JP2018173307A - Method for screening candidate compound presenting analgesic effect by specifically combining with film in peripheral nerve or spinal cord - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for screening using as an index a compound which specifically combines with a film in a peripheral nerve or in a spinal cord.SOLUTION: The method for screening a candidate compound with an analgesic effect includes the steps of: mixing a cyclic amine derivative shown by Chemical Formula (I) and a membrane fraction obtained by preparing from peripheral nerve or a spinal cord and obtaining a comparison sample; obtaining an evaluation sample in which the cyclic amine derivative and the membrane fraction are mixed with an evaluation compound; and comparing the physical property value of the comparison sample and that of the evaluation sample.SELECTED DRAWING: Figure 4

Description

  The present invention relates to a method for screening a candidate compound having an analgesic action by specifically binding to a membrane present in a peripheral nerve or spinal cord.

  Peripheral nerves are composed of somatic nerves such as sensory nerves and motor nerves, and autonomic nerves such as sympathetic nerves and parasympathetic nerves, and the impairment of the peripheral nerve functions causes various symptoms. For example, in metabolic peripheral neuropathy due to diabetes, when somatic nerve function is impaired, sensory dullness, chronic pain, numbness, etc. occur, and when the autonomic nervous system is impaired, diarrhea, constipation, Abnormal sweating occurs. Peripheral nerve dysfunction, such as trauma and surgical injury, viral infection, tumor, chemotherapeutic medication, autoimmune reactions (Guillain-Barre syndrome, etc.) and genetic diseases (Charcot-Marie-Tooth disease, etc.) It is known to cause In particular, pain induced by peripheral neuropathy is collectively referred to as neuropathic pain, and it is known that many patients suffer from daily life.

  As treatment of peripheral neuropathy, healing of causative diseases, removal of causative agents, and administration of drugs that protect neuropathies such as methylcobalamin or drugs that relieve symptoms associated with peripheral nerve disorders are performed. For example, for neuropathic pain, frequently used drugs include pregabalin, duloxetine, and amitriptyline, but common problems with these drugs include that there are many patients who are not effective. It is known that side effects such as drowsiness and light-headedness are likely to occur by suppressing the function of.

  On the other hand, many drugs that act on the nerve affect the function of the nerve by using receptors or ion channels on the cell membrane as molecular targets. Therefore, in the development of therapeutic drugs for the nervous system, the binding to specific molecular targets is used as an indicator. In addition, a method of screening a target candidate compound from an evaluation compound is widely performed.

  Specifically, a known compound known to specifically bind to the target molecular target is used as an index to compete with the evaluation compound, and the target compound is screened by comparing the binding ability to the target molecular target. The method of performing is common. For example, as a method of using the calcium channel α2δ-1 subunit, which is a molecular target of pregabalin, which is a drug frequently used for neuropathic pain, a comparison is performed using radiolabeled gabapentin as an index. Methods for screening for drugs that act on drugs have been reported (Patent Document 1 and Non-Patent Document 1). In addition, as a method using a membrane fraction obtained from a brain containing a γ-aminobutyric acid (GABA) receptor, which is a molecular target of a benzodiazepine compound having an anxiolytic action and hypnotic sedative action, radiolabeled A method has been reported in which comparison is performed using diazepam as an index and a drug that acts on nerves is screened from an evaluation compound (Patent Document 2 and Non-Patent Document 2).

  On the other hand, 1- (4- (dimethylamino) piperidin-1-yl) -3- (1-methyl-1H-imidazol-2-yl) is a compound that shows efficacy against rodent neuropathic pain models. ) Compounds represented by propan-1-one have been reported (Patent Document 3).

Patent Publication No. 2003-512602 US Pat. No. 5,597,920 International Publication No. 2013/147160

Gee NS, Brown JP, Dissanayake VU, Offord J, Thurlow R, Woodruff GN. J Biol Chem. 1996 Mar 8; 271 (10): 5768-76. Mohler H, Okada T. Science. 1977 Nov 25; 198 (4319): 849-51.

