CN114632156A - Use of Tim-3 for the prevention, treatment or alleviation of pain - Google Patents

Abstract

The invention discloses the use of Tim-3 in the prevention, treatment or alleviation of pain. In the specific embodiment of the invention, the Tim-3Ab is applied to intervene in an NTG mouse model, and the pre-drying of the Tim-3Ab is found to effectively slow down the decrease of the plantar pain threshold, so that the Tim-3 can be used for preventing, treating or relieving pain. The invention provides a new means for treating pain.

Description

Use of Tim-3 for the prevention, treatment or alleviation of pain

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to application of Tim-3 in preventing, treating or relieving pain.

Background

The rapid development of the society provides abundant material enjoyment for people and accelerates the pace of life rhythm. The nervous system disease, which is a highly stressful, environmentally induced migraine, affects nearly 10 million people's lives and studies worldwide. The ubiquitous nervous system disease with high disability rate is listed as the third most common disease and the second most disabling nervous system disease in the world by the world health organization.

Pain, an intrinsic response to trauma and disease, has been an important research point for various explorations. From physiology to pathology, the understanding of pain in humans is becoming more profound. With the development of research visual field and field, people find that pain not only affects physiology but also relates to psychological factors, and related psychological disorders are commonly existed in pain groups, so that the recovery of patients is greatly hindered. It has been found that depression, anxiety, PTSD, and cognitive disorders are all closely related to migraine. Chronic headache which repeatedly attacks for a long time seriously affects the mind and body of a patient. Current treatment of migraine is focused primarily on preventive relief and acute symptomatic treatment, with common medications including: antidepressants, non-steroidal anti-inflammatory drugs, ergot derivatives, opioids, triptans and the like. Not only has limited effect and has great addiction and tolerance risk, but also brings secondary pain to patients, and the research and development of new drugs with high efficiency and small side effect become problems and new targets of research which need to be solved urgently.

Disclosure of Invention

The invention aims to provide a new medicine for preventing, treating or relieving pain, and adopts the following technical scheme for realizing the aim:

in one aspect, the invention provides a pharmaceutical composition for preventing, treating or reducing pain, the pharmaceutical composition comprising a binding agent to a protein encoded by Tim-3.

In the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable buffer, carrier or excipient.

The phrase "pharmaceutically acceptable" is employed herein to refer to those agents, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

In the present invention, Tim-3 includes Gene with Gene ID 84868 and its encoded protein and its homologues, mutations, and isoforms.

The term "prevention" as used herein refers to prophylactic treatment or therapy to prevent one or more symptoms or conditions (e.g., pain) of a disease, disorder, or condition described herein. Prophylactic treatment can be initiated, for example, before (pre-exposure prevention) or after (post-exposure prevention) an event (e.g., pain) that precedes the onset of the disease, disorder, or condition. Prophylactic treatment comprising administration of the pharmaceutical compositions described herein may be acute, short-term or chronic.

In certain embodiments, the binding agent to a protein encoded by Tim-3 comprises a peptide, a peptide mimetic, a nucleic acid aptamer, a mirror image L-RNA aptamer, an ankyrin repeat protein, a Kunitz-type domain, or an antibody.

In certain embodiments, the binding agent to a protein encoded by Tim-3 is an antibody.

As used herein, the term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combination of the foregoing, through at least one antigen binding site. As used herein, the term encompasses intact polyclonal antibodies, intact monoclonal antibodies, single chain antibodies, antibody fragments (such as Fab, Fab ', F (ab') 2, and Fv fragments), single chain Fv (scfv) antibodies, multispecific antibodies (such as bispecific antibodies), monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen binding site of an antibody, and any other modified immunoglobulin molecule comprising an antigen binding site, so long as the antibody exhibits the desired biological binding activity. The antibody can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2). The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies may be naked or conjugated to other molecules, including but not limited to toxins and radioisotopes.

