Novel pharmaceutical composition for the treatment of dystonia
阅读说明：本技术 用于治疗肌张力障碍的新型药物组合物 (Novel pharmaceutical composition for the treatment of dystonia ) 是由 金大洙 金庭垠 于 2018-11-01 设计创作，主要内容包括：本发明涉及一种用于治疗肌张力障碍或缓解肌张力障碍患者由肌强直性疾病引起的疼痛的新型药物组合物。特别地,本发明提供了一种包含5-羟色胺受体5-HT<Sub>2A</Sub>抑制剂作为有效成分、用于治疗肌张力障碍或缓解由肌张力障碍引起的疼痛的药物组合物。(The present invention relates to a novel pharmaceutical composition for the treatment of dystonia or for relieving pain caused by myotonic disease in a dystonia patient. In particular, the invention provides a composition comprising 5-hydroxytryptamine receptor 5-HT 2A Inhibitor as active ingredient for treating or relieving dystoniaA pharmaceutical composition for treating pain.)
1. A pharmaceutical composition for treating dystonia or alleviating pain caused by myotonic disease comprising 5-hydroxytryptamine receptor 5-HT2AAn inhibitor as an active ingredient.
2. The pharmaceutical composition of claim 1, wherein the myotonic disorder is caused by dystonia, cerebral palsy, myotonic dystrophy, or spinal myopia.
3. The pharmaceutical composition of claim 1, wherein the 5-HT is2AThe inhibitor is 5-HT2AAntagonists or 5-HT2AAn inverse agonist.
4. The pharmaceutical composition of claim 1, wherein the 5-HT is2AThe antagonist is the same as the 5-HT2AAn antagonist antibody that binds specifically or a functional fragment of said antagonist antibody, clozapine, olanzapine, quetiapine, risperidone, ziprasidone, aripiprazole, asenapine, amitriptyline, clomipramine, cyproheptadine, eliserpentine, etoperidone, haloperidol, hydroxyzine, iloperidone, ketanserin, mexicam, mianserin, mirtazapine, nefazodone, pimaxoline, pizothidine, ritanserin, trazodone, or yohimbine.
5. The pharmaceutical composition of claim 3, wherein the 5-HT is2AThe antagonist is a selective 5-HT not acting on other types of 5-hydroxytryptamine receptors2AAntagonists or 5-HT2A/2CA dual antagonist.
6. The pharmaceutical composition of claim 5, wherein the 5-HT is2AThe selective antagonist is eliserin, 2-alkyl-4-aryl-tetrahydro-pyrimidine-azaAMDA (9-aminomethyl-9, 10-dihydroanthracene), hydroxyzine, benzothiadiadine, 5-methoxy-N- (4-bromobenzyl) tryptamine (5-MeO-NBpBrT), glaserine, nipramine, pimaxoline, florisiline or LY-367265.
7. The pharmaceutical composition of claim 5, wherein the 5-HT is2A/2CThe dual antagonist is ritanserin, ketanserin, cyproheptadine, AC-90179, trazodone or etoperidone.
8. The pharmaceutical composition of claim 4, wherein said functional fragment of an antagonist antibody is Fab, Fab ', F (ab')2scFv, diabody, triabody, sdAb, VHH. Nanobodies, single antibodies, Variable Lymphocyte Receptors (VLR), Affiln, Affimer, Affinin, Avimer, DARPin, Fynomer, or Affibody.
9. The pharmaceutical composition of claim 3, wherein the 5-HT is2AThe inverse agonist is AC-90179, pimaracin, netilmicin, flurliperin or eletriperin.
10. The pharmaceutical composition of claim 1, wherein the myotonic disorder is caused by excessive stress, an abnormal increase in 5-hydroxytryptamine following administration of the selective 5-hydroxytryptamine reuptake inhibitor, or an abnormal activation of the 5-hydroxytryptamine cycle.
11. The pharmaceutical composition of claim 10, wherein the selective 5-hydroxytryptamine reuptake inhibitor is citalopram, dapoxetine, escitalopram, fluvoxamine, paroxetine, fluoxetine, sertraline, zimelidine, or vortioxetine.
12. A method of screening for a therapeutic drug candidate for treating dystonia or alleviating pain caused by dystonia, comprising:
observation of whether a series of test compounds or natural products inhibit the 5-hydroxytryptamine receptor 5-HT2A(ii) a And
selected for identification as inhibiting 5-HT2AThe test compound or natural product of (1).
