JP2020026397A - PHARMACEUTICAL COMPOSITION COMPRISING TrkB ANTAGONIST - Google Patents

Abstract

To provide a pharmaceutical composition that is applicable to symptoms and diseases caused by abnormal interaction between a TrkB protein and a BDNF in vivo.SOLUTION: Provided is a pharmaceutical composition comprising, as an active ingredient, a peptide or protein containing a specific partial sequence selected from an extracellular region among amino acid sequences constituting a TrkB protein. Specifically, the pharmaceutical composition comprises, as an active ingredient, a peptide or protein that includes any one of the amino acid sequences shown by the following 1) or 2) and that interacts with a BDNF, and competitively with a TrkB protein. 1) An amino acid sequence represented by SEQ ID NO 4 or SEQ ID NO 10 in the sequence listing; 2) an amino acid sequence obtained by substituting, deleting, adding, or introducing one or more amino acids in the amino acid sequence shown in 1) above. Furthermore, the pharmaceutical composition of the present invention can exert its effect as a drug more continuously by forming it into a liposome-bound preparation.SELECTED DRAWING: Figure 11

Description

  The present invention relates to a pharmaceutical composition containing a Tyrosine receptor kinase B (TrkB) antagonist as an active ingredient. Specifically, the present invention relates to a pharmaceutical composition containing, as an active ingredient, a peptide or protein that acts competitively with TrkB protein in the interaction between BDNF and TrkB. Furthermore, the present invention relates to a liposome-bound protein preparation containing a TrkB antagonist as an active ingredient.

  Pain applied to the periphery is transmitted to the dorsal horn of the spinal cord through sensory nerves (primary neurons) located in the dorsal root ganglion (DRG), and to secondary neurons in the dorsal horn I and II layers. Transmitted via synapses, secondary neurons transmit their signals to the center (brain). Of the neurotrophic factor family, the expression of brain-derived neurotrophic factor (BDNF) is induced by pain stimulation. Tyrosine receptor kinase B (TrkB), a BDNF-specific receptor, binds BDNF to PI3K (phosphoinositide 3-kinase: PI3K), MAP kinase (extracellular signal-regulated kinase: ERK), phosphatidyl Pain is transmitted through inositol-specific phospholipase C (PLCγ) and Na ion channel (Nav1.9).

  Pain is classified by its duration into acute pain and chronic pain. Acute pain is pain associated with tissue damage and has a limited duration. Chronic pain persists after healing of the tissue disorder and often has no apparent organic cause. Acute exacerbation pain in chronic pain is similar to acute pain and is thought to be closely related to endogenous analgesics. Chronic pain includes intolerable chronic pain such as after-effects after a traffic accident or surgery, terminal cancer or diabetes. Pain is classified into nociceptive pain, neuropathic pain, and psychogenic pain according to the cause. These independent pains gradually merge over time, resulting in chronic refractory pain that is difficult to classify. If the neuropathic pain can be suppressed, it will be possible to alleviate the physical distress of the patient and improve the quality of life (QOL) in the clinical aspect.

  There is a report on a therapeutic agent for pain comprising a secretory TrkB, that is, a vector into which a gene encoding a protein consisting only of the extracellular region of TrkB is incorporated as an active ingredient (Patent Document 1: Patent No. 6105538). Patent Document 1 reported that BDNF could be captured after expression of an expression vector incorporating the above gene in a cultured cell system in vitro. Furthermore, it was also reported that administration of the above expression vector to a pain model animal in vivo reduced pain in a chronic pain animal. There is also a report that a TrkB-Fc fusion protein improves pain (Non-patent Document 1: Journal of Neurochemistry 93: 584-594 (2005)).

  In relation to TrkB, there is a report on a method for treating obesity or an overweight state containing a TrkB antagonist as an active ingredient (Patent Document 2: Japanese Patent Application Laid-Open No. 2009-525319). Here, an example of a TrkB antagonist is an anti-BDNF antibody, an anti-TrkB antibody or a TrkB-Fc fusion protein. There is a report that a TrkB-Fc fusion protein improves asthma (Non-patent document 2: Am J Physiol Lung Cell Mol Physiol 313: L360-L370 (2017)). There is also a report that the TrkB-Fc fusion protein improves epilepsy (Non-patent Document 3: Journal of Neuroscience, 19 (4), 1424-1436 (1999)). Furthermore, it has been reported that TrkB antagonist ANA-12 is effective for depression (Non-patent Document 4: J Clinical Investigation; 121, 1846-1857 (2011)). Regarding the interaction between BDNF and its receptor, TrkB, the function of TrkB is largely unknown, and the role of TrkB in various diseases is also elucidated.

Patent No.6105838 JP 2009-525319 A

Journal of Neurochemistry; 93, 584-594 (2005) Am J Physiol Lung Cell Mol Physiol; 313, L360-L370 (2017) Journal of Neuroscience; 19 (4), 1424-1436 (1999) Journal of Clinical Investigation; 121, 1846-1857 (2011)

  An object of the present invention is to provide a pharmaceutical composition containing a TrkB antagonist as an active ingredient. Specifically, it is an object of the present invention to find a peptide or protein that acts competitively with TrkB protein in the interaction between BDNF and TrkB, and to provide the peptide or protein as an active ingredient of a pharmaceutical composition. Furthermore, it is another object of the present invention to provide a preparation which can exert the action of a peptide or protein as an active ingredient more effectively and continuously.