The term "antibody fragment" refers to a portion of an intact antibody and refers to the epitope variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab ', F (ab') 2, and Fv fragments, linear antibodies, single chain antibodies, and multispecific antibodies formed from antibody fragments. As used herein, an "antibody fragment" comprises at least one antigen binding site or epitope binding site. The term "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, alone or in combination. The variable region of a heavy or light chain is typically composed of four Framework Regions (FRs) connected by three Complementarity Determining Regions (CDRs), also referred to as "hypervariable regions". The CDRs in each chain are held together in close proximity by the framework regions and contribute to the formation of the antigen-binding site of the antibody.

In certain embodiments, the antibody comprises a monoclonal antibody, a polyclonal antibody.

The term "monoclonal antibody" refers to a homogeneous population of antibodies that are involved in the highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies which typically comprise a mixture of different antibodies directed against a variety of different antigenic determinants. The term "monoclonal antibody" encompasses intact and full-length monoclonal antibodies as well as antibody fragments (e.g., Fab ', F (ab') 2, Fv), single chain (scFv) antibodies, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen binding site. In addition, "monoclonal antibody" refers to such antibodies prepared by a number of techniques including, but not limited to, hybridoma production, phage selection, recombinant expression, and transgenic animals.

In certain embodiments, the pharmaceutical composition is in the form of a tablet, capsule, granule, powder, syrup, injection, drop infusion, suppository, eye drop, or ophthalmic ointment.

The pharmaceutical compositions of the present invention may be administered by various means depending on their intended use. For example, if the pharmaceutical compositions of the present invention are to be administered orally, they may be formulated as tablets, capsules, granules, powders or syrups. Alternatively, the formulations of the present invention may be administered parenterally as injections (intravenous, intramuscular or subcutaneous), drop infusion formulations or suppositories. For administration by the ocular mucosal route, the pharmaceutical composition of the invention may be formulated as eye drops or an ophthalmic ointment. These formulations can be prepared in a conventional manner, and the composition can be mixed, if necessary, with any conventional additives such as excipients, binders, disintegrants, lubricants, flavoring agents, solubilizers, suspension aids, emulsifiers, or coating agents.

In the formulations of the present invention, wetting agents, emulsifying agents, and lubricating agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can be present in the formulated medicament.

The pharmaceutical compositions of the present invention may be suitable for oral, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. The amount of composition that can be combined with the carrier materials to produce a single dose will vary depending upon the subject being treated and the particular mode of administration.

The methods of making these formulations include the step of associating a binding agent to a protein encoded by Tim-3 of the present invention with a carrier and optionally one or more accessory ingredients. In general, the formulations are prepared by uniformly and finely associating the pharmaceutical agent with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product.

Formulations suitable for oral administration may be in the form of: capsules, cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), powders, granules or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil emulsion, or as an elixir or syrup, or as pastilles (using an inert base such as gelatin and glycerin, or sucrose and acacia), each containing a predetermined amount of its subject composition as an active ingredient. The pharmaceutical compositions of the invention may also be administered as a bolus, electuary or paste.

In solid dosage forms (capsules, tablets, pills, dragees, powders, granules, etc.) for oral administration, the binding agent for the protein encoded by Tim-3 is mixed with one or two 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) binding agents, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) dissolution retarders, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, acetol and glycerol monostearate; (8) absorbents such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof; and (10) a colorant. In the case of capsules, tablets and pills, the compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft-filled and hard-filled gelatin capsules using such excipients as lactose or milk sugar (milk sugar) as well as high molecular weight polyethylene glycols and the like.

Tablets may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binders (for example, gelatin or hydroxypropyl cellulose), lubricants, inert diluents, preservatives, disintegrating agents (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agents. Molded tablets may be prepared by molding in a suitable machine a mixture of the subject composition moistened with an inert liquid diluent. Tablets and other solid dosage forms such as dragees, capsules, pills and granules can optionally be scored or prepared with coating materials and capsule shells such as enteric coatings and other coatings well known in the pharmaceutical art.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the subject compositions, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, cyclodextrins and mixtures thereof.