13. The method of claim 12, further comprising:
confirmation of empirical inhibition of 5-HT2AWhether a test compound or natural product of (2) inhibits 5-HT2AThe function of the other 5-hydroxytryptamine receptor; and
selective removal of 5-HT2ATest compounds or natural products which do not inhibit the function of the 5-hydroxytryptamine receptor.
14. The method of claim 12, wherein the inhibition of 5-hydroxytryptamine receptor 5-HT by a series of test compounds or natural products is observed by various in vitro, in vivo or in silico assays2A。
15. The method of claim 14, wherein the in vitro assay is performed by contacting 5-hydroxytryptamine receptor 5-HT2AReacting a ligand thereof (e.g. 5-hydroxytryptamine) with a test compound or a natural product, and selecting for inhibition of the 5-hydroxytryptamine receptor 5-HT2AA test compound or a natural product that binds to the ligand.
16. The method of claim 14, wherein the in vitro assay comprises:
by expression of 5-hydroxytryptamine receptor 5-HT2AAfter treatment of the test compound or natural product with the cells of (3), 5-HT is assayed2AThe concentration or activity of downstream signaling molecules; and
selective inhibition of 5-HT2AA test compound or natural product of downstream signal molecule concentration or activity.
17. The method of claim 14, wherein the signal molecule is inositol triphosphate (IP)3) Diacylglycerol (DAG), Arachidonic Acid (AA), 2-arachidonic acid glycerol (2-AG), Ca2+Or a PKC.
18. The method of claim 14, wherein the in vivo analysis method comprises:
subjecting a toddler with a calcium channel mutation of type P/Q to stress to induce stress;
administering a test compound or natural product to a toddler under pressure; and
test compounds or natural products were selected that significantly reduced dystonia scores in toddlers.
19. The method of claim 14, wherein the in vivo analysis method comprises:
in addition to measuring dystonia scores for toddlers, genes encoding calcineurin proteins were locally transduced into the cerebellum of toddlers with P/Q type calcium channel mutations;
inducing stress in the toddler by placing the animal under stress;
administering a test compound or natural product to a toddler under pressure;
measuring the amount of calsenor protein that binds calcium in the cerebellum of a toddler; and
selecting a test compound or natural product that significantly reduces the amount of calcium sensor protein that binds calcium.
20. The method of claim 19, wherein the calcium sensor protein is Yellow Camelon (YC), Inverse-Pericam, camgro, TN-L15, SynapCam, or GcaMP.
21. The method of claim 20, wherein GcaMP is GcaMP1, GcaMP2, GcaMP3, GcaMP4, GcaMP5, or GcaMP 6.
22.5-Hydroxytryptamine receptor 5-HT2AUse of an inhibitor for the manufacture of a pharmaceutical formulation for the treatment of dystonia or for relieving pain caused by dystonia.
Technical Field
The present application claims priority from korean patent application No.2017-0160506, filed on 28/11/2017. The contents of the above-mentioned patent applications are incorporated herein by reference in their entirety.
The present invention relates to a novel pharmaceutical composition, and more particularly, to a novel pharmaceutical composition for the treatment of dystonia.
Background
Dystonia is a condition characterized by involuntary abnormal motor phenomena that, regardless of one's will, can result in incorrect posture or distorted muscle movement. This is a neurological disease. These symptoms result in involuntary contraction of muscles, resulting in abnormal movements and odd postures, such as muscle twisting or repetitive movements. Dystonia can occur as a secondary symptom to a genetic or specific cause disease. However, as to the pathogenesis of dystonia, similar symptoms to dystonia appear when GABA antagonists such as bicuculline are injected into animals, and therefore, dystonia is only assumed to develop when there is a problem with the neural network associated with the neurotransmitter GABA (Inase et al, J.Neuropysiol.75: 1087-1104,1996), but the exact pathogenesis is not clear.
In addition, at present, it is clinically used
On the other hand, as a method of treating dystonia, an electrical stimulation treatment apparatus has been proposed so far to perform local electrical stimulation to a muscle region where dystonia symptoms occur (US20160296756a 1). However, the prior art does not greatly affect the muscle tone caused by the stress factor.
Disclosure of Invention
The present invention has been made to solve a number of problems including the above problems, and more particularly, the present invention provides a novel pharmaceutical composition for treating dystonia, particularly dystonia due to stress, and relieving pain caused by dystonia. However, the scope of the present invention is not limited thereto.
In one aspect of the present invention, there is provided a pharmaceutical composition for treating dystonia or alleviating pain caused by myotonic disease comprising 5-hydroxytryptamine receptor 5-HT2AAn inhibitor as an active ingredient.
