M1 microglia-targeting fusion peptide and use thereof

Abstract

The purpose of the present invention is to provide a novel means that targets M1 microglia and is useful for the treatment of central nervous system diseases and the like. Provided is an M1 microglia-targeting fusion peptide in which an M1 microglia-targeting peptide and an apoptosis-inducing peptide are directly or indirectly linked to each other, wherein the M1 microglia-targeting peptide is a peptide mentioned in (1) or (2): (1) a peptide which has a structure such that at least one cysteine residue is directly linked to each of both ends of the amino acid sequence represented by HHSSSAR (SEQ ID NO: 1) or an amino acid sequence formed by linking a plurality of the sequences; and (2) a peptide which comprises an amino acid sequence having a structure such that one amino acid residue is or a plurality of amino acid residues are deleted, substituted or added in the amino acid sequence for the peptide (1) and which targets M1 microglia.

Description

M1 microglia-targeting fusion peptide and its utilization

[0001] Regarding M1 microglia-targeting fusion peptides and their applications.

[0002] Microglia are a type of glial cell in the central nervous system and are classified into types such as M1 and M2 depending on their characteristics. In particular, M1 microglia are known to be neurotoxic (inflammatory) microglia that release neurotoxic factors such as inflammatory cytokines, nitric oxide, and reactive oxygen species, while M2 microglia are known to be neuroprotective (anti-inflammatory) microglia that release anti-inflammatory cytokines, anti-inflammatory factors, and neuroprotective factors.

[0003] To date, a peptide capable of targeting M1 microglia (M1-Type-Specific Homing Peptide) has been reported, in which one cysteine ​​residue is directly linked to each end of the amino acid sequence HHSSSAR (SEQ ID NO: 1) by peptide bonds (Non-Patent Literature 1).

[0004] On the other hand, for example, neonatal hypoxic-ischemic encephalopathy (HIE) develops due to a hypoxic environment during delivery and causes neurological sequelae such as cerebral palsy. Conventionally, therapies such as cerebral hypothermia have been used to treat neonatal HIE, but their effectiveness is limited, and the incidence of cerebral palsy has not changed for the past 60 years, remaining at 1 to 3 per 1,000 births. Therefore, it is important to provide new methods that are useful for treating neonatal HIE. Furthermore, it is important to provide new methods that are useful not only for neonatal HIE but also for treating central nervous system diseases and other conditions involving M1 microglia.

[0005] Terashima T., et al., Gene Therapy for Neuropathic Pain through siRNA-IRF5 Gene Delivery with Homing Peptides to Microglia, Molecular Therapy: Nucleic Acids Vol. 11 June 2018, pp. 203-215

[0006] The objective is to provide a novel method that targets M1 microglia and is useful for treating central nervous system diseases and other conditions.

[0007] As a result of diligent research, the Disclosing Parties have found that an M1 microglia-targeting fusion peptide, which is a combination of an M1 microglia-targeting peptide and an apoptosis-inducing peptide, is useful in the treatment of neonatal HIE. This disclosure is the result of further research based on this finding and includes inventions such as those described below.

[0008] Item 1. An M1 microglia-targeting fusion peptide, wherein an M1 microglia-targeting peptide and an apoptosis-inducing peptide are directly or indirectly linked, wherein the M1 microglia-targeting peptide is a peptide represented by (1) or (2) below: (1) A peptide in which at least one cysteine ​​residue is directly linked to both ends of an amino acid sequence represented by HHSSSAR (SEQ ID NO: 1) or an amino acid sequence in which multiple such sequences are linked; (2) The peptide of (1), wherein the amino acid sequence consists of an amino acid sequence in which one or more amino acids are deleted, substituted, or added, and which targets M1 microglia. Item 2. The M1 microglia-targeting fusion peptide according to Item 1, wherein at the cysteine ​​residues at both ends, one cysteine ​​residue forms a disulfide bond with the cysteine ​​residue at the other end. Item 3. The M1 microglia-targeting fusion peptide according to item 1 or 2, wherein the apoptosis-inducing peptide is at least one peptide selected from the group consisting of a peptide comprising the amino acid sequence represented by KLAKLAKKLAKLAK (SEQ ID NO: 2), a peptide comprising the amino acid sequence represented by LLGDFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (SEQ ID NO: 20), a peptide comprising the amino acid sequence represented by GIGKFLHSAKKFGKAFVGEIMNS (SEQ ID NO: 21), and a peptide comprising the amino acid sequence represented by GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 22). Item 4. The M1 microglia-targeting fusion peptide according to any one of items 1 to 3, wherein the apoptosis-inducing peptide is a peptide comprising the amino acid sequence represented by KLAKLAKKLAKLAK (SEQ ID NO: 2). Item 5. An M1 microglia-targeting fusion peptide according to any one of claims 1 to 4, for the treatment of at least one central nervous system disorder selected from the group consisting of hypoxic-ischemic encephalopathy, Alzheimer's disease, Parkinson's disease, cerebral infarction, hypoxic encephalopathy, spinal cord injury, neuropathic pain, amyotrophic lateral sclerosis, and multiple sclerosis. Claim 6. A pharmaceutical composition containing an M1 microglia-targeting fusion peptide according to any one of claims 1 to 5.7. A pharmaceutical composition according to 6, for the treatment of at least one central nervous system disorder selected from the group consisting of hypoxic-ischemic encephalopathy, Alzheimer's disease, Parkinson's disease, cerebral infarction, hypoxic encephalopathy, spinal cord injury, neuropathic pain, amyotrophic lateral sclerosis, and multiple sclerosis. 8. A method for delivering an M1 microglia-targeted fusion peptide according to any one of 1 to 5 to M1 microglia, comprising the step of administering to a target subject an M1 microglia-targeted fusion peptide according to any one of 1 to 5 or a pharmaceutical composition according to 6 or 7.

