KR20110125424A - Pain relieving agents promoting activation of ryanodine receptor and screening method thereof - Google Patents

Abstract

PURPOSE: A method for screening a pain relieving agent is provided to block pain signal and to develop analgesics. CONSTITUTION: A method for screening a pain relieving agent comprises: a step of treating candidates material to nerve cells; a step of measuring ryanodine receptor(RyR) activation in the nerve cells; and a step of selecting a candidate which increases RyR activation comparing with a control group. The nerve cells are isolated from non-human test sample. An activator and an inhibitor of RyR are caffeine and ryanodine, respectively.

Description

Pain relieving agents promoting activation of ryanodine receptor and screening method

TECHNICAL FIELD The present invention relates to the field of neurobiology, and more particularly based on the study of the effect of the activity of the Lyanodine Receptor (RyR) on the signaling of pain in the regulation of intracellular calcium channels. The invention relates to a pain relief material and a method for screening the pain relief material.

Intracellular calcium ions (Ca ²⁺ ) are primarily derived from extracellular space or intracellular calcium reservoirs (Berridge et al., 2003). Among them, the calcium release from the intracellular calcium reservoir into the cell may be induced by the Lyanodine Receptor (RyR) or the Inositol-1,4,5-triphosphate receptor (IP). Occurs through intracellular calcium ion channels such as ₃ R). In particular, RyR is electrically conductive sikineunde rapidly release calcium ions in (conductance) is within the substrate (cytosolic matrix) large cell, which is the plasma membrane voltage operability, calcium ion channels (voltage gated Ca ^{2 +} channels) present in the (plasma membrane) Caused by influx of calcium ions through Thus, a calcium ion-induced calcium release by this ^{RyR (Ca 2 + -induced Ca 2+} -release, CICR) is an immediate response to an external stimulus, thereby promoting an increase in the intracellular calcium ion. RyR is expressed in a variety of neurons in the central nervous system (Furuichi et al., 1994; Ledbetter et al., 1994), and is also referred to as thalamocortical neurons (TC nerve cells) in rodents recently. There are reports that both RyR and IP ₃ R, both intracellular calcium ion channels, are expressed (Budde et al., 2000; Pape et al., 2004). Nevertheless, the physiological function of RyR in TC neurons is still unknown.

The thalamus plays an important role in sensory signal processing that integrates sensory inputs received from peripheral sensory organs and other brain regions and delivers them to the cerebral cortex (Sherman and Guillery, 1996). Firing patterns of TC neurons are largely divided into tonic firing and low-threshold burst firing, which deliver sensory information of terminal sensory organs. It is believed that this sensory form of TC neurons reflects the state of signal transduction from the thalamus to the cerebral cortex, which is believed to block sensory information generated by the -type calcium channel and delivered to the cerebral cortex (McCormick and von Krosigk, 1992). ). In addition, RyR is known to play a role in delivering pain information to the cerebral cortex in response to various noxious stimuli (Peschanski et al., 1983; Willis and Westlund, 1997), in the hypothalamus (ventroposteriomedial, VPM) or It is reported to be highly expressed in the ventroposteriolateral (VPL) nucleus (Furuichi et al., 1994).

On the other hand, analgesics are drugs used for the purpose of stimulating the central nervous system to remove or alleviate pain, and are generally classified into narcotic analgesics and non-narcotic analgesics. Conventional analgesics have limitations that do not alleviate neurological pain, and moreover, non-narcotic analgesics have a relatively poor efficacy than narcotic analgesics. Therefore, in the case of extreme pain, narcotic analgesics such as morphine (morphine), which are known to have good analgesic effects, have been used, and such narcotic analgesics have problems such as undesired side effects such as shortness of breath, constipation, and resistance to poisoning. . In particular, when long-term treatment is required, such as pain in the nervous system, the resistance resulting from the abuse of narcotic analgesics and the continued increase in the dose of analgesics resulting from such resistance can lead to addiction to analgesics, making the problem more serious. Do. Therefore, there is an urgent need for the development of painkillers that effectively block pain without the risk of poisoning.

Accordingly, the present inventors have studied the role of RyR in the regulation of TC neuronal ignition and the potential function of RyR in pain sensory transmission. As a result, RyR in VPM or VPL nuclei may exhibit excitability and pain in TC neurons. The present invention has been completed by confirming that it plays a decisive role in the regulation of signal processing.

It is an object of the present invention to provide a pain-relieving substance that enhances the activity of lyanodine receptor (RyR) and a screening method thereof.

In order to achieve the above object, the present invention comprises the steps of: 1) separating the neurons from the subject, selecting the neurons expressed Ryanodine receptor (Ryanodine Receptor, RyR);

2) measuring the tonic firing pattern and afterhyperpolarization (AHP) in the selected neurons of step 1);

3) treating the candidate substance to the selected neurons of step 1);

4) measuring a single ignition pattern and AHP in the treated neurons of step 3);

5) comparing the measurements of step 2) with the measurements of step 4); And

6) providing a method for screening pain relief substances comprising the step of selecting candidates when the single ignition is reduced and the AHP is increased in the measurement in step 4) as compared to the measurement in step 2). do.

