Application of calcium channel inhibitor in analgesia and itching relieving

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of a calcium channel inhibitor in analgesia and itching relieving. The calcium channel inhibitor is bromocriptine, it is identified that bromocriptine serves as a CaV2.2 selective inhibitor, and new application of bromocriptine in the aspects of analgesia and itching relieving is developed.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to an application of a calcium channel inhibitor in analgesia and antipruritic. Background Art

[0002] Pain is the most basic feeling of human beings. Under normal physiological conditions, it serves as one of the body's warning mechanisms, which can remind a certain part of the body that there are problems that need attention. However, under different pathological conditions, the pain becomes persistent and unbearable. This pain affects one-fifth of the world's population, and the cost of relieving pain is huge every year. According to relevant research reports, about 100 million people in the United States suffer from chronic pain, and the direct cost of medical expenses, reduced wages and reduced productivity caused by it have reached 560 to 635 billion US dollars. Research suggests that the incidence of chronic pain is high both internationally and domestically, and will further increase with the progress of aging. Chronic pain not only greatly affects people's physical and mental health, but also brings a heavy burden to patients and society.

[0003] At present, the most effective analgesics in clinical practice are morphine and its derivatives. Morphine inhibits pain-related neural circuits by activating opioid receptors expressed on neurons. However, the widespread expression of opioid receptors in the body and the side effects caused by their activation (such as tolerance, addiction, respiratory depression, etc.) have also brought new troubles. These side effects have greatly limited the clinical use of morphine. In order to overcome the side effects of the above-mentioned opioids, new opioids with subtype selectivity and excitatory bias are constantly being studied and prepared, but unfortunately these new drugs still have serious opioid side effects.

[0004] Voltage-gated calcium channels Ca V 2.2 is mainly expressed on the axon terminals of nociceptive neurons, mediating the influx of calcium ions caused by depolarization, thereby causing the release of neurotransmitters. The U.S. Food and Drug Administration approved the Ca V 2.2 Ziconotide, a specific peptide inhibitor, is clinically used for moderate to severe pain. Compared with opioids, ziconotide has significantly fewer side effects. However, as a natural peptide with a complex ring structure, ziconotide is difficult to prepare and cannot pass through the blood-brain barrier. This results in the high price of ziconotide and limited administration methods. Summary of the invention

[0005] The present invention aims to solve the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes bromocriptine as a specific small molecule calcium channel Ca V 2.2 Application of inhibitors in analgesia and antipruritic effects.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of the present invention provides use of bromocriptine in the preparation of a calcium channel inhibitor.

[0008] In some embodiments of the present invention, the calcium channel inhibitor is an N-type calcium channel Ca V 2.2 Inhibitors.

[0009] In some embodiments of the invention, bromocriptine specifically inhibits N-type calcium channels (e.g., Ca V 2.2), bromocriptine does not inhibit or substantially inhibits L-type calcium channels (e.g., Ca V 1.2 Ca V 1.3) and T-type calcium channels (e.g. Ca V 3.1 Ca V 3.2 Ca V 3.3).

[0010] The second aspect of the present invention provides the use of bromocriptine in the preparation of a drug for treating and / or preventing a disease targeting a calcium channel.

[0011] In some embodiments of the present invention, the calcium channel is an N-type calcium channel Ca V 2.2.

[0012] In some embodiments of the invention, bromocriptine specifically inhibits N-type calcium channels (e.g., Ca V 2.2), bromocriptine does not inhibit or substantially inhibits L-type calcium channels (e.g., Ca V 1.2 Ca V 1.3) and T-type calcium channels (e.g. Ca V 3.1 Ca V 3.2 Ca V 3.3).

[0013] Based on Ca V 2.2 It is mainly present in the presynaptic membrane of neurons and is closely related to a series of important physiological processes such as neurotransmitter release, pain and itch signal transmission. Therefore, it is understandable that bromocriptine can inhibit or block calcium channels (such as Ca V 2.2) thereby treating and / or alleviating and / or preventing symptoms such as pain, itching, etc. Therefore, in some embodiments of the present invention, the disease comprises pain and / or itching.

[0014] In some embodiments of the invention, bromocriptine treats and / or prevents pain and / or itch by blunting a subject's perception of pain and / or itch.

[0015] In some embodiments of the present invention, the pain includes acute / chronic inflammatory pain (e.g., caused by trauma, burns, scalds, infection, chemical stimulation, surgical operation, etc.), neuropathic pain (e.g., peripheral neuropathic pain (including but not limited to: back neuralgia, sciatica, diabetic peripheral neuropathy, postherpetic neuralgia), central neuropathic pain (including but not limited to: central neuralgia after stroke, spinal cord injury, multiple sclerosis, neurological dysfunction pain, fibromyalgia syndrome)), and pain induced by chemotherapeutic drugs (e.g., paclitaxel, vincristine, cisplatin).

[0016] In some embodiments of the present invention, the pruritus includes acute pruritus (including but not limited to serotonin-induced pruritus, histamine-induced pruritus, chloroquine-induced pruritus, opioid-induced pruritus), chronic pruritus (including but not limited to systemic diseases such as pruritus induced by cholestasis, metabolic diseases such as diabetic pruritus, skin-related pruritus such as pruritus caused by psoriasis, pruritus caused by atopic contact dermatitis, and pruritus induced by hematological malignancies such as Hodgkin's lymphoma).