  However, in order to find a new drug for treating peripheral neuropathy or treating or alleviating symptoms associated with peripheral neuropathy, a screening method using a conventionally used compound as an index is effective and safe. May not be enough.

  For example, the central side effects caused by pregabalin, duloxetine and amitriptyline prescribed for neuropathic pain, as described in Patent Document 1 and Non-Patent Document 1, are independent of the molecular target to show drug efficacy. It is thought that this is not the result of inhibiting the binding of the compound, but the result of inhibiting the molecular target itself to show the drug efficacy. Therefore, when these compounds are used as indicators, the ability to bind to molecules other than the target cannot be found from screening, and the concern of side effects cannot be dispelled.

  In addition, many of the drugs prescribed for existing neuropathic pain have the central nerve as an action point, whereas the number of drugs having the peripheral nerve as an action tissue is small. Moreover, the action point of the compound described in Patent Document 3 is unknown, and it has not been evaluated to which tissue it binds.

  Therefore, an object of the present invention is to provide a screening method for a candidate compound having an analgesic action using a compound that specifically binds to a membrane present in the peripheral nerve or spinal cord as an index.

  As a result of intensive studies to solve the above problems, the inventors of the present invention specifically bound the compound described in Patent Document 3 to a membrane present in the peripheral nerve or spinal cord, and the brain or other organs. The present invention has been completed by finding that it hardly or not binds to the membrane fraction obtained by preparing from the above.

That is, this invention provides invention of (1)-(3) for solving the said subject.
(1) A cyclic amine derivative represented by the following chemical formula (I) is mixed with a membrane fraction obtained by preparation from peripheral nerve or spinal cord, and the cyclic amine derivative is bound to a cell membrane contained in the membrane fraction. A control sample preparation step comprising: a binding step 1 to be removed; and a recovery step 1 to remove the cyclic amine derivative not bound to the cell membrane after the binding step 1 to obtain a membrane fraction after the removal;
The cyclic amine derivative, a membrane fraction obtained by preparation from peripheral nerves or spinal cord, and an evaluation compound are mixed to compete the cyclic amine derivative and the evaluation compound to form a cell membrane contained in the membrane fraction. An evaluation sample comprising: a binding step 2 for binding; and a recovery step 2 for removing the cyclic amine derivative not bound to the cell membrane and the evaluation compound after the binding step 2 to obtain a membrane fraction after removal. A method for screening a candidate compound having analgesic action by specifically binding to a membrane present in a peripheral nerve or spinal cord, comprising a preparation step and a step of comparing physical property values of the control sample and the evaluation sample.
(2) The screening method according to (1), wherein the cyclic amine derivative represented by the chemical formula (I) is labeled with a radioisotope.
(3) The screening method according to (1) or (2), wherein the peripheral nerve is a dorsal root ganglion, a trigeminal ganglion, or a sciatic nerve.

  The method of the present invention provides a novel method for screening a candidate compound having analgesic action that specifically binds to a molecular target in a membrane fraction existing in the peripheral nerve or spinal cord and does not bind to other molecular targets. Is done. The compounds screened by this method are expected to bind to peripheral nerves, and develop drugs for treating or alleviating symptoms associated with peripheral neuropathy or symptoms associated with peripheral neuropathy that have been difficult to date. It is useful in.

FIG. 1 is a list of proteins in which the cyclic amine derivative represented by the chemical formula (I) does not exhibit an inhibitory effect on known specific binding. FIG. 2 is a graph of specific binding (dpm / μg protein) of the cyclic amine derivative represented by the chemical formula (I) to various membrane fractions prepared from each mouse tissue. FIG. 3 is a graph of specific binding (dpm / μg protein) of the cyclic amine derivative represented by the chemical formula (I) to various membrane fractions prepared from each tissue of a neuropathic pain model rat. . FIG. 4 is a graph comparing the binding rates when various evaluation compounds are added. FIG. 5 is a graph showing the effect of the compound represented by the chemical formula (II) on the mouse partial sciatic nerve ligation model.