In addition to the binding agent for the protein encoded by Tim-3, the suspension may also contain suspension aids, such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Formulations for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing the binding agent for the protein encoded by Tim-3 with one or more suitable non-irritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or salicylate, and which is solid at room temperature, but liquid at body temperature and will therefore melt and release the active agent in the body cavity. Formulations suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be suitable.

Dosage forms for transdermal administration of the subject compositions include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, and patches.

For topical ocular administration, the pharmaceutical compositions of the present invention may take the form of solutions, gels, ointments, suspensions, or solid inserts that are formulated such that the unit dose contains a therapeutically effective amount of the active ingredient or, in the case of combination therapy, some multiple thereof.

Pharmaceutical compositions of the invention suitable for parenteral administration comprise a binding agent for a protein encoded by Tim-3 in combination with one or more of the following: pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers that can be employed in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, phytases

Mineral oils (such as olive oil), and injectable organic esters (such as ethyl oleate) and cyclodextrins. Proper fluidity can be maintained, for example, by the use of a coating material (e.g., lecithin), by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

The dosage of any pharmaceutical composition of the invention will vary depending on the symptoms, age and weight of the patient, the nature and severity of the condition to be treated or prevented, the route of administration, and the form of the subject composition. The dosage of the pharmaceutical composition used in the present invention can be readily determined by techniques known to those skilled in the art or as taught herein.

The therapeutically effective amount of a binding agent for a protein encoded by Tim-3 required in therapy varies with the form of the condition being treated, the length of treatment required, the age and condition of the patient, and is ultimately determined by the attending physician. The required dose may conveniently be administered in a single dose effective for two weeks, one week, 6, 5, 4, 3, 2 or 1 day, or as multiple doses administered at appropriate time intervals, for example as two, three, four or more sub-doses per day.

An effective dose or amount, as well as any possible effect on the time of administration of the formulation, may need to be identified for any particular pharmaceutical composition of the invention. This can be done by routine experimentation as described herein, using one or more groups of animals or, where appropriate, in human trials. The effectiveness of any pharmaceutical composition and method of treatment or prevention can be assessed by administering the pharmaceutical composition and assessing the effect of the administration by measuring one or more applicable indices, and comparing the post-treatment value of these indices to the value of the same index prior to treatment.

The precise time and amount of administration of any particular pharmaceutical composition that will result in the most effective treatment in a given patient will depend upon the activity, pharmacokinetics and bioavailability of the pharmaceutical composition, the physiological condition of the patient (including age, sex, type and stage of disease, general physical condition, responsiveness to the given drug dose and type), route of administration, and the like.

In treating a subject, the health of the patient can be monitored by measuring one or more correlation indices at predetermined times during the treatment period. Treatment (including composition, amount, number and formulation administered) may be optimized based on the results of such monitoring. The patient may be periodically re-evaluated to determine the degree of improvement by measuring the same parameters. Adjustments to the amount of pharmaceutical composition administered, and possibly the time of administration, may be made based on these re-evaluations.

Treatment may be initiated at smaller doses than the optimal dose of the compound. Thereafter, the dosage may be increased in smaller increments until the optimum therapeutic effect is achieved.

In another aspect, the invention provides the use of a pharmaceutical composition as hereinbefore described in the manufacture of a medicament for the prevention, treatment or alleviation of pain.

In certain embodiments, the pain is one or more selected from the group consisting of: nociceptive pain, psychogenic pain, inflammatory pain, and pathological pain. More specifically, the pain is one or more selected from the group consisting of: neuropathic pain, cancer pain, post-operative pain, trigeminal neuralgia-like pain, idiopathic pain, diabetic neuropathic pain, and migraine.

In certain embodiments, the pain is migraine.

In certain embodiments, the migraine comprises episodic migraine, chronic migraine, retinal migraine, ophthalmoplegic migraine, non-cephalic migraine, migraine-associated conditions, menstrual migraine, abdominal migraine, childhood periodic syndrome, cluster headache.