[0017] In some preferred embodiments of the present invention, the pruritus includes serotonin-induced pruritus, histamine-induced pruritus, chloroquine-induced pruritus, bile acid-induced pruritus, pruritus caused by psoriasis, and pruritus caused by atopic contact dermatitis.

[0018] In some embodiments of the present invention, bromocriptine, as a drug for treating and / or preventing diseases targeting calcium channels, does not produce significant drug tolerance, for example, it exhibits analgesic tolerance that is significantly weaker than that of opioids (such as morphine).

[0019] In some embodiments of the present invention, the medicament has bromocriptine as the only active ingredient.

[0020] In some embodiments of the present invention, the drug optionally contains other active ingredients, and the combined use of bromocriptine and other active ingredients should have the effects of synergistic enhancement or reduction of adverse reactions.

[0021] The drug of the present invention may also optionally include a pharmaceutically acceptable carrier. For example, the drug of the present invention may include, for example, 0.1-99.5% (more preferably 0.5-90%) of bromocriptine and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier includes, but is not limited to, diluents, adhesives, absorbents, disintegrants, dispersants, wetting agents, cosolvents, buffers, and surfactants; specifically, includes, but is not limited to, starch, dextrin, sucrose, milk powder, sweeteners, mannitol, lactose, cellulose and its derivatives, calcium carbonate, cyclodextrin, β-cyclodextrin, phospholipid materials, magnesium stearate, talc, and flavors.

[0022] The dosage forms of the medicine of the present invention include:

[0023] Solid dosage forms (e.g., hard or soft capsules, powders, tablets, granules, pills, dragees) may optionally contain one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as carboxymethylcellulose, alginate, gelatin, polyvinyl pyrrolidone, sucrose and / or gum arabic; (3) wetting agents, such as glycerol; (4) disintegrating agents, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution delay agents, such as paraffin; (6) absorption promoters, such as quaternary ammonium compounds; (7) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (8) absorbents, such as kaolin and bentonite; (9) lubricating agents, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents;

[0024] Liquid dosage forms (e.g., solutions, suspensions, syrups, injections, liniments, emulsions, microemulsions, tinctures, elixirs) In addition to the active ingredient, the liquid dosage form may optionally contain an inert diluent commonly used in the art, such as, for example, water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuran alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof; In addition to the active compound, the suspension may also contain suspending agents, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth gum and mixtures thereof;

[0025] Creams, ointments, plasters, gels, pastes, powders, patches and the like may optionally contain, in addition to the active compound, excipients such as animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof;

[0026] Aerosols and sprays etc. may optionally contain excipients, such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these substances, in addition to the active compound. Sprays may also contain conventional propellants, such as chlorofluorocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0027] In some embodiments of the present invention, the drug can be administered topically, orally, transdermally, rectally, vaginally, parenterally, intranasally, intrapulmonaryly, intraocularly, intravenously, intramuscularly, intraarterially, intrathecally, intracapsules, intraorbitally, intracardiacly, intradermally, intraperitoneally, transtracheally, subcutaneously, subcutaneously, intraarticularly, subcapsules, subarachnoidally, intraspinally, intrasternally, or by inhalation. Preferably, the drug is administered by a method selected from intrathecal injection, intravenous injection, intramuscular injection, subcutaneous injection, and intraperitoneal injection. More preferably, the drug is administered by intrathecal injection.

[0028] The amount of bromocriptine in the drug (generally referred to as the unit dose that produces a therapeutic effect) and the concentration (generally referred to as the concentration that produces a therapeutic effect) and the amount of the drug administered to the subject (generally referred to as the dosage that produces a therapeutic effect) can be selected based on clinically relevant factors, including but not limited to the medically relevant characteristics of the subject (such as age, weight, sex, other medical conditions, etc.), the solubility of bromocriptine contained in the drug, the efficacy and activity of bromocriptine, and the mode of administration of the drug.

[0029] In some embodiments of the present invention, the unit dose of bromocriptine in the drug is 0.02-200 μg, for example, 0.1-100 μg, 1-50 μg, 2-20 μg.

[0030] Generally, the suitable daily dose of bromocriptine is the amount of bromocriptine at the lowest dose that produces an effective therapeutic effect. Such effective doses generally depend on various factors, including bromocriptine or its salt, the activity of prodrugs, route of administration, administration time, excretion rate of bromocriptine, treatment period, other drugs, compounds and / or substances optionally used in combination with bromocriptine, age, sex, weight, condition, general health and prior medical history of the subject, and other well-known factors in the medical field. Typically, the intrathecal dose range of bromocriptine described herein for the subject is about 0.0001 to about 100 mg / kg body weight per day. For example, the dosage can be 1-1000 μg / kg, 2-50 μg / kg, 50-1000 μg / kg.

[0031] Optionally, the effective daily dose of bromocriptine may be divided into two, three, four, five, six or more sub-doses administered at appropriate intervals throughout the day, optionally in the form of unit dosage forms. Unit dosage forms may be prepared by any method well known in the art of pharmacy.

[0032] According to some embodiments of the present invention, there are at least the following beneficial effects:

[0033] Compared with classic analgesics in clinical practice, bromocriptine does not have side effects such as tolerance, addiction, and respiratory depression. Compared with the new peptide analgesic ziconotide that has entered clinical practice, bromocriptine is simpler and easier to obtain.