  The present inventors have conducted intensive studies in order to solve the above problems, and as a result, have found a site that most effectively interacts with BDNF in the amino acid sequence constituting the TrkB protein, and completed the present invention. The peptide or protein related to the site that most effectively interacts with BDNF acts competitively with the TrkB protein in the interaction between BDNF and TrkB, for example, effectively acts on pain. Furthermore, by using the above-mentioned peptide or protein as a liposome-bound peptide or protein, the action of the peptide or protein is more effectively and continuously exhibited.

That is, the present invention includes the following.
1. A pharmaceutical composition comprising, as an active ingredient, a peptide or protein that acts competitively with a TrkB protein in the interaction between BDNF and TrkB.
2. The peptide or protein that acts competitively with the TrkB protein comprises the amino acid sequence shown in 1) or 2) below and is a peptide or protein that interacts with BDNF competitively with the TrkB protein. The described pharmaceutical composition:
1) an amino acid sequence represented by SEQ ID NO: 4 or SEQ ID NO: 10 in the sequence listing;
2) An amino acid sequence obtained by substituting, deleting, adding or introducing one or more amino acids in the amino acid sequence shown in 1) above.
3. 2. The pharmaceutical according to the above item 1, wherein the peptide or protein that acts competitively with the TrkB protein comprises the amino acid sequence shown in 3) or 4) below, and is a peptide or protein that interacts with BDNF competitively with the TrkB protein. Composition:
3) an amino acid sequence represented by SEQ ID NO: 6 or SEQ ID NO: 11 in the sequence listing;
4) An amino acid sequence obtained by substituting, deleting, adding or introducing one or more amino acids in the amino acid sequence shown in 3) above.
4. The pharmaceutical composition according to any one of Items 1 to 3, wherein the peptide or protein as an active ingredient is used for amelioration or treatment of any symptom or disease selected from pain, obesity, asthma, and epilepsy. .
5. A liposome-binding protein preparation, wherein the peptide or protein as an active ingredient contained in the pharmaceutical composition according to any one of the above items 1 to 4, is a liposome-binding protein bound to a liposome.

  Among the amino acid sequences constituting the TrkB protein, a peptide or protein containing a specific partial sequence selected from the extracellular region of the present invention has a binding ability to BDNF, and the peptide or protein competes with the TrkB protein for BDNF. Interacts with. Thereby, the peptide or protein as an active ingredient in the pharmaceutical composition of the present invention is used for amelioration or treatment of symptoms or diseases caused by abnormal interaction between TrkB protein and BDNF in vivo. Can be. Specifically, it can be used for improving or treating any symptom or disease selected from pain, obesity, asthma and epilepsy. The peptide or protein as an active ingredient of the present invention has a particularly long pain-suppressing effect. Furthermore, when the peptide or protein as an active ingredient is a liposome-bound protein bound to a liposome, the pain-suppressing effect lasts longer.

It is a figure which shows the structure of TrkB protein and TrkB (eTrkB) protein which has only an extracellular domain. FIG. 2 is a diagram schematically showing the configuration of each vector for producing each eTrkB protein of Example 1. (Example 1) FIG. 2 is a diagram showing an amino acid sequence (SEQ ID NO: 1) constituting a rat TrkB protein and an amino acid sequence (SEQ ID NO: 2) constituting a rat TrkB partial protein prepared from the No. 1 vector shown in FIG. (Example 1) FIG. 2 is a view showing an amino acid sequence (SEQ ID NOs: 3 to 6) constituting a rat TrkB partial protein prepared from each of the vectors Nos. 2 to 5 shown in FIG. (Example 1) FIG. 2 is a view showing an amino acid sequence (SEQ ID NOS: 7 to 8) constituting a rat TrkB partial protein prepared from each of the vectors Nos. 6 to 7 shown in FIG. (Example 1) The amino acid sequence (SEQ ID NO: 9) constituting the human TrkB protein, the amino acid sequence (SEQ ID NO: 10) constituting the human TrkB partial protein prepared from the vector corresponding to No. 3 in FIG. FIG. 9 is a view showing an amino acid sequence (SEQ ID NO: 11) constituting a human TrkB partial protein contained in a corresponding vector. (Example 1) FIG. 9 is a view showing the results of confirming the binding ability to BDNF of eTrkB purified protein (No. 3) prepared using Escherichia coli by electrophoresis. (Example 1) FIG. 9 shows the results of electrophoretic confirmation of the binding ability to BDNF for eTrkB purified protein (No. 5) produced using cultured higher animal cells. (Example 2) It is a figure which shows the result of having confirmed the pain suppression effect of eTrkB refinement protein (No. 5) using the L5 pain model rat. (Experimental example 1) FIG. 9 shows the results of confirming the pain-suppressing effect of an inactivated eTrkB purified protein (No. 5) obtained by heating eTrkB purified protein (No. 5) at 95 ° C. for 4 minutes using an L5 pain model rat. (Experimental example 1) It is a figure which shows the result of having confirmed the pain suppression effect using the L5 pain model rat about eTrkB refinement | purification protein (No.3) and liposome binding type eTrkB protein (No.3). (Experimental example 2) FIG. 9 shows the results of confirming the pain-suppressing effect of eTrkB-purified various proteins (Nos. 3 and 5) and liposome-bound eTrkB protein (No. 3) using L5 pain model rats. (Experimental example 3) FIG. 3 shows the results of confirming the pain-suppressing effect of TrkB-Fc protein using L5 pain model rats. (Experimental example 4) FIG. 4 shows the results of confirming the pain-suppressing effect of a TrkB-Fc protein by changing the dose using L5 pain model rats. (Experimental example 4) It is a figure which shows the result of having confirmed the pain inhibitory effect about the TrkB antagonist (ANA-12) using the L5 pain model rat. (Comparative Example 1)

  The present invention relates to a pharmaceutical composition containing a TrkB antagonist as an active ingredient. In the above, a TrkB antagonist means a substance that acts on BDNF competitively with TrkB protein in the interaction between TrkB and BDNF. In this sense, examples of TrkB antagonists refer to peptides or proteins that act on BDNF competitively with TrkB proteins.