In certain embodiments, the migraine is a chronic migraine.

In another aspect, the invention also provides the application of the reagent for detecting the expression level of Tim-3 in the preparation of products for diagnosing migraine.

The terms "level of expression" or "expression level" are generally used interchangeably and generally refer to the amount of a polynucleotide, mRNA or amino acid product or protein in a biological sample. "expression" generally refers to the process by which information encoded by a gene is converted into structures present and operating in a cell. Thus, according to the present invention, "expression" of a gene may refer to transcription into a polynucleotide, translation into a protein, or even post-translational modification of a protein. Fragments of the transcribed polynucleotide, of the translated protein, or of the post-translationally modified protein should also be considered expressed, whether they are derived from transcripts generated or degraded by alternative splicing, or from post-translational processing of the protein (e.g., by proteolysis). "expressed gene" includes a gene that is transcribed into a polynucleotide (e.g., mRNA) and then translated into protein, and also a gene that is transcribed into RNA but not translated into protein (e.g., miRNA).

In certain embodiments, the products include nucleic acid membrane strips, chips, kits.

In the present invention, a nucleic acid membrane strip comprises a substrate and oligonucleotide probes immobilized on the substrate; the substrate may be any substrate suitable for immobilizing oligonucleotide probes, such as a nylon membrane, a nitrocellulose membrane, a polypropylene membrane, a glass plate, a silica gel wafer, a micro magnetic bead, or the like.

In the present invention, "chip", also referred to as "array", refers to a solid support comprising attached nucleic acid or peptide probes. Arrays typically comprise a plurality of different nucleic acid or peptide probes attached to the surface of a substrate at different known locations. These arrays, also known as "microarrays," can generally be produced using either mechanosynthesis methods or light-guided synthesis methods that incorporate a combination of photolithography and solid-phase synthesis methods. The array may comprise a flat surface, or may be nucleic acids or peptides on beads, gels, polymer surfaces, fibers such as optical fibers, glass, or any other suitable substrate. The array may be packaged in a manner that allows for diagnostic or other manipulation of the fully functional device.

In certain embodiments, the reagents include primers, probes, binding agents for proteins.

"primer" refers to an oligonucleotide that hybridizes to a sequence in a target nucleic acid (a "primer binding site") and can serve as a point at which synthesis is initiated along a complementary strand of the nucleic acid under conditions suitable for such synthesis

"Probe" refers to a molecule that binds to a particular sequence or subsequence or other portion of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that is capable of binding to another polynucleotide (often referred to as a "target polynucleotide") by complementary base pairing. Depending on the stringency of the hybridization conditions, a probe can bind to a target polynucleotide that lacks complete sequence complementarity to the probe. The probe may be directly or indirectly labeled. Hybridization modalities, including, but not limited to: solution phase, solid phase, mixed phase or in situ hybridization assays.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides the connection between Tim-3 and pain for the first time; by applying Tim-3Ab pre-drying, the pain can be effectively relieved, and a new means is provided for treating the pain.

Drawings

FIG. 1 is a diagram of the detection range of the VonFay Filement pain threshold test package;

FIG. 2 is a schematic diagram of Tim-3 intervention NTG mouse model-9 day modeling and pain threshold testing;

FIG. 3 is a schematic diagram of nitroglycerin chronic migraine mouse model-1 day modeling and pain threshold testing;

FIG. 4 is a graph showing the results of the change in plantar pain threshold in the NTG model (day 9);

FIG. 5 is a graph showing the effect of Tim-3Ab on endorphin expression levels, wherein Panel A is TimResults of the effect of-3 Ab on endorphins in peritoneal macrophages, Panel B is a graph of the effect of Tim-3Ab pre-intervention on endorphins in serum from NTG mouse models, Panel C is a graph of Tim-3Ab on BV₂Results of the effect of endorphins in the cells, and D is a graph of the effect of Tim-3Ab on endorphins in THP-1 cells.

