WO2024071509A1 - Pharmaceutical composition for prevention or treatment of alzheimer's disease - Google Patents

Pharmaceutical composition for prevention or treatment of alzheimer's disease Download PDF

Info

Publication number
WO2024071509A1
WO2024071509A1 PCT/KR2022/016873 KR2022016873W WO2024071509A1 WO 2024071509 A1 WO2024071509 A1 WO 2024071509A1 KR 2022016873 W KR2022016873 W KR 2022016873W WO 2024071509 A1 WO2024071509 A1 WO 2024071509A1
Authority
WO
WIPO (PCT)
Prior art keywords
disease
lnp
amyloid beta
cells
pharmaceutical composition
Prior art date
Application number
PCT/KR2022/016873
Other languages
French (fr)
Korean (ko)
Inventor
김병수
정문교
김영수
이송민
Original Assignee
서울대학교산학협력단
연세대학교 산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 서울대학교산학협력단, 연세대학교 산학협력단 filed Critical 서울대학교산학협력단
Publication of WO2024071509A1 publication Critical patent/WO2024071509A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the present invention relates to a pharmaceutical composition for preventing or treating Alzheimer's disease.
  • Korea is the world's fastest growing country to become a super-aged society, increasing the burden on the public to manage geriatric brain diseases and worsening the financial balance. It is estimated that approximately 10% of the population over 65 years of age in Korea suffers from dementia, and as we enter an aging society, personal and social burdens are increasing. After the age of 65, the prevalence of dementia tends to double every five years, and in developed countries, about 30% of people over the age of 85 suffer from dementia. Dementia has the second highest social cost after diabetes, and as it is the only disease with a continuously increasing mortality rate, the development of a treatment is very urgent.
  • Alzheimer's disease is a disorder that occurs in various cognitive functions that humans have, such as memory, attention, language function, frontal lobe executive function, including visuospatial ability and judgment, and as the disease progresses, it not only causes difficulties in one's daily life or social life. It is a disease that has a fatal impact on people around it, such as family members and local residents, and causes various social problems. Approximately 60-70% of dementia patients are caused by Alzheimer's disease (AD), and there is still no fundamental treatment drug, creating a high unmet demand in the global market. As the social and economic cost burden of AD continues to increase, the development of drugs that can fundamentally inhibit the progression of the disease is becoming more important.
  • AD Alzheimer's disease
  • AD amyloid beta
  • the purpose of the present invention is to provide a pharmaceutical composition for preventing or treating Alzheimer's disease.
  • amyloid beta peptide consists of a sequence containing at least part of the amino acid sequence of SEQ ID NO: 1.
  • amyloid beta peptide consists of a sequence selected from the group consisting of amino acid sequences of SEQ ID NO: 2 to SEQ ID NO: 8.
  • the carrier is a viral carrier, virus-like particle (VLP), positively charged polymer, liposome, lipid nanoparticle, gold, or semiconductor nanocrystal particle (quantum dot).
  • VLP virus-like particle
  • a pharmaceutical composition for preventing or treating Alzheimer's disease which is selected from the group comprising:
  • the carrier is 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioleoylphosphatidylethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol) )-2000] (PEG2000 PE) and a pharmaceutical composition for preventing or treating Alzheimer's disease, which are lipid nanoparticles containing cholesterol.
  • DOTAP 1,2-dioleoyl-3-trimethylammonium-propane
  • DOPE dioleoylphosphatidylethanolamine
  • PEG2000 PE 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol) )-2000]
  • a pharmaceutical composition for preventing or treating Alzheimer's disease which are lipid nanoparticles containing cholesterol.
  • Amyloid beta peptide of the present invention or nucleic acid encoding the same;
  • the pharmaceutical composition for preventing or treating Alzheimer's disease containing rapamycin or a derivative thereof not only inhibits the accumulation of amyloid beta by producing antibodies against amyloid beta, but also induces regulatory T cells specific for amyloid beta at the same time to prevent Alzheimer's disease. It can be used as an Alzheimer's vaccine composition with excellent therapeutic effect and few side effects by suppressing the inflammatory response in the diseased brain.
  • Figure 1 is a schematic diagram showing the mechanism of treatment for Alzheimer's disease of the pharmaceutical composition of the present invention.
  • Figure 2 analyzes the characteristics of lipid nanoparticles carrying amyloid beta peptide and rapamycin.
  • Figure 3 is an analysis of the induction of amyloid beta-specific antibodies by lipid nanoparticles carrying amyloid beta peptide and rapamycin and their effect on amyloid beta aggregation in an animal model of Alzheimer's disease.
  • Figure 4 is an analysis of the induction of immune-tolerant dendritic cells and regulatory T cells by lipid nanoparticles carrying amyloid beta peptide and rapamycin.
  • Figure 5 confirms the amyloid beta targeting efficiency of amyloid beta specific regulatory T cells induced by lipid nanoparticles carrying amyloid beta peptide and rapamycin.
  • Figure 6 analyzes the therapeutic effect of lipid nanoparticles carrying amyloid beta peptide and rapamycin on suppressing neuroinflammation in the brain.
  • Figure 7 analyzes the effect of lipid nanoparticles loaded with amyloid beta peptide and rapamycin on improving cognitive ability in an animal model of Alzheimer's disease.
  • the present invention relates to amyloid beta peptide or nucleic acid encoding it; and rapamycin or a derivative thereof. It relates to a pharmaceutical composition for preventing or treating Alzheimer's disease.
  • amyloid beta peptide contained in the pharmaceutical composition of the present invention can be recognized as an antigen in the human body and induce the formation of antibodies against the amyloid beta peptide through an immune response.
  • the induced antibodies against amyloid beta can travel to the brain and induce clearance of aggregated amyloid beta plaques.
  • amyloid beta peptide and rapamycin or its derivatives can be transfected together into dendritic cells with type 2 MHC molecules, thereby inducing the production of amyloid beta-specific regulatory T cells.
  • the amyloid beta-specific regulatory T cells can migrate to the brain, reduce cranial nerve inflammation occurring in the brain, and eliminate oxidative stress.
  • Alzheimer's disease can show a multifaceted treatment effect for Alzheimer's disease by removing amyloid beta plaques, the main cause of Alzheimer's disease, while also eliminating brain inflammation. Additionally, it can suppress brain inflammation, a typical side effect of vaccines that produce antibodies against amyloid beta.
  • the amyloid beta peptide may be a peptide containing the amino acid sequence of at least part of the amyloid beta protein.
  • the amyloid beta peptide has an epitope that binds to the MHC molecule of dendritic cells, and at least a portion of the amyloid beta peptide may contain a sequence that can be recognized as the epitope.
  • a sequence that can be recognized as an epitope refers to a sequence that functions as an epitope even if it consists of the entire epitope sequence, some amino acids are added to both ends of the sequence, or some amino acids are removed from both ends of the sequence.
  • the epitope may include at least a portion of the amino acid sequence of SEQ ID NO: 1, and more specifically, may be a sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 2 to SEQ ID NO: 8. However, it is not limited thereto, and any sequence that can be recognized as the epitope may be included without limitation, and a known epitope sequence may be used.
  • the length of the amyloid beta peptide is not limited as long as it can be recognized as an amyloid beta peptide by binding to an MHC molecule, for example, 0.5 kDa to 5 kDa, 1 kDa to 4 kDa, 1.5 kDa to 3 kDa. Or it may be 1.8 kDa to 2 kDa, etc. However, it is not limited to this.
  • amyloid beta peptide may preferably be derived from human amyloid beta peptide.
  • the nucleic acid encoding the amyloid beta peptide may be included within the scope of the present invention regardless of the type of nucleic acid as long as it encodes the above-mentioned amyloid beta peptide.
  • the type of nucleic acid may be DNA or RNA.
  • the nucleic acid encoding the amyloid beta peptide may include a nucleic acid encoding a sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 8. However, it is not limited to this.
  • the nucleic acid encoding the amyloid beta peptide can produce the amyloid beta peptide in the human body, and the derived peptide is introduced into antigen-presenting cells such as dendritic cells with type 2 MHC molecules, producing amyloid beta, as previously mentioned. It can induce peptide-specific regulatory T cells or generate antibodies specific for amyloid beta peptide.
  • rapamycin or a derivative thereof may be included in the scope of the present invention regardless of its type.
  • rapamycin can inhibit the growth of lymphocytes, promote differentiation, induce regulatory T cells specific for amyloid beta, and suppress the inflammatory response caused by amyloid beta peptide. Therefore, any derivative of rapamycin that can perform this function can be included in the scope of the present invention without limitation. Examples include benzoyl rapamycin, temsirolimus, everolimus, zotarolimus, biolimus, pimecrolimus, pimecrolimus, tacrolimus, and ridaporolimus. However, it is not limited to this.