  In the present specification, as the TrkB protein, specifically, a protein comprising the amino acid sequence represented by GenBank Accession number = M55291 (SEQ ID NO: 1) and the amino acid sequence represented by GenBank Accession number = Q16620.1 (SEQ ID NO: 9) And the like.

  In the pharmaceutical composition of the present invention, the peptide or protein contained as an active ingredient includes, for example, a peptide or protein selected from TrkB (eTrkB) protein having only the extracellular region of TrkB protein. For example, a peptide or protein consisting of the amino acid sequence of any one of the regions 1 to 6 of the extracellular region of the TrkB protein shown in FIG. 1, or one or more amino acids of these amino acid sequences are substituted, Peptides or proteins containing any amino acid sequence deleted, added or introduced and having a function of interacting with BDNF competitively with the TrkB protein are mentioned. In FIG. 1, region 1 is a signal peptide (Signal Peptide) sequence, region 2 is a leucine rich (Leucine rich repeat) sequence, region 3 is an immunoglobulin-like sequence, and region 4-5 is the fifth Trk protein. A domain (Fifth domain; immunoglobuline like) is shown, and region 7 shows a transmembrane region. In addition, in this specification, the difference between a peptide and a protein is generally interpreted by the difference in the number of amino acids constituting them, and is appropriately interpreted by those skilled in the art. In the present specification, a clear boundary line is not particularly defined for the difference in the number of amino acids constituting a peptide or protein.

  In the pharmaceutical composition of the present invention, the peptide or protein contained as an active ingredient is specifically any amino acid sequence selected from the amino acid sequences shown in SEQ ID NOs: 2 to 8, 10 and 11, or SEQ ID NO: 2 8, 10 and 11, one or more amino acids of any of the amino acid sequences selected from the amino acid sequence, substitution, deletion, including any of the amino acid sequence that is added or introduced, TrkB protein and Peptides or proteins having a function of competitively interacting with BDNF are mentioned.

In the pharmaceutical composition of the present invention, the peptide or protein contained as an active ingredient preferably has any one of the following amino acid sequences shown in 1) or 2), and is a peptide that interacts with BDNF competitively with TrkB protein: Or a protein.
1) an amino acid sequence represented by SEQ ID NO: 4 or SEQ ID NO: 10 in the sequence listing;
2) An amino acid sequence obtained by substituting, deleting, adding or introducing one or more amino acids in the amino acid sequence shown in 1) above.

In the pharmaceutical composition of the present invention, the peptide or protein contained as an active ingredient is a peptide or protein containing any one of the amino acid sequences shown in 3) or 4) below and interacting with BDNF competitively with TrkB protein. There may be.
3) an amino acid sequence represented by SEQ ID NO: 6 or SEQ ID NO: 11 in the sequence listing;
4) An amino acid sequence obtained by substituting, deleting, adding or introducing one or more amino acids in the amino acid sequence shown in 3) above.

  The present invention relates to a pharmaceutical composition containing, as an active ingredient, a peptide or protein that interacts with BDNF in a competitive manner with the TrkB protein, and can be used as a protein preparation.

  The peptide or protein contained as an active ingredient in the pharmaceutical composition of the present invention can be applied to any condition or disease caused by abnormal interaction between TrkB protein and BDNF in vivo. Examples of such symptoms and diseases include pain, obesity, asthma and epilepsy. The pharmaceutical composition comprising as an active ingredient a peptide or protein that interacts with BDNF competitively with the TrkB protein of the present invention is used for improving or treating any symptom or disease selected from pain, obesity, asthma and epilepsy. Used for

  Of all the symptoms and diseases caused by the abnormal interaction between the TrkB protein and BDNF in the living body described above, pain will be described in detail. Pain is classified by its duration into acute pain and chronic pain. Acute pain is pain associated with tissue damage and has a limited duration. Chronic pain persists after healing of the tissue disorder and often has no apparent organic cause. Acute exacerbation pain in chronic pain is similar to acute pain and is thought to be closely related to endogenous analgesics. Chronic pain includes intolerable chronic pain such as after-effects after a traffic accident or surgery, terminal cancer or diabetes. Pain is classified into nociceptive pain, neuropathic pain, and psychogenic pain according to the cause. These independent pains gradually merge over time, resulting in chronic refractory pain that is difficult to classify.

Neuropathic pain, unlike initial pain due to a wound (acute pain), is new pain that occurs after the wound has healed. As a neuropathic pain, a symptom that induces intense pain even by a stimulus that does not normally cause pain (called “allodynia”) is known. Conventionally, intractable pain such as neuropathic pain has been performed by combining existing narcotics, anti-inflammatory analgesics and the like with a nerve block injection (anesthetic). However, the effects of both methods are insufficient. For example, there is a report comparing the effects of neurotropin (NTP) and anti-inflammatory analgesics and antidepressants used for the treatment of pain diseases using L5 lumbar spinal nerve ligated rats, which are neuropathic pain models (Japan). Journal of Japan Society of Pain Clinicians Vol.15, No.14, 407-413 (2008)). Here, it has been reported that neurotropin ^(R) and milnacipran exhibited an analgesic effect only for 2 hours after drug administration, but the effect persistence is not sufficient.