Detailed Description

The invention is further illustrated below with reference to specific examples, which are intended to be purely exemplary of the invention and are not to be interpreted as limiting the same. Those of ordinary skill in the art will understand that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The following examples are examples of experimental methods not indicating specific conditions, and the detection is usually carried out according to conventional conditions or according to the conditions recommended by the manufacturers.

Experimental Material

First, principal drugs and reagents

Glycerin nitrate injection (beijing yimin pharmaceutical limited); 0.9% physiological saline (Shijiazhuang Siyao Co., Ltd.); tim-3Ab, Fc (control antibody) (both laboratory self-purification, Tim-3Ab antibody preparation method and sequence in application number 2022103375598 patent is described); PBS pH 7.4 buffer (Gibco, USA); TRIzon Reagent RNA extraction Reagent (kang century biotechnology limited); sodium pentobarbital, chloroform, isopropanol, absolute ethanol and xylene (all from the national pharmaceutical group chemical agents, ltd.); the reverse transcription reagent TransScript Uni All-in-One First-Strand cDNA Synthesis SuperMix (One-Step gDNA Removal) (Kyoto Kogyo Biotechnology Co., Ltd.); QPCR reagent GenStar 2 × RealStar Green Fast mix (Biotechnology, Inc., Kangruichen, Beijing); the primers were synthesized by Biotechnology engineering (Shanghai) Ltd; tissue fixing solution, citric acid (Ph 6.0) antigen repair solution, 3% hydrogen peroxide, BSA, hematoxylin staining solution, hematoxylin differentiation solution, hematoxylin rewet solution, neutral gum, c-fos primary antibody, HRP goat anti-rabbit secondary antibody and DAB color developing agent of a histochemical kit (all purchased from Wuhanseville Biotech, Ltd.).

Second, experimental instrument

Von frey file (UGO corporation, italy); PCR instrument (BIO-RAD C1000 Thermal Cycler, Rio-Rad, USA); real-time fluorescent quantitative PCR instrument (Roche LightCycler 96, Roche, Switzerland); high speed refrigerated centrifuge (Eppendorf, germany); vortex mixer (Ningbo Xinzhi Biotech Co., Ltd.); a dehydrator, an embedding machine and a freezing station (all from Junjie electronics, Inc., Wuhan); pathological microtomes (shanghai come instrument limited); tissue slide machine (Zhejiang Jinhuakedi instruments equipment ltd); oven (Shanghai Huitai Instrument manufacturing Co., Ltd.); coverslips (Jiangsu Shitai laboratory instruments Co., Ltd.); microwave ovens (grand microwave appliances, ltd); slides, decolorizing shaker, and palm centrifuge (all from Wuhan Seville Biotech Co., Ltd.).

Third, primer

The primer sequences are shown in Table 1

TABLE 1 primer sequences

Fourth, experimental animal

C57BL/6J mice, male, 6-8 weeks, 18-20g, purchased from Beijing Sibefu biotechnology Limited company (production permit: SCXK (Beijing) 2019-. The animal breeding is carried out by animal breeding center of military medical research institute under the conditions of 12 h day and night alternation, proper temperature and humidity (20 +/-2 ℃, 55 +/-5%) and sufficient water and food supply. Mice began modeling around one week of acclimation in the animal house. 4-6 in each group and randomly grouped.

Experimental method

First, establishing Tim-3Ab intervention nitroglycerin chronic migraine mouse model grouping and experimental process

The nitroglycerin chronic migraine mouse model is divided into two types, 9 days and 1 day. Modeling and pain threshold detection are carried out in the daytime (9: 00-16: 00).

Firstly, constructing a nitroglycerin chronic migraine mouse model for-9 days

Grouping mice: fc + NTG group and Tim-3Ab + NTG group.