The screening method for a candidate compound having analgesic action by specifically binding to a membrane present in the peripheral nerve or spinal cord of the present invention is prepared from a cyclic amine derivative represented by the following chemical formula (I) and the peripheral nerve or spinal cord. A coupling step 1 in which the cyclic amine derivative is bound to the cell membrane contained in the membrane fraction by mixing with the membrane fraction obtained in the step, and the cyclic amine derivative not bound to the cell membrane after the coupling step 1 A recovery step 1 for removing to obtain a membrane fraction after removal, and a control sample preparation step,
The cyclic amine derivative, a membrane fraction obtained by preparation from peripheral nerves or spinal cord, and an evaluation compound are mixed to compete the cyclic amine derivative and the evaluation compound to form a cell membrane contained in the membrane fraction. An evaluation sample comprising: a binding step 2 for binding; and a recovery step 2 for removing the cyclic amine derivative not bound to the cell membrane and the evaluation compound after the binding step 2 to obtain a membrane fraction after removal. It comprises a preparation step and a step of comparing the physical property values of the control sample and the evaluation sample.

In the present invention, a cyclic amine derivative represented by the following chemical formula (I) that specifically binds to a membrane present in the peripheral nerve or spinal cord (hereinafter referred to as the above cyclic amine derivative) is used as a competitive compound with the evaluation compound. By using it, candidate compounds can be screened. Here, the cyclic amine derivative represented by the following chemical formula (I) means one containing a salt of the cyclic amine derivative.

In the present invention, as a step of comparing the physical property values of the control sample and the evaluation sample, it is preferable to compare the radioactivity by a radioactive tracer method. Specifically, a control sample and an evaluation sample are prepared using the cyclic amine derivative labeled with a radioisotope. At this time, since the competition with the evaluation compound does not occur in the control sample, the binding of the cyclic amine derivative labeled with the radioisotope and the membrane fraction is maximized. Binding of the isotope-labeled cyclic amine derivative to the membrane fraction is reduced. As a result, the amount of the compound labeled with the radioisotope differs between the control sample and the evaluation sample. It can indicate how much specific binding occurs with membranes present in the peripheral nerve or spinal cord as compared to the derivative. Here, ³ H or ¹⁴ C is preferable as the radioisotope used for labeling.

  In the present invention, the membrane fraction is preferably prepared from animal tissues including dorsal root ganglia, trigeminal ganglia, sciatic nerves or other peripheral nerves or spinal cord, dorsal root ganglia, trigeminal nerves. More preferred are those prepared from mouse or rat tissue containing nodes, sciatic nerves or other peripheral nerves or spinal cord.

  In the present invention, as the membrane fraction, the tissue is crushed using a mechanical tissue crushing device, and the biological material is obtained from the crushed membrane fraction or surfactant obtained by fractionating with a centrifuge. The dissolved membrane fraction obtained by breaking and fractionating with a centrifuge can be used.

  In the present invention, the above-mentioned cyclic amine derivative and a membrane fraction obtained by preparation from peripheral nerve or spinal cord are mixed to bind the cyclic amine derivative to the cell membrane contained in the membrane fraction, A cyclic amine derivative, a membrane fraction obtained from peripheral nerve or spinal cord, and an evaluation compound are mixed to compete with the cyclic amine derivative and the evaluation compound to bind to the cell membrane contained in the membrane fraction As a condition of the binding step 2 to be performed, an arbitrary time from 5 minutes to 72 hours can be set under the temperature condition from 4 ° C. to 37 ° C. As the aqueous solution used in the binding steps 1 and 2, a buffer solution including a Krebs solution, a Tris buffer solution, a HEPES buffer solution and a phosphate buffer solution can be used. As a method for measuring the radioactivity of the cyclic amine derivative labeled with a radioisotope specifically bound to the membrane, a method of adsorbing a membrane fraction using a filter such as a glass filter, a scintillation proximity assay A method of separating a membrane fraction by centrifugation, a method of separating a membrane fraction solubilized with a surfactant by gel filtration or the like can be used.