In addition, the present invention comprises the steps of: 1) administering a candidate substance to the neuronal tissue of the subject is expressed in the lyanodine receptor (Ryanodine Receptor, RyR);

2) causing inflammation in the subject to which the candidate substance of step 1) has been administered;

3) analyzing the pain response in the treated subject of step 2);

4) comparing the pain response of the subject administered only the vehicle, the subject administered the RyR inhibitor, and the subject administered the RyR inhibitor and the candidate together with the pain response analyzed in step 3); And

5) As a result of the comparison in step 4), the pain response of the subjects to which the RyR inhibitor was administered and the subjects to which the RyR inhibitor and the candidate were administered were increased, compared to the pain response of the subjects to which the vehicle was only administered. However, the pain response of the subject to which the candidate was administered provides a method for screening a pain mitigating agent comprising selecting a candidate that has been reduced.

The present invention also provides a composition for pain relief comprising a lyanodine receptor (RyR) activator.

The pain relief material screening method of the present invention uses the same to screen pain relief materials that can replace conventional painkillers or narcotic painkillers that do not work effectively, thereby blocking pain signals at the thalamus level and thus reducing the risk of addiction. It can be usefully used in the development of painkillers that can effectively alleviate pain.

1 is a diagram showing the results of experiments that revealed the role of RyR expression and calcium ion channel of RyR in TC neurons,
A is a spectrum of experimental results showing that the calcium ions released from the neuronal reservoir increase the intracellular calcium concentration ([Ca ^{2 +} ] _i );
B is a spectrum of experimental results showing that the release of calcium ions from the reservoir in neurons is via RyR;
C and D are spectra (C) and graphs (D) of experimental results confirming the effect of RyR on [Ca ^{2 +} ] _i increase due to rapid depolarization of membrane potential of neurons;
E shows the results of experiments confirming RyR expression in neurons by reverse transcriptase-PCR, where M is DNA size marker, N is negative control group, B is whole brain cDNA and T is thalamic VPM / VPL nuclear cDNA do.
2 is a diagram showing the results of experiments that revealed the effect of RyR activity and activity inhibition on the pattern of single ignition in TC neurons,
VII and VII of A are spectra showing changes in neuronal single firing rate before and after lyanodine treatment;
Ii and VII of A are enlarged spectra of magnitude change of post-polarization (AHP) before and after lyanodine treatment;
B is a graph showing the quantification of neuronal single firing rate (i) and post-polarization size change (ii) before and after lyanodine treatment;
VII and VII of C are spectra showing changes in neuronal single firing rate before and after caffeine treatment;
Ii and VII of C are enlarged spectra of magnitude change of post-polarization (AHP) before and after caffeine treatment;
D is a graph showing the quantification of neuronal single firing rate (i) and post-polarization size change (ii) before and after caffeine treatment.
3 is a diagram showing the results of experiments that revealed the effect of RyR activity and activity inhibition on the pattern of multiple ignition in TC neurons,
VII and VII of A are spectra showing changes in neuronal cell ignition rate before and after lyanodine treatment;
Ii and VII of A are enlarged spectra of magnitude change of post-polarization (AHP) before and after lyanodine treatment;
B is a graph showing the numerical rate of neuronal cell ignition according to the prestimulus size before and after lyanodine treatment;
VII and VII of C are spectra showing changes in neuronal cell ignition rate before and after caffeine treatment;
Ii and VII of C are enlarged spectra of magnitude change of post-polarization (AHP) before and after caffeine treatment;
D is a graph showing the numerical rate of neuronal cell ignition according to the size of the preceding stimulus before and after caffeine treatment.
4 is a diagram showing the results of an experiment showing a change in acute pain response according to the activity of RyR in TC neurons,
A is a graph showing the change in response to mechanical stimuli;
B is a graph showing the change in response to heat stimulation.
5 is a diagram showing the results of experiments showing the change in inflammatory pain response according to the activity of RyR in TC neurons,
A is a graph showing the change in response to abdominal pain caused by acetic acid induced inflammation;
B is a graph showing the change in response to pain caused by formalin-induced inflammation.

Hereinafter, the present invention will be described in detail.

The present invention comprises the steps of: 1) treating the candidate material to neurons;

2) measuring the activity of the Lyanodine Receptor (RyR) in the treated neurons of step 1); And

3) It provides a pain relief screening method comprising the step of selecting a candidate substance that increased the activity of RyR, compared to the control group not treated with the candidate substance.