In another aspect of the present invention, there is provided a method of screening a therapeutic substance candidate for treating dystonia or alleviating pain caused by dystonia, comprising:
observation of whether a series of test compounds or natural products inhibited the 5-hydroxytryptamine receptor 5-HT2A(ii) a And
selected for identification as inhibiting 5-HT2AThe test compound or natural product of (1).
In another aspect of the invention, there is provided a method of treating dystonia in a subject with symptoms of dystonia due to stress, the method comprising administering to the subject a therapeutically effective amount of 5-hydroxytryptamine receptor 5-HT2AAnd (3) an inhibitor.
In another aspect of the invention, there is provided a method of reducing pain caused by dystonia in a subject with symptoms of dystonia caused by stress, the method comprising administering to the subject a therapeutically effective amount of 5-hydroxytryptamine receptor 5-HT2AAnd (3) an inhibitor.
In another aspect of the invention, there is provided a method of preventing dystonia in a subject associated with dystonia caused by stress, the method comprising administering to the subject a therapeutically effective amount of 5-hydroxytryptamine receptor 5-HT2AAnd (3) an inhibitor.
In another aspect of the invention, 5-hydroxytryptamine receptor 5-HT is provided2AUse of an inhibitor for the manufacture of a medicament for the treatment of dystonia or for relieving pain caused by dystonia.
Advantageous effects
As described above, according to the embodiments of the present invention, dystonia caused by extreme stress can be effectively treated and prevented in addition to genetic factors. Of course, the scope of the present invention is not limited to these effects.
Drawings
FIG. 1 shows the results of observing the symptoms of dystonia in a dystonia model animal by various analytical methods according to a specific embodiment of the present invention. (A) A photographic (lower) overview of the animal experiments (upper) and hind limb hyperextension of experimental animals in dystonic posture in accordance with a particular embodiment of the present invention; (B) recording a graph of dystonia score for toddlers placed in an open field box as a function of time of exposure to ambient pressure; (C) measuring Electromyograms (EMG) of extensors (gastrocnemius) and tibialis anterior muscles in a normal state and in dystonia respectively; (D) comparison of mean RMS of extensor and tibialis anterior (reflecting muscle contraction or muscle tone) at dystonia and at rest; (E) a series of graphs showing the co-contraction rate of two muscles (gastrocnemius and tibialis anterior) following administration of vehicle (control) and three types of 5-hydroxytryptamine receptor antagonists (MDL100907, Way100135, and ondansetron) to a dystonic model animal; (F) a series of graphs showing the number of dystonic events (left) and dystonic score (right) after administration of various 5-hydroxytryptamine receptor antagonists; (G) a graph showing the relationship after administration of various 5-hydroxytryptamine receptor antagonists is shown.
Figure 2 shows a venn diagram for each of the "antagonist" and "inverse agonist". Wherein the "antagonist" inhibits activation of the receptor by interfering with binding to the agonist but does not affect the activation/inactivation balance; an "inverse agonist" is a drug that stabilizes a target molecule with an inactive structure.
FIG. 3 shows the 5-HT of various 5-hydroxytryptamine receptors2AAdministration of the antagonist to measure the results of the symptoms of dystonia in a dystonia model animal. (A) A series of graphs showing the results of measuring the number of dystonic events (left) and dystonic score (right) after dose-dependent administration of MDL100907 to dystonic model animals, (B) a series of graphs showing the results of showing the dystonic score (right)Disorder model animals were administered vehicle (control) and three types of 5-hydroxytryptamine receptor 5-HT2AGraphs of the results of dystonic events (left) and dystonic scores (right) following antagonists (1mg MDL100907, pimaxaline, and glaserin); and (C) is a graph showing the results obtained by changing the concentration of MDL100907 administered to group B to 2mg alone.
Figure 4 shows the results of real-time fluorescence analysis in the cerebellum of mice locally transduced with GCaMP6 according to one embodiment of the present invention. (A) Overview of fluorescence micrographs for the real-time fluorescence analysis system of the invention (left) and cerebellar sections extracted from experimental animals (right); (B) a graph showing the rate of change of fluorescence according to the behavioral state of the experimental animal; (C) hit plots of initial partial oriented fluorescence changes associated with dystonia in the control group; (D, E) a series of graphs showing the rate of change in fluorescence in the control group and the MDL 100907-administered group, respectively, indoors and after transfer to an open-field chamber; and (F) shows a graph of the rate of change of fluorescence according to exposure time to ambient pressure after the control group and the drug-administered group were indoors and moved to an open-field chamber.
Detailed Description
Definition of terms
The term "dystonia" as used herein is an involuntary abnormal movement that results in a sustained posture or a distorted muscle movement, regardless of the person's will. As a neurological disease characterized by symptoms, there is known familial or stress-related dystonia. In particular, stress-related dystonia may be caused by extreme stress prior to performance or presentation reporting, even without other genetic predisposition.