[0034] The terms used in this invention are:

[0035] The term "bromocriptine" in the present invention includes bromocriptine (CAS: 25614-03-3, structural formula ) or a pharmaceutically acceptable salt or prodrug thereof (which can be bioconverted into bromocriptine in vivo and thus be active).

[0036] The term "pharmaceutically acceptable" in the present invention refers to those compounds, substances, compositions and / or dosage forms that are suitable for contact with human and animal tissues within the scope of reasonable medical judgment but without excessive toxicity, irritation, allergic response or other problems or complications, and they are matched with a reasonable benefit / risk ratio.

[0037] The pharmaceutically acceptable salt of bromocriptine in the present invention can be selected from hydrochloride, phosphate, hydrogen phosphate, sulfate, hydrogen sulfate, sulfite, acetate, oxalate, malonate, valerate, glutamate, oleate, palmitate, stearate, laurate, borate, p-toluenesulfonate, methanesulfonate, isethionate, maleate, malate, tartrate, benzoate, pamoate, salicylate, vanillate, mandelate, succinate, gluconate, lactobionate or lauryl sulfonate, etc.

[0038] The term "Ca" in the present invention V 2.2" refers to N-type (Neuron, neuron type) voltage-gated calcium channels (Ca V ), most of these channels are present in nerve cells.

[0039] The term "Ca" in the present invention V 1.2", "Ca V 1.3" refers to the L-type (long-lasting) voltage-gated calcium channel (Ca V ), which are mainly expressed in skeletal muscle, cardiac muscle and vascular smooth muscle.

[0040] The term "Ca" in the present invention V 3.1", "Ca V 3.2", "Ca V 3.3" refers to the T-type (Transient) voltage-gated calcium channel (Ca v ), which are mainly expressed in neurons.

[0041] The term "non-inhibitory" in the present invention refers to the effect of bromocriptine on N-type calcium channels (e.g., Ca V 2.2) Compared with the inhibitory effect of bromocriptine on L-type calcium channels (e.g. Ca V 1.2 CaV 1.3) and T-type calcium channels (e.g. Ca V 3.1 Ca V 3.2 Ca V 3.3) is less than 10%, such as less than 8%, less than 6%, less than 4%, less than 2%, less than 1%.

[0042] The term "substantially does not inhibit" in the present invention refers to the effect of bromocriptine on N-type calcium channels (e.g., Ca V 2.2) Compared with the inhibitory effect of bromocriptine on L-type calcium channels (e.g. Ca V 1.2 Ca V 1.3) and T-type calcium channels (e.g. Ca V 3.1 Ca V 3.2 Ca V 3.3) has an inhibitory effect of 10-50%, for example 10-45%, 10-40%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 15-50%, 15-45%, 15-40%, 15-30%, 15-25%, 15-20%, 20-50%, 20-45%, 20-40%, 20-30%, 20-25%, 25-50%, 25-45%, 25-40%, 25-30%, 30-50%, 30-45%, 30-40%, 40-50%, 40-45%.

[0043] The term "subject" in the present invention refers to a human or an animal (especially a mammal such as a rat, a mouse, a guinea pig, a rabbit, a dog, a pig, a cat).

[0044] The tolerance of humans and animals to the same drug is quite different. Generally speaking, animals have greater tolerance than humans. Generally, it can be converted according to the following ratio: the human dosage is 1, mice and rats are 25-50, rabbits and guinea pigs are 15-20, and dogs and cats are 5-10. In addition, the surface area calculation method for humans and animals can be used for conversion, generally using the Xu Wen formula (Chinese Journal of Physiology, 12, 327, 1937) and the Mech-Rubner formula. The above method can be applied to the conversion of drug dosages between humans and different types of animals in the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The present invention will be further described below in conjunction with the accompanying drawings and embodiments, wherein:

[0046] Figure 1 The effect of bromocriptine on heterologous expressed Ca V 2.2 Calcium current inhibition effect diagram, where A is the inhibitory effect of different concentrations of bromocriptine on heterologous expression of Ca VB is the inhibitory effect of bromocriptine.

[0047] Figure 2 The effect of bromocriptine on heterologously expressed other Ca V Figures A and B show that 10 μM bromocriptine significantly inhibited the heterologous expression of Ca channels. V 2.2-mediated calcium currents without affecting calcium currents mediated by other family members.

[0048] Figure 3 This is a diagram showing the analgesic effect of intrathecal bromocriptine injection in mice in the hot plate test in Example 3 of the present invention, wherein A is the effect of different concentrations of bromocriptine on the paw withdrawal threshold of mice over time; and B is the concentration-effect curve of bromocriptine.

[0049] Figure 4 The figure is a graph showing the analgesic effect of intrathecal bromocriptine in Example 4 of the present invention in the mouse formalin pain model, wherein A is the effect of different concentrations of bromocriptine on the pain of formalin injection into the plantar of mice over time; B is the effect of different concentrations of bromocriptine on the total paw licking time of the two phases of formalin pain; and C is the dose-effect dependence curve of bromocriptine on the two phases of formalin pain.

[0050] Figure 5 : This is a graph showing the analgesic effect of intrathecal bromocriptine in Example 5 of the present invention on CFA-induced chronic inflammatory pain in mice, wherein A is the effect of different concentrations of bromocriptine on the mechanical pain threshold of the plantar of mice treated with CFA over time; B is the dose-effect dependence curve of bromocriptine analgesia in the CFA-induced chronic inflammatory pain model.