The neuron of step 1) is a) separating the neuron from the subject;

b) sequentially treating the neuronal cells of step a) with a RyR activator and a RyR inhibitor;

c) measuring the change in the concentration of calcium ion ([Ca ^{2 +} ] _i ) in the neuron during the treatment of step b); And

d) as a result of the measurement in step c), the concentration of the calcium ion is increased by the RyR activator, may be selected by the step of selecting a neuron is inhibited by the RyR activity inhibitor increases the concentration of the calcium ion, The neuron of step 1) is a) separating the neuron from the non-human subject;

b) treating the neuron of step a) with a RyR activator and a RyR inhibitor, followed by treating the RyR activator;

c) measuring the change in the concentration of calcium ion ([Ca ^{2 +} ] _i ) in the neuron during the treatment of step b); And

d) As a result of the measurement in step c), the concentration of the calcium ion is increased by the RyR activator, may be selected by the step of selecting a neuron is inhibited by the RyR activity inhibitor increases the concentration of the calcium ion, It is not limited to this. In addition, the RyR activator is preferably any one of caffeine (caffeine), N-methyl-D-aspartic acid (NMDA) or derivatives thereof, and more preferably, the RyR activator is caffeine, but is not limited thereto. Not. In addition, the RyR activity inhibitor is preferably any one of ryanodine, dantrolene or derivatives thereof, and the RyR activity inhibitor is more preferably lyanodine, but is not limited thereto. .

The nerve cell may be derived from a mammal, and the nerve cell may be derived from any one of a mouse, a rat, a pig, a rabbit or a monkey, and the nerve cell may be a mouse or a rat. More preferably, it is derived from any one of the rats, and the neurons are most preferably derived from a mouse, but are not limited thereto.

The subject may be a mammal, and the subject may be any one of a mouse, a rat, a pig, a rabbit or a monkey, and the subject may be a mouse or a rat. It is more preferably any one of, the subject is most preferably, but not limited to a mouse.

Preferably, the selected neurons of step 1) are thalamic cortical neurons (TC neurons), and the selected neurons of step 1) may be intrathalamic (ventroposteriomedial, VPM) or ventral lateral (ventroposteriolateral). , VPL) is more preferably a neuron of the nucleus, but is not limited thereto.

Determination of the activity of the lyanodine receptor of step 2) is preferably performed by measuring a tonic firing aspect, or by measuring afterhyperpolarization (AHP), but is not limited thereto.

The candidate substance which increased the activity of RyR in step 3) is preferably a candidate substance which reduced single ignition of neurons or increased AHP of neurons, but is not limited thereto.

The pain is preferably neurological pain, but is not limited thereto.

In a specific embodiment of the present invention, by using whole-cell patch clamping, the analysis of the ignition before and after treatment with RyR activator and activity inhibitor in TC neurons in mouse brain slices, The multiple ignition rate of TC neurons was not affected by either lyanodine and caffeine, whereas the single ignition rate was dependent on RyR activity (see FIGS. 2 and 3). In addition, it was confirmed that the change in single ignition rate according to the RyR activity affects the inflammatory pain signal transmission (see FIGS. 4 and 5).

In addition, the present invention comprises the steps of: 1) administering a candidate substance to the neural tissue of a non-human subject expressing a Lyanodine Receptor (RyR);

2) causing inflammation in the subject to which the candidate substance of step 1) has been administered;

3) analyzing the pain response in the treated subject of step 2);

4) comparing the pain response of the subject administered only the vehicle, the subject administered the RyR inhibitor, and the subject administered the RyR inhibitor and the candidate together with the pain response analyzed in step 3); And

5) As a result of the comparison in step 4), the pain response of the subjects to which the RyR inhibitor was administered and the subjects to which the RyR inhibitor and the candidate were administered were increased, compared to the pain response of the subjects to which the vehicle was only administered. However, the pain response of the subject to which the candidate was administered provides a method for screening a pain mitigating agent comprising selecting a candidate that has been reduced.

The subject may be a mammal, and the subject may be any one of a mouse, a rat, a pig, a rabbit or a monkey, and the subject may be a mouse or a rat. It is more preferably any one of, the subject is most preferably, but not limited to a mouse.

The neuronal tissue of the subject in which the lyanodine receptor (RyR) is expressed in step 1) is preferably in the hypothalamus of the subject's hypothalamus (ventroposteriomedial, VPM) or the ventrotropiniolateral (VPL) nucleus. It is not limited.

Inflammation of step 2) may be caused by administering acetic acid to the peritoneum of the subject, and inflammation of step 2) may be caused by subcutaneous injection of formalin in the subject's feet. When the inflammation of step 2) is caused by injecting acetic acid into the peritoneum of the subject, the pain response is preferably a writhing response, and the pain response is stretching and constriction of the subject. More preferably, but not limited to, behavior. When the pain response is the kidney and contraction behavior of the subject, the analysis of the kidney and contraction behavior is preferably performed by measuring the number of kidney and contraction behavior of the subject, but is not limited thereto. In addition, when the inflammation of step 2) is caused by subcutaneous injection of formalin into the subject's foot, the pain response is preferably a licking, biting and shaking action of the subject. This is not limitative. If the pain response is a subject's licking, biting, and shivering behavior, the analysis of the licking, biting, and shivering behavior is performed by measuring the time taken until the subject develops a licking, biting, and shivering response. But is not limited thereto.