Before modeling on day 1, performing basic pain threshold (Baseline) detection by using a VonFery file (figure 1), placing a mouse on an iron frame with a horizontal mouse cage, after the emotion of the mouse is stabilized, vertically stimulating the soles of hind feet of the mouse sequentially by using VonFery fiber wires according to the gram number of the fiber wires from low to high, avoiding the parts of foot pads, stimulating for 5 times at each intensity, and enabling the toes of the mouse to be opened, feet to shake or feet to be lifted for 3 times, namely the basic pain threshold of the soles of the feet of the mouse. The VonFrey fiber filaments are bent into a C shape by the sole of a foot during measurement, and the detection of a mouse during standing and excretion is avoided. And then respectively carrying out Fc (0.1 mL/10g, i.p.) and Tim-3Ab (5 mg/kg, i.p.) intervention, giving an NTG working solution (10 mg/kg, i.p.) to the mouse after 0.5h, and detecting the plantar pain threshold of the mouse by applying VonFrey finish at an interval of 1.5 h. The modeling was completed for 9 days, and the above operations were performed on days 1, 3, 5, 7, and 9, respectively (fig. 2), and the basic pain threshold test was performed only before modeling on day 1.

Establishment of a nitroglycerin chronic migraine mouse model in 1 day

Grouping mice: fc + NTG group, Tim-3Ab + NTG group, NTG + Tim-3Ab group

Before modeling, carrying out basic pain threshold (Baseline) detection on three groups of mice by applying VonUrey files; then, the pain threshold detection is carried out on the three groups by applying Fc (0.1 mL/10g, i.p.) and Tim-3Ab (5 mg/kg, i.p.) respectively for the first group and the second group and applying VonUrey finish at an interval of 0.5 h. Immediately giving three groups of NTG working solution (10 mg/kg, i.p.) at an interval of 0.5h, and applying VonFrey Filament again for pain threshold detection, immediately giving three groups of Tim-3Ab (5 mg/kg, i.p.) at an interval of 0.5h, and applying VonFrement at an interval of 0.5h for pain threshold detection of the three groups; after which three groups were again tested for pain threshold using von frey finish at 0.5h intervals. The experimental procedure was completed within 1 day (fig. 3).

Two, real-time fluorescence quantitative PCR and immunohistochemistry

Cardiac perfusion acquisition of TNC and spleen

a, immediately carrying out intraperitoneal injection of 0.7% pentobarbital sodium according to the dose of 0.1 mL/10g to anesthetize the mouse after modeling is finished;

b, after the mouse is anesthetized, fixing the mouse on the back, cutting the chest cavity, and exposing the heart;

c, inserting an infusion needle connected with 0.9% physiological saline into the left ventricle of the mouse, immediately cutting off the right auricle of the mouse, opening an infusion switch, perfusing until the liver becomes white, immediately cutting off the head to obtain TNC, and respectively taking a proper amount of TNC to be stored in TRIzon Reagent and tissue fixing solution;

d, laying the mouse on the stomach, and separating the peripheral connective tissues at the lower part of the left chest diaphragm to obtain the spleen;

e cut portions and store in TRIzon Reagent.

RNA extraction and real-time fluorescent quantitative PCR

The method a is the same as the method.

TNC Paraffin section c-fos immunohistochemistry

a after TNC is stored in a tissue fixative for >24 hours, trimming TNC in a fume hood;

b, placing the trimmed TNC into a dehydration box, sequentially carrying out gradient alcohol dehydration in a dehydration machine, wherein 75% ethanol is 4 hours, 85% ethanol is 2 hours, 90% ethanol is 2 hours, 95% ethanol is 1 hour, absolute ethanol I is 0.5 hour, absolute ethanol II is 0.5 hour, alcohol benzene is 5-10 min, xylene I is 5-10 min, xylene II is 5-10 min, 65% melted paraffin I is 1 hour, 65% melted paraffin II is 1 hour, and 65% melted paraffin III is 1 hour;

embedding the tissues soaked with the wax, selecting a proper position, and freezing at-20 ℃;

d, slicing the cooled wax block with the thickness of 4 microns, flattening and floating in a spreading machine, taking out the glass slide, putting the glass slide into a 60 ℃ oven, baking the glass slide, and drying for 1-2 hours;