[0009] The M1 microglia-targeted fusion peptide disclosed herein is useful for the treatment of neonatal HIE. Furthermore, according to this disclosure, the M1 microglia-targeted fusion peptide can be specifically delivered to M1 microglia. For this reason, the M1 microglia-targeted fusion peptide disclosed herein is useful for the treatment of central nervous system diseases and other conditions involving M1 microglia.

[0010] This paper presents a model morphology of an M1 microglia-targeting fusion peptide (MG1-KLA fusion peptide) formed by linking an M1 microglia-targeting peptide with an apoptosis-inducing peptide via a linker. It also demonstrates the in vitro M1 microglia apoptosis-inducing effect of the MG1-KLA fusion peptide, the administration method and binding rate of the MG1-KLA fusion peptide to HIE mice, the effect of reducing M1 microglia, the evaluation effect on neurological disorders, the evaluation effect on behavioral function, and the evaluation effect of cytokines by q-PCR.

[0011] The embodiments included in this disclosure will be described in further detail below. In this disclosure, “contains” also means “substantially consists of” or “consists of.”

[0012] This disclosure includes M1 microglia-targeting fusion peptides, which are formed by directly or indirectly linking an M1 microglia-targeting peptide with an apoptosis-inducing peptide. In the fusion peptide, the M1 microglia-targeting peptide is a peptide represented by (1) or (2) below: (1) A peptide in which at least one cysteine ​​residue is directly linked to both ends of an amino acid sequence represented by HHSSSAR (SEQ ID NO: 1) or an amino acid sequence in which multiple such sequences are linked; (2) The peptide of (1), wherein the amino acid sequence consists of an amino acid sequence in which one or more amino acids are deleted, substituted, or added, and which targets M1 microglia.

[0013] Hereinafter, the M1 microglia-targeting fusion peptide may be referred to as "the fusion peptide of this disclosure" in this disclosure.

[0014] In this disclosure, the M1 microglia-targeting peptide may also be referred to as the M1 microglia-homing peptide, and is the peptide represented by (1) or (2) above.

[0015] The above (1) is a peptide in which at least one cysteine ​​residue is directly linked to both ends of the "amino acid sequence represented by HHSSSAR (SEQ ID NO: 1)" or "an amino acid sequence in which multiple such sequences are linked together". The "amino acid sequence in which multiple such sequences are linked together" is, in other words, an "amino acid sequence in which multiple HHSSSAR (SEQ ID NO: 1) sequences are linked together". The linkage is not particularly limited, as long as it can target M1 microglycemic in the same way as the "amino acid sequence represented by HHSSSAR (SEQ ID NO: 1)", and preferably an example is one in which the amino acid sequence HHSSSAR is directly linked by a peptide bond. The range of "multiple" preferably includes two or three amino acid sequences, and more preferably two amino acid sequences.

[0016] In (1) above, at least one cysteine ​​residue is directly linked to each end of the amino acid sequence ("the amino acid sequence represented by HHSSSAR (SEQ ID NO: 1)" or "an amino acid sequence formed by linking multiple such sequences"). This linkage is not particularly limited as long as the fusion peptide of this disclosure can target M1 microglia, and preferably an example is one in which a cysteine ​​residue is directly linked to the end of the amino acid sequence by a peptide bond. The number of cysteine ​​residues at both ends may be the same or different, and the number of cysteine ​​residues at each end is exemplified as 1 to 3, preferably 1 or 2, and more preferably 1.

[0017] The peptide in (1) above can take any form, such as linear or loop-shaped, and is preferably loop-shaped, and more preferably a loop-shaped form in which the cysteine ​​residue at one end forms a disulfide bond with the cysteine ​​residue at the other end. A model form of a loop-shaped peptide is shown in Figure 1. The model form shown in Figure 1 is a loop-shaped M1 microglia-targeting peptide consisting of a total of nine amino acid residues, with one cysteine ​​residue (C) linked to each end.

[0018] The number of amino acid residues in the peptide (1) is not particularly limited, as long as the peptide can target M1 microglia, but is preferably 9 to 16, more preferably 9 to 14, and even more preferably 9.

[0019] The peptide in (2) above is a peptide in which the amino acid sequence ("the amino acid sequence represented by HHSSSAR (SEQ ID NO: 1)" or "an amino acid sequence in which multiple such sequences are linked together") consists of an amino acid sequence in which one or more amino acids are deleted, substituted, or added, and which targets M1 microglia.

[0020] In (2) above, the range of "one or more amino acids" is not particularly limited, as long as the peptide in (2) can target M1 microglia, and includes, for example, 1 to 5, preferably 1 to 4, more preferably 1 to 3, even more preferably 1 or 2, and particularly preferably 1. In particular, when the "amino acid sequence" described in (2) above is the "amino acid sequence represented by HHSSSAR (SEQ ID NO: 1)", one is preferably given as an example of the range of "one or more amino acids". Techniques for deleting, substituting, or adding one or more amino acids in a specific amino acid sequence are known. In this disclosure, amino acids are defined in accordance with common technical knowledge in the art and typically include alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V).

[0021] In this disclosure, "targeting M1 microglia" and "M1 microglia targeting" mean that the peptides in this disclosure have a higher affinity for M1 microglia compared to M2 microglia, or in other words, that they can recognize M1 microglia with higher efficiency compared to M2 microglia. For example, Non-Patent Literature 1 reports that a peptide in which one cysteine ​​residue is directly linked to each end of the amino acid sequence represented by HHSSSAR (Sequence ID 1) by peptide bonds can target M1 microglia. Therefore, in this disclosure, "targeting M1 microglia" and "M1 microglia targeting" mean that the peptides have a high affinity for M1 microglia to a degree equivalent to or greater than that of a peptide in which one cysteine ​​residue is directly linked to each end of the amino acid sequence represented by Sequence ID 1 by peptide bonds.