The RyR activity inhibitor is preferably any one of ryanodine, dantrolene or derivatives thereof, and the RyR activity inhibitor is more preferably lyanodine, but is not limited thereto.

The pain is preferably neurological pain, but is not limited thereto.

In a specific embodiment of the present invention, by using whole-cell patch clamping (whole-cell patch clamping), it was confirmed that the single ignition rate of TC neurons depends on the RyR activity (see Figs. 2 and 3). In a specific embodiment of the present invention, a mouse, caffeine or lyanodine is injected into the VPM / VPL nucleus of the mouse, respectively, and the mouse is subjected to mechanical stimulation, heat stimulation and inflammatory stimulation by acetic acid or promalin. As a result of measuring the acute pain response, both lyanodine and caffeine had no effect on the acute pain response by mechanical stimulation and heat stimulation (see FIG. 4), while in the case of inflammatory pain, the pain response by the cyanine RyR activator It was confirmed that this significantly reduced (see Fig. 5).

In addition, the present invention provides a composition for pain relief comprising a lyanodine receptor (RyR) activator.

The composition may further comprise a carrier capable of crossing the cerebrovascular barrier, the composition may be mixed with a physiologically acceptable carrier medium, and the physiologically acceptable carrier medium may be water , Buffered water, general saline, 0.4% saline, 0.3% glycine and hyaluronic acid, but are not limited thereto.

In addition, the pharmaceutical composition may include customary pharmaceutical excipients and / or additives. Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents. Suitable additives include the addition of physiologically biocompatible buffers (eg tromethamine hydrochloride), chelants (such as DTPA or DTPA-bisamide), Or calcium chelate complexes (eg calcium DTPA, CaNaDTPA-bisamide), or optionally, addition of calcium or sodium salts (eg calcium chloride, calcium ascorbate, calcium gluconate). (calcium gluconate) or calcium lactate. The pharmaceutical compositions of the invention may be packaged in liquid form for use or may be lyophilized under reduced pressure.

For solid compositions, customary nontoxic solid carriers can be used, which are pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin (sodium saccharin), talc (talcum), cellulose, glucose, sucrose and magnesium carbonate (magnesium carbonate) and the like are preferred, but are not limited thereto.

In addition, the pharmaceutical composition may be administered by oral administration and parenteral administration such as intravenous, intramuscular, and transdermal administration. When formulated into preparations for oral and parenteral administration that can be used in practice in the clinic, conventional fillers, extenders, binders, wetting agents, diluents or excipients such as disintegrants, surfactants and the like can be used. Solid form preparations for oral administration include tablets, pills, powders, granules and capsules, and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or It is prepared by mixing lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium, styrate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups, and the like, and may include various excipients such as wetting agents, sweeteners, fragrances, and preservatives in addition to the commonly used simple diluents, water and liquid paraffin. Parenteral administration is by subcutaneous injection, intravenous injection or intramuscular injection. Formulations for parenteral administration are prepared as a solution or suspension by mixing the composition with water in a stabilizer or buffer and formulated into a unit dosage form of ampoules or vials. In addition, the compositions of the present invention can be formulated in oral administration into pills, pills and other forms by conventional methods used in herbal medicine.

In addition, the RyR activator is preferably a caffeine derivative, but is not limited thereto, and the pain is preferably inflammatory pain, but is not limited thereto.

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples are intended to specifically illustrate the present invention, the content of the present invention is not limited by the following examples.

< Example  1> Brain Slice ( slices ) And immediately isolated neurons ( acutely - dissociated  neuronal cells ) Production

<1-1> experimental animals

In a specific embodiment of the present invention, according to the ethical guidelines of the Institutional Animal Care and Use Committee of the Korea Institute of Science and Technology, two inbred strains, 129 / sv Only males were used in the first generation hybrid mice (F1 hybrid mice) obtained by hybridizing with C57BL / 6J.

<1-2> Preparation of Brain Slice

The brains of the mice of 4 weeks old were subjected to cold slicing solution (124 mM NaCl, 26 mM NaHCO ₃ , 1.25 mM NaH ₂ PO ₄ , 5 mM MgCl ₂ , 1 mM CaCl ₂ , 3 mM KCl and 10 mM). Of glucose) was cut into a coronal plane and then incubated at room temperature for 1 hour.