Example 1: Inhibitory effect of cyclic amine derivative represented by chemical formula (I) on known specific binding (hereinafter referred to as Compound 1) Compound for specific binding of biological sample containing known protein and corresponding various ligands The inhibitory action of 1 was comprehensively examined. Specifically, a sample was prepared which was a mixture of 88 kinds of known receptors, channels and transporters shown in FIG. Further, 10 μmol / L of Compound 1 was added, and a step of competing Compound 1 for specific binding between the ligand and the protein was performed to examine the inhibitory action. As a result, the inhibitory action of the cyclic amine derivative was less than 30% on the specific binding of 88 kinds of known receptors, channels and transporters shown in FIG. 1 and the corresponding ligands. This suggested that Compound 1 has binding to an unknown molecular target.

Example 2: Evaluation of binding of membrane fraction obtained from mouse tissue and compound 1 labeled with ³ H Membrane fraction obtained from mouse tissue and compound labeled with ³ H By assessing the binding of 1, the tissue containing the unknown molecular target of Compound 1 was revealed.

  As the mouse, an ICR male mouse (manufactured by Charles River Japan Co., Ltd.) received at 5 weeks of age was used at 6 weeks of age. The mice were anesthetized with isoflurane, and perfused and bled from the left ventricle using room temperature phosphate buffered saline (hereinafter, PBS) (Takara Bio Inc.) containing heparin (1000 U / L; manufactured by Ajinomoto Co., Inc.). After decapitation, the following tissues were collected from the head. The eyes were isolated and then cut on the equator plane, and the retinal side was collected. After the skull was cooled with cold PBS for 1 minute, the skull was removed and the whole brain was removed. In addition, the cerebellum and brain stem were collected from the whole brain, and the remaining part of the whole brain was collected as the cerebrum. The pituitary and trigeminal ganglia were collected from the skull after the whole brain was removed. The spinal column from the shoulder to the L6 spinal cord was removed from the body, and the spinal cord and dorsal root ganglion (hereinafter referred to as DRG) were collected. The liver and kidney were collected after removing the membrane. Fat was collected around the testis. The small intestine was collected after removing the contents with a syringe. For skeletal muscle, a part of the thigh was collected. The skin was collected by cutting the thigh into 5 mm squares. In addition, sciatic nerve, heart, lung, adrenal gland, spleen, bladder and pancreas were collected according to a standard method. The collected tissue was immediately frozen on dry ice and stored at -80 ° C. The pituitary, trigeminal ganglion, DRG, and sciatic nerve were treated as one sample by mixing tissues collected from three individuals. The other tissues were treated as one sample from one animal. Each group consisted of 2 groups, and a total of 6 mice were used in this study.

The membrane fraction was obtained according to the method of Cox et al. (B. Cox, A. Emili, Nature Protocol, 1, 1872-78 (2006)). A homogenized buffer (sucrose (250 mmol / L), Tris-HCl (50 mmol / L), 1 × protease inhibitor cocktail (manufactured by Sigma Aldrich); homogenized buffer pH: 7.6) was added to the collected tissue, and a polytron homogenizer was used. It was crushed. After removing nuclear and mitochondrial components as precipitates by centrifugation at 6000 × g, microsomes in the supernatant were precipitated by ultracentrifugation at 100,000 × g. The precipitate was added to a Krebs solution (NaCl (117 mmol / L), KCl (3.6 mmol / L), CaCl ₂ (2.5 mmol / L), MgCl ₂ (1.2 mmol / L), NaH ₂ PO ₄ (1.2 mmol / L). L), NaHCO ₃ (2.5 mmol / L), glucose (11 mmol / L); pH of Krebs solution: 7.4), suspended in BCA protein assay kit (manufactured by Thermo Fisher Scientific), protein mass Was stored at −80 ° C. as a quantified membrane fraction.