  • amyloid beta peptide of the present invention or a nucleic acid encoding the same; and rapamycin or a derivative thereof; may be carried and delivered in a carrier, and are preferably amyloid beta peptide or a nucleic acid encoding the same; and rapamycin or a derivative thereof; may be carried and delivered together so as to be included in each individual delivery vehicle.
  • the delivery vehicle greatly improves the delivery efficiency of amyloid beta peptide and rapamycin or its derivatives to dendritic cells, resulting in the induction of tolerant dendritic cells that present amyloid beta peptide to type 1 or type 2 MHC molecules.
  • Efficiency can be increased, and thus the production efficiency of amyloid beta peptide-specific antibodies and the induction efficiency of amyloid beta peptide-specific regulatory T cells can be greatly increased.
  • the increase in the induction efficiency of amyloid beta-specific regulatory T cells is advantageous because it can suppress cranial nerve inflammation and protect nerves by suppressing oxidative stress.
  • the delivery vehicle includes amyloid beta peptide or a nucleic acid encoding the same; and rapamycin or a derivative thereof, and can be selected without limitation by those skilled in the art as long as it can be delivered to the target cell and release the substance therein.
  • the lipid nanoparticles can be selected and used by those skilled in the art regardless of their components.
  • phospholipid molecules constituting lipid nanoparticles neutral lipids, anionic and/or cationic phospholipids can all be used, and lipid nanoparticles containing cationic phospholipids are preferably used.
  • Examples of the neutral lipid include L- ⁇ -phosphatidylcholine (PC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (1,2-dioleoyl-sn-glycero- 3-phosphocholine, DOPC), 1,2-distearoyl-sn-glycero-3-phophocholine (DSPC), 1,2-dipalmi Toyl-sn-glycero-3-phosphocholine (1,2-dipalmitoylsn-glycero-3-phophocholine, DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (1, 2-dimyristoyl-sn-glycero-3-phosphocholine, DMPC), and the PC may be PC derived from soybeans, eggs, hydrogenated soybeans or eggs, but is not limited thereto.
  • PC may be PC derived from soybeans, eggs, hydrogenated soybeans or eggs, but is not limited thereto.
  • anionic lipid examples include L- ⁇ -phosphatidic acid, L- ⁇ -phosphatidyl-DL-glycerol, cardiolipin, L- ⁇ -phosphatidylinositol, L- ⁇ -phosphatidylserine, 1,2-dilauroyl-sn-glycero-3-[phospho-rac-( 1-glycerol)](1,2-dilauroyl-sn-glycero-3-[phospho-rac-(1-glycerol)], DLPG), 1,2-dilauroyl-sn-glycero-3-[ phospho-L-serine](1,2-dilauroyl-sn-glycero-3-[phospho-L-serine], DLPS), 1,2-dilauroyl-sn-glycero-3-phosphate (1 ,2-dilauroyl-sn-glycero-3-phosphate, DLPA), 1,2-dimyristoyl-sn-glycero-3-[phosphor
  • cationic lipid examples include 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC), 1,2- 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), dioleoyl glutamide, distearoylglutamide, dipalmitoyl Dipalmitoyl glutamide, dioleoylaspartamide, 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), ß-[ N-(N',N'-dimethylaminoethane-carbamoyl], (3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl], DC-Chol), dimethyldiochtadecylammonium bromide , DDAB), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (1,2-dioleoyl)
  • the phospholipid surface of the lipid particle may be PEGylated with polyethylene glycol (PEG), etc. to improve the fluidity, loading efficiency, and/or stability of the particle, and cholesterol, 1,2-Dioleoyl-3 -trimethylammoniumpropane (DOTAP) may be further included.
  • the lipid particles can be used to include the aforementioned cationic lipids such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOTAP, PEG2000, and cholesterol.
  • DOTAP 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • the average diameter of the carrier is within the scope of the present invention without limitation, and may be selected by a person skilled in the art depending on conditions such as the amount of peptide, nucleic acid, and drug to be carried.
  • the average diameter may be 50 nm to 600 nm.
  • the lower limit of the average particle diameter can be, for example, 50 nm, 80 nm, 110 nm, 140 nm or 170 nm
  • the upper limit can be, for example, 600 nm, 500 nm, 400 nm, 350 nm, 300 nm. there is. However, it is not limited to this.
  • composition of the present invention may additionally contain one or more active ingredients that exhibit the same or similar function in relation to the prevention or treatment of Alzheimer's disease, or a compound that maintains/increases the solubility and/or absorption of the active ingredient. .
  • the appropriate dosage of the composition of the present invention may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity. You can. However, it is not limited to this.
  • concentration of the active ingredient included in the composition of the present invention can be determined considering the purpose of treatment, patient's condition, necessary period, etc., and is not limited to a specific concentration range.
  • the composition of the present invention is administered in a pharmaceutically effective amount.
  • the effective dosage level depends on factors including the type and severity of the patient's disease, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the field of medicine. can be decided.
  • the pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.
  • the method of administering the composition of the present invention can be selected without limitation.
  • intramuscular injection IM
  • SC subcutaneous injection
  • IV intravenous injection
  • ID intradermal injection
  • IP intraperitoneal injection
  • subcutaneous injection may be advantageous as it is easily absorbed by dendritic cells located in the lower layer of the skin.
  • it is not limited thereto.
  • the dosage range of the composition of the present invention varies greatly depending on the patient's weight, age, gender, health condition, diet, administration time, administration method, excretion rate, and severity of the disease.
  • the appropriate dosage can be determined by, for example, the patient. It may vary depending on the amount of drug accumulated in the body and/or the specific efficacy of the delivery vehicle of the present invention used. For example, it may be 0.01 ⁇ g to 1 g per kg of body weight, and may be administered in divided doses, such as daily, weekly, monthly, or yearly units, once or several times per unit period, or administered continuously over a long period of time using an infusion pump. It can be. The number of repeated doses is determined by considering the time the drug stays in the body and the concentration of the drug in the body. Even after treatment, depending on the course of disease treatment, the composition may be administered for recurrence.
  • LNP-R/A ⁇ prepared in Preparation Example 1 was observed under a transmission electron microscope and confirmed to be in the form of spherical nanoparticles with a phospholipid bilayer.
  • EDS Energy Dispersive X-ray Spectroscopy
  • carbon, oxygen, nitrogen contained in amyloid protein, and phosphorus contained in lipids were detected.
  • LNP-R/A ⁇ of the present invention is a nanoparticle with a size of 232.7 ⁇ 84.0 nm, and its size maintained its stability when observed for 7 days. It was confirmed that the nanoparticles had a slight positive charge due to DOTAP, the main lipid component of this lipid nanoparticle ( Figure 2a).
  • lipids nanoparticles rapamycin antigen Loading amount ( ⁇ g) Loading efficiency (%) Loading amount ( ⁇ g) Loading efficiency (%) LNP-R 18.36 ⁇ 0.60 26.23 ⁇ 0.86 N/A N/A LNP-A ⁇ N/A N/A 106.18 ⁇ 12.3 53.09 ⁇ 6.17 LNP-R/A ⁇ 18.73 ⁇ 0.83 26.75 ⁇ 1.19 103.78 ⁇ 9.25 51.89 ⁇ 4.63
  • Example 2 Induction of A ⁇ -specific antibodies by LNP-R/A ⁇ and verification of effect on A ⁇ aggregate accumulation in the brain of an Alzheimer's disease animal model
  • LNP-R/A ⁇ Since LNP-R/A ⁇ is expected to be uptaken by dendritic cells after intradermal injection into mice and move to the lymph nodes to act, dendritic cells were treated with LNP-R/A ⁇ at the cellular level and observed over time. It was confirmed that the antigen was processed by dendritic cells and presented on the surface over time. As a result of comparing the antigen presentation ability of dendritic cells with a mixture of rapamycin and antigen without lipid nanoparticles, it was confirmed that the efficiency was significantly increased when lipid nanoparticles were used.
  • E ⁇ peptide rather than amyloid beta was used as the antigen (LNP-R/E ⁇ ), and an antibody that detects E ⁇ -MHC class II was used.
  • IVIS 24 hours after intradermal injection of LNP-R/A ⁇ confirmed that it had migrated to the lymph nodes ( Figure 3a).
  • LNP-R/A ⁇ was detected in dendritic cells.
  • Tfh antigen-specific folicular helper T
  • LNP-R/A ⁇ The antibody-inducing effect of LNP-R/A ⁇ in vivo was confirmed using 5XFAD mice, a mouse model of Alzheimer's disease.
  • LNP-R/A ⁇ or its control group was injected intradermally five times at 1-week intervals into 5-month-old 5
  • a ⁇ -specific antibodies were observed in the serum and brain tissue of the LNP-A ⁇ and LNP-R/A ⁇ administration groups containing A ⁇ antigen.