[0022] This affinity can be confirmed by a procedure similar to the in vitro culture and staining procedure described in the "Analysis of Binding of Homing Peptides to Astrocytes and Microglia In Vitro and In Vivo" section of Non-Patent Literature 1. A peptide that shows a higher positive (staining) rate in M1 microglia than in M2 microglia, and is equivalent to or better than the peptide in which one cysteine ​​residue is directly linked to each end of the amino acid sequence represented by SEQ ID NO: 1 by peptide bonds (hereinafter sometimes referred to as the reference peptide), can be determined to have a high affinity for M1 microglia. More specifically, the determination procedure involves contacting the peptide (FITC labeled) with microglia (primary cultured M1 microglia, primary cultured M2 microglia), culturing at 37°C for 24 hours, and then fixing the cells with 4% paraformaldehyde at 20°C for 10 minutes. After fixation, the cells are incubated with a primary antibody (rabbit anti-Iba-1 antibody (for microglia), rabbit anti-CD86 antibody (for M1 microglia), or rabbit anti-CD206 antibody (for M2 microglia)), and then incubated again with a secondary antibody (Alexa Fluor 555). Next, the stained cells are counted using a confocal laser microscope, and the ratio of M1 microglia to microglia (M1 microglia positivity rate) and the ratio of M2 microglia to microglia (M2 microglia positivity rate) are calculated and compared. If the M1 microglia positivity rate relative to the M2 microglia positivity rate is equal to or higher than the positivity rate for the standard peptide, it can be determined that the peptide has high affinity for M1 microglia. Unless otherwise specified, the procedure is the same as in the test examples described later.

[0023] Apoptosis-inducing peptides are not particularly limited, and antimicrobial peptides (AMPs) are exemplified. Examples of antimicrobial peptides include those with approximately 6 to 40 amino acid residues. Although not limiting to this disclosure, examples of apoptosis-inducing peptides include, preferably, peptides consisting of the amino acid sequence represented by KLAKLAKKLAKLAK (SEQ ID NO: 2), LL-37, Magainin-2, Melittin (bee venom peptide), and other antimicrobial peptides.

[0024] Without limiting this disclosure, a more preferred example of an apoptosis-inducing peptide is a peptide consisting of the amino acid sequence represented by KLAKLAKKLAKLAK (SEQ ID NO: 2). The peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is known as a peptide that induces cell apoptosis by disrupting the mitochondrial membrane, and is also known by names such as KLA and helical peptide KLA. The peptide KLA may be either the D-form or the L-form, and is preferably the D-form. The apoptosis-inducing peptide is commercially available.

[0025] Furthermore, LL-37 is a human-derived AMP (amphidopropyl Aminopropyl Algebra) and is known as a peptide that induces apoptosis in cells by disrupting the mitochondrial membrane, and is used in cancer and inflammation models (Bankell et al., Biochemistry and Biophysics Reports 2022, 29:101192, Mader et al., Mol. Cancer Res. 2009, 7(5):689-702). An example of LL-37 is the peptide consisting of the amino acid sequence represented by LLGDFRFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (SEQ ID NO: 20). This apoptosis-inducing peptide is also commercially available.

[0026] Furthermore, Maginin-2 is known to possess an amphipathic α-helix structure and induce cell death by transmembrane penetration (Imura et al., Biophys J. 2008, 95(12):5757-5765). An example of Maginin-2 is a peptide consisting of the amino acid sequence represented by GIGKFLHSAKKFGKAFVGEIMNS (SEQ ID NO: 21). This apoptosis-inducing peptide is also commercially available.

[0027] Furthermore, Melittin (a bee venom peptide) is known to possess strong membrane-disrupting properties due to its amphiphilic structure, and can disrupt cell membranes and mitochondrial membranes (GM Kong et al., World J Gastroenterol., 2016, 22(11):3186-3195). An example of Melittin is the peptide consisting of the amino acid sequence represented by GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 22). This apoptosis-inducing peptide is also commercially available.

[0028] In the fusion peptides of this disclosure, the M1 microglia-targeting peptide and the apoptosis-inducing peptide are linked directly or indirectly. When linked indirectly, they are preferably linked via a linker. The linker is not limited to the extent that it does not impede the effects of this disclosure, and examples include a linker consisting only of glycine (G), a linker consisting only of serine (S), a GS linker, an EAAAK linker, etc. The length of the linker is not particularly limited, and examples of the number of amino acid residues of the linker include preferably 2 to 10, more preferably 2 to 6, even more preferably 2 to 5, and particularly preferably 2, 3, or 4. As a GS linker, (G4S)n is preferably exemplified, where n is an integer of 1 or 2. As an EAAAK linker, (EAAAK)n is exemplified, where n is an integer of 1 or 2 (EAAAK is shown in Sequence ID No. 3). A linker consisting only of glycine (G) is preferably exemplified as a linker. Linkers may be used individually or in combination of two or more types.

[0029] A preferred example of the fusion peptide of this disclosure is one in which an apoptosis-inducing peptide is linked to the terminal cysteine ​​residue of the M1 microglia-targeting peptide via a linker. In this case, the apoptosis-inducing peptide may be linked to only one cysteine ​​residue or to both cysteine ​​residues, as long as the effects of this disclosure are obtained. A preferred example of the fusion peptide of this disclosure is one in which the apoptosis-inducing peptide is linked via a linker to a cysteine ​​residue that is directly linked to an arginine residue (R) constituting the M1 microglia-targeting peptide, and this model configuration is shown in Figure 1.

[0030] The M1 microglia-targeting peptides, apoptosis-inducing peptides, and fusion peptides described herein can all be produced according to conventionally known chemical synthesis methods, genetic engineering techniques, etc. For example, peptides may be synthesized and purified by conventionally known chemical synthesis methods according to the amino acid sequence or nucleotide sequence information encoding the peptide. Chemical synthesis methods include liquid-phase and solid-phase peptide synthesis methods. Alternatively, the polynucleotide encoding the peptide may be inserted into a vector, and then the desired peptide may be obtained after culturing a transformant incorporating the vector.

[0031] Without limiting this disclosure, the base sequence represented by Sequence ID No. 4 is given as an example of a polynucleotide encoding the amino acid sequence represented by Sequence ID No. 1. Thus, polynucleotides encoding various amino acid sequences constituting the fusion peptide of this disclosure can be analyzed and obtained using conventionally known methods.