  The reaction of the membrane fraction and Compound 1 was all carried out in the 96-well plate using the above Krebs solution. 25 μL of various membrane fractions were added to the Krebs solution so that the protein mass after addition was approximately 2 to 10 μg. To these various membrane fractions, 25 μL of 30 μmol / L (final concentration 10 μmol / L) of Compound 1 (unlabeled) was added as sample A group, and 25 μL of solvent (distilled water) was added as sample. Group B.

³ H of 30 nmol / L (final concentration of 10 nmol / L) containing n-octyl-β-D-thioglucopyranoside (final concentration of 0.1% (w / v)) for the above samples A and B 25 μL of the labeled compound 1 was added, and the reaction was quickly started by moving into a 37 ° C. incubator. After incubation for 1 hour, the reaction was stopped by cooling at 4 ° C. for 5 minutes. The membrane fraction was adsorbed on a glass filter plate (Perkin Elmer Co., Ltd.). After washing the glass filter plate (6 times), 20 μL of Microscint 20 (manufactured by PerkinElmer Co., Ltd.) was added, and the decay rate (hereinafter referred to as dpm) of the radioactivity of the bound ³ H-labeled compound 1 was determined by liquid scintillation method. It was measured by. The difference obtained by subtracting the average dpm measurement value for the sample A group from the average dpm measurement value for the sample B group and dividing by the protein mass was calculated as specific binding (dpm / μg protein). It was shown to.

  As a result, the specific binding of Compound 1 to various membrane fractions was prominent in membrane fractions obtained from peripheral nerves such as dorsal root ganglia, trigeminal ganglia or sciatic nerves. The membrane fraction obtained from the spinal cord was 19% of the membrane fraction obtained from the dorsal root ganglion. The membrane fraction obtained from the brain stem is less than 15% of the membrane fraction obtained from the dorsal root ganglion, and the membrane fraction obtained from other tissues including the brain. In less than 5% of the membrane fraction obtained from dorsal root ganglia. Since Compound 1 specifically binds to a membrane fraction obtained from peripheral nerves or spinal cord, the molecular target of Compound 1 is present in the peripheral nerve or spinal cord and is present in the peripheral nerve or spinal cord It has been found to act in association with the membrane.

Example 3: image neuropathic pain model rat tissues were obtained by prepared from the membrane fraction and the ^{3 H-labeled} membrane fraction obtained was prepared from coupling assessment neuropathic pain model rat tissues of Compound 1 The binding of Compound 1 labeled with ³ H was evaluated.

  Sprague Dawley male rats (manufactured by Charles River Japan Co., Ltd.) received at 5 weeks of age were used at 6 weeks of age. The neuropathic pain model was prepared according to the method of Chung et al. (S. Kim, J. Chung, Pain, 50, 355-63 (1992)). A neuropathic pain model was prepared by incising the right lumbar dorsal part of the rat under isoflurane inhalation anesthesia, excising the spinal vertebral arch and completely ligating the L5 and L6 nerve roots of the spinal nerve. A von Frey test was performed on rats one week after the nerve ligation, and individuals exhibiting allodynia were used in the following experiments.

  Rats were anesthetized with isoflurane, and about 5 mL of blood was collected from the abdominal vena cava. Subsequently, the blood was perfused and removed from the left ventricle using a cold PBS solution (Takara Bio Inc.). Thereafter, the cerebrum, spinal cord, dorsal root ganglion (ligation side L4 to L6), sciatic nerve (ligation side), skin (cut from the back side of the hind limb on the ligation side) and liver are collected and washed with cold PBS. Cut. Blood was added with 10 U heparin per mouse and allowed to stand at room temperature, and then a 1/5 volume of HetaSep (manufactured by Stem Cell Technologies) was added to use a fraction rich in white blood cells and platelets. The cerebrum and liver were sampled for one rat, and the other tissues were combined for two rats. Two groups, the unlabeled compound 1 addition group and the solvent addition group, used one tissue divided and used a total of 6 rats.

