Application of agents that promote DOCK11 expression as drugs for the treatment of neuropathic pain

Abstract

The present invention discloses the use of agents that promote DOCK11 expression as drugs for treating neuropathic pain. This study found that mice with neuropathic pain experience significant mechanical allodynia and thermal hyperalgesia, and that DOCK11 expression is closely associated with mechanical and thermal allodynia in CCI mice. Overexpression of a DOCK11 vector can effectively promote DOCK11 expression in the spinal cord and significantly suppress neuropathic pain. These findings provide potential therapeutic targets for the clinical treatment of neuropathic pain.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to the use of a reagent that promotes DOCK11 expression as a drug for treating neuropathic pain. Background Art

[0002] Neuropathic pain (NP) is a chronic condition secondary to peripheral or central nervous system damage. It presents with severe symptoms and a high prevalence. Data from Lancet Neurology indicate that the prevalence of NP in the general population is approximately 8.0%. my country has one of the highest rates of NP, with an annual incidence rate of 8.7%, far exceeding the global average. Based on these data, it is estimated that the number of patients with NP in my country currently reaches 90 million. It is reasonable to believe that this prevalence will continue to rise with the aging population, the increasing incidence of diabetes, and the prolonged survival of cancer patients. In October 2020, data from the Global Burden of Disease (GBD) published by The Lancet indicated that neuropathic pain remains a leading cause of global disease burden, with chronic pain costing patients $100 billion annually in medical expenses, not including the loss of work due to pain. Studies have shown that nearly half of patients with NP experience concurrent depression, which significantly impacts their quality of life. There are currently no effective treatments for neuropathic pain. Even with evidence-based treatment, only 50% of patients achieve 40% pain relief. Existing medications also have significant side effects with long-term use. Therefore, finding effective treatments for neuropathic pain remains a daunting challenge in modern anesthesia and pain medicine. Summary of the Invention

[0003] In order to overcome the deficiencies of the prior art, the present invention aims to provide a pharmaceutical composition for treating neuropathic pain and its application.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions:

[0005] The present invention provides use of an agent for promoting DOCK11 expression in preparing a pharmaceutical composition for treating neuropathic pain.

[0006] An agent that promotes DOCK11 expression refers to any substance that can increase the activity of DOCK11 protein, improve the stability of DOCK11 gene or protein, upregulate the expression of DOCK11 protein, increase the effective action time of DOCK11 protein, or promote the transcription and translation of DOCK11 gene. These substances can be used in the present invention as substances useful for upregulating DOCK11, thereby being used to treat neuropathic pain. For example, the promoting agent includes a nucleic acid promoter and a protein promoter. In some embodiments, the agent upregulates the expression of DOCK11 protein, including but not limited to a vector that overexpresses DOCK11, DOCK11 protein, or its active peptide.

[0007] Furthermore, the reagent for promoting DOCK11 expression is a DOCK11 overexpression vector or DOCK11 protein.

[0008] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0009] The present invention also provides a pharmaceutical composition for treating neuropathic pain, wherein the pharmaceutical composition comprises an effective amount of an agent that promotes DOCK11 expression.

[0010] The effective amount of the pharmaceutical composition of the present invention will vary depending on the desired effect. Thus, one skilled in the art can readily determine the optimal dosage to be administered, and the optimal dosage will vary with the specific drug used, the route of administration, the strength of the formulation, and the progression of the disease condition. In addition, factors related to the specific subject being treated, including the subject's age, weight, diet, and time of administration, will result in the need to adjust the dosage to an appropriate therapeutic level.

[0011] According to specific embodiments, an effective amount or effective dose refers to a therapeutic amount sufficient to achieve one, two, three, four, or more of the following effects: (i) reducing or ameliorating the severity of the disease, disorder, or condition being treated or the symptoms associated therewith; (ii) reducing the duration of the disease, disorder, or condition being treated or the symptoms associated therewith; (iii) preventing the development of the disease, disorder, or condition being treated or the symptoms associated therewith; (iv) causing the regression of the disease, disorder, or condition being treated or the symptoms associated therewith; (v) preventing the development or onset of the disease, disorder, or condition being treated or the symptoms associated therewith. (vi) prevent recurrence of the disease, disorder or condition being treated, or symptoms associated therewith; (vii) reduce hospitalization of a subject suffering from the disease, disorder or condition being treated, or symptoms associated therewith; (viii) reduce the length of hospital stay of a subject suffering from the disease, disorder or condition being treated, or symptoms associated therewith; (ix) improve survival of a subject suffering from the disease, disorder or condition being treated, or symptoms associated therewith; (xi) inhibit or reduce symptoms of the disease, disorder or condition being treated, or symptoms associated therewith, in a subject; and / or (xii) enhance or improve the prophylactic or therapeutic effect of another therapy.