[0032] Furthermore, the M1 microglia-targeting peptide of this disclosure may be linked to an apoptosis-inducing substance other than the apoptosis-inducing peptide (hereinafter sometimes simply referred to as "apoptosis-inducing substance"). Thus, this disclosure can also be said to encompass M1 microglia-targeting fusion peptides, in which the M1 microglia-targeting peptide and the apoptosis-inducing substance are directly or indirectly linked. In such fusion peptides, the M1 microglia-targeting peptide is described in the same manner as described above.

[0033] Examples of such apoptosis-inducing substances include agents that induce apoptosis (programmed cell death) in mitochondria. For example, substances that directly stimulate the mitochondrial pathway (intrinsic apoptosis system) are exemplified. As such substances, BH3 mimetics (BH3 mimetics, BCL-2 inhibitors) are exemplified, and small molecule substances such as ABT-737, ABT-263 (navitoclax), Venetoclax (ABT-199), etc., which directly induce apoptosis by mitochondrial outer membrane permeabilization (MOMP), are exemplified. Further, the apoptosis-inducing substance may be an inhibitor such as Bcl-xL (BCL-XL inhibitor, etc.) known as an anti-apoptotic protein similar to BCL-2. Examples of such inducing substances, as well as other apoptosis-inducing substances, include cisplatin, camptothecin, fenbendazole, elesclomol, staurosporine, gossypol, MT-21, darinaparsin, etc.

[0034] For example, cisplatin (molecular formula Cl2H6N2Pt), camptothecin (C

[0036] , 19 , 20 H 16 N2O4) is a compound known as a chemotherapeutic agent that causes DNA damage, and it is known that part of its cell death-inducing effect activates apoptosis in the mitochondrial pathway through signal transduction such as p53.

[0035] For example, fenbendazole (C 15 H 13 N3O2S) is an anthelmintic for animals, but is a compound known to induce apoptosis in cancer cells through mitochondrial damage and inhibition of glucose metabolism, etc.

[0036] For example, elesclomol (C 19 H 20N4O2S2) is a copper ionophore that targets mitochondria, destroys iron-sulfur clusters, and induces mitochondrial outer membrane permeabilization (MOMMP) and apoptosis by causing a burst of reactive oxygen species (ROS).

[0037] For example, staurosporine (C 28 H 26 N4O3) is a non-specific agent that inhibits various protein kinases and is a compound known to have a strong apoptosis-inducing effect including the mitochondrial pathway.

[0038] For example, gossypol (C 30 H 30 O8) is a natural compound that targets Bcl-2 family proteins and promotes apoptosis. In particular, the R-(−)-enantiomer of gossypol is known to induce apoptosis by activating caspase-9 and is known to exhibit cytotoxicity against multiple myeloma and drug-resistant cell lines.

[0039] For example, MT-21 (C 16 H 27 NO3) is an apoptosis inducer that targets mitochondria, causes the release of cytochrome c from mitochondria, and is known to induce apoptosis by activating caspase-9.

[0040] For example, dacinaparsin (C 12 H 22 AsN3O6S) is a compound known as an anti-tumor drug that targets mitochondria, causes disorders of mitochondrial function and an increase in the production of reactive oxygen species, and is known to induce apoptosis.

[0041] The linkage of the apoptosis inducer to the M1 microglia-targeting peptide of the present disclosure is not limited as long as they can be linked, and according to the conventional known linking means between a peptide and a compound or the like, reagents, linkers, etc. can be appropriately used for linking according to the type of the apoptosis inducer.

[0042] The apoptosis-inducing peptide or any other apoptosis-inducing substance may be used alone or in combination of two or more, as needed. While not limiting this disclosure, preferably the apoptosis-inducing peptide is linked to the M1 microglia-targeting peptide.

[0043] When the fusion peptides of this disclosure are used in vivo, the route of administration to the target organism (subject, target animal) is not particularly limited, and parenteral administration such as intravenous administration, nasal administration, subcutaneous administration, intramuscular administration, intrathecal administration (including intraspinal cavity), and direct administration to tissue (ventricular administration, etc.) are examples. The target organisms for which the peptides of this disclosure are not particularly limited, and mammals such as humans and non-human primates are examples. The dosage, administration interval, etc. of the fusion peptides of this disclosure should be appropriately determined according to the target organism, route of administration, type of disease the target organism suffers from, expected effect, etc. Furthermore, the fusion peptides of this disclosure may be further modified or inserted with sequences known to promote blood-brain barrier passage, or linked with small molecules known to promote blood-brain barrier passage, as needed. When the fusion peptides of this disclosure are used in vitro or ex vivo, the fusion peptides of this disclosure may be administered (contacted) to the target organism (e.g., cells, tissues, organs, etc.), and incubated as needed. The dosage (contact amount), administration interval, incubation conditions, etc., of the fusion peptide disclosed herein may be determined as appropriate.

[0044] The fusion peptide of this disclosure can target M1 microglia due to the M1 microglia-targeting peptide that constitutes the fusion peptide, and therefore can selectively deliver the fusion peptide to cells in which M1 microglia are present and / or accumulate (target cells). As a result, the apoptosis-inducing peptide or the apoptosis-inducing substance that constitutes the fusion peptide of this disclosure can act to induce cell death in the target cells. Thus, the fusion peptide of this disclosure can directly induce apoptosis in M1 microglia that produce inflammatory cytokines and induce neurological damage.

[0045] The inventors, in conducting tests as shown in the test examples described below, have confirmed that M1 microglia accumulate in the brain in hypoxic-ischemic encephalopathy. As shown in the test examples described below, the fusion peptide of this disclosure can selectively target M1 microglia and directly induce apoptosis in target cells (i.e., M1 microglia). Therefore, the fusion peptide of this disclosure can be said to be useful in the treatment of central nervous system diseases associated with M1 microglia, such as hypoxic-ischemic encephalopathy (including neonatal hypoxic-ischemic encephalopathy), Alzheimer's disease, Parkinson's disease, cerebral infarction, hypoxic encephalopathy, spinal cord injury, neuropathic pain, amyotrophic lateral sclerosis, and multiple sclerosis. In this disclosure, treatment includes inhibiting disease progression and improvement.