  When the pharmaceutical composition of the present invention is applied to pain, a peptide or protein as an active ingredient is released from BDNF acting on the dorsal horn of the spinal cord among BDNF, more specifically from sensory nerves and / or glial cells. Peptides or proteins capable of competitively inhibiting the interaction between BDNF and TrkB protein are preferred. The active ingredient in the pharmaceutical composition of the present invention competitively acts with BDNF acting on the dorsal horn of the spinal cord, more specifically BDNF released from sensory nerves and / or glial cells and the like, whereby BDNF and TrkB protein Interactions can be blocked and subsequent transmission of pain can be suppressed. BDNF is a secretory protein having a molecular weight of 13.5 kD and forms a homodimer. BDNF binds with high affinity to its receptor, the specific receptor TrkB on the surface of target cells, and exerts its physiological effects via intracellular signaling mechanisms. BDNF is also known to be present in high concentrations in the hippocampus. Since BDNF may have a function to enhance pain transmission in the dorsal horn of the spinal cord, in the present invention, it is possible to suppress pain by competitively inhibiting the interaction between BDNF and TrkB protein. Conceivable.

  The amount of the peptide or protein as an active ingredient contained in the pharmaceutical composition of the present invention depends on the severity of each symptom or disease such as pain, obesity, asthma or epilepsy, the administration site, the number of administrations, the desired treatment period, and the age of the patient. , Weight, etc., and can be determined as appropriate. When the pharmaceutical composition containing the peptide or protein of the present invention as an active ingredient is a solution preparation, generally, 0.01 μg to 200 mg / mL, preferably 0.5 μg to 100 mg / It can contain mL of peptide or protein.

  In the pharmaceutical composition of the present invention, the peptide or protein contained as an active ingredient can be bound to liposomes or microcapsules, and more preferably a liposome-bound protein preparation. By binding the liposome or microcapsule to the peptide or protein as the active ingredient in the present invention, the action of the active ingredient in the pharmaceutical composition can act more continuously.

  The pharmaceutical compositions of the present invention can include a pharmaceutically acceptable carrier. In formulating the protein preparation, a stabilizer is preferably added. Examples of the stabilizer include albumin, globulin, gelatin, mannitol, glucose, dextran, ethylene glycol and the like. Further, the preparation of the present invention may be blended with additives necessary for preparation, for example, excipients, solubilizers, antioxidants, soothing agents, tonicity agents and the like. In the case of a liquid preparation, it is preferable to store the liquid preparation by removing the moisture by cryopreservation or lyophilization. The freeze-dried agent is used by adding distilled water for injection or the like to the business and redissolving. When a sustained release agent is used, as a carrier for sustained release, for example, soluble collagen or a soluble collagen derivative, proteins such as gelatin, porous ceramics, polyamino acid, polylactic acid, chitin or a chitin derivative, a water-swellable polymer gel Etc. can be used.

  The pharmaceutical composition of the present invention can be usually administered by a parenteral administration route, for example, administered as an injection (subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, spinal intrathecal injection, etc.). In addition, it can be administered transdermally, transmucosally, nasally, pulmonarily, etc. Such a pharmaceutical composition may be in the form of a solution or may be lyophilized for reconstitution before use. As an excipient for freeze-drying, for example, sugar alcohols such as mannitol and glucose and sugars can be used. In the case of solution preparations, they can be supplied in fixed volume containers such as sealed, sterilized plastic or glass vials, ampoules, syringes, and large volume containers such as bottles. it can.

  EXAMPLES The present invention will be specifically described with reference to Examples and Experimental Examples to facilitate understanding of the present invention, but it is needless to say that the present invention is not limited to these.

(Example 1) Preparation of eTrkB protein using Escherichia coli To prepare a TrkB (eTrkB) protein having only an extracellular region, an expression plasmid for Escherichia coli was constructed, introduced into Escherichia coli, and then purified. Based on the amino acid sequence of the rat TrkB gene (GenBank Accession number = M55291) (SEQ ID NO: 1), the extracellular region and the transmembrane region are divided into seven (1 to 7), and four types of modifications having a plurality of regions are provided. A cDNA expression vector was designed for each of the type proteins eTrkB (see FIG. 1).

In addition, the area | region divided into seven includes the following.
Region 1 = Signal Peptide sequence region 2 = Leucine rich repeat sequence region 3 = Immunoglobulin like sequence region 4-5 = Fifth domain; immunoglobuline like )
Region 7 = transmembrane region

Each cDNA was prepared by DNA synthesis. Each cDNA was fused with an S-tag sequence for purification and a FLAG-tag sequence, and introduced into a pHAT-10 plasmid having a lac promoter and a HAT-tag sequence. Sequencing analysis confirmed the TrkB gene and HAT-tag, FLAG-tag, and S-tag sequences.
No. 1: TrkB partial region including region 234567 shown in FIG. 1 (SEQ ID NO: 2)
No. 2: TrkB partial region including region 23456 shown in FIG. 1 (SEQ ID NO: 3)
No. 3: TrkB partial region including region 456 shown in FIG. 1 (SEQ ID NO: 4)
No. 4: TrkB partial region including region 45 shown in FIG. 1 (SEQ ID NO: 5)

  Escherichia coli BL21 (DE3) pLysS transfected with pHAT-10 plasmid (Nos. 1-4) is cultured, and lac promoter transcription is performed by adding IPTG (isopropyl β-D-thiogalactopyranoside, final concentration 2 mM) to the culture solution. Was induced to perform cDNA protein expression. Six hours later, Escherichia coli was collected from the culture solution, a portion was adjusted with an SDS-sample buffer, and SDS gel electrophoresis was performed. Protein expression was detected from the four plasmids. All were confirmed to be near the target estimated molecular weight. The remaining cells were stirred with PBS and sonicated to separate them into a solubilized fraction and an insoluble fraction, and the target protein was confirmed on an SDS gel. The target protein was present in the solubilized fraction only in the culture supernatant of No. 3 plasmid-containing cells (culture supernatant No. 3).