<1-3> Fabrication of Immediately Isolated Neurons

The brain of the mouse of <1-1>, 2 to 3 weeks old, was prepared as brain slices as in <1-2>. Brain slices prepared as described above were aerobic HEPES-buffered solution (oxygenated HEPES-buffered solution) containing 2 mg / ml of protease XIV (Sigma) of 150 mM NaCl, 3 mM KCl, 2 mM CaCl ₂ , 10 mM HEPES, 2 mM MgCl ₂ and 10 mM glucose, pH-7.4 and 320-330 mOsm) for 10 minutes at 37 ℃, then incubated for 5 minutes at room temperature. Then, slices containing the ventroposteriomedial (VPM) or ventroposteriolateral (VPL) nuclei are detailed with a scalpel, and the cells are crushed into a fire-polished Pasteur pipette. It was.

Observation of the morphology of the cells produced as described above under a microscope, and treated with NMDA (60 μM) in the same manner as in <2-1> to increase the concentration of intracellular calcium ions ([Ca ^{2 +} ] _i ). It was confirmed (B of Fig. 1).

As a result, it was confirmed that the cells produced as described above were neurons, and the neurons were sufficiently intact.

< Example  2> of mouse VPM Of VPL  Nuclear TC  In neurons RyR Expression and function

<2-1> intracellular Calcium ion  Imaging ( intracellular Ca ^{2 +} imaging Intracellular) Calcium ion  density([ Ca ^{2 +} ]) Check for changes

Immediately isolated neurons prepared as in <1-3> were treated with 5 μg of Fura-2AM (Molecular Probes) mixed with 5 μl of pluronic acid in HEPES-buffered solution, followed by reversed phase. The buffer solution was perfused with the neurons at a flow rate of 2 ml / min while plated on poly-D-lysine-coated coverslips in a chamber of a microscope (TE3000, Nikon). The cells were irradiated with an excitation wavelength of 340/380 nm with an IonOptix chopper (PTI) equipped with a xenon light source and a computer-controlled electronic shutter, followed by a microscope and a combined enhanced CCD camera (PTI-). IC200, PTI) to measure the concentration of intracellular calcium ions of the cells. N-methyl-D-aspartic acid (NMDA), lyanodine, dantrolene and caffeine used in this example were obtained from Sigma.

Treatment of high concentrations of caffeine (10 mM) on the neurons resulted in an increase in the concentration of intracellular calcium ions ([Ca2 +] i) in calcium-free buffers as well as in general extracellular buffers containing 2 mM calcium ions. Caffeine-induced [Ca2 +] i increase was attributed to intracellular storage (FIG. 1A), and this caffeine-induced [Ca2 +] i increase was blocked by pretreatment with lyanodine (50 μM) It was confirmed that it was released through the lyanodine receptor, the calcium channel of the membrane. The increase in [Ca2 +] i induced by subsequent treatment of NMDA (60 μM) in the same cell shows that the cell is intact due to an increase in calcium ions coming from the NMDA receptor from outside (FIG. 1B). In addition, as a result of treating the nerve cells with a high concentration of KCl solution (30 mM), a sudden depolarization of the membrane potential of the nerve cells temporarily increased [Ca ^{2 +} ] _i , but treated with low concentration of caffeine (0.5 mM). in one case it was confirmed that the increase in [Ca ^{2 +]} _i alone caffeine treatment is not induced (C and D in Fig. 1). However, [Ca ^{2 +} ] _i by high concentrations of KCl was further increased by low concentrations of caffeine (0.5 mM) pretreatment, and this [Ca ^{2 +} ] _i increase was completely blocked by pretreatment with lyanodine (50 μM). It was confirmed (C and D of FIG. 1).

These results indicate that the increase in caffeine-induced [Ca ^{2 +} ] _i was caused by the release of calcium ions through RyR from the intracellular reservoir, and the larger increase in [Ca ^{2 +} ] _i by low concentration of caffeine pretreatment resulted in RyR. Implied by the promotion of calcium ion release through.

<2-2> Reverse transcriptase - PCR through RyR Expression Check

Total RNA was isolated from both the brain and VPM and VPL nuclei of the first-generation wild type mice of <1-1> using an RNA separation reagent (Tri reagent, MRC). To generate cDNA, 1 μg total RNA was reverse transcribed with M-MLV reverse transcriptase (Invitrogen) using the primers and oligo (dT) priming of Table 1. Primer pairs of SEQ ID NOs: 1 and 2 were used to generate 462 bp of actin cDNA, primer pairs of SEQ ID NOs: 3 and 4 were used to generate 330 bp of RyR type 1 cDNA, and primer pairs of SEQ ID NOs: 5 and 6 Was used to generate 315 bp RyR type 2 cDNA, and primer pairs of SEQ ID NOs: 7 and 8 were used to generate 344 bp RyR type 3 cDNA. The PCR reaction was performed by reaction at 94 ° C. for 5 minutes, 40 cycles at 94 ° C., 40 seconds at 56 ° C., 40 seconds at 72 ° C., and finally at 72 ° C. for 5 minutes.