[0046] Without limiting this disclosure, an example of a central nervous system disease is described in more detail below. Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive cognitive decline, memory impairment, and the accumulation of amyloid-beta plaques and tau neurofibrillary tangles. Inflammatory cytokines are thought to play an important role in the pathophysiology of AD, and high levels of TNF-α, IL-1β, and IL-6 have been detected in the blood and cerebrospinal fluid of AD patients. Microglia that accumulate around amyloid-beta plaques recognize and phagocytose amyloid-beta as a foreign substance, but excessive accumulation of amyloid-beta plaques activates microglia, which leads to the excessive release of inflammatory cytokines such as TNF-α and nerve cell damage, and this cycle of further accumulation of amyloid-beta plaques accelerates the progression of AD (Hickman et al., The Journal of Neurosci., 2008, 28(33):8354-8360).

[0047] Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by a decrease in dopaminergic neurons and Lewy bodies composed of α-synuclein aggregates. It causes motor symptoms such as tremors, muscle rigidity, and bradykinesia, as well as non-motor symptoms such as cognitive decline and sleep disturbances. Inflammatory cytokines such as TNF-α, released in large quantities from microglia, not only damage dopaminergic neurons but also cause further activation of microglia, neuroinflammation, and neurodegeneration. Postmortem brain analysis of PD patients has detected high levels of activated microglia and inflammatory cytokines such as TNF-α, and inflammatory cytokines have also been found in the serum of Parkinson's disease patients (Collins et al., Neuropharmacol., 2012, 62(7):2154-2168).

[0048] Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that primarily targets motor neurons, causing muscle weakness, speech and / or swallowing difficulties, spasticity and / or paralysis, and neuroinflammation plays a significant role in disease progression. High levels of TNF-α have been detected in the plasma and serum of ALS patients and in animal models of ALS. In ALS mice, M2 microglia protect motor neurons at the onset of the disease, but M1 microglia exacerbate motor neuron damage in the chronic phase (Liao et al., Exp. Neurol., 2012, 237(1): 147-152).

[0049] Multiple sclerosis (MS) is an autoimmune disease in which the immune system attacks the myelin sheath covering the central nervous system, including the brain and spinal cord, resulting in demyelination. In the acute phase of MS, microglia are activated as M1 type, and in the chronic phase, they shift to M2 type. In the acute phase of MS, microglia produce inflammatory cytokines that damage glial cells and nerves, causing demyelination. In the early stages of remyelination, microglia change from M1 type to M2 type (Miron et al., Nature Neurosci., 2013,16(9):1211-1220).

[0050] Inflammatory cytokines such as TNF-α, which play a crucial role in the progression of neuroinflammation, are central to the progression of many neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), and multiple sclerosis. For example, chronic increases in TNF-α in the central nervous system contribute to disease progression through nerve cell damage and the accumulation of toxic proteins. Therefore, regulating inflammatory cytokines such as TNF-α is a promising therapeutic strategy for neurodegenerative diseases.

[0051] Based on these findings, the fusion peptide of this disclosure can be said to be useful not only for neonatal HIE but also for the treatment of central nervous system diseases involving M1 microglia, by inducing apoptosis in M1 microglia that release inflammatory cytokines such as TNF-α.

[0052] Pharmaceutical Composition Thus, the fusion peptide of this disclosure can be said to be useful in the treatment of diseases related to M1 microglia. For this reason, this disclosure also includes pharmaceutical compositions containing the fusion peptide of this disclosure. Hereinafter, such pharmaceutical compositions may be referred to as "pharmaceutical compositions of this disclosure".

[0053] The content of the fusion peptide in the pharmaceutical composition of this disclosure is not limited and may be appropriately determined depending on the form of the pharmaceutical composition, the route of administration to the target subject, etc. The amount and number of applications of the pharmaceutical composition of this disclosure to the target subject may also be appropriately determined depending on the form of the pharmaceutical composition, the route of administration to the target subject, the expected effect, etc. The form of the pharmaceutical composition of this disclosure may be solid (powder, granules, tablets, etc.), semi-solid, or liquid. In addition, for example, in the case of a solid (solid dosage form), it may be dissolved by adding distilled water for injection, sterile water, etc. at the time of use.

[0054] The pharmaceutical compositions of this disclosure may further contain any pharmaceutically acceptable components as necessary, provided that they do not impair the effects of this disclosure. Examples of such components include carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, colorants, fragrances, chelating agents, pH adjusters, and preservatives. These components may be used individually or in combination of two or more, and their amounts may be determined as appropriate.

[0055] Method: Based on these considerations, the present disclosure can be said to further encompass a method for delivering the fusion peptide of the present disclosure to M1 microglia, comprising the step of administering the fusion peptide of the present disclosure or the pharmaceutical composition of the present disclosure to a target.

[0056] The fusion peptides, pharmaceutical compositions, target populations, and delivery methods of the disclosed herein will be described in the same manner as described above. The dosage of the fusion peptides or pharmaceutical compositions of the disclosed herein to the target population should be determined appropriately according to the target population, route of administration, type of disease the target population suffers from, expected effect, etc., provided that the fusion peptides of the disclosed herein are administered to the target population in an amount effective for inducing apoptosis.

[0057] According to the delivery method of this disclosure, the fusion peptide of this disclosure can be selectively delivered to M1 microglia, and therefore, the apoptosis-inducing effect caused by the apoptosis-inducing peptide or the apoptosis-inducing substance constituting the fusion peptide of this disclosure can be efficiently exerted in M1 microglia.