[0051] Figure 6 The diagram is a graph showing the analgesic effect of intrathecal bromocriptine in the neuropathic pain of mice induced by partial sciatic nerve ligation in Example 6 of the present invention, wherein A is the effect of different concentrations of bromocriptine on the plantar mechanical pain threshold of mice after partial sciatic nerve ligation over time; B is the dose-effect dependence curve of bromocriptine analgesia in the partial sciatic nerve ligation model.

[0052] Figure 7 The diagram shows the antipruritic effect of intrathecal bromocriptine in the histamine-induced mouse pruritus model in Example 7 of the present invention, wherein A is a curve showing the change of histamine-induced mouse scratching over time after administration of different concentrations of bromocriptine; B is the effect of different concentrations of bromocriptine on histamine-induced pruritus; and C is a dose-effect dependence curve of bromocriptine antipruritic in the histamine-induced pruritus model.

[0053] Figure 8The diagram shows the antipruritic effect of intrathecal bromocriptine in the chloroquine-induced mouse pruritus model in Example 8 of the present invention, wherein A is a curve showing the change of chloroquine-induced scratching over time after administration of different concentrations of bromocriptine; B is the effect of different concentrations of bromocriptine on chloroquine-induced pruritus; and C is a dose-effect dependence curve of bromocriptine antipruritic in the chloroquine-induced pruritus model.

[0054] Fig. 9 Graph showing the antipruritic effect of intrathecal bromocriptine in Example 9 of the present invention in a mouse psoriasis itch model, wherein A is the effect of administering different concentrations of bromocriptine on psoriasis-induced itch; and B is the dose-effect dependence curve of bromocriptine antipruritic in a psoriasis-induced itch model.

[0055] Fig.10 1 is a graph showing the antipruritic effect of intrathecal bromocriptine in an atopic contact dermatitis itch model in Example 10 of the present invention, wherein A is the effect of administering different concentrations of bromocriptine on the itch induced by atopic contact dermatitis; B is a dose-effect dependence curve of bromocriptine in the itch model induced by atopic contact dermatitis.

[0056] Fig.11 1 is a graph showing the analgesic tolerance test results of bromocriptine in Example 11 of the present invention during continuous intrathecal administration of bromocriptine in mice, wherein A is the change in the efficacy of bromocriptine and morphine over time during continuous administration; B is a comparison of the efficacy of bromocriptine and morphine on the first and sixth days.

[0057] “*” represents p≤0.05, “**” represents p≤0.01, “***” represents p≤0.001, “****” represents p≤0.0001, and “ns” represents no significant difference. DETAILED DESCRIPTION

[0058] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0059] In the description of the present invention, the terms "include" and "have" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or apparatus comprising a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to these processes, methods, products or apparatuses.

[0060] When a numerical range is disclosed herein, the above range is considered to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0061] The present invention is further described below by way of specific examples. Unless otherwise specified, the various chemical reagents used in the examples of the present invention are obtained through conventional commercial channels.

[0062] The term "patch clamp" in the present invention refers to the patch clamp technique, which is a microelectrode technique that uses a clamping voltage or current method to record the electrical activity of ion channels on biological membranes.

[0063] In the embodiments of the present invention, adult mice with C57BL / 6J background over 8 weeks old and weighing 20-30 g were used for animal experiments. Mice were kept in a specific pathogen-free (SPF) feeding room at the Experimental Animal Center of Southern University of Science and Technology, with free access to food and water and a 12-hour: 12-hour light: dark cycle. All experimental protocols were approved by the Experimental Animal Welfare Ethics Committee of Southern University of Science and Technology (SUSTech-JY202409029).

[0064] In the examples of the present invention, bromocriptine was directly dissolved in DMSO to obtain a bromocriptine stock solution with a concentration of 30 mM, which was stored in a -80°C refrigerator and diluted as needed when used.

[0065] Example 1 Effect of Bromocriptine on Ca V 2.2 Inhibitory effect

[0066] The patch clamp method was used to record the effect of bromocriptine and cell bath solution on Ca V 2.2 The inhibitory effect of current, the specific steps are as follows.

[0067] Preparation of bromocriptine solution:

[0068] Take a 30mM bromocriptine stock solution, dissolve it in the cell bath solution, and dilute it to the target concentration. The formula of the cell bath solution is as follows: 135mM tetraethylammonium chloride (TEA)-Cl), 4mM MgCl 2 , 3 mM CaCl 2 , 10 mM HEPES, pH = 7.4.

[0069] Cell culture:

[0070] HEK293T cells were seeded in 6-well plates and placed in a sterile incubator at 37°C and 5% carbon dioxide. When the cells grew to a density of 60%, the culture medium was replaced with 2 mL of serum-free Opti-MEM for 30 minutes of starvation induction. At the same time, the liposome-encapsulated cells expressing Ca V 2.2 The three required plasmids (wherein, the three plasmids are used to express α subunit (addgene#62574), β3 subunit (addgene#26574), and α2δ subunit (addgene#26575) respectively). The specific steps are as follows: add the three plasmids (1.5 μg each) and 9 μL Lipofectamine 2000 transfection reagent (Thermo Fisher Cat#11668019) to two small centrifuge tubes containing 150 μL Opti-MEM, let them stand for a while, mix the two, let them stand for 15 minutes again, add them to the 6-well plate and incubate for 4-6 hours, and then replace the culture medium with complete culture medium.