[0012] Furthermore, the reagent for promoting DOCK11 expression is a DOCK11 overexpression vector or DOCK11 protein.

[0013] The pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0014] In the present invention, the term "pharmaceutically acceptable carrier" refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposomal encapsulation, or other material known in the art for use in pharmaceutical formulations. It should be understood that the characteristics of the carrier will depend on the route of administration for a specific application. As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic material that does not interfere with the efficacy or biological activity of the pharmaceutical composition according to the present invention. The use of pharmaceutically acceptable carriers to formulate active pharmaceutical ingredients is known in the art, for example, as described in Remington: The Science and Practice of Pharmacy (e.g., 21st edition (2005), and any later editions). Non-limiting examples of pharmaceutically acceptable carriers include salts (e.g., acid salts / anionic salts, basic salts / cationic salts), excipients, buffers, diluents, solubilizers, tonicity modifiers, surfactants, preservatives, isotonicity agents, stabilizers, and chelating agents. One or more pharmaceutically acceptable carriers can be used to formulate the pharmaceutical composition of the present invention.

[0015] In one embodiment of the present invention, the pharmaceutically acceptable carrier comprises an acid salt / anionic salt. Non-limiting examples of acid salts / anionic salts include, but are not limited to, acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camphorsulfonate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, glucoheptonate, gluconate, glutamate, glycolylaminophenylarsonic acid salt, hexylresorcinate, hydralamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, methanesulfonate, methylbromide, methylnitrate, methylsulfate, mucate, naphthenate, nitrate, pamoate, pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, theoclate, tosylate, and triethiodide.

[0016] In one embodiment of the invention, pharmaceutically acceptable carriers include alkali / cationic salts. Non-limiting examples of alkali / cationic salts include, but are not limited to, aluminum, 2-amino-2-hydroxymethyl-propane-1,3-diol (also known as tris(hydroxymethyl)aminomethane, tromethamine, or "TRIS"), ammonia, benzathine penicillin, tert-butylamine, chloroprocaine, choline, cyclohexylamine, diethanolamine, ethylenediamine, lithium, L-lysine, magnesium, meglumine, N-methyl-D-glucamine, piperidine, potassium, procaine, quinine, sodium, triethanolamine, or zinc.

[0017] In one embodiment of the present invention, the pharmaceutically acceptable carrier comprises a buffer. Non-limiting examples of buffers include, but are not limited to, arginine, aspartic acid, bicine, citrate, disodium hydrogen phosphate, fumaric acid, glycine, glycylglycine, histidine, lysine, maleic acid, malic acid, sodium acetate, sodium carbonate, sodium dihydrogen phosphate, sodium phosphate, succinate, tartaric acid, triazine, and tris(hydroxymethyl)aminomethane, and mixtures thereof.

[0018] In one embodiment of the present invention, the pharmaceutically acceptable carrier includes a preservative. Non-limiting examples of preservatives include, but are not limited to, benzethonium chloride, benzoic acid, benzyl alcohol, bromonitropropylene glycol, butyl 4-hydroxybenzoate, chlorobutanol, chlorocresol, chlorhexidine, chlorphenesin, o-cresol, m-cresol, p-cresol, ethyl 4-hydroxybenzoate, imidurea, methyl 4-hydroxybenzoate, phenol, 2-phenoxyethanol, 2-phenylethanol, propyl 4-hydroxybenzoate, sodium dehydroacetate, thimerosal, and mixtures thereof.

[0019] In one embodiment of the invention, pharmaceutically acceptable carriers include isotonic agents. Non-limiting examples of isotonic agents include but are not limited to amino acids (such as glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan and threonine), sugar alcohols (such as glycerol, 1,2-propylene glycol, propylene glycol), 1,3-propylene glycol and 1,3-butylene glycol), polyethylene glycol (for example, PEG400) and mixtures thereof. Another example of isotonic agents includes sugar. Non-limiting examples of sugar can be monosaccharides, disaccharides or polysaccharides, or water-soluble glucans, including for example fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, glucan, amylopectin, dextrin, cyclodextrin, α and β-HPCD, soluble starch, hydroxyethyl starch and sodium carboxymethyl cellulose. Another example of isotonic agents is sugar alcohol, wherein the term "sugar alcohol" is defined as a C (4-8) hydrocarbon with at least one -OH group. Non-limiting examples of sugar alcohols include mannitol, sorbitol, inositol, galactitol, hexasol, xylitol, and arabitol.Medicaments comprising each of the isotonic agents listed in this paragraph constitute alternative embodiments of the present invention.