As a result, it was confirmed that all RyR types were expressed in brain neurons, but RyR types 2 and 3 were expressed in VPM and VPL nuclei.

object SEQ ID NO: Forward primer SEQ ID NO: Reverse primer actin One 5'-AGATCTGGCACCACACCTTC-3 ' 2 5'-TCTCCAGGGAGGAAGAGGAT-3 ' RyR type 1 3 5'-TGAAGGAATTCACGGACCTC-3 ' 4 5'-CTGGTCCAAGTCCCACAACT-3 ' RyR type 2 5 5'-TGAAGACGGGCCTAGACAGT-3 ' 6 5'-GCCTCTCCACGTAGATGCTC-3 ' RyR type 3 7 5'-GGAAGCAAGAGCTTGGAAGA-3 ' 8 5'-AATAAGCAGCAGTCGCCCTA-3 '

From the results of <2-1> and <2-2>, it can be seen that RyR is expressed in TC neurons of the VPM / VPL nucleus of the mouse, and RyR serves as a pathway for calcium ion in the cells.

< Example  3> RyR Depends on whether TC  Analysis of ignition pattern of neurons

<3-1> Whole Cell Patch Whole-cell patch clamping  Single speech analysis

ACSF solution foamed with 95% O ₂ /5% CO ₂ (124 mM NaCl, 26 mM NaHCO ₃ , 1.25 mM NaH ₂ PO ₄ , 1.3 mM MgSO ₄ , 2.4 mM CaCl ₂ , 3 mM KCl and 10 mM glucose) was used to analyze the ignition pattern before and after treatment with RyR activator and activity inhibitor in TC neurons in the brain slice prepared as in <1-2>. Patch electrodes (4-6Ω) made of borosilicate glass were prepared using an intratrapetette solution (140mM K-gluconate, 10mM KCl, 1mM MgCl ₂ , 10mM HEPES, 0.02mM EGTA). , 4mM of Mt-ATP and 0.3mM of Na ₂ -GTP, pH 7.35), flowing a range of depolarization currents to TC neurons maintained at -60mV to induce a single ignition, Amplification was performed with a Multiclamp 700-A amplifier (Molecular devices) and analyzed with pCLAMP 9.2 and MiniAnalysis software (Synaptosoft). N-methyl-D-aspartic acid (NMDA), lyanodine, dantrolene and caffeine used in this example were obtained from Sigma.

As a result, when 50 μM of lyanodine was treated, a single ignition of TC neurons increased by about 22% from 46.5 ± 1.9 to 56.5 ± 2.5 per second due to the inflow of 400 pA of depolarized current (Fig. 2A, And B) and afterhyperpolarization (AHP) of A decreased about 54% from -4.52 ± 0.43mV to -2.06 ± 0.52mV (Fig. 2A, ⅱ, Aⅳ) And ii) of B. In addition, 0.5 mM caffeine was treated to determine the role of activated RyR without changing the basal level of intracellular calcium ions or depleting internal calcium ion reservoirs (based on Example <2-1>). Single ignition of TC neurons was reduced by approximately 26% from 48.9 ± 2.4 to 36.4 ± 2.7 (C in Fig. 2, C in C and D in D), and AHP size was -8.15 at -5.45 ± 0.46 mV. An increase of about 50% was observed at ± 0.28 mV (ii in C, VII in C and ii in D).

<3-2> Whole Cell Patch Whole-cell patch clamping  Multiple ignition analysis using

ACSF solution foamed with 95% O ₂ /5% CO ₂ (124 mM NaCl, 26 mM NaHCO ₃ , 1.25 mM NaH ₂ PO ₄ , 1.3 mM MgSO ₄ , 2.4 mM CaCl ₂ , 3 mM KCl and 10 mM glucose) was used to analyze the ignition pattern before and after treatment with RyR activator and activity inhibitor in TC neurons in the brain slice prepared as in <1-2>. Patch electrodes (4-6Ω) made of borosilicate glass were prepared using an intratrapetette solution (140mM K-gluconate, 10mM KCl, 1mM MgCl ₂ , 10mM HEPES, 0.02mM EGTA). , 4mM Mt-ATP and 0.3mM Na ₂ -GTP, pH 7.35), which occurs when the membrane potential returns to the holding potential (-60mV) after 1 _{second of} prepolarization pre-pulse. The signal of multiple ignition was amplified with a Multiclamp 700-A amplifier (Molecular devices) and analyzed by pCLAMP 9.2 and MiniAnalysis software (Synaptosoft). N-methyl-D-aspartic acid (NMDA), lyanodine, dantrolene and caffeine used in this example were obtained from Sigma.

As a result, not only the treatment with 50 μM lyanodine (A and B in Fig. 3), but also the treatment with 0.5 mM caffeine (C and D in Fig. 3), had no effect on the rate of multiple ignition of TC neurons. It was confirmed that there was no.

From the results of <3-1> and <3-2>, it can be seen that the release of calcium ions through RyR regulates the single ignition rate of TC neurons.

