerve was lifted, the nerve was ligated, the skin was sutured, and the wound was treated with antibiotics to prevent infection.

[0108] After modeling the mice, the PWT value of the paw of the mice on the surgical side was continuously observed using Von frey to determine whether the modeling was successful (the model was generally stable 12-17 days after surgery). During the modeling, the surgical group ligated the sciatic nerve, while the sham-operated group completed the modeling process, but did not perform nerve ligation to form a control group to determine whether the neuralgia modeling was successful. After determining that the modeling of the mice in the surgical group was successful, subsequent drug administration can be performed. This experiment was intraperitoneal injection, and the pain response of the mice was observed 0.5, 1, 2, 3, 4, 6, and 7 hours after administration, and the PWT of the mice in each experimental group was statistically analyzed and compared.

[0109] 1. Vehicle control group (n=12): Mice were intraperitoneally injected with the vehicle in a volume of 200 μl.

[0110] 2. Sham operation group (n=12): Mice were intraperitoneally injected with the vehicle in a volume of 200 μl.

[0111] 3. Test drug + visceral pain model group (n=12): After the model was stabilized, 200 μl of drug was injected intraperitoneally.

[0112] Note: When the number of animals is even, half are male and half are female.

[0113] Drug dosage:

[0114] Z944: 10 mg / kg, intraperitoneal injection

[0115] Buxusemine L: 2.5, 5, 10 mg / kg, intraperitoneal injection.

[0116] Drug vehicle: physiological saline solution containing 20% ​​(v / v) β-cyclodextrin and 0.4% DMSO

[0117] CCI: sciatic nerve ligation model group; Sham: sham operation group; 10: Buxusemine L

[0118] The method for significant difference between groups was Brown-Forsythe and Welch ANOVA test with TamhaneT2MCT.*P<0.05,**P<0.01,****P<0.0001(vs.Vehicle+Sham group);ns indicates no significant difference. All data are mean±SD.

[0119] Example 6

[0120] Buxusemine H relieves acetic acid-induced visceral pain.

[0121] like Figure 5 As shown in A, mice showed visceral pain response after intraperitoneal injection of acetic acid, showing writhing reactions with abdomen concave, anterior abdominal wall close to cage bottom, hip twisting and hind limb extension. Intraperitoneal injection of Buxusemine H (20 mg / kg) can significantly reduce the writhing reaction induced by acetic acid in mice ( Figure 5 Unlike Buxusemine L, Buxusemine H (20 mg / kg) had no significant effect on the time to the first writhing reaction in mice ( Figure 5 (middle B).

[0122] Experimental methods:

[0123] SPF grade C57BL / 6 experimental mice aged 8-10 weeks were adapted to the SPF barrier system environment for 3 days before the experiment.

[0124] The acetic acid writhing model was selected as the visceral pain model. 0.5 h after intraperitoneal injection, the mice were intraperitoneally injected with 0.6% acetic acid (0.1 mL / 10 g) to establish the model, and the number of writhing times of the mice within 20 min after acetic acid administration and the time of the first writhing were observed.

[0125] Experimental groups:

[0126] 1. Visceral pain model group (n=8): Mice were intraperitoneally injected with 200 μl of 0.6% acetic acid solution.

[0127] 2. Test drug + visceral pain model group (n=8): 200 μl of the test drug was injected intraperitoneally 30 minutes before modeling.

[0128] Note: When the number of animals is even, half are male and half are female.

[0129] Drug dosage:

[0130] Buxusemine H: 20 mg / kg, intraperitoneal injection.

[0131] Drug vehicle: physiological saline solution containing 20% ​​(v / v) β-cyclodextrin and 0.4% DMSO

[0132] AA: Acetic acid; 9: Buxusemine H

[0133] T-test was used for the significant difference between the two groups, ***P<0.001; ns indicates no significant difference. All data are mean ± standard deviation.

[0134] Formulation Examples

[0135] 1 Tablets: Take the compounds Buxusemine H and Buxusemine L, and salts made from organic acids (tartaric acid, citric acid, formic acid, oxalic acid, etc.) or inorganic acids (hydrochloric acid, sulfuric acid, phosphoric acid, etc.), add excipients at a weight ratio of 1:5-1:10 to the excipients, and granulate and tablet.

[0136] 2. Oral solution: Take the compounds Buxusemine H and Buxusemine L, and salts made from organic acids (tartaric acid, citric acid, formic acid, oxalic acid, etc.) or inorganic acids (hydrochloric acid, sulfuric acid, phosphoric acid, etc.), and prepare oral solution according to conventional oral solution preparation methods.

[0137] 3. Capsules, granules or powders: Take the compounds Buxusemine H and Buxusemine L, and salts made from organic acids (tartaric acid, citric acid, formic acid, oxalic acid, etc.) or inorganic acids (hydrochloric acid, sulfuric acid, phosphoric acid, etc.), add excipients at a weight ratio of 5:1 to the excipients to prepare capsules, granules or powders.

[0138] 4. Ointment: Take the compounds Buxusemine H and Buxusemine L, and salts made from organic acids (tartaric acid, citric acid, formic acid, oxalic acid, etc.) or inorganic acids (hydrochloric acid, sulfuric acid, phosphoric acid, etc.), add excipients at a weight ratio of 5:1 to the excipients to prepare an ointment.

[0139] 5. After the compounds Buxusemine H and Buxusemine L are dissolved in DMSO, injection water is added according to the conventional method, fine filtration is performed, and filling and sterilization are performed to prepare an injection solution, wherein the concentration of the injection solution is 0.5-5 mg / mL.

[0140] 6. Dissolve the compounds Buxusemine H and Buxusemine L in DMSO, dissolve them in sterile water for injection, stir to dissolve, filter with a sterile suction funnel, filter aseptically, dispense into ampoules, freeze-dry at low temperature, and aseptically seal to obtain powder injections.

[0141] 7. Compounds Buxusemine H and Buxusemine L were added to the excipient at a mass ratio of 9:1 to the excipient to prepare a powder.

[0142] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of Buxusemine H and Buxusemine L, alkaloid compounds of boxwood, in the preparation of drugs for treating inflammatory pain, visceral pain and neuropathic pain.

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