  • the antibodies produced in both administration groups were confirmed to have an epitope within the 8 amino acid sequence of the N-terminus of the A ⁇ antigen (Figure 3c).
  • LNP-R/OVA was created using OVA as an antigen.
  • wild-type C57BL/6 mouse bone marrow-derived dendritic cells were treated and analyzed through flow cytometry and qRT-PCR, and low levels of MHC class II and co-stimulatory molecule expression, known as characteristics of immune-tolerant dendritic cells, were observed. and high levels of CCR7, TGF-beta1, IL-10, HO-1, IDO, and PD-L1 expression were confirmed.
  • Example 4 Confirmation of A ⁇ targeting efficiency of A ⁇ -specific regulatory T cells induced by LNP-R/A ⁇ .
  • Example 3 wild-type C57BL/6 mice were injected intradermally twice at one-week intervals, and 5 days later, the spleens were harvested and restimulated with OVA antigen for an additional 3 days. Afterwards, it was analyzed through qRT-PCR, FACS, and ELISA, and it was confirmed that antigen-specific regulatory T cells were induced. In particular, tetramer staining using a tetramer that recognizes OVA-specific TCR confirmed that the number of antigen-specific regulatory T cells was significantly higher than that of other groups (Figure 5a).
  • Adoptive Treg cell transfer was performed to confirm that A ⁇ -specific regulatory T cells induced by LNP-R/A ⁇ in the body target A ⁇ in the brain more efficiently than non-specific regulatory T cells and suppress the accumulation of A ⁇ plaques. did. This is a test to see only the effect of regulatory T cells, excluding the effect of anti-A ⁇ antibodies formed by injection of LNP-R/A ⁇ .
  • LNP and LNP-R/A ⁇ were administered twice at 1-week intervals to a 6-week-old wild-type mouse model. After 1 week, the same number of regulatory T cells were isolated from both groups and intravenously injected into 5-month-old 5XFAD mice, respectively. Two weeks later, the effect was confirmed by immunohistostaining (Figure 5b).
  • the transferred regulatory T cells were stained with CFSE dye and the presence of transferred regulatory T cells in the brain was observed using a fluorescence microscope. Additionally, to confirm changes in A ⁇ plaques, immunohistostaining was performed using an anti-A ⁇ antibody. As a result, it was confirmed that A ⁇ plaques were significantly reduced in the brains of mice administered regulatory T cells induced by LNP-R/A ⁇ compared to the control group. Unlike the control group, regulatory T cells transferred around the A ⁇ plaques were found. . Therefore, it was confirmed that A ⁇ -specific regulatory T cells were induced by LNP-R/A ⁇ and showed more efficient A ⁇ targeting and removal compared to non-A ⁇ -specific regulatory T cells (Figure 5b).
  • Example 5 Confirmation of the therapeutic effect of LNP-R/A ⁇ on suppressing neuroinflammation in the brain.
  • LNP-R/A ⁇ can alleviate neuroinflammation in the brain
  • splenocytes derived from OT-2 mice were treated with LNP-R/OVA and cultured for 4 days to induce regulatory T cells. .
  • the cells were analyzed by co-culturing them with LPS-treated Raw264.7 cells or glial cells, respectively, which mimic microglial cells.
  • iNOS and inflammatory cytokine (TNF-alpha) markers of M1 microglial cells, were decreased by LNP-R/OVA
  • Arg- a marker of M2 microglial cells
  • 1I anti-inflammatory cytokine increased.
  • IL-10 1I anti-inflammatory cytokine
  • polarization into M2 microglial cells was induced by LNP-R/OVA ( Figure 6a).
  • LNP-R/OVA increased the expression of growth factors in glial cells, and in particular, compared to LNP-OVA, LNP-R/OVA secreted cytokines known to be expressed by reactive astrocytes. It was confirmed that can be reduced.
  • regulatory T cells induced by LNP-R/OVA can protect nerves by themselves (Figure 6b).
  • LNP-R/A ⁇ In order to confirm the effect of LNP-R/A ⁇ on chronic neuroinflammation, one of the major lesions of Alzheimer's disease, activated microglia and glial cells, which are major biomarkers of inflammatory response, were tested in the 5XFAD mouse brain of Example 2. (Iba1, GFAP) were observed through immunohistostaining. Microglial cells, which show a close correlation with the amount of A ⁇ accumulated in the brain, were significantly reduced in both the LNP-A ⁇ and LNP-R/A ⁇ administration groups, and in the case of glial cell activation, LNP-R/A ⁇ compared to the LNP-A ⁇ administration group. It decreased significantly in the administration group.
  • the LNP-R/A ⁇ administration group like the LNP-A ⁇ administration group, reduces A ⁇ accumulation in the brain through antibody formation, suppressing chronic microglial activity, and further LNP-R/A ⁇ administration group.
  • a ⁇ administration group it was confirmed that activation of glial cells was reduced by inducing A ⁇ -specific regulatory T cells ( Figures 3f, 6d).
  • LNP-R/A ⁇ also restored synaptic function in the cortex of 5XFAD mice ( Figure 6d).
  • Example 6 Confirmation of the effect of LNP-R/A ⁇ on improving cognitive ability in an Alzheimer's disease mouse model.
  • LNP-R/A ⁇ The effect of LNP-R/A ⁇ on an Alzheimer's disease mouse model was evaluated through a Morris water maze experiment that evaluates spatial learning and memory abilities.
  • LNP-R/A ⁇ and the control group were intradermally injected into a 7-month-old 5XFAD mouse model 5 times at weekly intervals. Learning and memory abilities were assessed starting 2 weeks after injection and on day 6 following a 5-day learning period.
  • rats were trained to find an invisible platform in a water tank with a diameter of 1 m and a water depth of 60 cm, with four quadrants marked using cues.
  • Opaque white paint was added to the water to make the platform invisible, and the temperature of the water was maintained at 21-22°C. Mice repeated four trials starting from each quadrant per day over a learning period of 5 days, and were allowed to swim freely for 60 seconds in each trial to find the hidden platform. If the platform was not found within the time limit, the space was learned through peripheral cues for 15 seconds after the experimenter guided the participant onto the platform. If the mouse found the platform within the time limit and stayed on the platform for more than 6 seconds, the mouse was taken out and the time to reach the platform was measured. On the last evaluation day, the platform was removed and the animals were taken out after swimming freely for 60 seconds starting from one quadrant.
  • mice in the LNP-R/A ⁇ administration group reached the platform significantly faster than those in the LNP administration group, found the platform location faster and within a shorter distance on the evaluation day, and performed at a level similar to that of the wild-type control group. showed. Therefore, it was confirmed that the spatial learning and memory abilities of mice were restored to wild-type levels by administration of LNP-R/A ⁇ (Figure 7).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Veterinary Medicine (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Biomedical Technology (AREA)
  • Neurosurgery (AREA)
  • Neurology (AREA)
  • Immunology (AREA)
  • Hospice & Palliative Care (AREA)
  • Nanotechnology (AREA)
  • Mycology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Optics & Photonics (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Psychiatry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Marine Sciences & Fisheries (AREA)

Abstract

The present invention may provide a pharmaceutical composition for the prevention or treatment of Alzheimer's disease, the composition containing: an amyloid-beta peptide or a nucleic acid encoding same; and rapamycin or a derivative thereof. The composition can generate antibodies against amyloid-beta to suppress the accumulation of amyloid-beta while inducing amyloid-beta-specific regulatory T cells to suppress inflammatory reactions in the brain affected by Alzheimer's disease. Therefore, the composition can be utilized as an Alzheimer's vaccine composition that is therapeutically excellent and preferably has few side effects.

Description

알츠하이머 예방 또는 치료용 약학 조성물Pharmaceutical composition for preventing or treating Alzheimer's disease
본 발명은 알츠하이머 예방 또는 치료용 약학 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for preventing or treating Alzheimer's disease.
한국은 세계에서 가장 빠르게 초 고령사회에 진입하는 국가로서, 노인성 뇌질환 관리를 위한 국민 부담이 가중되고 재정수지 악화를 초래하고 있다. 우리나라 65세 이상 인구의 약 10 %가 치매환자로 추정되며, 고령화 사회에 접어들며 개인적/사회적 부담이 가중되고 있다. 65 세가 지나면 치매 유병률은 5 년마다 두 배씩 증가하는 추세를 보이며, 선진국의 경우 85 세 이상 연령층의 약 30 %가 치매를 앓고 있는 실정이다. 치매는 당뇨병에 이어 두 번째로 높은 사회적 비용이 발생하고 있으며, 무엇보다 유일하게 사망률이 지속적으로 증가하는 질환으로써 치료제 개발이 매우 시급한 실정이다.Korea is the world's fastest growing country to become a super-aged society, increasing the burden on the public to manage geriatric brain diseases and worsening the financial balance. It is estimated that approximately 10% of the population over 65 years of age in Korea suffers from dementia, and as we enter an aging society, personal and social burdens are increasing. After the age of 65, the prevalence of dementia tends to double every five years, and in developed countries, about 30% of people over the age of 85 suffer from dementia. Dementia has the second highest social cost after diabetes, and as it is the only disease with a continuously increasing mortality rate, the development of a treatment is very urgent.