[0020] In one embodiment of the present invention, the pharmaceutically acceptable carrier comprises a chelating agent. Non-limiting examples of chelating agents include, but are not limited to, salts of citric acid, aspartic acid, ethylenediaminetetraacetic acid (EDTA), and mixtures thereof.

[0021] In one embodiment of the present invention, the pharmaceutically acceptable carrier includes a stabilizer. Non-limiting examples of stabilizers include carboxy- / hydroxycellulose and its derivatives (such as HPC, HPC-SL, HPC-L and HPMC), cyclodextrin, 2-methylthioethanol, polyethylene glycol (such as PEG 3350), polyvinyl alcohol (PVA), polyvinyl pyrrolidone, salts (such as sodium chloride), sulfur-containing substances such as monothioglycerol or thioglycolic acid.

[0022] In one embodiment of the present invention, the pharmaceutically acceptable carrier comprises one or more surfactants, preferably one surfactant, at least one surfactant, or two different surfactants. The term "surfactant" refers to any molecule or ion consisting of a water-soluble portion (hydrophilic) and a fat-soluble portion (lipophilic). For example, the surfactant is selected from the group consisting of anionic surfactants, cationic surfactants, nonionic surfactants, and / or zwitterionic surfactants.

[0023] In one embodiment of the present invention, the drug is a liquid formulation. A preferred example of a liquid formulation is an aqueous formulation, i.e., a formulation comprising water. Liquid formulations include, but are not limited to, solutions, suspensions, emulsions, microemulsions, or gels. Aqueous formulations typically comprise at least 50% w / w water, or at least 60% w / w, 70% w / w, 75% w / w, 80% w / w, 85% w / w, 90% w / w, or at least 95% w / w water.

[0024] In one embodiment, the drug can be formulated as an injectable, which can be injected, for example, via an injection device (eg, a syringe or an infusion pump). The injection can be delivered, for example, subcutaneously, intramuscularly, intraperitoneally, or intravenously.

[0025] In another embodiment, the drug is a solid formulation, such as a freeze-dried or spray-dried drug, which can be used as is, or a solvent and / or diluent can be added by the physician or patient before use. Solid dosage forms can include tablets, such as compressed and / or coated tablets, and capsules (e.g., hard gelatin capsules or soft gelatin capsules). The pharmaceutical composition can also be in the form of a sachet, dragee, powder, granule, lozenge, or powder, for example, for reconstitution.

[0026] The present invention also provides a kit for treating neuropathic pain, comprising the aforementioned pharmaceutical composition, optionally a container and instructions.

[0027] The kit of the present invention comprises separate containers, dividers or compartments for the drug and the informational material. For example, the drug can be contained in a bottle, vial or syringe, and the informational material can be contained in conjunction with the container. In some embodiments, the separate elements of the kit are contained in a single, undivided container. For example, the drug is contained in a bottle, vial or syringe (with the informational material attached in the form of a label). In some embodiments, the kit comprises a plurality (e.g., a pack) of separate containers, each container containing one or more unit dosage forms (e.g., the dosage forms described herein) of active substance.

[0028] The present invention also provides the use of DOCK11 in screening candidate drugs for treating neuropathic pain.

[0029] Furthermore, the method for screening candidate drugs for treating neuropathic pain is as follows: treating a culture system expressing or containing the DOCK11 gene or a protein encoded by it with a substance to be screened; and detecting the expression or activity of the DOCK11 gene or a protein encoded by it in the system; wherein,

[0030] When the substance to be screened promotes the expression level or activity of the DOCK11 gene or the protein encoded by it, the substance to be screened is a candidate drug for treating neuropathic pain.

[0031] The present invention also provides a method for screening candidate drugs for treating neuropathic pain, which is as follows: treating a culture system expressing or containing the DOCK11 gene or a protein encoded by it with a substance to be screened; and detecting the expression or activity of the DOCK11 gene or a protein encoded by it in the system; wherein, when the substance to be screened promotes the expression level or activity of the DOCK11 gene or a protein encoded by it, the substance to be screened is a candidate drug for treating neuropathic pain.