[0058] Thus, according to the fusion peptide, pharmaceutical composition, and delivery method of this disclosure, the apoptosis-inducing peptide or apoptosis-inducing substance constituting the fusion peptide can be selectively delivered to target M1 microglia, thereby directly and selectively inducing apoptosis in M1 microglia. Therefore, according to the fusion peptide, pharmaceutical composition, and delivery method of this disclosure, the apoptosis-inducing effect caused by the apoptosis-inducing peptide or apoptosis-inducing substance constituting the fusion peptide can be efficiently exerted in target cells, namely M1 microglia. Furthermore, as shown in the test examples described later, it was confirmed that the fusion peptide of this disclosure can improve brain atrophy and behavioral dysfunction in in vivo tests. From this, the fusion peptide, pharmaceutical composition, and delivery method of this disclosure are also useful in that they can reduce or avoid side effects such as brain atrophy and behavioral dysfunction.

[0059] The embodiments of this disclosure will be described in more detail below with examples, but the embodiments of this disclosure are not limited to the examples below. All animal experiment protocols have been approved by the Shiga University of Medical Science Animal Welfare and Use Committee, and comply with the guidelines of the U.S. National Institutes of Health and the ARRIVE Guidelines (approval numbers 2020-6-12 and 2023-6-6).

[0060] Test Example Test Procedure: Preparation of KLA-linked M1 microglia-targeting fusion peptide. M1 microglia-targeting peptide C-HHSSSAR-C (hereinafter referred to as MG1 (a peptide in which one cysteine ​​residue is directly linked to each end of the amino acid sequence represented by SEQ ID NO: 1 via peptide bonds)) and apoptosis-inducing peptide D [KLAKLAK] 2 The MG1-KLA fusion peptide (hereinafter referred to as MG1-KLA, Example 1) was synthesized by contract (Invitrogen, Carlsbad, CA) by linking it with (hereinafter referred to as KLA (amino acid sequence represented by Sequence ID No. 2, composed of D amino acids)) via a linker.

[0061] Except for using the peptide C-SASHRSH-C (hereinafter referred to as SCR) obtained by scrambling the amino acid sequence of MG1 in place of MG1, the same procedure as described above was used to link SCR and KLA via a linker to synthesize an SCR-KLA fusion peptide (hereinafter referred to as SCR-KLA, Comparative Example 1), which was used as a negative control. SASHRSH is shown in Sequence ID No. 5. These fusion peptides were labeled by attaching a biotin molecule to the N-terminus. The structure of MG1-KLA is shown in Figure 1.

[0062] The animals used were male C57BL / 6 neonatal mice. All mice were housed in a 12-hour light-dark cycle, and food and water were provided ad libitum. Mice that performed a rotarod test for less than 5 seconds, did not eat or drink, or lost more than 20% of their body weight within one week were excluded from the experiment (no mice met these criteria).

[0063] • Culture of primary microglia: Primary microglia were cultured according to a known procedure. Specifically, six male C57BL6 / J neonatal mice (P1) were decapitated, and mixed glial cells were dissociated from the cerebral cortex. By culturing them at 37°C for two weeks in DMEM / F12 medium (Thermo Fisher, Waltham, MA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher), astrocytes appeared in layers, followed by primary microglia cells. Microglia cells were collected by shaking the flask. 48 hours after collection, the medium was replaced with a medium containing 1 μg / mL lipopolysaccharide (LPS) or 40 ng / mL IL-4 (Figure 2).

[0064] - Microglia isolated from primary cultures (primary culture microglia) were stained in vitro using immunohistochemistry, with 6 × 10⁶ samples per well. 2 Cells were seeded in 96-well chamber slides and cultured with the fusion peptide MG1-KLA or SCR-KLA. After culturing, the culture medium was removed and the cells were fixed in 4% paraformaldehyde in 0.1M phosphate buffer at 20°C (room temperature) for 10 minutes. After fixation, the cells were incubated overnight at 4°C with primary antibodies of rabbit anti-Iba-1 antibody (1:1,000; Wako, Osaka, Japan), rabbit anti-CD86 antibody (1:1,000; Abcam, Cambridge, UK), or rabbit anti-CD206 antibody (1:1,000; Abcam). To visualize the biotinylated peptides, sections were incubated with TRITC-labeled rhodamine avidin D (Vector Laboratories, Newark, CA). Sections were mounted using VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories) and observed with a confocal laser microscope (TCS SP8 X; Leica, Wetzlar, Germany) (Figure 2). The binding rates of each fusion peptide to M1 and M2 microglia were also calculated and compared (Figure 2).

[0065] - Cell viability assay: Microglia cells were placed in a 96-well plate at a rate of 1 x 10⁶ cells per well. 4Cells were seeded at a density of 100% and cultured for 24 hours in DMEM / F12 medium supplemented with 10% FBS prior to the assay. Cells were incubated with MG1-KLA or SCR-KLA at different concentrations (50, 100, and 150 μM) for 48 hours. Cell viability was measured and calculated according to the protocol using the WST-8 assay (Dojindo, Kumamoto, Japan) (Figure 2).

[0066] For the annexin-V apoptosis assay, the FITC Annexin-V Apoptosis Detection Kit (Becton, Dickinson and Company, Franklin Lakes, NJ) was used according to the manufacturer's protocol. Fluorescence was measured by flow cytometry (BD FACS Canto™ II, Becton, Dickinson and Company) (Figure 2). The apoptosis induction rates of each fusion peptide for M1 and M2 microglia were calculated and compared (Figure 2). In vitro studies on primary microglia were performed using at least three samples per study.

[0067] • HI (Hypoxic-Ischemic) Procedure: Using a modified protocol based on the known Rice-Vannucci model, male mice at P9 (9 days postnatal) were subjected to hypoxic-ischemic treatment including left common carotid artery occlusion and hypoxic exposure. Specifically, the mice were sedated with isoflurane (4% induction, 1.5% maintenance) and locally anesthetized with 0.25% Marcaine (Aspen, Tokyo, Japan). The mice were then placed on their backs, and the skin of the left neck was incised. After widening the field of view of the left side of the neck, the left CCA was isolated and coagulated using bipolar electronic forceps (7W). The skin incision was ligated with 6-0 silk, and 0.25% Marcaine (Aspen, Tokyo, Japan) was injected as an analgesic. The pup (neonatal) mice were returned to their mothers for 1 hour and placed in a hypoxic chamber (BioSpherix, Parish, NY) at 36.5°C, 10% O2, and 90% N2 for 50 minutes. In the sham procedure (placebo group), the left common carotid artery was exposed by widening the left side of the neck, but blood flow was not blocked by coagulation or exposed to hypoxia (Figure 3).