  Therefore, after adsorbing the solubilized fraction of the protein present in the culture supernatant No. 3 onto a TALON-Resin column (TAKARA BIO Inc. Siga, Japan) that specifically binds to the HAT-tag sequence, 10 mM imidazol And eluted with Imidazol (150-1000 mM) for purification.

  Using a part of the eTrkB protein solution obtained by purifying from the culture supernatant No. 3, add 0.5 μg of BDNF (Abcam ab9794, mature type; Cambridge) and Proteinase inhibitor (10 μL; Sigma) for 30 minutes. Mix at 4 ° C. Using the S-tag sequence of eTrkB protein, the eTrkB protein complex was adsorbed with S-protein agarose beads (50 μL; Novagen), pulled down, and washed five times with phosphate buffer. If eTrkB protein retains the ability to bind to BDNF, BDNF should be detected in the complex in which eTrkB protein has been precipitated. After adding SDS-sample buffer to the obtained complex, the mixture was heated at 95 ° C for 5 minutes, subjected to SDS gel electrophoresis, and analyzed by Western blot using an anti-BDNF antibody (ab9793). BDNF was detected. (FIG. 6, lane 1). It was confirmed that eTrkB protein purified from No. 3 culture solution (hereinafter, “eTrkB purified protein (No. 3)” can bind to BDNF.

(Example 2) Preparation of eTrkB protein using cultured cells of higher animals In order to prepare TrkB (eTrkB) protein having only an extracellular region, an expression plasmid for higher animals was constructed and introduced into cultured cells, followed by protein purification. Was. Based on the amino acid sequence of the rat TrkB gene (GenBank Accession number = M55291), three types of modified protein eTrkB cDNA expression vectors having extracellular regions were designed (FIG. 1).

No. 5: TrkB partial region including region 123456 shown in FIG. 1 (SEQ ID NO: 6)
No. 6: TrkB partial region including region 1456 shown in FIG. 1 (SEQ ID NO: 7)
No. 7: TrkB partial region including region 15 shown in FIG. 1 (SEQ ID NO: 8)

  Each cDNA was prepared by DNA synthesis. Each cDNA was fused with a purification His-tag sequence, S-tag sequence and FLAG-tag sequence, and introduced into a pCMVscript plasmid having a CMV promoter and a G418 drug resistance gene. Sequencing analysis confirmed the TrkB gene and the His-tag, S-tag, and FLAG-tag sequences.

  After introducing plasmids (Nos. 5 to 7) into human HEK293 cells (10 T-175TC flasks) using Lipofectamin 2000 (Invitrogen), the medium was replaced with a serum-free medium the next day, and 1 L of the culture supernatant was recovered two days later did. 1 mL of the culture supernatant was collected, eTrkB protein was pulled down with S-protein agarose beads (50 μL; Novagen), and analyzed by Western blot using anti-His antibody and anti-FLAG antibody. It was confirmed that eTrkB protein was secreted only in the culture supernatant of the plasmid-carrying cells (culture supernatant No. 5).

  Therefore, 0.5 μg of BDNF (Abcam ab9794, mature type) and proteinase inhibitor (Sigma) were added to 1 mL of culture supernatant No. 5 and mixed at 4 ° C. for 30 minutes, and eTrkB protein complex was added with S-tag agarose beads. The body was pulled down and washed 5 times with phosphate buffer. After adding an SDS-sample buffer to the obtained complex, the mixture was heated at 95 ° C. for 5 minutes, subjected to SDS gel electrophoresis, and analyzed by Western blot using an anti-BDNF antibody (Abcam ab9793). As a result, it was confirmed that eTrkB protein purified from the No. 5 culture solution (hereinafter, “eTrkB purified protein (No. 5)”) can bind to BDNF (FIG. 7, lane 2). The desired His-tag fusion protein was purified from the remaining culture supernatant using a Ni-NTA column (QIAGEN, Hilden, Germany).

(Experimental example 1) Experiment of administration of eTrkB protein to pain model rat In this experimental example, eTrkB purified protein (No. 3) prepared in Example 1 and eTrkB purified protein (No. 5) prepared in Example 2 were used. Was administered to a neuropathic pain model rat, and a test for determining the effect of suppressing pain was performed. With respect to the purified eTrkB protein (No. 5), a determination test of the pain-suppressing effect was also performed on the inactivated eTrkB, which was inactivated by heating the eTrkB purified protein (No. 5) at 95 ° C. for 4 minutes.

1) Preparation of pain model rat
Pain model rats were prepared according to the method of Chung JM et al. (Methods Mol Med 2004; 99, 35-45). Seven-week-old male Wistar rats were subjected to general anesthesia with sevoflurane. After making a midline incision in the back to identify and cut the L6 transverse process, the left L5 lumbar spinal nerve (peripheral to the dorsal root ganglion) was ligated with a 5-0 silk thread (hereinafter referred to as the “L5 pain model”). Rat "). The membrane and skin were sutured and disinfected. The model was made day0.