[0032] The present invention also provides a method for screening drugs for treating neuropathic pain, which is as follows:

[0033] 1) Establish an animal model of neuropathic pain;

[0034] 2) administering a test drug to the animal model of step 1); when the test drug promotes the expression level or activity of the DOCK11 gene or the protein encoded by it, the test drug is a candidate drug for treating neuropathic pain. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Figure 2 shows the effect of overexpression of DOCK11 on neuropathic pain, where A: paw withdrawal threshold; B: paw withdrawal latency; n=6; *P<0.05, compared with the Sham group; #P<0.05, compared with the CCI group; two-way ANOVA;

[0036] Figure 2 The results of DOCK11 expression are shown in Figure 1, where A: immunoblotting; B: bar graph; *P<0.05, compared with the Sham group; #P<0.05, compared with the CCI group. DETAILED DESCRIPTION

[0037] The present invention will be further described in detail below in conjunction with the accompanying drawings and Examples. The following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. Simple modifications to the present invention made according to the essence of the present invention all fall within the scope of protection claimed in the present invention.

[0038] Example 1 Study on the correlation between DOCK11 expression and neuropathic pain

[0039] 1. Experimental Materials

[0040] (1) Experimental Grouping: Adult male C57BL / 6J mice, 6-8 weeks old, weighing 20-25 g at the time of surgery, were purchased from the Experimental Animal Center of the Academy of Military Medical Sciences of the Chinese People's Liberation Army. They were randomly divided into 4 groups (n=6):

[0041] ① Sham operation group (Sham group), only the right sciatic nerve of mice was exposed without ligation;

[0042] ② In the CCI group (CCI group), the right sciatic nerve of mice was exposed and loosely ligated with 4-0 catgut without nerve contraction;

[0043] ③ NC-overexpressing adenovirus (control adenovirus) + CCI group (N+CCI group): 1 μl of NC-overexpressing adenovirus (Shanghai Genema Gene Co., Ltd.) was injected into the L4-5 dorsal root ganglion one month before model establishment;

[0044] Construction process of adenovirus overexpressing NC: His-tag, FLAG-tag, and Bio-tag sequences were amplified from pET101 / D-TOPO, pCMV6-LIPG, and BioEase vectors, respectively, and then cloned into pcDNA3.1 to generate empty vectors named BioFLAGHis-pcDNA or HisBioFLAG-pcDNA for use as NC control.

[0045] ④ Adenovirus overexpressing DOCK11 + CCI group (D+CCI group): 1 μl of adenovirus overexpressing DOCK11 (Shanghai Genema Company) was injected into the L4-5 dorsal root ganglion one month before model establishment.

[0046] Construction of adenovirus overexpressing DOCK11: DOCK11 DNA was cloned. The His-tag, FLAG-tag, and Bio-tag sequences were amplified from pET101 / D-TOPO, pCMV6-LIPG, and BioEase vectors, respectively, and cloned into pcDNA3.1 to generate empty vectors designated BioFLAGHis-pcDNA or HisBioFLAG-pcDNA. The full-length DOCK11 gene or the amino acid sequence from 1516 to 2073 was amplified by PCR and cloned into the BioFLAGHis-pcDNA vector to generate DOCK11-BioFLAGHis-pcDNA or DOCK11-DHR2-BioFLAGHis-pcDNA.

[0047] Table 1 Sequence information

[0048]

[0049]

[0050] (2) Preparation of neuropathic pain model: Mice were anesthetized by inhalation of 2% sevoflurane and immobilized in the prone position. The skin of the upper hind limb was incised, the muscles were separated, and the sciatic nerve trunk was exposed. Catgut was wrapped around the sciatic nerve and fixed with a single knot. The catgut was preferably able to slide over the sciatic nerve trunk. The sham operation group was operated on the same procedures as above, except that the sciatic nerve was not ligated.

[0051] (3) Behavioral experiment: The thermal stimulation paw withdrawal latency (PWL) and mechanical stimulation paw withdrawal threshold (PWT) were measured 24 h before modeling (0 day) and 1 day, 3 days, 5 days, 7 days, and 14 days after modeling. The laboratory temperature was 18-22°C and the environment was quiet. PWL was measured using a YLS-6B intelligent hot plate instrument (Huaibei Zhenghua Biological Instrument Equipment Co., Ltd.) with a set temperature of 52°C. The time from the right hind paw contacting the hot plate to the occurrence of any reaction such as withdrawal, tiptoeing, struggling, screaming, or licking the paw was recorded as PWL. The measurement was repeated three times with an interval of 5 minutes, and the average value was taken as PWL (sec). To prevent scalding of the mouse paw, the upper limit of PWL was set to 30 seconds. Mice were placed in a 10 cm × 10 cm × 20 cm metal cage. After 30 minutes, BSEVF3 von Frey fibers (Harvard Apparatus, USA) were used to stimulate the space between the second and third phalanges of the right hind paw. Vertical pressure was applied and the pressure at the time of rapid paw withdrawal, licking of the right paw, or neighing was recorded. The pressure was measured three times with 5-minute intervals, and the average value was taken as PWT (g).