< Example  4> TC  In nerve cells RyR  Analysis of changes in physiological pain response according to activity

<4-1> micro osmotic pump ( micro - osmotic pump Drug administration via insertion

In order to administer a drug such as a vehicle (vehicle), caffeine, lyanodine and the like to the brain of the mouse obtained as described above <1-1>, using a stereotaxic device (Kopf Instruments), the micro osmotic pump The tip of the tip was directed to the sagittal VPM / VPL nucleus (1.6mm dislocation, ± 1.7mm lateral, 3.4mm rear). The insertion dosing position was confirmed by post necropsy.

<4-2> Analysis of pain response by mechanical and thermal stimulation

Pain testing for mechanical stimuli was performed according to the methods described in the prior arts Chapman et al., 1985 and Kim et al., 2001, and by the method of <4-1>, solvents, caffeine or lyanodine Were injected into the VPM / VPL nuclei of mice, respectively. The mechanical threshold was measured using metered von Frey fiber (Stoelting) and the bending force with which the mouse shrugs the hind paws was measured in g. The response score was assessed by the total number of hindpaws shrunk by trying 10 times with each fiber.

In addition, the pain test for thermal stimulation, after injecting a vehicle (vehicle), caffeine or lyanodine into the VPM / VPL nucleus of the mouse by the method of <4-1>, respectively, prior art document Hargreaves et al., According to the Hargreaves method described in 1988 and Kim et al., 2001, it was performed using a plantar test apparatus. After 1 hour of acclimation, 40 intensity levels (any unit in the device) were applied to the mouse's hind paw five times at 5 minute intervals, after which time the mouse took to shrunk the hind paw. N-methyl-D-aspartic acid (NMDA), lyanodine, dantrolene and caffeine used in this example were obtained from Sigma, and the pain test and data analysis proceeded in a double-blinded manner. It was. For the reliability of the data, mice were administered without information about the drug, and analysis of the data was performed by at least two people. For statistical analysis of the data, a two-tailed t-test method was used and all measurements were expressed as mean ± standard deviation. If the p value is less than 0.05, it means that the data is reliable.

As a result, it was confirmed that when treated with 50μM lyanodine and treated with 0.5mM caffeine, there was no effect on the pain response (B in Fig. 4) to mechanical stimulation and heat stimulation (Fig. 4).

<4-3> Analysis of pain response by inflammatory stimulus

Abdominal pain test by inflammatory stimulation, after injecting the vehicle (vehicle), caffeine, lyanodine, dantrolene and caffeine / lyanodine by the method of <4-1> to the mouse VPM / VPL nucleus, respectively , Prior art Kim et al., 2003. To observe the mouse's feet and abdomen without any obstructions, place the mouse in an acrylic cylinder on a mirror at a 45 ° angle, inject saline containing 0.6% acetic acid into the peritoneum at a rate of 6 ml / kg, The abdominal pain response was measured. The measurement of the abdominal pain response was performed by measuring the number of writhing responses, such as stretching and constriction behavior in mice.

In addition, formalin-induced pain test, after injecting the vehicle (vehicle), caffeine, lyanodine, dantrolene and caffeine / lyanodine by the method of <4-1> to the mouse VPM / VPL nucleus, respectively , Prior art Kim et al., 2001. 10 μl of saline containing 2.0% formalin was subcutaneously injected into the left inferior foot of the mouse, and the measurement of formalin-induced pain response was measured by the mouse in response to pain such as licking, biting, and shaking. This was done by measuring the time taken to produce the. N-methyl-D-aspartic acid (NMDA), lyanodine, dantrolene and caffeine used in this example were obtained from Sigma, and the pain test and data analysis proceeded in a double-blinded manner. It was. For the reliability of the data, mice were administered without information about the drug, and analysis of the data was performed by at least two people. For statistical analysis of the data, a two-tailed t-test method was used and all measurements were expressed as mean ± standard deviation. If the p value is less than 0.05, it means that the data is reliable.

As a result, both the abdominal pain test (A of FIG. 5) and the formalin-induced pain test (B of FIG. 5) showed the same pattern of reaction, compared to 50 μM of lyanodine or dantrol compared to mice injected only with a vehicle. It was confirmed that the pain response increased in the mouse injected with len, while the pain response was significantly reduced in the mouse injected with caffeine. In addition, mice injected with caffeine and lyanodine showed similar pain response to mice injected with lyanodine or dantrolene, respectively.

From the results of <4-2> and <4-3>, the single ignition increased or decreased by the RyR activator had no effect on the pain response by the mechanical or thermal stimulus, while increased by the RyR activator It can be seen that the inflammatory pain response is increased due to a single ignition and the inflammatory pain response is reduced due to a single ignition reduced by the RyR activator.