알츠하이머병이란 인간이 가진 여러 가지 인지 기능인 기억력, 주의력, 언어기능, 시공간능력과 판단력을 포함한 전두엽 집행 기능 등의 장애가 발생하여 질환이 진행됨에 따라 본인의 일상생활이나 사회생활을 하는데 어려움을 초래할 뿐만 아니라 가족이나 지역 주민 등 주변인에게도 치명적 영향을 주며 다양한 사회적 문제를 유발하는 질병에 해당한다. 치매 환자의 약 60~70 %는 알츠하이머병(Alzheimer's disease, AD)으로 인한 치매로 아직까지 근본적인 치료약물이 없어 글로벌 시장의 미충족 수요가 높다. AD로 인한 사회/경제적 비용 부담이 점점 증가함에 따라 병의 진행을 근원적으로 억제할 수 있는 의약품 개발이 더욱 중요해지고 있다.Alzheimer's disease is a disorder that occurs in various cognitive functions that humans have, such as memory, attention, language function, frontal lobe executive function, including visuospatial ability and judgment, and as the disease progresses, it not only causes difficulties in one's daily life or social life. It is a disease that has a fatal impact on people around it, such as family members and local residents, and causes various social problems. Approximately 60-70% of dementia patients are caused by Alzheimer's disease (AD), and there is still no fundamental treatment drug, creating a high unmet demand in the global market. As the social and economic cost burden of AD continues to increase, the development of drugs that can fundamentally inhibit the progression of the disease is becoming more important.
AD를 일으키는 단일 질병기전은 명확하게 밝혀지지 않았으며, 뇌에서 아밀로이드 베타(Aβ) 플라크의 축적, 콜린성 신경세포의 사멸, 미토콘드리아의 에너지 대사 이상, 신경세포 수상돌기의 이상, 산화스트레스, 뇌신경 염증 등이 다양한 발병원인 이론으로 제시되고 있다. 질병의 진행을 멈추거나 치료하기 위해 Aβ 또는 Tau 단백질을 타겟으로 하는 6가지 접근 방식(Aβ 제거, Aβ 분해/파괴, Aβ 생산 방지/지연, Tau 제거, Tau 분해/파괴, Tau 생산 방해/지연)이 가장 많이 시도되어 왔으며, 현재 대부분의 임상 치료제 개발은 이와 같은 접근법에 초점을 맞추어 왔다. 개발 초기에는 APP 분비효소 억제제 와 Aβ 응집 억제제 중심의 신약 개발이 이루어졌으며, 최근에는 Aβ 제거 기전의 항체 후보 물질이 다수 개발되고 있는 실정이나, 현재까지 개발된 AD 치료제의 경우, 실패율이 매우 높다.The single disease mechanism that causes AD has not been clearly identified, and includes accumulation of amyloid beta (Aβ) plaques in the brain, death of cholinergic neurons, abnormalities in mitochondrial energy metabolism, abnormalities in neuronal dendrites, oxidative stress, and brain nerve inflammation. Various causative theories have been proposed. Six approaches targeting Aβ or Tau proteins to halt or treat disease progression: Aβ removal, Aβ degradation/destruction, prevention/delay of Aβ production, Tau removal, Tau degradation/destruction, and interference/delay of Tau production. This has been the most attempted, and most current clinical treatment development has focused on this approach. In the early stages of development, new drug development focused on APP secretase inhibitors and Aβ aggregation inhibitors, and recently, many antibody candidates with Aβ removal mechanisms have been developed. However, the failure rate of AD treatments developed to date is very high.
Aβ 또는 Tau 기전을 조절하는 후보물질이 지속적으로 임상에서 실패하면서, 최근 AD에서 신경염증 및 장내 미생물의 변화 등이 관찰되면서 새로운 기전을 타겟으로 하는 약물이 다양하게 시도되고 있으나, 아직까지 확실한 치료제 개발은 제시되고 있지 않은 실정이다. 이에 AD의 궁극적인 치료를 위하여 다면적 치료 전략의 개발이 필요한 실정이다. As candidate substances that regulate the Aβ or Tau mechanism continue to fail in clinical trials, and as neuroinflammation and changes in intestinal microorganisms have recently been observed in AD, various drugs targeting new mechanisms are being tried, but no definitive treatment has yet been developed. is not currently being presented. Accordingly, the development of a multifaceted treatment strategy is necessary for the ultimate treatment of AD.
본 발명은 알츠하이머병의 예방 또는 치료용 약학 조성물을 제공하는 것을 목적으로 한다.The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating Alzheimer's disease.
1. 아밀로이드 베타 펩타이드 또는 이를 코딩하는 핵산; 및 라파마이신 또는 그 유도체;를 포함하는 알츠하이머병 예방 또는 치료용 약학 조성물.1. Amyloid beta peptide or nucleic acid encoding it; and rapamycin or a derivative thereof; a pharmaceutical composition for preventing or treating Alzheimer's disease.
2. 위 1에 있어서, 상기 아밀로이드 베타 펩타이드는 서열번호 1의 아미노산 서열의 적어도 일부를 포함하는 서열로 이루어진 것인 알츠하이머병 예방 또는 치료용 약학 조성물.2. The pharmaceutical composition for preventing or treating Alzheimer's disease according to 1 above, wherein the amyloid beta peptide consists of a sequence containing at least part of the amino acid sequence of SEQ ID NO: 1.
3. 위 1에 있어서, 상기 아밀로이드 베타 펩타이드는 서열번호 2 내지 서열번호 8의 아미노산 서열로 이루어진 군에서 선택되는 서열로 이루어진 것인 알츠하이머병 예방 또는 치료용 약학 조성물.3. The pharmaceutical composition for preventing or treating Alzheimer's disease according to 1 above, wherein the amyloid beta peptide consists of a sequence selected from the group consisting of amino acid sequences of SEQ ID NO: 2 to SEQ ID NO: 8.
4. 위 1에 있어서, 상기 아밀로이드 베타 펩타이드 또는 이를 코딩하는 핵산; 및 라파마이신 또는 그 유도체;는 전달체에 담지된 것인 알츠하이머병 예방 또는 치료용 약학 조성물.4. The method of 1 above, wherein the amyloid beta peptide or the nucleic acid encoding it; and rapamycin or a derivative thereof; a pharmaceutical composition for preventing or treating Alzheimer's disease carried in a carrier.
5. 위 4에 있어서, 상기 전달체는 바이러스 전달체, 바이러스 유사 입자(virus-like particle, VLP), 양전하성 폴리머, 리포좀, 지질나노입자(lipid nanoparticle), 금 또는 반도체 나노결정 입자(quantum dot)를 포함하는 군에서 선택되는 것인 알츠하이머병 예방 또는 치료용 약학 조성물.5. In item 4 above, the carrier is a viral carrier, virus-like particle (VLP), positively charged polymer, liposome, lipid nanoparticle, gold, or semiconductor nanocrystal particle (quantum dot). A pharmaceutical composition for preventing or treating Alzheimer's disease, which is selected from the group comprising:
6. 위 4에 있어서, 상기 전달체는 1,2-dioleoyl-3-trimethylammonium-propane(DOTAP), dioleoylphosphatidylethanolamine(DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000](PEG2000 PE) 및 콜레스테롤을 포함하는 지질나노입자인 알츠하이머병 예방 또는 치료용 약학 조성물.6. In item 4 above, the carrier is 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioleoylphosphatidylethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol) )-2000] (PEG2000 PE) and a pharmaceutical composition for preventing or treating Alzheimer's disease, which are lipid nanoparticles containing cholesterol.
7. 위 4에 있어서, 상기 전달체의 직경은 50 nm 내지 600 nm인 알츠하이머병 예방 또는 치료용 약학 조성물.7. The pharmaceutical composition for preventing or treating Alzheimer's disease according to 4 above, wherein the diameter of the carrier is 50 nm to 600 nm.