The term "cerebral palsy" as used herein refers to a state in which motor function is paralyzed due to damage to the brain, and is currently known to be caused by brain damage at birth, symptomatic jaundice of a newborn, and meningitis. The most important symptom is the improper control of the neuromuscular system, and about 70% to 80% of cerebral palsy patients are known to suffer spastic cerebral palsy, which results in muscle stiffness and difficulty in movement, resulting in abnormal muscle tone.
The term "myotonic dystrophy" also known as steinernemia disease, as used herein, is a disease in which muscles are gradually weakened. Symptoms of this disease can occur in various types in various parts of the body. The symptom is muscle stiffness. Generally, muscle relaxation occurs after muscle contraction. In patients with this disease, muscle relaxation progresses slowly and the overall muscle is stiff.
The term "spinal bulbar muscular atrophy", also known as kennedy's disease, as used herein, is a sex-related genetic disorder caused by mutations in the androgen receptor gene on the X chromosome. When gene duplication occurs, the function of androgen receptor does not work well, resulting in the degeneration of motor neurons. Symptoms do not appear before the age of 30 years, but after 10-20 years, muscle disorders such as muscle stiffness begin to have severe attacks, require wheelchairs, and speech or swallowing become impaired.
The term "5-hydroxytryptamine receptor" as used herein refers to a receptor that binds to the neurotransmitter 5-hydroxytryptamine (5-HT) and causes physiological activity in cells. In mammals, it is present in high concentrations in the peripheral nervous system and brain nuclei, substantia nigra, globus pallidus, basal ganglia and choroid plexus, and it is known that they modulate K by either G-protein or cAMP as secondary signaling molecule+Or Ca2+And (3) ion channels. As is well known, 5-hydroxytryptamine receptors include 5-HT1A、5-HT1B、5-HT1D、5-HT1E、5-HT1F、5-HT2A、5-HT2B、5-HT2C、5-HT3、5-HT4、5-HT5A、5-HT5B、5-HT6And 5-HT7These 14 subtypes. These subtypes are known to differ slightly in function, antagonist, agonist, and the like.
As used herein, the term "antagonist" refers to a ligand or drug that acts to block or attenuate a biological response by binding to a target molecule and blocking its function, rather than activating the target molecule as an agonist.
As used herein, the term "selective antagonist" refers to a ligand or drug that selectively blocks only the function of a particular subtype among target molecules having various subtypes.
As used herein, the term "dual antagonist" refers to an antagonist that inhibits two or more subtypes that are structurally closely related to a target molecule having various subtypes. For example, 5-HT as used in the present invention2AAnd 5-HT2CAre very close receptors in structure, and there are differences that inhibit only these two subtypes by distinguishing them from the other subtypes without distinguishing them. However, while they have the advantage of fewer side effects than non-discriminatory non-selective antagonists, they have more side effects than selective antagonists.
As used herein, the term "inverse agonist" refers to a drug that stabilizes a target molecule to an inactive structure. In general, a target molecule such as a receptor has an activation structure and an inactivation structure in an equilibrium state, and the equilibrium in the activation structure tends to depend on an agonist. Antagonists interfere with activation by interfering with agonist binding, but do not affect the activation/inactivation balance. Inverse agonists act by tilting the equilibrium state to an inactive structure and thus may permanently inhibit the activity of the receptor.
As used herein, the term "antagonist antibody" refers to an antibody that binds to a particular target protein and inhibits the physiological activity of the target protein.
The term "functional fragment of an antibody" as used herein refers to a fragment of an antibody, e.g., Fab ', F (ab')2They have a conserved antigen binding site created by cleavage of the antibody with a protein cleaving enzyme, or scFv, diabody, triabody, which are single chain antibody analogs that connect the antibody heavy chain variable region (VH) and the antibody light chain variable region (VL) through a linker, or more generally, other single chain antibody analogs include sdAb, VHH. Nanobodies, monobodies (monobodies), Variable Lymphocyte Receptors (VLR), Affilin, Affimer, Affitin, affinity multimers (Avimer), DARPin, Fynomer, affinity antibodies (affibodies).
The term "Fab" as used herein is an antigen-binding fragment, a fragment produced by cleavage of an antibody molecule with a protease, papain, which isFrom two peptides VH-CH1 and VL-CL(iii) a constituent heterodimer. Another fragment produced by papain is called the crystallizable fragment (Fc).