[0071] Patch clamp assay:

[0072] Cells were taken 24 hours after transfection and spread on cell slides, and cell baths containing different concentrations (0, 0.1μM, 1μM, 2.5μM, 5μM, 10μM, 50μM) of bromocriptine were perfused for patch clamp recording, with -70mV as the clamping voltage, and depolarization induced by increasing to 10mV within 100ms to generate current, and the trajectory of the obtained membrane current was recorded. At the same time, the relative current was calculated, and the calculation formula was: relative current = current after administration / current before administration.

[0073] like Figure 1 As shown in A and B in Figure 1, bromocriptine dose-dependently inhibited Ca V 2.2 Current, half inhibitory concentration (IC 50 ) was 2.94 μM. The above results prove that bromocriptine has an V 2.2 Inhibitory effect.

[0074] Example 2 Effect of Bromocriptine on Ca V 2.2 Specificity

[0075] The preparation steps of bromocriptine solution, cell culture method and patch clamp detection method in this embodiment are all referred to Example 1, except that: the Ca2+ expressed in HEK293T cells was transfected and V 2.2 are replaced by Ca V 1.2(addgene#26572), Ca V 1.3(addgene#26576), Ca V 3.1(addgene#197324), Ca V3.2 (addgene#45809), Ca V 3.3 (addgene#45810).

[0076] like Figure 2 As shown in A and B in Figure 1, 10 μM bromocriptine did not have a significant inhibitory effect on other calcium current subtypes expressed heterologously in HEK293T cells. V 2.2 has good subtype selectivity.

[0077] Example 3 Analgesic effect of intrathecal bromocriptine solution in hot plate test

[0078] Bromocriptine stock solution (30 mM) was dissolved in sterile saline to dilute concentrations of 0.3 mM, 1 mM and 3 mM respectively.

[0079] The mice were randomly divided into a control group (normal saline group, n=8), a low-dose drug group (3nmol bromocriptine, n=7), a medium-dose drug group (10nmol bromocriptine, n=7), and a high-dose drug group (30nmol bromocriptine, n=8).

[0080] The mice in the control group were intrathecally injected with 10 μL of normal saline, and the mice in the low-, medium-, and high-dose drug groups were intrathecally injected with 10 μL of 0.3 mM bromocriptine solution (total drug amount was 3 nmol), 10 μL of 1 mM bromocriptine solution (total drug amount was 10 nmol), and 10 μL of 3 mM bromocriptine solution (total drug amount was 30 nmol), respectively.

[0081] In the hot plate test, mice are placed on a hot plate at 53°C, and the time when escape reactions such as licking the hind paws and jumping appear, that is, the pain threshold of the hot plate, is recorded. The later the time of escape, the less sensitive the mouse is to pain. Before drug injection, the basal threshold is measured three times, with an interval of 5 minutes each time. The average of the three measurements is taken as the basal value, and mice that deviate from the average level (10-15 seconds) are discarded. The thresholds at different time points are recorded after administration. In order to prevent burns, the longest threshold is set to 40 seconds. To calculate the maximum analgesic effect, the formula is: (paw retraction threshold after administration - paw retraction threshold before administration) / (longest threshold - paw retraction threshold before administration) * 100%.

[0082] The experimental results are as follows Figure 3 As shown. During the continuous test, the pain threshold of the control group mice (10 μL saline solution injected intrathecally) remained basically unchanged, indicating that neither the mechanical stimulation of the injection nor the repeated measurement affected the pain perception of the mice. After intrathecal injection of an equal volume of bromocriptine solution, the pain threshold of the hot plate test of the mice increased significantly, reaching the maximum analgesic effect 1 hour after administration, and the higher the concentration of bromocriptine administered, the more obvious the analgesic effect, showing a dose-effect dependence, and its half-effective dose ED50 The value was 8.848 nmol. No difference was observed in the analgesic effect of bromocriptine between female and male mice. The above results prove that intrathecal injection of bromocriptine can effectively inhibit the pain response caused by heat.

[0083] Example 4 Study on the analgesic effect of intrathecal injection of bromocriptine solution in formalin-induced acute inflammatory pain

[0084] The mouse grouping, preparation of drug solution and administration method refer to Example 3.

[0085] The mice were placed in a transparent plexiglass box (20cm×20cm×30cm) and adapted to the environment for 10-15 minutes. After the adaptation, each group of mice was intrathecally injected with normal saline (n=8) or low (3nmol, n=7), medium (10nmol, n=8), and high (30nmol, n=7) doses of bromocriptine solution. 30 minutes after administration, 20μL of 5% formalin solution was subcutaneously injected into the toe of the right hind foot of the mouse, and it was quickly returned to the observation box. A high-definition camera was used to record the behavior of the mice within 40 minutes after the injection of formalin solution. The time of pain reactions such as licking, biting, and shaking of the injected foot in the recorded video was analyzed. Among them, the formalin pain of mice is divided into two phases, 0-10 minutes is the first phase, which is the acute pain induced by chemical stimulation; 10-40 minutes is the second phase, which is the inflammatory pain induced by inflammatory response. The maximum analgesic effect was calculated using the formula: (paw licking time before drug administration-paw licking threshold after drug administration)*100%.