  The membrane fraction was obtained in the same manner as the method shown in Example 2. 150 μL of the membrane fraction was added to give approximately 40 μg of protein. To these various membrane fractions, 500 μmol / L (final concentration 100 μmol / L) of Compound 1 (unlabeled product) 50 μL was added as sample A group, and solvent (distilled water) 50 μL was added as sample. Group B.

With respect to the above samples A group and B group, ³ H of 50 nmol / L (final concentration 10 nmol / L) containing n-octyl-β-D-thioglucopyranoside (final concentration 0.1% (w / v)) 50 μL of the labeled compound 1 was added and immediately moved into a 37 ° C. incubator to start the reaction. After incubation for 1 hour, the membrane fraction was adsorbed on a glass filter (brandel) treated with 0.03% (w / v) polyethyleneimine. After washing the glass filter (six times), it was inserted into a polyethylene vial, and the dpm was measured by the liquid scintillation method for the radioactivity of ³ H-labeled compound 1 added by adding Clearsol I (manufactured by Nacalai Tesque). did. The difference obtained by subtracting the average of the measured values of dpm for the sample A group from the average of the measured values of the dpm for the sample B group was calculated as the specific binding (dpm / μg protein) as shown in FIG. It was shown to.

  As a result, the specific binding of Compound 1 is found only in membrane fractions obtained from peripheral nerves or spinal cords such as the trigeminal ganglion, dorsal root ganglion and sciatic nerve, and prepared from the brain and other tissues. The specific binding to the membrane fraction obtained was less than 5% of the membrane fraction prepared from dorsal root ganglia. That is, in the experiment using the peripheral nerve tissue of the neuropathic pain model rat, as in Example 2, the molecular target of Compound 1 exists in the peripheral nerve or spinal cord and binds to the membrane present in the peripheral nerve or spinal cord. Has been found to work.

Example 4: establish a screening method using the compound 1 impact assessment radiolabeled of various evaluations compound in was membrane fraction and ^{3 H} obtained by prepared from rat tissues for the specific binding of the labeled compound 1 Therefore, to evaluate the effect of various evaluations compound at the membrane fraction and ^{3 H} obtained by prepared from rat tissues for specific binding of labeled compound 1.

  Sprague Dawley male rats (manufactured by Charles River Japan Co., Ltd.) received at 5 weeks of age were used at 6 weeks of age. The rats were euthanized by exsanguination under isoflurane anesthesia, and dorsal root ganglia were collected, washed with cold PBS and cut. Acquisition of various membrane fractions was performed in the same manner as the method shown in Example 2. 150 μL of the membrane fraction was added to make approximately 40 μg of protein. A sample A group was prepared by adding 50 μL of various evaluation compounds (non-labeled substances) so that the final concentration (10 nmol / L or 100 nmol / L) shown in FIG. Samples added with 50 μL of (dimethyl sulfoxide or distilled water) were used as Sample B group.

The sample B group was labeled with ³ nm of 50 nmol / L (final concentration 10 nmol / L) containing n-octyl-β-D-thioglucopyranoside (final concentration 0.1% (w / v)). 50 μL of Compound 1 was added, and the reaction was quickly started by moving into a 37 ° C. incubator. Here, a binding step 1 for binding Compound 1 to the cell membrane contained in the membrane fraction by incubating for 1 hour was performed. After incubation, the membrane fraction was adsorbed to a glass filter (brandel) treated with 0.03% (w / v) polyethyleneimine, and the glass filter was washed (six times) to give compound 1 not bound to the cell membrane. A recovery step was performed to obtain a membrane fraction after removal, and a control sample group was prepared.