The term "F (ab')2"is a fragment containing the antigen binding site generated by cleavage of an antibody with protease, pepsin, and refers to a tetrameric form in which two fabs are linked by a disulfide bond. Another fragment produced by pepsin is called pFC'.
The term "Fab '" as used herein is an antibody fragment which shares similarities with Fab and is produced by reduction of F (ab')2Resulting in a heavy chain portion slightly longer than the Fab.
The term "scFv" as used herein refers to a single chain variable fragment which is the variable region (V) between the Fab of an antibody by a linker peptideHAnd VL) Recombinant antibody fragments prepared as single chains were ligated.
The term "sdAb (single domain antibody)" as used herein, referred to as nanobody, is an antibody fragment comprising a single antibody variable region fragment. Heavy chain-derived sdabs have been used in large numbers, but it has been reported that a single variable region fragment derived from the light chain also binds specifically to an antigen.
The term "V" as used hereinHH "is a variable region fragment of an IgG heavy chain that consists only of dimers of the heavy chain found in camelids and has the smallest size (-15 kD) among antibody fragments that specifically bind antigen. It is marketed by Ablynix under the trademark Abynix
As used herein, "Fv (fragment variable)" is a variable chain consisting of only the heavy and light chains of an antibody (V)HAnd VL) A composed dimeric antibody fragment of a size between the sdAb and the Fab (about-25 kD). By hydrolyzing proteins or by subjecting V to specific conditionsHAnd VLThe coding gene is inserted into an expression vector and expressed.
As used herein, "diabodies"is V by shortening scFvHAnd VLThe length of the linkage between (5a.a) so that the two scfvs form a dimer with higher antigen specificity than conventional scfvs.
The term "calcineurin" as used herein is a fluorescent protein genetically engineered to measure changes in calcium ion concentration in cells by changes in fluorescence, also known as fluorescent Ca2+Indicator Protein (FCIP). It is primarily a fusion protein in which a calcium ion binding protein (e.g., calmodulin) is linked to a fluorescent protein (e.g., GFP) via a linker. It is used to detect the concentration of intracellular calcium ions by detecting fluorescence emitted only when calcium ions are bound by a direct structural change, or by a Fluorescence Resonance Energy Transfer (FRET) phenomenon between two fluorescent proteins according to a structural change upon binding to calcium ions.
Detailed Description
In one aspect of the present invention, there is provided a pharmaceutical composition for treating dystonia or alleviating pain caused by myotonic disease comprising 5-hydroxytryptamine receptor 5-HT2AAn inhibitor as an active ingredient.
As used herein, the concept of the term "treatment" encompasses the elimination of pathological causes and the amelioration or alleviation of symptoms.
In the composition, the myotonic disorder can be caused by dystonia, cerebral palsy, myotonic dystrophy, or spinal bulbar atrophy. Tonic symptoms include not only dystonia, but also various neurological diseases, especially cerebral palsy (Asagai et al, Laser Therapy,14(4): 171-. Myotonic disorders may be caused by overstressing, an abnormal increase in 5-hydroxytryptamine or an abnormal activation of the 5-hydroxytryptamine cycle following administration of a selective 5-hydroxytryptamine reuptake inhibitor. In particular, myotonia may be caused by the side effect of a selective 5-hydroxytryptamine reuptake inhibitor ('SSRI') which is an antidepressant drug that treats depression by increasing the concentration of 5-hydroxytryptamine at extracellular levels by inhibiting reuptake of cells (presynaptic cells). However, in some patients, as a side effect of SSRI, there is a problem of muscle tension due to an excessive increase in 5-hydroxytryptamine in synapses, and therefore, patients with muscle tension often stop taking SSRI (Mossavi et. al., Global. J. health Sci.6(6):295-299, 2014). Thus, the pharmaceutical compositions of the present invention are useful for preventing or alleviating the symptoms of dystonia in depression patients prescribed with SSRI. In this case, the selective 5-hydroxytryptamine reuptake inhibitor may comprise citalopram (citalopram), dapoxetine (dapoxetine), escitalopram (escitalopram), fluvoxamine (fluvoxamine), paroxetine (parooxetine), fluoxetine (fluoxetine), sertraline (sertraline), zimelidine (zimeldin) or vortioxetine (vortioxetine).