[0086] The experimental results are as follows Figure 4 As shown. In the control group of mice (10 μL saline injected intrathecally), formalin injection induced a significant two-phase pain response. After intrathecal injection of an equal volume of bromocriptine solution (dissolved in saline), the duration of the pain response of mice in the first and second phases was significantly reduced, and the higher the dose given, the more obvious the relief of pain behavior, showing a dose-effect dependence. The half-effective concentration ED of bromocriptine in the first and second phases was 50 The analgesic effect of bromocriptine was not different between female and male mice. The above results prove that bromocriptine can effectively inhibit the pain response induced by acute inflammatory conditions.

[0087] Example 5 Study on the analgesic effect of intrathecal injection of bromocriptine solution in chronic inflammatory pain induced by complete Freund's adjuvant. Refer to Example 3 for the mouse grouping, preparation of drug solution and administration method.

[0088] After the mouse foot is injected with complete Freund adjuvant (CFA), a strong inflammatory response is induced at the injection site, which further induces hyperalgesia in the mouse foot. This hyperalgesia is a common pain model for pain circuit research and analgesic drug development.

[0089] Before the experiment, the basal pain threshold of normal mice was first measured, and then 20 μL CFA was injected subcutaneously into the mice's toes. Three days later, each group of mice was intrathecally injected with normal saline (n=6) or low (3nmol, n=6), medium (10nmol, n=6), and high doses (30nmol, n=6) of bromocriptine solution, and the mechanical pain sensitivity of the injected feet of the mice and the mechanical pain thresholds at different time points (0.5h, 1h, 1.5h, 2h, 3h, 5h) after intrathecal injection of various concentrations of bromocriptine were measured.

[0090] The mechanical stimulation threshold of the mouse plantar was evaluated by the Von-Frey method. This method uses a series of Von-Frey fibers (0.16g, 0.4g, 0.6g, 1.0g, 2.0g) with logarithmic stiffness to pierce the center of the mouse's hind paw at a vertical angle to observe whether the mouse has pain reactions such as shaking paws and licking paws. The mechanical threshold of mechanical pain in the mouse plantar was estimated by Dixon's up-down method. The maximum analgesic effect was calculated using the following formula: (paw withdrawal threshold after drug administration - paw withdrawal threshold before drug administration) / (paw withdrawal threshold before modeling - paw withdrawal threshold before drug administration)*100%.

[0091] The experimental results are as follows Figure 5 As shown. During the continuous test, the pain threshold of the control group mice (10 μL saline injected intrathecally) remained basically unchanged, indicating that neither the mechanical stimulation of the injection nor the repeated measurement affected the pain perception of the mice. After intrathecal injection of an equal volume of bromocriptine solution (dissolved in saline), the mechanical pain threshold of the mice increased significantly, and the higher the dose, the more obvious the analgesia, showing a dose-effect dependence, and its half-effective concentration ED 50 The analgesic effect of bromocriptine was 7.656 nmol. No difference was observed between female and male mice. The above results prove that bromocriptine can effectively inhibit the hyperalgesia caused by chronic inflammatory conditions.

[0092] Example 6 Study on the analgesic effect of intrathecal injection of bromocriptine solution in neuropathic pain caused by sciatic nerve injury

[0093] The mouse grouping, preparation of drug solution and administration method refer to Example 3.

[0094] The sciatic nerve is a mixed nerve that innervates the mouse leg. Artificial partial ligation can cause compression and injury to the sciatic nerve, and one of the phenotypes after injury is mechanical hypersensitivity in the sole of the mouse foot.

[0095] The basic pain threshold of mechanical pain in normal mice was measured before surgery. The mice were then anesthetized with isoflurane (2%), fixed in a prone position, the right sciatic nerve was exposed, and half of the sciatic nerve was ligated using a 6-0 braided suture to construct a partial sciatic nerve ligation model (pSNL). One week after the operation, the mechanical pain sensitivity value of the sole of the mouse on the surgical side was first measured, and then the drug was injected, and the mechanical pain threshold at different time points after administration (0.5h, 1h, 1.5h, 2h, 3h, 5h) was recorded. The mechanical pain threshold was evaluated using the Von-Frey method in Example 5. The maximum analgesic effect was calculated, and the calculation formula was: (paw retraction threshold after administration-paw retraction threshold before administration) / (paw retraction threshold before modeling-paw retraction threshold before administration)*100%.

[0096] The experimental results are as follows Figure 6 As shown. During the continuous test, the pain threshold of the control group mice (10 μL saline injected intrathecally) remained basically unchanged, indicating that neither the mechanical stimulation of the injection nor the repeated measurement affected the pain perception of the mice. After intrathecal injection of an equal volume of bromocriptine solution (dissolved in saline), the mechanical pain threshold of the mice increased significantly, and the higher the dose, the more obvious the analgesia, showing a dose-effect dependence, and its half-effective concentration ED 50 The analgesic effect of bromocriptine was 5.814 nmol. No difference was observed between female and male mice. The above results prove that bromocriptine can effectively inhibit the hyperalgesia caused by nerve injury.

[0097] Example 7 Study on the antipruritic effect of intrathecal injection of bromocriptine solution in histamine-induced pruritus model

[0098] The bromocriptine stock solution was dissolved in sterile physiological saline to dilution concentrations of 0.1 mM, 0.3 mM, 1 mM and 3 mM respectively.