The sample A group was labeled with ³ nm of 50 nmol / L (final concentration 10 nmol / L) containing n-octyl-β-D-thioglucopyranoside (final concentration 0.1% (w / v)). 50 μL of Compound 1 was added, and the reaction was quickly started by moving into a 37 ° C. incubator. Here, by performing incubation for 1 hour, a binding step 2 in which the compound 1 and the evaluation compound compete with each other and bind to the cell membrane contained in the membrane fraction was performed. After the incubation, the membrane fraction was adsorbed on a glass filter (manufactured by Brandel) treated with 0.03% (w / v) polyethyleneimine. The glass filter was washed (6 times) to remove the compound 1 not bound to the cell membrane and the evaluation compound, and a recovery step for obtaining a membrane fraction after removal was performed to prepare an evaluation sample group.

The above control sample group and evaluation sample group were each inserted into a polyethylene vial, clear sol I (manufactured by Nacalai Tesque Co., Ltd.) was added, and the radioactivity of ³ H-labeled compound 1 bound to various membrane fractions, dpm was measured by the liquid scintillation method. A value obtained by dividing the average of the measured values of dpm for the evaluation sample group by the average of the measured values of the control sample group was calculated as a binding rate (%) (FIGS. 4A to 4C).

The μ opioid receptor agonist DAMGO (manufactured by Sigma-Aldrich), diazepam (manufactured by Sigma-Aldrich) acting on the GABAA receptor, and the cannabinoid receptor agonist WIN55, 212-2 (sigma) used as evaluation compounds, respectively. Aldrich), thioperamide (Sigma Aldrich) which is an antagonist of histamine H3 and H4 receptors, and pregabalin acting on the calcium channel α2δ-1 subunit are each ³ even under conditions of high concentration (100 μmol / L). The binding rate of Compound 1 labeled with H is 75% or more (FIGS. 4A and B), and since the specific binding of Compound 1 was not inhibited, these molecular targets are different from those of Compound 1 It has been shown.

Further, the compound represented by the following chemical formula (II) (hereinafter referred to as Compound 2) has a binding rate of Compound 1 labeled with ³ H of less than 40% even under the condition of 100 μmol / L (FIG. 4C). 1 specific binding was inhibited. From this, it was found that Compound 2 binds to the same molecular target as Compound 1 and specifically binds to a membrane present in the peripheral nerve.

Example 5: Effect on mouse partial sciatic nerve ligation model Using a mouse partial sciatic nerve ligation model (Seltzer model) capable of evaluating neuropathic pain, the analgesic action of Compound 2 found from this screening method in Example 4 was demonstrated. investigated.

  The mouse partial sciatic nerve ligation model was prepared according to the method of Seltzer et al. (Malberg et al., Pain, 1998, Vol. 76, p. 215-222).

  As the mouse, an ICR male mouse (manufactured by Charles River Japan Co., Ltd.) received at the age of 4 was used at the age of 5 weeks. The mouse was anesthetized with sodium pentobarbital (70 mg / kg, intraperitoneal administration), the sciatic nerve of the right hind limb thigh was exposed, and 8-0 silk thread (manufactured by Natsume Seisakusho Co., Ltd.) was used under a stereomicroscope. The group in which the sciatic nerve was triple-ligated with a half circumference strength was defined as the sciatic nerve partial ligation group, and the group that had not been ligated only by exposing the sciatic nerve was defined as the sham operation group.

  Evaluation of neuropathic pain (hereinafter referred to as von Frey test) was performed by acclimatizing a mouse for at least 2 hours in a measurement acrylic cage (manufactured by Shinano Manufacturing Co., Ltd.) placed on a net, and then applying 0.16 g of pressure. Using such a filament (manufactured by Neuroscience), mechanical tactile stimulation that presses the filament against the sole of the right hind limb for 3 seconds is repeated 3 times at intervals of 3 seconds, and the strength of escape behavior when mechanical tactile stimulation is applied (0: no response, 1: slow and slight escape behavior with respect to the stimulus, 2: quick escape behavior with respect to the stimulus without flinching (behavior shaking the foot quickly) or licking (foot licking behavior) 3: fast escape behavior with flinching or licking), and the total of the three scores (hereinafter, total score) was used as an index of pain.