2) Pain evaluation method The pain was evaluated by the von Frey test. Specifically, the rats were placed in a transparent plastic case (10 × 13 × 15 cm ³ ) with a wire mesh pulled down, acclimated for 30 minutes, and then used with von Frey filaments (TACTILE TEST AESTHSIO, Murimachi Kikai Co.). Von Frey test. The pain threshold was calculated as a 50% PWT (paw withdrawal threshold) according to the method of Chaplan SR et al. (J Neurosci Methods 1994; 53, 55-63). Changes in the pain threshold were confirmed by a Wilcoxn rank-sum test adjusted by the Bonferroni method. p <0.05 was taken as a statistically significant difference.

3) Pain inhibitory action of each eTrkB protein One week after the L5 pain model rat prepared in 1) above was prepared (day 7), the pain threshold was measured. After the measurement of the pain threshold, general anaesthesia of the L5 pain model rat was performed with sevoflurane on day 7, and a catheter was inserted into the spinal cord of the rat. A 23G needle was punctured and inserted into the lumen through a catheter to a depth where the tip was centered on the L5 vertebral body. Each purified eTrkB protein was administered through a catheter and the pain threshold was measured.

・ Purified eTrkB protein (No.5)
After administration of 100 ng / 3 μL of eTrkB purified protein (No. 5), 20 μL of physiological saline was additionally administered (n = 9). As a control, 10 μL (n = 9) of physiological saline was administered. Before administration (day 7), there was no significant difference from 1.56 vs 1.01 (eTrkB group vs Control group, p = 1.00). On the other hand, two days after administration (day 9), a significant pain-suppressing effect was observed between 10.73 vs 1.19 (eTrkB group vs Control group, p = 0.0068) and the eTrkB purified protein (No. 5) administration group. Five days after administration (day 12), there was a tendency of suppression of 4.78 vs 1.32 (eTrkB group vs Control group, p = 0.179), but there was no statistically significant difference (FIG. 8).

・ Inactivated eTrkB purified protein (No.5)
The pain suppression effect was similarly confirmed for the inactivated eTrkB purified protein (No. 5) obtained by heating the above eTrkB purified protein (No. 5) at 95 ° C. for 4 minutes. After administration of 100 ng / 3 μL, a physiological saline solution was additionally administered at 20 μL (n = 6). As a control, 10 μL (n = 6) of physiological saline was administered. There was no difference in pain threshold between the inactivated eTrkB administration group (n = 6) and the control group (n = 6) (FIG. 9). These results indicate that the active heat-treated purified eTrkB protein (No. 5) has a strong pain-suppressing effect.

・ Purified eTrkB protein (No.3)
10 μL (n = 3) of 10 ng / μL of eTrkB purified protein (No. 3) was administered, and 20 μL (n = 5) of physiological saline was administered as a control. At 4 hours after administration (day 7.5), a very strong pain-suppressing effect was observed, and it continued until around day 12 (FIG. 10).

  The eTrkB purified protein (No. 5) is composed of 425 amino acids of TrkB shown in SEQ ID NO: 6 in the sequence listing, whereas the eTrkB purified protein (No. 3) is composed of 131 amino acids shown in SEQ ID NO: 4 in the sequence listing. It is configured. The above results show that the partial sequence related to the extracellular region of TrkB, and even the peptide identified by 131 amino acids shown in SEQ ID NO: 4 in the sequence listing has a pain-suppressing effect. It was suggested that it is possible to reduce the molecular weight of the peptide.

(Example 3) Preparation of eTrkB protein-bound liposome In this example, using the eTrkB purified protein (No. 3) prepared in Example 1, eTrkB protein-bound liposome (hereinafter referred to as "eTrkB protein-bound liposome") was prepared. , “ETrkB protein (No. 3) -bound liposome”).

  High purity phospholipids COATSOME NC-21 (hydrogenated soy phosphatidylcholines, 90% hydrogenated soy phosphatidylcholine) and COATSOME FE-8181SU5 (N- (Succinimidyloxy-glutaryl) -L-α-phosphatidyl ethanolamine, Dioleoyl) in chloroform-methanol solution After dissolution, the solvent was distilled off by vacuum drying to prepare a lipid thin film. The thin film was dissolved in a buffer (10 mM sodium phosphate (pH 8.0), 150 mM NaCl) and treated with a bath-type sonicator for 30 minutes to form liposomes (Katayama Chemical Industry Co., Ltd.).

  As a preliminary study, human serum albumin (HSA) and liposomes were processed and fractionated with an ultrafiltration membrane. After measuring the particle diameter and potential of the purified liposome, the purified liposome was decomposed and quantified for lipid and protein.

  Next, the eTrkB purified protein (No. 3) was processed with the liposome under these conditions, and fractionated with an ultrafiltration membrane. The particle size, potential, and polydispersity index (Polydispersity Index: PDI, an index for evaluating the width of the particle size distribution) of the obtained eTrkB protein (No. 3) -bound liposome are Zetasizer Nano ZSP (Malvern Instruments Ltd, Worcestershire, UK). For protein quantification, BCA Protein Assay Reagent (Thermo Fisher Scientific) was used, and for lipid measurement, phospholipid C-Test Wako (Wako Pure Chemical Industries, Ltd.) was used. The particle size of the eTrkB protein (No. 3) -bound liposome was 201 nm on average, and the potential was -16 mV. Since the polydispersity index DPI was 0.3 or less, it is considered that the preparation comprising the present eTrkB protein (No. 3) -bound liposome has a narrow size distribution. The lipid concentration was 1.7 mg / mL, the protein concentration was 57 μg / mL, and the amount of protein per lipid was 33.5 μg / mg. This preparation is composed of high-purity unsaturated phospholipid using olein, and is considered to exhibit remarkably high oxidative stability (FIG. 10).