[0099] The mice were randomly divided into a control group (normal saline group, n=8), a lower dose drug group (1 nmol bromocriptine, n=5), a low dose drug group (3 nmol bromocriptine, n=8), a medium dose drug group (10 nmol bromocriptine, n=6), and a high dose drug group (30 nmol bromocriptine, n=6).

[0100] The control group mice were intrathecally injected with 10 μL of normal saline, and the mice in the lower, low, medium, and high dose drug groups were intrathecally injected with 10 μL of 0.1 mM bromocriptine solution (total drug amount was 1 nmol), 10 μL of 0.3 mM bromocriptine solution (total drug amount was 3 nmol), 10 μL of 1 mM bromocriptine solution (total drug amount was 10 nmol), and 10 mL of 3 μM bromocriptine solution (total drug amount was 30 nmol), respectively.

[0101] Intradermal injection of histamine at the back of the neck can significantly induce a scratching reaction. This example investigates the effect of bromocriptine on the itch caused by histamine.

[0102] Construction of histamine-induced pruritus model: Three days before the experiment, the hair on the right back of the neck of the mice was removed using an electric shaver. One hour before the experiment, the mice were randomly divided into five groups. The mice in the control group were injected with 10 μL of normal saline intrathecally, and the mice in the experimental group were injected with 1 nmol, 3 nmol, 10 nmol, and 30 nmol of bromocriptine solution intrathecally, and 50 μL of histamine (100 μM) was injected intradermally at the shaved parts of the mice in the control group and the experimental group. The number of itching of the mice within 30 minutes was observed and counted. The scratching behavior was counted as one time when the front paw or hind paw of the mouse left the ground and scratched the drug-treated part of the right back of the neck until the paw was put back on the ground. The mean number of scratches of each group of mice was compared and statistically processed. The maximum antipruritic effect was calculated, and the calculation formula was: 1-(number of scratches in the drug-treated group-number of scratches in the non-drug-treated group)*100%.

[0103] The experimental results are as follows Figure 7 Compared with the control group (10 μL saline injected intrathecally), intrathecal injection of equal volumes of bromocriptine solution (dissolved in saline) with different concentrations inhibited the scratching response induced by histamine in a dose-dependent manner, with the half-effective concentration ED 50 The antipruritic effect of bromocriptine was 4.603 nmol. No difference was observed between female and male mice. The above results prove that bromocriptine can effectively inhibit the itching caused by histamine.

[0104] Example 8 Study on the antipruritic effect of intrathecal injection of bromocriptine solution in chloroquine-induced pruritus model

[0105] The mouse grouping, preparation of drug solution and administration method refer to Example 3.

[0106] Intradermal injection of chloroquine at the back of the neck can also significantly induce a scratching response, and it is believed that it can activate a neural circuit different from histamine. This example investigates the effect of bromocriptine on chloroquine-induced itch.

[0107] Construction of chloroquine-induced pruritus model: The hair on the neck of mice was shaved with an electric razor, and the mice were used for the experiment after adapting to the experimental environment for 3 days. Half an hour before the experiment, the mice were randomly divided into four groups. The control group mice (normal saline group, n = 8) were intrathecally injected with 10 μL normal saline, and the low-dose drug group (n = 9), medium-dose drug group (n = 8), and high-dose drug group (n = 8) were intrathecally injected with 3nmol, 10nmol, and 30nmol bromocriptine solutions, respectively. 50 μL chloroquine (8mM) was intradermally injected into the shaved parts of the control group and experimental group mice. The number of itching of mice within 30 minutes was observed and counted. The scratching behavior was counted as one time when the front paw or hind paw of the mouse left the ground and scratched the drug-treated part of the right back neck until the paw was put back on the ground. The mean number of scratches of each group of mice was compared and statistically processed. The maximum antipruritic effect was calculated, and the calculation formula was: 1-(number of scratches in the drug-treated group-number of scratches in the non-drug-treated group)*100%.

[0108] The experimental results are as follows Figure 8 Compared with the control group (10 μL saline injected intrathecally), intrathecal injection of equal volumes of bromocriptine solution (dissolved in saline) with different concentrations inhibited the scratching response caused by chloroquine in a dose-dependent manner, with the half-effective concentration ED 50 The antipruritic effect of bromocriptine was 8.386 nmol. No difference was observed between female and male mice. The above results prove that bromocriptine can effectively inhibit the itching caused by histamine.

[0109] Example 9 Study on the antipruritic effect of intrathecal injection of bromocriptine solution in imiquimod-induced psoriasis model. Refer to Example 3 for mouse grouping, preparation of drug solution and administration method.

[0110] Continuously applying imiquimod on the back of the neck of mice is often used to construct a mouse psoriasis model, which can cause skin lesions and persistent and significant scratching reactions. This example investigates the effect of bromocriptine on itching caused by skin lesions under psoriasis conditions.

[0111] The specific method of model construction in this embodiment is as follows: 62.5 mg of 5% imiquimod ointment was applied to the shaved back skin (2.5×2 cm) of mice every day for 7 consecutive days, and on the 8th day, the control group (normal saline group, n=11), low-dose drug group (n=12), medium-dose drug group (n=6), and high-dose drug group (n=10) mice were intrathecally injected with normal saline, low (3nmol), medium (10nmol), and high-dose (30nmol) bromocriptine solution, respectively, and the efficacy test was performed after 1h and 5h, respectively. The maximum antipruritic effect was calculated by the formula: 1-(number of scratches in the drug group-number of scratches in the non-drug group)*100%.