본 발명의 아밀로이드 베타 펩타이드 또는 이를 코딩하는 핵산; 및 라파마이신 또는 그 유도체를 포함하는 알츠하이머병의 예방 또는 치료용 약학 조성물은 아밀로이드 베타에 대한 항체를 생성하여 아밀로이드 베타의 축적을 억제할 뿐만 아니라, 동시에 아밀로이드 베타에 특이적인 조절 T 세포를 유도하여 알츠하이머병 뇌에서의 염증 반응을 억제하여 치료효과가 우수하고 부작용이 적은 알츠하이머 백신 조성물로서 활용할 수 있다.Amyloid beta peptide of the present invention or nucleic acid encoding the same; And the pharmaceutical composition for preventing or treating Alzheimer's disease containing rapamycin or a derivative thereof not only inhibits the accumulation of amyloid beta by producing antibodies against amyloid beta, but also induces regulatory T cells specific for amyloid beta at the same time to prevent Alzheimer's disease. It can be used as an Alzheimer's vaccine composition with excellent therapeutic effect and few side effects by suppressing the inflammatory response in the diseased brain.
도 1은 본 발명의 약학 조성물의 알츠하이머병 치료 기전을 나타낸 모식도이다.Figure 1 is a schematic diagram showing the mechanism of treatment for Alzheimer's disease of the pharmaceutical composition of the present invention.
도 2는 아밀로이드 베타 펩타이드 및 라파마이신을 담지한 지질나노입자의 특성을 분석한 것이다.Figure 2 analyzes the characteristics of lipid nanoparticles carrying amyloid beta peptide and rapamycin.
도 3은 아밀로이드 베타 펩타이드 및 라파마이신을 담지한 지질나노입자에 의한 아밀로이드 베타 특이적 항체 유도 및 알츠하이머병 동물 모델에서 아밀로이드 베타 응집에 미치는 영향을 분석한 것이다.Figure 3 is an analysis of the induction of amyloid beta-specific antibodies by lipid nanoparticles carrying amyloid beta peptide and rapamycin and their effect on amyloid beta aggregation in an animal model of Alzheimer's disease.
도 4는 아밀로이드 베타 펩타이드 및 라파마이신을 담지한 지질나노입자에 의한 면역관용 수지상세포 및 조절 T 세포 유도 여부를 분석한 것이다.Figure 4 is an analysis of the induction of immune-tolerant dendritic cells and regulatory T cells by lipid nanoparticles carrying amyloid beta peptide and rapamycin.
도 5는 아밀로이드 베타 펩타이드 및 라파마이신을 담지한 지질나노입자에 의해 유도된 아밀로이드 베타 특이적 조절 T 세포의 아밀로이드 베타 타겟 효율성을 확인한 것이다.Figure 5 confirms the amyloid beta targeting efficiency of amyloid beta specific regulatory T cells induced by lipid nanoparticles carrying amyloid beta peptide and rapamycin.
도 6은 아밀로이드 베타 펩타이드 및 라파마이신을 담지한 지질나노입자가 뇌에서 신경염증 억제에 미치는 치료 효과를 분석한 것이다.Figure 6 analyzes the therapeutic effect of lipid nanoparticles carrying amyloid beta peptide and rapamycin on suppressing neuroinflammation in the brain.
도 7은 아밀로이드 베타 펩타이드 및 라파마이신을 담지한 지질나노입자가 알츠하이머병 동물 모델의 인지능력 향상에 미치는 영향을 분석한 것이다.Figure 7 analyzes the effect of lipid nanoparticles loaded with amyloid beta peptide and rapamycin on improving cognitive ability in an animal model of Alzheimer's disease.
이하 본 발명을 상세히 설명한다. 특별한 정의가 없는 한 본 명세서의 모든 용어는 본 발명이 속하는 기술분야의 통상의 지식을 가진 기술자가 이해하는 당해 용어의 일반적인 의미와 동일하고 만약 본 명세서에 사용된 용어의 의미와 충돌하는 경우에는 본 명세서에 사용된 의미를 따른다.Hereinafter, the present invention will be described in detail. Unless otherwise specified, all terms in this specification have the same general meaning as understood by a person skilled in the art to which the present invention pertains, and if there is a conflict with the meaning of the terms used in this specification, this specification Follow the meaning used in the specification.
본 발명은 아밀로이드 베타 펩타이드 또는 이를 코딩하는 핵산; 및 라파마이신 또는 그 유도체;를 포함하는 알츠하이머병 예방 또는 치료용 약학 조성물에 관한 것이다.The present invention relates to amyloid beta peptide or nucleic acid encoding it; and rapamycin or a derivative thereof. It relates to a pharmaceutical composition for preventing or treating Alzheimer's disease.
본 발명의 상기 약학 조성물에 포함된 아밀로이드 베타 펩타이드는 인체 내에서 항원으로써 인식되어 면역반응에 의하여 아밀로이드 베타 펩타이드에 대한 항체 형성을 유도할 수 있다. 유도된 아밀로이드 베타에 대한 항체는 뇌로 이동하여 응집된 아밀로이드 베타 플라크의 제거를 유도할 수 있다. 동시에, 아밀로이드 베타 펩타이드 및 라파마이신 또는 그 유도체는 함께 제 2형 MHC 분자를 가진 수지상세포에 이입될 수 있고, 그로 인하여 아밀로이드 베타 특이적인 조절 T 세포의 생성을 유도할 수 있다. 해당 아밀로이드 베타 특이적 조절 T 세포는 뇌로 이동하여 뇌에서 발생하는 뇌신경 염증을 감소시키고, 산화적 스트레스를 제거할 수 있다. 즉, 알츠하이머병의 주요 원인인 아밀로이드 베타 플라크를 제거하면서도 뇌 염증을 제거할 수 있어 알츠하이머병에 대한 다면적인 치료 효과를 보일 수 있는 것에 해당한다. 또한, 아밀로이드 베타에 대한 항체를 생성하는 백신의 대표적인 부작용인 뇌 염증을 억제할 수 있다. The amyloid beta peptide contained in the pharmaceutical composition of the present invention can be recognized as an antigen in the human body and induce the formation of antibodies against the amyloid beta peptide through an immune response. The induced antibodies against amyloid beta can travel to the brain and induce clearance of aggregated amyloid beta plaques. At the same time, amyloid beta peptide and rapamycin or its derivatives can be transfected together into dendritic cells with type 2 MHC molecules, thereby inducing the production of amyloid beta-specific regulatory T cells. The amyloid beta-specific regulatory T cells can migrate to the brain, reduce cranial nerve inflammation occurring in the brain, and eliminate oxidative stress. In other words, it can show a multifaceted treatment effect for Alzheimer's disease by removing amyloid beta plaques, the main cause of Alzheimer's disease, while also eliminating brain inflammation. Additionally, it can suppress brain inflammation, a typical side effect of vaccines that produce antibodies against amyloid beta.
본 발명에서 상기 아밀로이드 베타 펩타이드는 아밀로이드 베타 단백질 중 적어도 일부의 아미노산 서열을 포함하는 펩타이드일 수 있다. 아밀로이드 베타 펩타이드는 수지상 세포의 MHC 분자에 결합하는 에피토프를 가지고 있어, 상기 적어도 일부는 상기 에피토프로서 인식될 수 있는 서열을 포함하는 것일 수 있다.In the present invention, the amyloid beta peptide may be a peptide containing the amino acid sequence of at least part of the amyloid beta protein. The amyloid beta peptide has an epitope that binds to the MHC molecule of dendritic cells, and at least a portion of the amyloid beta peptide may contain a sequence that can be recognized as the epitope.
에피토프로서 인식될 수 있는 서열은 에피토프 서열 전체로 이루어지거나, 그 서열의 양 말단에 일부 아미노산이 추가되거나, 양 말단에서 일부 아미노산이 제거되어도 에피토프로서 작동하는 서열을 의미한다.A sequence that can be recognized as an epitope refers to a sequence that functions as an epitope even if it consists of the entire epitope sequence, some amino acids are added to both ends of the sequence, or some amino acids are removed from both ends of the sequence.
상기 에피토프는 서열번호 1의 아미노산 서열의 적어도 일부를 포함할 수 있고, 보다 구체적으로는 서열번호 2 내지 서열번호 8의 아미노산 서열로 이루어진 군에서 선택되는 서열일 수 있다. 다만 이에 제한되는 것은 아니고, 상기 에피토프로서 인식될 수 있는 서열이라면 제한 없이 포함될 수 있고 공지된 에피토프 서열을 사용할 수도 있다.The epitope may include at least a portion of the amino acid sequence of SEQ ID NO: 1, and more specifically, may be a sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 2 to SEQ ID NO: 8. However, it is not limited thereto, and any sequence that can be recognized as the epitope may be included without limitation, and a known epitope sequence may be used.
상기 아밀로이드 베타 펩타이드의 길이는 앞서 언급하였듯, MHC 분자에 결합하여 아밀로이드 베타 펩타이드로 인식될 수 있는 것이라면 제한되지 않으며, 예를 들면 0.5 kDa 내지 5 kDa, 1 kDa 내지 4 kDa, 1.5 kDa 내지 3 kDa 또는 1.8 kDa 내지 2 kDa 등일 수 있다. 다만 이에 제한되는 것은 아니다.As mentioned above, the length of the amyloid beta peptide is not limited as long as it can be recognized as an amyloid beta peptide by binding to an MHC molecule, for example, 0.5 kDa to 5 kDa, 1 kDa to 4 kDa, 1.5 kDa to 3 kDa. Or it may be 1.8 kDa to 2 kDa, etc. However, it is not limited to this.