(Experimental Example 2) Administration experiment of liposome eTrkB (No. 3) to pain model rat In this experimental example, the pain suppressing effect of the eTrkB protein (No. 3) -bound liposome prepared in Example 3 was examined. Evaluation was performed by the von Frey test using L5 pain model rats by the same method as in Example 1.

  As in Experimental Example 1, one week after the L5 pain model rat was prepared (day 7), the pain threshold was measured, and then the eTrkB protein (No. 3) -bound liposome was administered to the spinal subarachnoid space. The eTrkB protein (No. 3) -bound liposome was administered at a protein concentration of 10 ng / μL at 10 μL (n = 4). The eTrkB protein (No. 3) -bound liposome showed a tendency to suppress pain 4 hours after administration (day 7.5), and a pain suppressing effect was observed for about 10 days until day 16. When compared with the result of eTrkB purified protein (No. 3) of Experimental Example 1 in which the pain-suppressing effect was confirmed under the same conditions, the eTrkB-protein-bound liposome has a longer-lasting effect than the single administration of eTrkB protein. This was confirmed (FIG. 10).

(Experimental Example 3) Evaluation of Pain Suppressing Effect by Escape Reaction Time Measurement (Plantar Test Method) In this experimental example, eTrkB purified protein (No. 3) (Example 1), eTrkB purified protein (No. 5) (Example) 2) and eTrkB-purified protein (No. 3) -bound liposome (Example 3) were administered to L5 pain model rats, and a test for determining the pain-suppressing effect by measuring the escape reaction time (Plantar test method) was performed.

1) Preparation of a Pain Model Rat An L5 pain model rat was prepared according to the method of Experimental Example 1.

2) Pain evaluation method Pain was evaluated by measuring escape reaction time (Plantar test method). Specifically, after placing rats one by one in a plastic cage and acclimating to the environment, a thermal stimulus was applied to the hind footpad of the rat from under the glass plate, and the latency until the hind limb escape response was shown. It was measured and evaluated using a planter-type analgesic effect measuring device (MODEL37370, UgoBasile).

3) Pain-suppressing action of each eTrkB protein One week after the L5 pain model rat prepared in 1) above was prepared (day 7), the pain threshold was measured, and 10 ng / μL of the purified eTrkB protein (No. .5) in 10 μL (n = 6), 10 μL of 10 ng / μL purified eTrkB protein (No. 3) (n = 7), and 10 μL of eTrkB protein No. 3 binding liposome (protein concentration 10 ng / μL) (N = 7), and 10 μL (n = 3) of physiological saline was administered as Control.

  In the group treated with eTrkB purified protein (No. 5), there was almost no difference between the latency before the administration and the control group before the escape response, but the latency until the escape response in the eTrkB administration group 2 days after administration (day 9). An extension of time was seen. This indicates that administration of eTrkB suppresses hyperalgesia to thermal stimulation. FIG. 11 (A)

  Similarly, in the group treated with eTrkB purified protein (No. 3), there was almost no difference in latency until the escape response was observed before administration compared with the control group, but the escape response of the eTrkB administration group was observed 2 days after administration (day 9). An extension of the latency was seen. This indicates that administration of eTrkB suppresses hyperalgesia to thermal stimulation. FIG. 11 (B)

  In the eTrkB purified protein (No. 3) administration group and the eTrkB protein No. 3 binding type liposome administration group, hyperalgesia was suppressed 2 days after administration (day 9) in the protein-administered rats. From day 1 to at least 9 days after administration (day 16), statistically significant suppression of hyperalgesia is observed. This tendency of suppressing hyperalgesia in the liposome-administered rats was observed until 14 days after administration (day 21). FIG. 11 (C)

(Experimental example 4) Pain suppression effect by administration of TrkB-Fc protein In this experimental example, the same method as in Experimental example 1 was used for commercially available TrkB-Fc (Recombinant human TrkB-Fc Chimera protein, 688-TK-100, R & D systems). Was administered to an L5 pain model rat, and a pain suppression effect determination test was performed by a von Frey test.

  One week after the preparation of the L5 pain model rat shown in Experimental Example 1 (day 7), the pain threshold was measured, and then 100 ng / 5 μL of TrkB-Fc was administered to the spinal subarachnoid space by the same method as in Experimental Example 1 (n = 6). As a control, 10 μL (n = 6) of physiological saline was administered. As a result of measuring the pain threshold, no significant difference was observed between the TrkB-Fc group and the control group (FIG. 12). Although the amount of TrkB-Fc protein was administered at 100 ng in the same manner as the amount of eTrkB protein, the effect of improving the pain threshold was low because the molecular weight of TrkB-Fc increased and the number of moles of TrkB protein administered as an active ingredient was low It was thought to be.

  From the above results, it was confirmed whether the pain threshold was improved by increasing the dose of TrkB-Fc. The pain threshold was measured under exactly the same conditions as above except that 1 μg / 5 μL of TrkB-Fc was administered. As a result, the effect of improving the pain threshold was confirmed (FIG. 13). From the above results, it was considered that the eTrkB protein can exert an analgesic effect with a smaller dose than TrkB-Fc.