[0112] The experimental results are as follows Fig. 9 Compared with the control group (10 μL saline injected intrathecally), intrathecal injection of different concentrations of bromocriptine solution (dissolved in saline) inhibited the scratching response under the skin lesions induced by imiquimod in a dose-dependent manner, with the half effective concentration ED 50 The antipruritic effect of bromocriptine was 4.405 nmol. No difference was observed between female and male mice. The above results prove that bromocriptine can effectively relieve the itching caused by skin lesions under psoriasis conditions.

[0113] Example 10 Study on the analgesic effect of intrathecal injection of bromocriptine solution in a DNFB-induced atopic contact dermatitis model

[0114] The mouse grouping, preparation of drug solution and administration method refer to Example 3.

[0115] Continuously applying DNFB (2,4-dinitrofluorobenzene) on the back of the neck of mice can be used to construct a mouse atopic contact dermatitis model, which can cause skin inflammatory lesions and persistent and significant scratching reactions. This example investigates the effect of bromocriptine on itching caused by skin lesions under atopic contact dermatitis conditions.

[0116] The specific method of constructing the model in this example is as follows: First, DNFB was dissolved in a mixed solvent of acetone and olive oil (v / v = 4:1) to prepare a DNFB solution. The mice were depilated on the abdomen one day before the experiment, and the depilated area was about 1×1 cm 2 24 hours after depilation, 0.5% DNFB solution (50 μL / mouse) was evenly applied to the depilated area of ​​the abdomen of each group of mice for sensitization. On the 5th, 7th, 9th, and 11th days after the first sensitization, the depilated area on the back of the mice (approximately 1×1 cm) was coated with DNFB solution. 2 ) was applied with 0.25% DNFB solution (30 μL / mouse) to stimulate skin inflammation. The control group mice (normal saline group, n=10) were intrathecally injected with 10 μL normal saline, and the low-dose drug group (n=7), medium-dose drug group (n=7), and high-dose drug group (n=7) were intrathecally injected with 3nmol, 10nmol, and 30nmol bromocriptine solution, respectively. The efficacy test was performed on the 12th day. The mean number of scratches of each group of mice was compared and statistically processed. The maximum antipruritic effect was calculated, and the calculation formula was: 1-(number of scratches in the drug group-number of scratches in the non-drug group)*100%.

[0117] The experimental results are as follows Fig.10 Compared with the control group (10 μL saline injected intrathecally), intrathecal injection of equal volumes of different concentrations of bromocriptine (dissolved in saline) inhibited the scratching response under DNFB-induced skin lesions in a dose-dependent manner, with the half-effective concentration ED 50The antipruritic effect of bromocriptine was 6.802 nmol. No difference was observed between female and male mice. The above results prove that bromocriptine can effectively relieve the itching caused by skin lesions under the condition of atopic contact dermatitis.

[0118] Example 11 Bromocriptine analgesia is not highly tolerated

[0119] Dissolve the bromocriptine stock solution in sterile saline and dilute to a concentration of 3 mM. Prepare 0.1 mM morphine solution in the same manner.

[0120] The mice were randomly divided into a bromocriptine-administered group (n=12) and a morphine-administered group (n=7).

[0121] The mice in the bromocriptine group were intrathecally injected with 10 μL of bromocriptine solution, and the mice in the morphine group were intrathecally injected with 10 μL of morphine solution.

[0122] Refer to Example 6 to construct a sciatica model.

[0123] The analgesic tolerance of bromocriptine was tested in a partial sciatic nerve injury model of neuropathic pain.

[0124] The experimental results are as follows Fig.11 As shown in the figure, during the 6-day treatment, the therapeutic effect of morphine continued to decline until it disappeared, while the efficacy of bromocriptine remained basically unchanged. The above results indicate that the analgesic tolerance of bromocriptine is better than that of morphine.

[0125] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of bromocriptine in the preparation of calcium channel inhibitors.

2. Use of bromocriptine in the preparation of drugs for treating and / or preventing diseases targeting calcium channels.

3. The use according to claim 1 or 2, characterized in that Bromocriptine specifically inhibits N-type calcium channels.

4. The use according to claim 3, characterized in that Bromocriptine specifically inhibits N-type calcium channels Ca V 2.

2.

5. The use according to claim 2, characterized in that: The symptom includes pain and / or itching.

6. The use according to claim 5, characterized in that Bromocriptine blunts an individual's perception of pain and / or itch.

7. The use according to claim 5, characterized in that The pain includes acute / chronic inflammatory pain, neuropathic pain, and pain induced by chemotherapy drugs.

8. The use according to claim 5, characterized in that The pruritus includes serotonin-induced pruritus, histamine-induced pruritus, chloroquine-induced pruritus, opioid-induced pruritus, systemic disease-induced pruritus, metabolic disease-induced pruritus, skin-derived pruritus, pruritus caused by atopic contact dermatitis, and pruritus induced by hematological malignancies.

9. The use according to claim 2, characterized in that: The drug contains bromocriptine as the only active ingredient.

10. The use according to claim 2, characterized in that: The dosage form of the drug is selected from capsules, powders, tablets, granules, pills, dragees, solutions, suspensions, syrups, injections, liniments, emulsions, microemulsions, tinctures, elixirs, creams, ointments, plasters, gels, pastes, powders, patches, aerosols or sprays.

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