e, sequentially putting the baked slices into dimethylbenzene I, dimethylbenzene II and dimethylbenzene III for 15 min respectively, then putting absolute ethyl alcohol I and absolute ethyl alcohol II for 5min respectively, then putting 85% alcohol and 75% alcohol for 5min respectively, and then cleaning with distilled water;

f, putting the slices into citric acid antigen repairing buffer solution (pH 6.0), heating with a microwave oven for 9min to boil, stopping heating for 7 min and heating for 6 min, naturally cooling, and washing with PBS for 3 times and 5 min/time;

g, putting the mixture into a 3% hydrogen peroxide solution, incubating the mixture for 25 min at room temperature in a dark place, and then putting the mixture into PBS for washing for 3 times and 5 min/time;

dropwise adding 3% BSA, and sealing at room temperature for 0.5 h;

i, throwing off confining liquid, dropwise adding the prepared primary antibody, and keeping the temperature in a wet box overnight at 4 ℃;

j PBS wash 3 times, 5 min/time; slightly spin-drying the slices, dripping secondary antibody, and incubating at room temperature for 50 min;

washing with PBS for 3 times and 5 min/time, slightly drying the slices, dripping the ready-prepared DAB color developing solution, observing under a microscope to control the color developing time, and washing with tap water to stop color development;

l, counter-staining hematoxylin for about 3 min, gently rinsing with tap water, differentiating hematoxylin differentiation solution for several seconds, gently rinsing with tap water, returning hematoxylin to blue, and rinsing with running water;

sequentially adding slices m into 75% alcohol, 85% alcohol, anhydrous ethanol I, anhydrous ethanol II and xylene for 5min respectively until the slices are transparent, slightly drying, and sealing with neutral gum;

and (n) performing microscopic examination under a microscope, and acquiring and analyzing images.

Third, statistical analysis

Data analysis and processing were performed using GraphPad Prism 8.0 software. Data are expressed in x ± s. The data were analyzed using t-test and Two-way ANOVA test, and differences of P <0.05 were statistically significant.

Example 1 Tim-3Ab effectively reduces the decrease in plantar pain threshold in NTG mice

According to the invention, a chronic migraine mouse model (9 days) is established by injecting Nitroglycerin (NTG) into the abdominal cavity, and the expression of Tim-3 in the NTG mouse is detected, so that the relation between Tim-3 and migraine is determined. The results found that the expression of Tim-3 was elevated in a Nitroglycerin (NTG) chronic migraine mouse model, suggesting that there is a link between Tim-3 and migraine.

The prophase data shows that Tim-3 is up-regulated in NTG mouse models, following which the present invention gives 6-8 week old, male mice (WT + Fc, WT + Tim-3 Ab) to NTG working fluid (10 mg/kg, i.p.) for modeling and testing plantar pain threshold using von frey fiber. As a result, compared with the basal pain threshold (Baseline) before modeling, the pain threshold of the Fc group is reduced most obviously, and Tim-3Ab intervention obviously slows down the reduction of the plantar pain threshold of the mice (as shown in figure 4, Fc in the figure represents an Fc + NTG group, Tim-3Ab represents a Tim-3Ab + NTG group, and the base line is 3 +/-0.33 (Mean +/-Sem)P<0.05, denotesP<0.01, denotesP<0.001）。

In subsequent tissue sample assays, it was observed that Tim-3Ab predrying reduced the expression of inflammatory factors and c-fos protein in a mouse model of NTG. Pain-associated inflammatory factors such as IL-6, IL-1 β and the nociceptive-stimulating marker c-fos protein were detected by QPCR and immunohistochemistry, and as a result, it was found that the expression of Tim-3Ab group was reduced compared to the control group.