상기 아밀로이드 베타 펩타이드는 바람직하게는 인간의 아밀로이드 베타 펩타이드에서 유래한 것 일 수 있다.The amyloid beta peptide may preferably be derived from human amyloid beta peptide.
본 발명에서 상기 아밀로이드 베타 펩타이드를 코딩하는 핵산은 앞서 언급한 아밀로이드 베타 펩타이드를 코딩하고 있는 것이라면 그 핵산의 종류에 무관하게 본 발명의 범위에 포함될 수 있다. 핵산의 종류의 예를 들면, DNA 또는 RNA 일 수 있다. 상기 아밀로이드 베타 펩타이드를 코딩하는 핵산의 예를 들면 서열번호 1 내지 서열번호 8의 아미노산 서열로 이루어진 군에서 선택되는 서열을 코딩하는 핵산을 포함하는 것일 수 있다. 다만 이에 제한되는 것은 아니다.In the present invention, the nucleic acid encoding the amyloid beta peptide may be included within the scope of the present invention regardless of the type of nucleic acid as long as it encodes the above-mentioned amyloid beta peptide. For example, the type of nucleic acid may be DNA or RNA. Examples of the nucleic acid encoding the amyloid beta peptide may include a nucleic acid encoding a sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 8. However, it is not limited to this.
상기 아밀로이드 베타 펩타이드를 코딩하는 핵산은 인체 내에서 상기 아밀로이드 베타 펩타이드를 생성할 수 있고, 유도된 상기 펩타이드는 앞서 언급되었듯 제 2형 MHC 분자를 가진 수지상세포 등의 항원제시세포에 유입되어 아밀로이드 베타 펩타이드 특이적인 조절 T 세포를 유도하거나, 아밀로이드 베타 펩타이드에 특이적인 항체를 생성할 수 있다.The nucleic acid encoding the amyloid beta peptide can produce the amyloid beta peptide in the human body, and the derived peptide is introduced into antigen-presenting cells such as dendritic cells with type 2 MHC molecules, producing amyloid beta, as previously mentioned. It can induce peptide-specific regulatory T cells or generate antibodies specific for amyloid beta peptide.
본 발명에서 상기 라파마이신 또는 그 유도체는 그 종류에 무관하게 본 발명의 범위에 포함될 수 있다.In the present invention, rapamycin or a derivative thereof may be included in the scope of the present invention regardless of its type.
본 발명에서 라파마이신은 림프구의 성장을 억제하고, 분화를 촉진하여 아밀로이드 베타에 대해 특이적인 조절 T세포를 유도할 수 있고, 아밀로이드 베타 펩타이드에 의하여 유발되는 염증 반응을 억제할 수 있다. 따라서 이러한 기능을 수행할 수 있는 라파마이신의 유도체라면 제한 없이 본 발명의 범위에 포함될 수 있다. 그 예를 들면, 벤조일 라파마이신, 템시로리무스, 에베로리무스, 조타롤리무스, 바이오리무스, 피메크로리무스, 피메크로리무스, 타크로리무스, 리다포로리무스 등을 들 수 있다. 다만, 이에 제한되는 것은 아니다.In the present invention, rapamycin can inhibit the growth of lymphocytes, promote differentiation, induce regulatory T cells specific for amyloid beta, and suppress the inflammatory response caused by amyloid beta peptide. Therefore, any derivative of rapamycin that can perform this function can be included in the scope of the present invention without limitation. Examples include benzoyl rapamycin, temsirolimus, everolimus, zotarolimus, biolimus, pimecrolimus, pimecrolimus, tacrolimus, and ridaporolimus. However, it is not limited to this.
본 발명의 상기 아밀로이드 베타 펩타이드 또는 이를 코딩하는 핵산; 및 라파마이신 또는 그 유도체;는 전달체에 담지되어 전달될 수 있고, 바람직하게는 아밀로이드 베타 펩타이드 또는 이를 코딩하는 핵산; 및 라파마이신 또는 그 유도체;가 함께 개별 전달체마다 포함되도록 담지되어 전달될 수 있다.The amyloid beta peptide of the present invention or a nucleic acid encoding the same; and rapamycin or a derivative thereof; may be carried and delivered in a carrier, and are preferably amyloid beta peptide or a nucleic acid encoding the same; and rapamycin or a derivative thereof; may be carried and delivered together so as to be included in each individual delivery vehicle.
본 발명에서 상기 전달체는 아밀로이드 베타 펩타이드 및 라파마이신 또는 그 유도체의 수지상세포에의 전달효율을 크게 향상시키므로, 결과적으로 아밀로이드 베타 펩타이드를 제 1형 또는 제 2형 MHC 분자에 제시하는 관용 수지상세포의 유도 효율을 증가시킬 수 있고, 따라서 아밀로이드 베타 펩타이드 특이적인 항체의 생성 효율 및 아밀로이드 베타 펩타이드 특이적 조절 T 세포의 유도 효율을 크게 높일 수 있다. 상기 아밀로이드 베타 특이적 조절 T 세포의 유도 효율의 증가는 앞서 언급하였듯, 뇌신경 염증을 억제하고, 산화적 스트레스를 억제하여 신경을 보호할 수 있어 유리하다.In the present invention, the delivery vehicle greatly improves the delivery efficiency of amyloid beta peptide and rapamycin or its derivatives to dendritic cells, resulting in the induction of tolerant dendritic cells that present amyloid beta peptide to type 1 or type 2 MHC molecules. Efficiency can be increased, and thus the production efficiency of amyloid beta peptide-specific antibodies and the induction efficiency of amyloid beta peptide-specific regulatory T cells can be greatly increased. As mentioned above, the increase in the induction efficiency of amyloid beta-specific regulatory T cells is advantageous because it can suppress cranial nerve inflammation and protect nerves by suppressing oxidative stress.
본 발명에서 상기 전달체는 내부에 아밀로이드 베타 펩타이드 또는 이를 코딩하는 핵산; 및 라파마이신 또는 그 유도체;를 담지하고, 타깃 세포에 전달하여 내부의 물질을 방출할 수 있는 것이라면, 당업자에 의하여 제한 없이 선택될 수 있다. 예를 들면, 바이러스 전달체, 바이러스 유사 입자(virus-like particle, VLP), 양전하성 폴리머, 리포좀, 지질나노입자(lipid nanoparticle), 금 및 반도체 나노결정 입자(quantum dot) 를 포함하는 군에서 선택되는 것일 수 있고, 바람직하게는 지질나노입자일 수 있다. 다만 이에 제한되는 것은 아니다.In the present invention, the delivery vehicle includes amyloid beta peptide or a nucleic acid encoding the same; and rapamycin or a derivative thereof, and can be selected without limitation by those skilled in the art as long as it can be delivered to the target cell and release the substance therein. For example, selected from the group including viral carriers, virus-like particles (VLP), positively charged polymers, liposomes, lipid nanoparticles, gold, and semiconductor nanocrystal particles (quantum dots). It may be a lipid nanoparticle, preferably a lipid nanoparticle. However, it is not limited to this.
본 발명에서 상기 지질나노입자는 그 성분에 무관하게 당업자에 의하여 선택하여 사용할 수 있다. 지질나노입자를 구성하는 인지질 분자로서, 중성지질, 음이온성 및/또는 양이온성 인지질 모두가 사용이 가능하고, 바람직하게는 양이온성 인지질을 포함하는 지질나노입자를 사용할 수 있다.In the present invention, the lipid nanoparticles can be selected and used by those skilled in the art regardless of their components. As phospholipid molecules constituting lipid nanoparticles, neutral lipids, anionic and/or cationic phospholipids can all be used, and lipid nanoparticles containing cationic phospholipids are preferably used.
상기 중성지질의 예를 들면, L-α포스파티딜콜린(L-α-phosphatidylcholine, PC), 1,2-디올레오일-sn-글리세로-3-포스포콜린(1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC), 1,2-디스테아로일-sn-글리세로-3-포스포콜린(1,2-distearoyl-sn-glycero-3-phophocholine, DSPC), 1,2-디팔미토일-sn-글리세로-3-포스포콜린(1,2-dipalmitoylsn-glycero-3-phophocholine, DPPC), 1,2-디미리스토일-sn-글리세로-3-포스포콜린(1,2-dimyristoyl-sn-glycero-3-phosphocholine, DMPC)으로 이루어진 군에서 선택된 적어도 하나일 수 있고, 상기 PC는 콩, 계란, 수소화된 콩 또는 계란으로부터 유래된 PC일 수 있으나, 이로 제한되지 않는다. Examples of the neutral lipid include L-α-phosphatidylcholine (PC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (1,2-dioleoyl-sn-glycero- 3-phosphocholine, DOPC), 1,2-distearoyl-sn-glycero-3-phophocholine (DSPC), 1,2-dipalmi Toyl-sn-glycero-3-phosphocholine (1,2-dipalmitoylsn-glycero-3-phophocholine, DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (1, 2-dimyristoyl-sn-glycero-3-phosphocholine, DMPC), and the PC may be PC derived from soybeans, eggs, hydrogenated soybeans or eggs, but is not limited thereto.