<110> Korea Institute of Science and Technology <120> Pain relieving agents promoting activation of ryanodine receptor          and screening method <130> 10P-02-44 <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for actin <400> 1 agatctggca ccacaccttc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for actin <400> 2 tctccaggga ggaagaggat 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for RyR Type 1 <400> 3 tgaaggaatt cacggacctc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for RyR Type 1 <400> 4 ctggtccaag tcccacaact 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for RyR type2 <400> 5 tgaagacggg cctagacagt 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for RyR type 2 <400> 6 gcctctccac gtagatgctc 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for RyR type 3 <400> 7 ggaagcaaga gcttggaaga 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for RyR type 3 <400> 8 aataagcagc agtcgcccta 20

Claims (27)

1) treating the candidate with the neuron;
2) measuring the activity of the Lyanodine Receptor (RyR) in the treated neurons of step 1); And
3) A method for screening pain relief materials comprising screening for candidates with increased activity of RyR compared to the control without treatment of candidates.
The method of claim 1, wherein the neurons of step 1) are selected by the following method:
a) separating neurons from non-human subjects;
b) treating the neurons of step a) with a RyR activator followed by treating the RyR activator and the RyR activator together;
c) measuring the change in the concentration of calcium ion ([Ca ^{2 +} ] _i ) in the neuron during the treatment of step b); And
d) selecting the neurons in which the concentration of the calcium ions is increased by the RyR activator and the increase in the concentration of the calcium ions is suppressed by the RyR activator as a result of the measurement in step c).
The method of claim 1, wherein the neurons of step 1) are selected by the following method:
a) separating neurons from non-human subjects;
b) treating the neuron of step a) with a RyR activator and a RyR inhibitor, followed by treating the RyR activator;
c) measuring the change in the concentration of calcium ion ([Ca ^{2 +} ] _i ) in the neuron during the treatment of step b); And
d) selecting the neurons in which the concentration of the calcium ions is increased by the RyR activator and the increase in the concentration of the calcium ions is suppressed by the RyR activator as a result of the measurement in step c).
4. The method of claim 2 or 3, wherein said RyR activator is caffeine.
4. The method of claim 2 or 3, wherein said RyR activity inhibitor is ryanodine.
The method of claim 1, wherein the neurons are derived from a mouse.
The method of claim 1, wherein the neurons are thalamic cortical neurons (TC neurons).
The method of claim 1, wherein the neurons are neurons of the hypothalamus of ventral vesicles (ventroposteriomedial, VPM) or of the ventral lateral (VPL) nucleus.
The method of claim 1, wherein the activity measurement of the lyanodine receptor in step 2) is performed by measuring a tonic firing pattern or afterhyperpolarization (AHP).
The method of claim 1, wherein the candidate for increasing the activity of RyR in step 3) is a candidate for reducing the single ignition of neurons, or increased the AHP of the neurons.
The method of claim 1, wherein said pain is inflammatory pain.
1) administering a candidate substance to neural tissues of the subject except humans in which the ryanodine receptor (RyR) is expressed;
2) causing inflammation in the subject to which the candidate substance of step 1) has been administered;
3) analyzing the pain response in the treated subject of step 2);
4) comparing the pain response of the subject administered only the vehicle, the subject administered the RyR inhibitor, and the subject administered the RyR inhibitor and the candidate together with the pain response analyzed in step 3); And
5) As a result of the comparison in step 4), the pain response of the subjects to which the RyR inhibitor was administered and the subjects to which the RyR inhibitor and the candidate were administered were increased, compared to the pain response of the subjects to which the vehicle was only administered. Wherein the pain response of the subject to which the candidate has been administered comprises selecting a candidate that has reduced pain.
The method of claim 12, wherein the subject is a mouse.
The neuronal tissue of the subject in which the lyanodine receptor (RyR) is expressed in step 1) is the intrathalamic ventroposteriomedial (VPM) or the ventroposteriolateral (VPL) nucleus. A method for screening pain relief substances, characterized in that
13. The method of claim 12, wherein the inflammation of step 2) is caused by administering acetic acid to the peritoneum of the subject.
16. The method of claim 15, wherein said pain response is writhing responses.
18. The method of claim 16, wherein the writhing responses are stretching and constriction behavior of the subject.
18. The method of claim 17, wherein said analysis of renal and contractile behavior is performed by measuring the number of renal and contractile behaviors of a subject.
The method of claim 12, wherein the inflammation of step 2) is caused by subcutaneous injection of formalin into the subject's foot.
20. The method of claim 19, wherein the pain response is a licking, biting, and shaking behavior of the subject.
21. The method of claim 20, wherein the analysis of the licking, biting, and shaking behavior is performed by measuring the time taken until the subject develops a licking, biting, and shaking response.
13. The method of claim 12, wherein said RyR inhibitor is Ryanodine or Dantrolene.
13. The method of claim 12, wherein said pain is inflammatory pain.
Pain relief composition comprising a lyanodine receptor (RyR) activator.
25. The composition of claim 24, wherein the composition further comprises a carrier capable of crossing the cerebrovascular barrier.
25. The composition of claim 24, wherein said RyR activator is a caffeine derivative.
25. The composition of claim 24, wherein the pain is inflammatory pain.

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