In the pharmaceutical composition, 5-HT2AThe inhibitor may be 5-HT2AAntagonists or 5-HT2AInverse agonists, and 5-HT2AAntagonists specifically bind to 5-HT2A。5-HT2AThe antagonist may be an antagonistic antibody or a functional fragment of an antagonistic antibody or clozapine (clozapine), olanzapine (olanzapine), quetiapine (quetiapine), risperidone (risperidone), ziprasidone (ziprasidone), aripiprazole (aripiprazole), asenapine (asepinene), amitriptyline (amitriptyline), clomipramine (clomipramine), cyproheptadine (cyproheptadine), elicline (eplavastin), etoperidone (etoposide), haloperidol (haloperidol), hydroxyzine (hydroxyzine), ilopipinone (iloperidine), ketanserin (tryptiline), mexiletine (methyserpin), chromacline (chromacline), thiamine (tryptiline), thiamethoxazole (triazepine), or triazepine (azone). Or, 5-HT2AThe antagonist may be a selective antagonist that does not act on other types of 5-hydroxytryptamine receptors or 5-HT2A/2CA dual antagonist. 5-HT2AThe selective antagonist may be an antagonist ofLissenilin (eplivanserin), 2-alkyl-4-aryl-tetrahydro-pyrimidine-aza
At the same time, 5-HT2AThe inverse agonist may be AC-90179, pimavanserin (pimavanserin), netanotryptin (nelotanserin), fluranserin (volinanserin) or elinserin (eplivanserin).
The functional fragment of the antagonist antibody can be Fab, Fab ', F (ab')2scFv, diabody, triabody, sdAb, VHH. Nanobodies, single antibodies, Variable Lymphocyte Receptors (VLR), Affiln, Affimer, Affinin, Avimer, DARPin, Fynomer, or Affibody.
The pharmaceutical compositions of the present invention may comprise at least one pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a variety of oral or parenteral formulations, but is preferably a parenteral formulation. In the case of formulations, they are prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants and surfactants. The oral solid preparation comprises tablets, pills, powder, granules, capsules and the like. These solid formulations can be prepared by mixing at least one compound and at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, and the like. It is prepared by mixing. In addition, lubricants such as magnesium stearate and talc may be used in addition to simple excipients. Liquid preparations for oral administration include suspensions, oral liquids, emulsions, syrups and the like, and these liquid preparations may contain, in addition to water and paraffin, other various excipients such as wetting agents, sweeteners, flavors and preservatives, which are generally used as diluents. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations and suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As the base of the suppository, there can be used vebisol, polyethylene glycol, tween 61, cacao oil, bay oil, glycerogelatin, and the like.
The pharmaceutical composition of the present invention may be any one selected from the group consisting of: tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilisates and suppositories.
The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, administration can be carried out by various routes such as intravenous injection, intranasal inhalation, intramuscular administration, intraperitoneal administration, transdermal absorption, and the like.
The pharmaceutical compositions of the present invention may be administered in a pharmaceutically effective amount.
The term "pharmaceutically effective amount" as used herein refers to an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and an effective dose may be determined according to factors including type, severity, age, sex, pharmaceutical activity, drug sensitivity of a subject, administration time, administration route, excretion, duration of treatment, drug used at the same time, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered at a dose of 0.1mg/kg to 1g/kg, more preferably 1mg/kg to 500 mg/kg. Meanwhile, the administration dose may be appropriately adjusted according to the age, sex and condition of the patient.
In another aspect of the present invention, there is provided a method of screening a therapeutic substance candidate for treating dystonia or alleviating pain caused by dystonia, comprising:
observation of whether a series of test compounds or natural products inhibited the 5-hydroxytryptamine receptor 5-HT2A(ii) a And
selection identified as inhibiting 5-HT2AThe test compound or natural product of (1).
In another aspect of the invention, a method of treating dystonia is provided, comprising administering 5-hydroxytryptamine receptor 5-HT to a subject suffering from symptoms of dystonia caused by stress2AAnd (3) an inhibitor.
The screening method may comprise determining that inhibition of 5-HT has been confirmed2AWhether the test compound or natural product of (2) inhibits 5-HT2AThe function of the other 5-hydroxytryptamine receptor; and selectively remove 5-HT2AA test compound or natural product that does not inhibit 5-hydroxytryptamine receptor function.
The determination has confirmed that 5-HT2 is inhibitedAWhether the test compound or natural product of (2) inhibits 5-HT2AThe function of the other 5-hydroxytryptamine receptor can be performed by various methods, such as in vitro, in vivo, or in silico analysis. In vitro methods may use the 5-hydroxytryptamine receptor 5-HT2A、5-HT2AWith a test compound or natural product and selectively inhibits the 5-hydroxytryptamine receptor 5-HT2AA test compound or natural product that binds to a ligand. Determining whether a test compound or natural product inhibits the 5-hydroxytryptamine receptor 5-HT2ABinding to ligands, interactions between various target proteins and their ligands can be analyzed by a variety of methods, including radioisotope-labeled cellular complex chromatography, Drug Affinity Reaction Target Stability (DARTS), oxidation rate protein Stability (SPROX), Differential Static Light Scattering (DSLS) analysis, differential scanning fluorescence measurement (DSF), and differential radial capillary ligand analysis (DraCALA), and such analytical methods have been described in detail by McFedries et al (chem.biol., 20: 667-. The above documents are incorporated herein by reference.