[0068] • Intraventricular (ICV) injection of MG1-KLA fusion peptide: MG1-KLA, SCR-KLA, or PBS (control) dissolved in 1 μL of PBS at a concentration of 2,890 ng was administered stereotactically into the left ventricle of mice 24 hours after HI treatment using a 30-gauge needle. Specifically, mice were sedated with isoflurane (4% for induction, 1.5% for maintenance), locally anesthetized with 0.25% Marcaine (Aspen, Tokyo, Japan), and then injected with MG1-KLA, SCR-KLA, or PBS into the HI-treated mice (HIE mice) using the stereotactic method. At this time, the skin of the head was cut to confirm the location of the bregma. Stereotactic injection was performed over 30 seconds using a stereotactic fixation device (Narishige, Tokyo, Japan) (1 mm to the left and 1 mm posterior to the bregma, 1.5 mm deep). The skin incision was ligated with 6-0 silk (Figure 3). Histological analysis was performed on page 26, and behavioral function assessments were performed on pages 23-25. All tests were blinded to the mouse group.

[0069] In vivo immunohistochemical evaluation: Brain tissue was extracted by perfusion with 4% paraformaldehyde in 0.1M phosphate buffer, and then isolated into P10, 12, and 26. Brains from P10 and P12 were frozen and sectioned to a thickness of 20 μm using a cryostat (CM3050 S, Leica). Brains from P26 were embedded in paraffin blocks and sectioned to a thickness of 5 μm using a microtome (REM-710, YAMATO, Asaka, Japan). The slice positions corresponded to the bregma of adult mice at +0.6 mm and -1.8 mm. Frozen sections from P10 and P12 were incubated overnight at 4°C with rabbit anti-Iba-1 antibody (1:1,000; Wako) and rat anti-CD86 antibody (1:1,000; eBioscience, San Diego, CA) as primary antibodies. Subsequently, the sections were incubated with anti-rabbit Alexa 488 antibody (1:1,000; Life Technologies), anti-rabbit Alexa 555 antibody (1:1,000; Life Technologies), and anti-rat Alexa 488 (1:1,000; Life Technologies) as secondary antibodies at 20°C (room temperature) for 4 hours. Images were captured using a confocal laser microscope (TCS SP8 X; Leica, Wetzlar, Germany) in the same manner as described above (Figures 3 and 4). In addition, the number of stained cells was measured in the striatum and hippocampus after administration of the fusion peptide or PBS, and the binding rate to M1 microglia, the reduction rate of M1 microglia, etc. were calculated and compared (Figures 3 and 4). The paraffin sections of P26 were deparaffinized, and the antigen was activated using HistoVT One (pH 7.0; Nacalai Tesque, Kyoto, Japan). After blocking, sections were incubated with mouse anti-MAP2 antibody (Sigma-Aldrich, St.), then incubated with a secondary antibody (anti-mouse IgG antibody), and stained using the ABC kit (Vector Laboratories) and 3,3′-diaminobenzidine (DAKO, Glostrup, Denmark) according to the manufacturer's protocol. Images were captured using an all-in-one fluorescence microscope (BZ-X800; KEYENCE, Osaka, Japan) (Figure 5).To quantify the rate of volume reduction, the areas of the striatum and hippocampus were compared between the affected and unaffected sides using ImageJ software (NIH). MAP2-positive areas and signal intensity were automatically calculated using the hybrid cell count application (BZ-H4C, KEYENCE) of BZ-X Analyzer software (BZ-H4A, KEYENCE) (Figure 5).

[0070] • Behavioral function assessment (rotarod test) Motor function assessment was performed using the rotarod test (Ugo Basile, Comerio-Varese, Italy) two weeks after HI treatment. The rotarod test was performed in a speed range of 5-50 rpm / min and 9 rpm / min 2 The test was conducted for 5 minutes at a specified acceleration, and the time it took for the mouse to fall was measured. Five tests were performed for each mouse, and the average of the three medians was calculated and used for median analysis. All tests were conducted blinded to the group to which the mice belonged (Figure 6).

[0071] • Behavioral function assessment (open field test) Emotional behavior assessment was performed using the open field test two weeks after HI treatment. The open field test was conducted for 5 minutes in a 50cm x 50cm area. Motor data was recorded and analyzed using a video tracking system (Muromachi Kikai, Tokyo, Japan). Average speed, total distance, and time to reach the center were measured and compared. All tests were conducted blinded to the group to which the mice belonged (Figure 6).