Example 2 mechanism of Tim-3 Effect on migraine

Previous data have shown the effect of Tim-3 on pain, and the present invention further explores how Tim-3 affects pain. In humans, endorphin-neurons play an important role in the regulation of pain stimuli, altering pain perception through related neurotransmitters and receptors. The invention observes the influence of a cell line, mouse abdominal cavity macrophages and NTG model serum samples on the expression of Tim-3Ab endorphin, and finds that the expression level of beta-endorphin is obviously increased when Tim-3Ab exists (as shown in figure 5, Con represents an abdominal cavity macrophage group, Tim-3Ab represents an abdominal cavity macrophage + Tim-3Ab 20 ug/mL group in figure A, Con represents a WT + PBS + NTG group in figure B, Tim-3Ab represents a WT + Tim-3Ab 5mg/kg + NTG group in figure C, and Con represents BV₂Cell group, Tim-3Ab for BV₂+ Tim-3Ab 20 ug/mL, panel D, Con for THP-1 cell group, Tim-3Ab for THP-1+ Tim-3Ab 20 ug/mL). Inner partThe enkephalin has the analgesic effect, which indicates that the intervention of Tim-3Ab for relieving the reduction of the pain threshold of the mice is possibly related to the generation of the endorphin, and indicates that Tim-3Ab acts on Tim-3 for promoting the generation of the beta-endorphin, thereby playing the role of relieving the pain.

In addition, the result of the detection of the beta-endorphin on the Tim-3 knockdown cell line shows that after Tim-3 is knocked down, the intervention of the Tim-3Ab does not play a significant role in promoting the secretion of the beta-endorphin, and the Tim-3Ab plays a role in relieving pain by acting on Tim-3 to promote the generation of the beta-endorphin. In the following experiments, the invention also uses opioid antagonist naltrexone for pre-intervention to construct an NTG model, and the results of the pain threshold detection and later-stage related tissue immunohistochemical analysis show that the intervention of naltrexone ensures that the Tim-3Ab group does not show obvious advantages when compared with a control group, thereby defining the relationship between Tim-3 and beta-endorphin in pain regulation.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that it would be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit of the invention, and these modifications and variations also fall within the scope of the claims of the present invention.

Sequence listing

<110> military medical research institute of military science institute of the people's liberation army of China

<120> use of Tim-3 for preventing, treating or alleviating pain

<141> 2022-05-11

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gcaccaccac cacggaatcg 20

<210> 2

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ttgacggaag ggcaccacca g 21

<210> 3

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gatggatgct accaaactgg a 21

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tctgaaggac tctggctttg 20

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ggtctgggcc atagaactga 20

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

tgaagcagct atggcaactg 20

Claims (10)

1. A pharmaceutical composition for preventing, treating or reducing pain, comprising a binding agent to a protein encoded by Tim-3.

2. The pharmaceutical composition of claim 1, wherein the binding agent for the protein encoded by Tim-3 comprises a peptide, a peptide mimetic, a nucleic acid aptamer, a mirror image L-RNA aptamer, an ankyrin repeat protein, a Kunitz-type domain, or an antibody.

3. The pharmaceutical composition of claim 2, wherein the binding agent to the protein encoded by Tim-3 is an antibody.

4. The pharmaceutical composition of claim 3, wherein the antibody comprises a monoclonal antibody or a polyclonal antibody.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the pharmaceutical composition is in the form of a tablet, capsule, granule, powder, syrup, injection, drop infusion, suppository, eye drop or ophthalmic ointment.

6. Use of a pharmaceutical composition according to any one of claims 1 to 5 in the manufacture of a medicament for the prevention, treatment or alleviation of pain.

7. The use according to claim 6, wherein the pain is migraine.

8. The use as claimed in claim 7, wherein the migraine headache comprises paroxysmal migraine, chronic migraine, retinal migraine, ophthalmoplegic migraine, non-headache migraine, migraine disorders, menstrual migraine, abdominal migraine, childhood periodic syndrome, cluster headache.

9. The use of claim 8, wherein the migraine is chronic migraine.

10. The application of the reagent for detecting the expression level of Tim-3 in the preparation of products for diagnosing migraine.

Images