[0052] (4) Western blot: After the last behavioral test, the mice were killed and the L3 6 dorsal root ganglion was taken and the protein expression was determined by Western blot. The L3 6 dorsal root ganglion tissue was added to the pre-cooled tissue protein lysis buffer and ground into a tissue homogenate. The homogenate was centrifuged at 4°C for 30 minutes, 15000 rpm, and a centrifugal radius of 10 cm. The supernatant was the total protein of the spinal cord tissue. The membrane protein was extracted using a membrane protein extraction kit (Thermo, USA) according to the instructions. Anti-DOCK11 (A301-638A, 1:1000, Bethyl) was used to determine the expression of DOCK11 according to the instructions.

[0053] (5) Statistical analysis: SPSS 18.0 statistical software was used for analysis. Normally distributed quantitative data were expressed as mean ± standard deviation. One-way analysis of variance was used to compare quantitative data of randomized block design. Analysis of variance of repeated measurement design was used to compare quantitative data of repeated measurement design. P < 0.05 was considered statistically significant.

[0054] 2. Experimental results

[0055] (1) CCI mice have mechanical allodynia and thermal hyperalgesia

[0056] Compared with the Sham group, the thermal stimulation paw withdrawal latency (PWL) and mechanical stimulation paw withdrawal threshold (PWT) of the CCI group mice were significantly reduced, which appeared on the first day after surgery and remained until the 14th day after surgery. These results indicate that the neuropathic pain mice have obvious mechanical allodynia and thermal hyperalgesia (P < 0.05, Figure 1 A and 1B).

[0057] (2) Neuropathic pain significantly inhibited the expression of DOCK11

[0058] Fourteen days after successful modeling, the mice were killed and the spinal cord L3-L6 segments were obtained for Western Blot analysis of DOCK11 expression. WB results showed that the expression of DOCK11 protein in the CCI group was significantly reduced compared with that in the Sham group (P < 0.05, Figure 2 ), which suggests that the decreased expression of DOCK11 protein may be related to the pathogenesis of neuropathic pain.

[0059] (3) Overexpression of DOCK11 can significantly reverse mechanical allodynia and thermal hyperalgesia in CCI mice

[0060] WB detection results showed that the expression of DOCK11 in the spinal cord increased significantly after administration of DOCK11 overexpression vector (P < 0.05, Figure 2 On this basis, behavioral tests after model establishment revealed that the thermal stimulation paw withdrawal latency (PWL) and mechanical stimulation paw withdrawal threshold (PWT) of CCI mice were significantly increased after overexpression of DOCK11 (P < 0.05, Figure 1 A and 1B), these symptoms appeared on the first day after surgery and lasted until the 14th day after surgery, indicating that DOCK11 has potential therapeutic effects on neuropathic pain.

[0061] The above embodiments are only provided for understanding the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by a person skilled in the art, and such improvements and modifications shall fall within the scope of protection of the claims of the present invention.

Claims

1. Use of an agent that promotes DOCK11 expression in the preparation of a pharmaceutical composition for treating neuropathic pain, wherein the agent that promotes DOCK11 expression is a DOCK11 overexpression vector.

2. The use according to claim 1, characterized in that The pharmaceutical composition further includes a pharmaceutically acceptable carrier.

3. Application of DOCK11 in screening candidate drugs for treating neuropathic pain, the method for screening candidate drugs for treating neuropathic pain is as follows: treating a culture system expressing or containing the DOCK11 gene or a protein encoded by it with a substance to be screened; and detecting the expression of the DOCK11 gene or a protein encoded by it in the culture system; wherein, When the substance to be screened promotes the expression level of the DOCK11 gene or the protein encoded by it, the substance to be screened is a candidate drug for treating neuropathic pain.

4. A method for screening candidate drugs for treating neuropathic pain, characterized in that: The method is as follows: treating a culture system expressing or containing the DOCK11 gene or a protein encoded by it with a substance to be screened; and detecting the expression of the DOCK11 gene or the protein encoded by it in the system; wherein, when the substance to be screened promotes the expression level of the DOCK11 gene or the protein encoded by it, the substance to be screened is a candidate drug for treating neuropathic pain.

5. A method for screening drugs for treating neuropathic pain, characterized in that: The method is as follows: 1) Establish an animal model of neuropathic pain; 2) administering a test drug to the animal model of step 1); when the test drug promotes the expression level of the DOCK11 gene or the protein encoded by it, the test drug is a candidate drug for treating neuropathic pain.

Images