[0072] Quantitative PCR: The effect of the fusion peptide on inflammatory cytokine production was evaluated. Specifically, brain tissue was isolated after blood removal at P10 (before ICV injection and placebo group), 8 hours after ICV injection (P10 + 8 hours), and P11. Total RNA was extracted from the ipsilateral brain using the RNeasy® Plus Universal Kit (QIAGEN, Hilden, Germany). After RNA extraction, reverse transcription and real-time PCR were performed according to the manufacturer's protocol using TAKARA PrimeScript® RT Reagent Kit with gDNA Eraser (Takara Bio Inc., Kusatsu, Japan), Luna® Universal qPCR Master Mix (New England Biolabs, Ipswich, MA), and LightCycler® 480 (Roche Diagnostics, Basel, Switzerland).The following primers were used: Cd86, forward 5′-CACGAGCTTTGACAGGAACA-3′ (SEQ ID NO: 6) and reverse 5′-TTAGGTTTCGGTGACCTTG-3′ (SEQ ID NO: 7); Cd206, forward 5′-CTATGCAGGCCACTGCTACA-3′ (SEQ ID NO: 8) and reverse 5′-GTTCTCATGGCTTGGCTCTC-3′ (SEQ ID NO: 9); Tnfa, forward 5′-CACGTCGTAGCAAACCACCAAGTGG-3′ (SEQ ID NO: 10) and reverse 5′-GATAGCAAATCGCTGACGGTGTGG-3′ (SEQ ID NO: 11); Il1b, forward 5′-CAACCAACAAGTGATATTCTCCA TG-3′ (SEQ ID NO: 12) and reverse 5′-GATCCACACTCTCCAGCTGCA-3′ (SEQ ID NO: 13); Tgfb, forward 5′-CAGAGCTGCGCTTGCAGAG-3′ (SEQ ID NO: 14) and reverse 5′-GTCAGCAGCCGGTTACCAAG-3′ (SEQ ID NO: 15); Il10, forward 5′-CCAAGCCTTATCGGAAATGA-3′ (SEQ ID NO: 16) and reverse 5′-TTTTCACAGGGAGAAATCG-3′ (SEQ ID NO: 17); and Actb, forward 5′-CGTGCGTGACATCAAAGAGAA-3′ (SEQ ID NO: 18) and reverse 5′-TGGATGCCACAGGATTCCAT-3′ (SEQ ID NO: 19). β-actin (Actb) was used as a housekeeping gene to determine the relative expression level of target genes using the comparative cycle threshold method (Figure 7).

[0073] Statistical analysis data are presented as mean ± standard deviation (SD), and all statistical analyses were performed using GraphPad Prism software version 9 (GraphPad Software Inc.). For parametric analyses, Student's t-test was used to compare two groups. For multiple datasets, one-way analysis of variance (ANOVA) was used, followed by appropriate post-hoc analysis. Differences were considered statistically significant at p<0.05.

[0074] The results are shown in Figure 2 for in vitro studies and in Figures 3-7 for in vivo studies. As shown in Figure 2, when primary cultures of microglia were performed from neonatal mouse brains and examined, it was confirmed that MG1-KLA selectively recognizes M1 microglia, and that the number of viable M1 microglia is selectively reduced, with this reduction being MG1-KLA concentration-dependent. When SCR-KLA was used, no such selectivity was observed, nor was there a significant reduction in cells. From this, it was found that MG1-KLA has a selective apoptotic effect on M1 microglia.

[0075] Similar to the in vitro results, in vivo studies confirmed that MG1-KLA selectively recognizes M1 microglia, and selectively reduces the number of viable M1 microglia. This selectivity was not observed with SCR-KLA, nor was there a significant reduction in cell count. Therefore, MG1-KLA exhibits a selective apoptotic effect on M1 microglia. Furthermore, suppression of M1 microglia accumulation was observed in the MG1-KLA administration group. Improvements in brain atrophy were also observed in both the striatum and hippocampus in the MG1-KLA administration group. Additionally, MG1-KLA administration resulted in improvements in behavioral dysfunction.

[0076] Thus, the selective apoptotic effect of MG1-KLA on M1 microglia was confirmed. Furthermore, it was confirmed that reducing M1 microglia using apoptosis-inducing peptides is useful in the treatment of neonatal HIE. In addition, it was revealed that M1 microglia-targeted apoptosis-inducing peptides improve brain damage by selectively removing M1 microglia and suppressing the production of inflammatory cytokines. Improvement in brain atrophy and behavioral dysfunction were also observed. The treatment with the fusion peptide disclosed herein is completely different from existing therapies and is expected to be a novel molecular therapy for neonatal HIE. Furthermore, since the selective apoptotic effect on M1 microglia was confirmed, it is suggested that it may also be useful in the treatment of central nervous system diseases and other diseases related to M1 microglia. Furthermore, since the selective apoptotic effect on M1 microglia was confirmed, it is suggested that a selective apoptotic effect on M1 microglia can also be obtained when the apoptosis-inducing peptide or the apoptosis-inducing substance is used instead of KLA, and that it may also be useful in the treatment of central nervous system diseases and other diseases related to M1 microglia.

Claims

1. An M1 microglia-targeting fusion peptide, wherein an M1 microglia-targeting peptide and an apoptosis-inducing peptide are directly or indirectly linked, wherein the M1 microglia-targeting peptide is a peptide represented by (1) or (2) below: (1) A peptide in which at least one cysteine ​​residue is directly linked to both ends of an amino acid sequence represented by HHSSSAR (SEQ ID NO: 1) or an amino acid sequence in which multiple such sequences are linked; (2) The peptide of (1), wherein the amino acid sequence consists of an amino acid sequence in which one or more amino acids are deleted, substituted, or added, and which targets M1 microglia.

2. The M1 microglia-targeting fusion peptide according to claim 1, wherein at both terminal cysteine ​​residues, one cysteine ​​residue forms a disulfide bond with the other cysteine ​​residue.

3. The M1 microglia-targeting fusion peptide according to claim 1, wherein the apoptosis-inducing peptide is a peptide consisting of an amino acid sequence represented by KLAKLAKKLAKLAK (SEQ ID NO: 2).

4. The M1 microglia-targeting fusion peptide according to claim 1, for the treatment of at least one central nervous system disorder selected from the group consisting of hypoxic-ischemic encephalopathy, Alzheimer's disease, Parkinson's disease, cerebral infarction, hypoxic encephalopathy, spinal cord injury, neuropathic pain, amyotrophic lateral sclerosis, and multiple sclerosis.

5. A pharmaceutical composition containing the M1 microglia-targeting fusion peptide described in claim 1.

6. The pharmaceutical composition according to claim 5, for the treatment of at least one central nervous system disorder selected from the group consisting of hypoxic-ischemic encephalopathy, Alzheimer's disease, Parkinson's disease, cerebral infarction, hypoxic encephalopathy, spinal cord injury, neuropathic pain, amyotrophic lateral sclerosis, and multiple sclerosis.

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