상기 음이온성 지질의 예를 들면, L-α포스파티딕산(L-α-phosphatidic acid), L-α포스파티딜-DL-글리세롤(L-α-phosphatidyl-DL-glycerol), 카디오리핀(cardiolipin), L-α포스파티딜이노시톨(L-α-phosphatidylinositol), L-α-포스파티딜세린(L-α-phosphatidylserine), 1,2-디라우로일-sn-글리세로-3-[포스포-rac-(1-글리세롤)](1,2-dilauroyl-sn-glycero-3-[phospho-rac-(1-glycerol)], DLPG), 1,2-디라우로일-sn-글리세로-3-[포스포-L-세린](1,2-dilauroyl-sn-glycero-3-[phospho-L-serine], DLPS), 1,2-디라우로일-sn-글리세로-3-포스페이트(1,2-dilauroyl-sn-glycero-3-phosphate, DLPA), 1,2-디미리스토일-sn-글리세로-3-[포스포-rac-(1-글리세롤)]1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], DMPG), 1,2-디미리스토일-sn-글리세로-3-[포스포-L-세린] (1,2-dimyristoyl-sn-glycero-3-[phospho-L-serine], DMPS), 1,2-디미리스토일-sn-글리세로-3-포스페이트(1,2-dimyristoyl-sn-glycero-3-phosphate, DMPA), 1,2-디올레오일-sn-글리세로-3-[포스포-rac-(1-글리세롤)](1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], DOPG), 1,2-디올레오일-sn-글리세로-3-[포스포-L-세린](1,2-dioleoyl-sn-glycero-3-[phospho-L-serine], DOPS), 1,2-디올레오일-sn-글리세로-3-포스페이트(1,2-dioleoyl-sn-glycero-3-phosphate, DOPA), 1,2-디팔미토일-sn-글리세로-3-[포스포-L-세린](1,2-dipalmitoyl-sn-glycero-3-[phospho-L-serine], DPPS), 1,2-디팔미토일-sn-글리세로-3-포스페이트(1,2-dipalmitoyl-sn-glycero-3-phosphate, DPPA), 1,2-디스테로일-sn-글리세로-3-[포스포-rac-(1-글리세롤)](1,2-distearoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], DSPG), 1,2-디스테로일-sn-글리세로-3-[포스포-L-세린](1,2-distearoyl-sn-glycero-3-[phospho-L-serine], DSPS), 1,2-디스테로일-sn-글리세로-3-포스페이트(1,2-distearoyl-sn-glycero-3-phosphate, DSPA), 1,2-디스테로일-sn-글리세로-3-포스포에탄올아민-N-[카복시(폴리에틸렌글리콜2000](1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)2000]), 1,2-디스테로일-sn-글리세로-3-포스포에탄올아민-N-[말레이미드(폴리에틸렌글리콜)2000](1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)2000]), 1,2-디스테로일-sn-글리세로-3-포스포에탄올아민-N-[PDP(폴리에틸렌글리콜)2000](1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[PDP(polyethylene glycol)2000]), 1-팔미토일-2-올레일-sn-글리세로-3-[포스포-rac-(1-글리세롤)](1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], POPG), 1-팔미토일-2-올레일-sn-글리세로-3-[포스포-L-세린](1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine], POPS), 1-팔미토일-2-올레일-sn-글리세로-3-포스페이트(1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate, POPA) 및 올레산(oleicacid)으로 이루어진 군에서 선택된 적어도 하나일 수 있으나, 이로 제한되지는 않는다.Examples of the anionic lipid include L-α-phosphatidic acid, L-α-phosphatidyl-DL-glycerol, cardiolipin, L-α-phosphatidylinositol, L-α-phosphatidylserine, 1,2-dilauroyl-sn-glycero-3-[phospho-rac-( 1-glycerol)](1,2-dilauroyl-sn-glycero-3-[phospho-rac-(1-glycerol)], DLPG), 1,2-dilauroyl-sn-glycero-3-[ phospho-L-serine](1,2-dilauroyl-sn-glycero-3-[phospho-L-serine], DLPS), 1,2-dilauroyl-sn-glycero-3-phosphate (1 ,2-dilauroyl-sn-glycero-3-phosphate, DLPA), 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]1,2-dimyristoyl- sn-glycero-3-[phospho-rac-(1-glycerol)], DMPG), 1,2-dimyristoyl-sn-glycero-3-[phospho-L-serine] (1,2- dimyristoyl-sn-glycero-3-[phospho-L-serine], DMPS), 1,2-dimyristoyl-sn-glycero-3-phosphate (1,2-dimyristoyl-sn-glycero-3-phosphate , DMPA), 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)](1,2-dioleoyl-sn-glycero-3-[phospho-rac-( 1-glycerol)], DOPG), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine](1,2-dioleoyl-sn-glycero-3-[phospho-L- serine], DOPS), 1,2-dioleoyl-sn-glycero-3-phosphate (1,2-dioleoyl-sn-glycero-3-phosphate, DOPA), 1,2-dipalmitoyl-sn- Glycero-3-[phospho-L-serine](1,2-dipalmitoyl-sn-glycero-3-[phospho-L-serine], DPPS), 1,2-dipalmitoyl-sn-glycero- 3-phosphate (1,2-dipalmitoyl-sn-glycero-3-phosphate, DPPA), 1,2-disteroyl-sn-glycero-3-[phospho-rac-(1-glycerol)](1 ,2-distearoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], DSPG), 1,2-distearoyl-sn-glycero-3-[phospho-L-serine]( 1,2-distearoyl-sn-glycero-3-[phospho-L-serine], DSPS), 1,2-distearoyl-sn-glycero-3-phosphate (1,2-distearoyl-sn-glycero- 3-phosphate, DSPA), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy (polyethylene glycol 2000](1,2-distearoyl-sn-glycero-3-phosphoethanolamine -N-[carboxy(polyethylene glycol)2000]), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide (polyethylene glycol) 2000](1,2-disteroyl -sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)2000]), 1,2-disteroyl-sn-glycero-3-phosphoethanolamine-N-[PDP(polyethylene glycol)2000 ](1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[PDP(polyethylene glycol)2000]), 1-palmitoyl-2-oleyl-sn-glycero-3-[phospho-rac -(1-glycerol)](1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], POPG), 1-palmitoyl-2-oleoyl-sn-glycerol ro-3-[phospho-L-serine](1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine], POPS), 1-palmitoyl-2-oleoyl-sn- It may be at least one selected from the group consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA) and oleic acid, but is not limited thereto.
상기 양이온성 지질의 예를 들면, 1,2-디올레오일-sn-글리세로-3-에틸포스포콜린(1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, EDOPC), 1,2-디올레오일-3-트리메틸암모늄-프로판(1,2-dioleoyl-3-trimethylammoniumpropane, DOTAP), 디올레오일 글루타마이드(dioleoyl glutamide), 디스테아로일 글루타마이드(distearoylglutamide), 디팔미토일 글루타마이드(dipalmitoyl glutamide), 디올레오일 아스파르타마이드(dioleoylaspartamide), 1,2-디올레오일-3-디메틸암모늄-프로판(1,2-dioleoyl-3-dimethylammonium-propane, DODAP), ß-[N-(N',N'-디메틸아미노에탄-카바모일], (3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl], DC-Chol), 메틸디옥타데실암모늄 브로마이드 (dimethyldiochtadecylammonium bromide, DDAB), 1,2-디올레오일-sn-글리세로-3-포스포에탄올아민(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE), 1,2-디스테아로일-sn-글리세로-3-포스포에탄올아민(1,2-distearoyl-sn-glycero-3-phophoethanolamine, DSPE) 및 1,2-디팔미토일-sn-글리세로-3-포스포에탄올아민 (1,2-dipalmitoyl-sn-glycero-3-phophoethanolamine, DPPE)으로 이루어진 군에서 선택된 적어도 하나일 수 있으나, 이로 제한되지는 않는다.Examples of the cationic lipid include 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC), 1,2- 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), dioleoyl glutamide, distearoylglutamide, dipalmitoyl Dipalmitoyl glutamide, dioleoylaspartamide, 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), ß-[ N-(N',N'-dimethylaminoethane-carbamoyl], (3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl], DC-Chol), dimethyldiochtadecylammonium bromide , DDAB), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE), 1,2-distearoyl- sn-glycero-3-phosphoethanolamine (1,2-distearoyl-sn-glycero-3-phophoethanolamine, DSPE) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (1 , 2-dipalmitoyl-sn-glycero-3-phophoethanolamine, DPPE), but is not limited thereto.