(Comparative Example 1) Pain Suppressing Effect by Administration of TrkB Antagonist (ANA-12) In this experimental example, TrkB antagonist ANA-12 (SML 0209, N- [2-[[(Hexahydro-2-oxo-1H-azepin) -3-yl) amino] carbonyl] phenyl] -benzo [b] thiophene-2-carboxamide (igma-Aldrich) was administered to L5 pain model rats by the same method as in Experimental Example 1, and the pain was suppressed by the von Frey test. Was carried out.

  One week after the L5 pain model rat shown in Experimental Example 1 was prepared (day 7), the pain threshold was measured, and then ANA-12 was administered to the spinal subarachnoid space by 100 ng / 5 μL in the same manner as in Experimental Example 1 (n = 7). As a control, 10 μL (n = 6) of physiological saline was administered.

  Before administration (day 7), the ratio was 0.74 vs 0.78 (ANA-12 group vs control group, p = 1.00), and there was no difference between the two groups, but 11.83 4 hours after administration of ANA-12 (day 7.5). There was a significant difference with vs 1.01 (ANA-12 group vs Control group, p = 0.0132). On day 8, the ratio was 3.47 vs 1.19 (ANA-12 group vs Control group, p = 0.0704), indicating that ANA-12 administration tended to show a pain-suppressing effect, but there was no statistically significant difference . Two days after administration (day 9), the ratio was 1.60 vs 1.32 (ANA-12 group vs Control group, p = 0.88), and there was no difference between the two groups (FIG. 14).

  It has been reported that ANA-12 acts as an antagonist of TrkB and can inhibit the BDNF / TrkB pathway by competing with BDNF (Cazorla M. et al., J Clinical Investigation 2011; 121, 1846-1857). Regarding ANA-12, there have been some reports on depression (Zhang JC et al., Int J Neuropsychopharmacol 2015; 18, 1-9; Shirayama Y et al., Eur Neuropsychopharmacol 2015; 25, 2449-2458). On the other hand, there is no report on neuropathic pain, and the result of this comparative example is the first report that the pain threshold was improved by administration to the spinal subarachnoid space. However, the improvement effect of pain observed by the administration of ANA-12 was confirmed 4 hours after administration, but on the next day, the L5 pain model rat was in a state of hypersensitivity. Since the eTrkB protein had an effect of improving the pain threshold even after 2 days, it was considered that the eTrkB protein lasted longer than ANA-12 by a single administration.

  As described in detail above, among the amino acid sequences constituting the TrkB protein, a peptide or protein containing a specific partial sequence selected from the extracellular region of the present invention has a binding ability to BDNF, and the peptide or protein is Interacts with BDNF competitively with TrkB protein. Thereby, the peptide or protein as an active ingredient in the pharmaceutical composition of the present invention can be used for improving or treating a symptom or disease caused by abnormal interaction between TrkB protein and BDNF in vivo. it can. Specifically, it can be used for improving or treating any symptom or disease selected from pain, obesity, asthma and epilepsy. The peptide or protein as an active ingredient of the present invention has a particularly long pain-suppressing effect. Furthermore, when the peptide or protein as an active ingredient is a liposome-bound protein bound to a liposome, the pain-suppressing effect lasts longer.

  Among the above-mentioned symptoms and diseases, in particular, pain is presently insufficient in terms of the sustainability of the effect even with existing pharmaceuticals at present. It has been confirmed that by using a peptide or protein as an active ingredient in the pharmaceutical composition of the present invention, the effect can be more sustained, and by using a liposome-bound preparation, the effect can be further maintained. Be expected. Chronic pain includes intolerable chronic pain such as after-effects after a traffic accident or surgery, terminal cancer or diabetes. The number of patients with cancer and diabetes is large. If the symptoms of chronic pain associated with these patients can be suppressed or reduced, clinical pain can be alleviated and QOL can be improved, and industrial applicability is excellent.

Claims (5)

A pharmaceutical composition comprising, as an active ingredient, a peptide or protein that acts competitively with a TrkB protein in the interaction between BDNF and TrkB. The peptide or protein that competitively interacts with the TrkB protein is a peptide or protein that includes any of the following amino acid sequences 1) or 2) and that interacts with BDNF competitively with the TrkB protein: Pharmaceutical composition according to:
1) an amino acid sequence represented by SEQ ID NO: 4 or SEQ ID NO: 10 in the sequence listing;
2) An amino acid sequence obtained by substituting, deleting, adding or introducing one or more amino acids in the amino acid sequence shown in 1) above. The peptide or protein which competitively interacts with the TrkB protein, wherein the peptide or protein has any one of the amino acid sequences shown in the following 3) or 4) and is a peptide or protein which interacts with BDNF competitively with the TrkB protein: Pharmaceutical composition according to:
3) an amino acid sequence represented by SEQ ID NO: 6 or SEQ ID NO: 11 in the sequence listing;
4) An amino acid sequence obtained by substituting, deleting, adding or introducing one or more amino acids in the amino acid sequence shown in 3) above. The pharmaceutical composition according to any one of claims 1 to 3, wherein the peptide or protein as an active ingredient is used for amelioration or treatment of any symptom or disease selected from pain, obesity, asthma and epilepsy. object. A liposome-bound protein preparation, wherein the peptide or protein as an active ingredient contained in the pharmaceutical composition according to any one of claims 1 to 4 is a liposome-bound protein bound to a liposome.