  Seven days after sciatic nerve ligation surgery, compound 2 (0.1 mg / kg) or pregabalin (3 mg / kg; manufactured by Kenprotech) as a positive control was dissolved in physiological saline to mice in the sciatic nerve partial ligation group. Administration was via the tail vein. The group in which compound 2 was administered to mice in the sciatic nerve partial ligation group was referred to as “sciatic nerve partial ligation + compound 2” group, and the group in which pregabalin was administered was referred to as “sciatic nerve partial ligation + pregabalin” group. In addition, the group in which physiological saline was intravenously administered to mice in the sciatic nerve partial ligation group was referred to as the “sciatic nerve partial ligation + physiological saline” group, and the group in which physiological saline was intravenously administered to mice in the sham operation group And “sham surgery + saline solution” group. The von Frey test was performed before intravenous administration of the test compound (pre value), 30 minutes after intravenous administration and 120 minutes after.

  The results are shown in FIG. In the figure, the vertical axis represents the total score of the von Frey test (mean value ± standard error; n = 5 to 6), and the higher the value, the stronger the pain. The horizontal axis shows the time (hr) after administration of the test compound. For the evaluation of drug efficacy, for the group administered pregabalin (“sciatic nerve partial ligation + pregabalin” in the figure), the “sciatic nerve partial ligation + physiological saline” group (“sciatic nerve partial ligation + Statistical treatment was performed by two unmatched Welch's tests using “saline solution”) as a control. On the other hand, for the group administered with Compound 2 (“sciatic nerve partial ligation + compound 2” in the figure), the “sciatic nerve partial ligation + physiological saline” group (“sciatic nerve partial ligation + Statistical treatment was performed by Welch's test using “saline solution”) as a control. The * mark in the figure indicates that it is statistically significant (p <0.05) compared with the “sciatic nerve partial ligation + saline solution” group (Welch test of two groups without correspondence).

  According to the results of the von Frey test, intravenous administration of compound 2 (“sciatic nerve partial ligation + compound 2” in the figure) is positive control pregabalin (“sciatic nerve partial ligation + pregabalin” in the figure) Similarly, it showed a statistically significant analgesic effect. From this, it was found that this screening method is useful as a screening method for a candidate compound having an analgesic action by specifically binding to a membrane present in the peripheral nerve or spinal cord.

  By developing a drug that acts on peripheral nerves by using a method that screens candidate compounds that bind to membranes mainly present in peripheral nerves, it reduces side effects on the central nervous system and suppresses the progression of peripheral nerve damage or It is possible to provide a drug capable of reinforcing or restoring the function of the impaired peripheral nerve.

Claims (3)

A binding step in which a cyclic amine derivative represented by the following chemical formula (I) is mixed with a membrane fraction obtained by preparation from peripheral nerves or spinal cord to bind the cyclic amine derivative to a cell membrane contained in the membrane fraction. 1 and a recovery step 1 for removing the cyclic amine derivative not bound to the cell membrane after the binding step 1 to obtain a membrane fraction after removal, a control sample preparation step,
The cyclic amine derivative, a membrane fraction obtained by preparation from peripheral nerves or spinal cord, and an evaluation compound are mixed to compete with the cyclic amine derivative and the evaluation compound to form a cell membrane contained in the membrane fraction. An evaluation sample comprising: a binding step 2 for binding; and a recovery step 2 for removing the cyclic amine derivative and the evaluation compound not bound to the cell membrane after the binding step 2 to obtain a membrane fraction after removal. A preparation process;
Comparing the physical property values of the control sample and the evaluation sample;
A method for screening a candidate compound having analgesic action by specifically binding to a membrane present in a peripheral nerve or spinal cord.   The screening method according to claim 1, wherein the cyclic amine derivative represented by the chemical formula (I) is labeled with a radioisotope. The screening method according to claim 1 or 2, wherein the peripheral nerve is a dorsal root ganglion, a trigeminal ganglion, or a sciatic nerve.