Alternatively, the in vitro method may comprise treating a sample expressing 5-hydroxytryptamine receptor 5-HT with a test compound or a natural product2AAfter the cells of (4), 5-HT was analyzed2AThe concentration or activity of downstream signaling molecules; selective inhibition of 5-HT2ATest compounds or naturally occurring products of downstream signal molecule concentration or activityA compound (I) is provided.
The signal molecule may be a triphosphate (IP)3) Diacylglycerol (DAG), Arachidonic Acid (AA), 2-arachidonic acid glyceride (2-AG), Ca2+Or a PKC.
In a screening method, the in vivo method comprises inducing stress by subjecting a toddler having a P/Q type calcium channel mutation to stress; administering a test compound or natural product to a toddler under pressure; test compounds or natural products were selected that significantly reduced dystonia scores in toddlers.
The term "stress" as used herein refers to the psychological and physical stress felt by a subject when exposed to a poorly adapted environment. Thus, "stress state" refers to a state in which a subject is exposed to strange and strange environments. These stress states are when a person encounters a new environment, such as contacting a new person, emerging at a new location, undertaking a new task, moving to a new area, or going to a higher school. If a person develops myodystonia under stress, his/her social life is significantly hampered.
Optionally, the in vivo method may comprise, in addition to measuring dystonia score for a toddler, locally transducing a gene encoding a calcineurin into the cerebellum of the toddler having a mutation in a P/Q type calcium channel; inducing stress in a toddler by placing the animal under stress; administering a test compound or natural product to a toddler under pressure; measuring the amount of calsensorin protein bound to calcium in the cerebellum of the toddler; and selecting a test compound or natural product that significantly reduces the amount of calcium sensor protein that binds calcium.
In the screening method, the calsenor protein is Yellow Cameleon (YC), reverse-Pericam (Nagaiet al, Proc. NatI. Acad. Sci. USA.98(6): 3197. times. 3202,2001), Camgoroo (Baird et al, Proc. NatI. Acad. Sci. USA.,96(20): 11246, 1999; Griesbeck et al, J.biol. chem.276(31): 29188. times. 29194,2001), TN-L15(Heim and Griesbeck, J.biol. chem. 279(14): 14280. times. chem. 14286,2004), SynapCam (Guerrere 118et al, Nat. Neurosci. 8(9): 8,2005) or GCaMP 1196. The YC may be YC 2.1, YC 3.1, YC 2.12, YC 3.12 or YC 3.60, the Camgoroo may be Camgoroo-1 or Camgoroo-2, and the GCaMP may be GCaMP1, GCaMP2, GCaMP3, GCaMP4, GCaMP5 or GCaMP 6. Among them, GcaMP is a calcium sensor originally developed by Junichi Nakai, which is a fusion protein consisting of cpEGFP, calmodulin and M13, and M13 is a peptide sequence derived from myosin light chain kinase, and has a property of exhibiting fluorescence in proportion to the intracellular calcium ion level. It can be used to measure intracellular calcium ion levels and is particularly useful for measuring the concentration of calcium ions in particular tissues or cells in living cells or in vivo in animals in real time (Nakai et al, Nat. Biotehonol.,19:137-141, 2001).
In another aspect of the invention, there is provided a method of treating dystonia in a subject suffering from symptoms of dystonia caused by stress, the method comprising administering to the subject a therapeutically effective amount of 5-hydroxytryptamine receptor 5-HT2AAnd (3) an inhibitor.
In this method, the 5-hydroxytryptamine receptor 5-HT2AThe inhibitor is as described above.
In another aspect of the invention, there is provided a method of reducing pain caused by dystonia in a subject suffering from symptoms of dystonia caused by stress, the method comprising administering to the subject a therapeutically effective amount of 5-hydroxytryptamine receptor 5-HT2AAnd (3) an inhibitor.
In this method, the 5-hydroxytryptamine receptor 5-HT2AThe inhibitor is as described above.
In another aspect of the invention, there is provided a method for preventing dystonia in a subject associated with dystonia caused by stress, the method comprising administering to the subject a therapeutically effective amount of the 5-hydroxytryptamine receptor 5-HT2AAnd (3) an inhibitor.
In this method, the 5-hydroxytryptamine receptor 5-HT2AThe inhibitor is as described above.
In another aspect of the invention, there is provided 5-hydroxytryptamine receptor 5-HT2AInhibitor for treating dystonia or relieving pain caused by dystoniaUse in an agent.
In this use, the 5-hydroxytryptamine receptor 5-HT2AThe inhibitor is as described above.
The inventors used toddlers prepared by inducing a mutation in a P/Q type calcium channel that when sustained pressure was applied to the experimental animals, caused the symptoms of dystonia to worsen with the severity of muscle tone. By measuring dystonia score and Electromyography (EMG), it was confirmed that dystonia symptoms worsened with time, and when 5-HT was administered to the experimental animals1A、5-HT2AAnd 5-HT3When the antagonist is selective, only selective 5-HT is confirmed2AAntagonists may alleviate the symptoms of dystonia (see figure 1). In addition, by selective 5-HT2AAntagonist pair 5-HT2AThe binding affinity of the receptor was compared with the degree of remission of dystonia to screen the effect of the drug (see figure 2 and table 1). In particular, pimaxoline and MDL100907 can act as both antagonist and inverse agonist, significantly reducing the symptoms of dystonia compared to glaserine acting only as antagonist (see figure 3).
TABLE 1 Primary Selective 5-HT2ASeveral Properties of the antagonist
In addition, 5-HT is considered2AIn the fact that it is involved in calcium release into cells, the present inventors measured the concentration of calcium ions in the cerebellum of a stress-induced dystonia model animal using the GCaMP6 method. As a result, the calcium ion concentration in the cerebellum of an animal exhibiting directly the symptoms of dystonia was significantly increased, but it was confirmed that in 5-HT2AIn the group to which the selective antagonist was administered, the increase in the concentration of calcium ions in the cerebellum was suppressed (see FIG. 4).
As can be seen from the above experimental results, the inclusion of selective 5-HT according to one embodiment of the present invention2AThe pharmaceutical compositions of the antagonists can be effectively used in patients suffering from dystonia, in particular for dystonia caused by stress conditionsA patient. In addition, an effective option for preventing the onset of dystonia is to prescribe to the patient, prior to the stress condition, a pharmaceutical composition according to the invention which can prevent the symptoms of dystonia and improve the quality of life of the patient. In addition, by reducing the influence of the muscle tone of the speaker to be presented, the performer to be shown, the player before the race in which many people are competing, and the dystonia patient, it is possible to suppress the disturbance due to the abnormal muscle tone. Generally, in case of tension, a slight muscle tone is transferred to mental burden, which is then progressed to a larger muscle tone, and the pharmaceutical composition of the present invention is considered to be effective for treatment or prevention. Typically, it is useful for vocal tremor or muscle tension when a person is presenting a presentation. In addition, it is considered that by taking the pharmaceutical composition of the present invention before and after muscle tone, it is possible to prevent and treat muscle dysfunction caused by excessive muscle tone not only in a dystonic patient but also in a normal person at an appropriate concentration.
In addition, it is believed that the discovery of therapeutic agents is effective not only for dystonia, but also for motor diseases such as parkinson's disease and tic disorder aggravated by stress.
In addition, the experimental animal model used in the present invention can confirm the efficacy of the candidate therapeutic agent by observing the myoelectric analysis and by observing whether the muscle tone is relieved by showing calcium-specific fluorescence in brain neurons. The calsenor protein can be used for screening a drug candidate for dystonia by quantitatively confirming the efficacy of the drug candidate by changing the concentration of calcium ions in a specific brain region while a living animal is moving.
In addition, to date, there is a lack of reliable relevant biomarkers for dystonia and, therefore, there is no way to differentiate between various disease mechanisms in a patient population. This may reduce the therapeutic effect or lead to a wrong diagnosis. Accordingly, the present invention provides a method for confirming 5-HT in the apical lobe nucleus region by brain imaging techniques (MRI, PET)2AA method of screening for activity of a relevant biomarker in a population of patients suffering from dystonia. It is aThe method comprises regulating 5-hydroxytryptamine content with drug and using 5-HT2AReceptor-specific radioligands [11C]NMSP confirms the receptor activity by PET imaging (Nordstrom AL et AL, int.J.Neuropsychopharmacol.11(2): 163-. 5-HT may be used2AThe receptor agonists escitalopram, citalopram or DOI modulate the content of 5-hydroxytryptamine. In this way, patients known as "task-specific dystonia" may be specifically selected, who develop symptoms at the onset of a particular activity in a patient population, and may provide effective conditions for administration and treatment.