본 발명에서 상기 지질입자는 입자의 유동성, 담지 효율 및/또는 안정성 등을 개선하기 위하여 추가적으로 폴리에틸렌글리콜(polyethylene glycol, PEG) 등으로 인지질 표면이 PEGylation 될 수 있고, 콜레스테롤, 1,2-Dioleoyl-3-trimethylammoniumpropane (DOTAP)을 더 포함할 수도 있다. 바람직하게는 앞서 언급한 양이온성 지질인 1,2-디올레오일-sn-글리세로-3-포스포에탄올아민, DOTAP, PEG2000 및 콜레스테롤을 포함하도록 지질 입자를 사용할 수 있다. 다만 이에 제한되는 것은 아니다.In the present invention, the phospholipid surface of the lipid particle may be PEGylated with polyethylene glycol (PEG), etc. to improve the fluidity, loading efficiency, and/or stability of the particle, and cholesterol, 1,2-Dioleoyl-3 -trimethylammoniumpropane (DOTAP) may be further included. Preferably, the lipid particles can be used to include the aforementioned cationic lipids such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOTAP, PEG2000, and cholesterol. However, it is not limited to this.
본 발명에서 상기 전달체의 평균 직경은 제한 없이 본 발명의 범위에 포함되고, 담지되는 펩타이드, 핵산 및 약물의 양 등의 조건에 따라 당업자에 의해 선택될 수 있다. 전달체의 직경이 클수록 더 많은 양의 물질의 담지가 가능하나, 전달체의 혈중 안정성, 혈중 이동속도 등에 영향을 줄 수 있어 적절한 범위의 평균 직경을 선택할 수 있다. 평균 직경의 예를 들면, 50 nm 내지 600 nm일 수 있다. 평균 입자 직경의 하한의 예를 들면, 50 nm, 80 nm, 110 nm, 140 nm 또는 170 nm 일 수 있고, 상한의 예를 들면, 600 nm, 500 nm, 400 nm, 350 nm, 300 nm 일 수 있다. 다만, 이에 제한되는 것은 아니다.In the present invention, the average diameter of the carrier is within the scope of the present invention without limitation, and may be selected by a person skilled in the art depending on conditions such as the amount of peptide, nucleic acid, and drug to be carried. The larger the diameter of the delivery vehicle, the more it can carry a larger amount of material, but it can affect the stability of the delivery vehicle in the blood and the speed of movement through the blood, so an average diameter within an appropriate range can be selected. For example, the average diameter may be 50 nm to 600 nm. The lower limit of the average particle diameter can be, for example, 50 nm, 80 nm, 110 nm, 140 nm or 170 nm, and the upper limit can be, for example, 600 nm, 500 nm, 400 nm, 350 nm, 300 nm. there is. However, it is not limited to this.
본 발명의 상기 조성물은 상기 알츠하이머병의 예방 또는 치료와 관련하여 동일 또는 유사한 기능을 나타내는 유효성분을 1종 이상 또는 유효성분의 용해성 및/또는 흡수성을 유지/증가 시키는 화합물을 추가로 함유할 수 있다.The composition of the present invention may additionally contain one or more active ingredients that exhibit the same or similar function in relation to the prevention or treatment of Alzheimer's disease, or a compound that maintains/increases the solubility and/or absorption of the active ingredient. .
본 발명의 상기 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있다. 다만, 이에 제한되지 않는다. 또한, 본 발명의 조성물에 포함되는 유효성분의 농도는 치료 목적, 환자의 상태, 필요기간 등을 고려하여 결정할 수 있으며, 특정 범위의 농도로 한정되지 않는다. The appropriate dosage of the composition of the present invention may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity. You can. However, it is not limited to this. In addition, the concentration of the active ingredient included in the composition of the present invention can be determined considering the purpose of treatment, patient's condition, necessary period, etc., and is not limited to a specific concentration range.
본 발명의 상기 조성물은 약학적으로 유효한 양으로 투여한다. 유효용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 약학적 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The composition of the present invention is administered in a pharmaceutically effective amount. The effective dosage level depends on factors including the type and severity of the patient's disease, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the field of medicine. can be decided. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.
본 발명의 상기 조성물의 투여 방법은 제한 없이 선택될 수 있다. 예를 들면, 근육주사(intramuscular injection, IM), 피하주사(subcutaneous injection, SC), 정맥주사(intravenous injection, IV), 피내주사(intradermal injection, ID), 복강주사(intraperitoneal injection, IP) 할 수 있고, 바람직하게는 피부하층에 위치하는 수지상세포에 의하여 흡수되기 용이한 피하주사가 유리할 수 있다 .다만 이에 제한되는 것은 아니다.The method of administering the composition of the present invention can be selected without limitation. For example, intramuscular injection (IM), subcutaneous injection (SC), intravenous injection (IV), intradermal injection (ID), and intraperitoneal injection (IP) can be performed. Preferably, subcutaneous injection may be advantageous as it is easily absorbed by dendritic cells located in the lower layer of the skin. However, it is not limited thereto.
본 발명의 상기 조성물의 투여량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설률 및 질환의 중증도 등에 따라 그 범위가 매우 다양하며, 적정한 투여량은 예를 들면 환자의 체내에 축적된 약물의 양 및/또는 사용되는 본 발명의 전달체의 구체적 효능정도에 따라 달라질 수 있다. 예를 들면 체중 1 kg당 0.01 μg 내지 1 g 일 수 있으며, 일별, 주별, 월별 또는 연별의 단위 기간으로, 단위 기간 당 일회 내지 수회 나누어 투여될 수 있으며, 또는 인퓨전 펌프를 이용하여 장기간 연속적으로 투여될 수 있다. 반복투여 횟수는 약물이 체내 머무는 시간, 체내 약물 농도 등을 고려하여 결정된다. 질환 치료 경과에 따라 치료가 된 후라도, 재발을 위해 조성물이 투여될 수 있다.The dosage range of the composition of the present invention varies greatly depending on the patient's weight, age, gender, health condition, diet, administration time, administration method, excretion rate, and severity of the disease. The appropriate dosage can be determined by, for example, the patient. It may vary depending on the amount of drug accumulated in the body and/or the specific efficacy of the delivery vehicle of the present invention used. For example, it may be 0.01 μg to 1 g per kg of body weight, and may be administered in divided doses, such as daily, weekly, monthly, or yearly units, once or several times per unit period, or administered continuously over a long period of time using an infusion pump. It can be. The number of repeated doses is determined by considering the time the drug stays in the body and the concentration of the drug in the body. Even after treatment, depending on the course of disease treatment, the composition may be administered for recurrence.
이하, 본 발명을 구체적으로 설명하기 위해 실시예를 들어 상세하게 설명하기로 한다.Hereinafter, the present invention will be described in detail with reference to examples.
제조예 1. LNP-R/Aβ의 제조Preparation Example 1. Preparation of LNP-R/Aβ
846 μg의 18:1 TAP (DOTAP, 1,2-dioleoyl-3-trimethylammonium-propane), 675 μg의 18:1 (△9-Cis) PE (DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), 358 μg 의 18:0 PEG2000 [1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]], 115 μg의 콜레스테롤, 70 μg의 라파마이신을 공기 중에서 건조시켜 thin film으로 만들었다. 그 후 200 μg의 아밀로이드 단백질을 함유한 1mL의 PBS로 1시간 동안 수화시켰다. 수화된 용액은 polycarbonate membrane filters (pore sizes of 1 μm, 400 nm, and 200 nm) 를 통과시켜 centrifugal filter unit (100 kDa)를 이용해 농축하였다.846 μg of 18:1 TAP (DOTAP, 1,2-dioleoyl-3-trimethylammonium-propane), 675 μg of 18:1 (△9-Cis) PE (DOPE, 1,2-dioleoyl-sn-glycero-3) -phosphoethanolamine), 358 μg of 18:0 PEG2000 [1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]], 115 μg of cholesterol, and 70 μg of rapamycin. It was dried in air and made into a thin film. Afterwards, it was hydrated with 1 mL of PBS containing 200 μg of amyloid protein for 1 hour. The hydrated solution was passed through polycarbonate membrane filters (pore sizes of 1 μm, 400 nm, and 200 nm) and concentrated using a centrifugal filter unit (100 kDa).
실시예 1. 지질 나노입자의 특성 확인Example 1. Confirmation of properties of lipid nanoparticles
상기 제조예 1에 의해 만들어진 LNP-R/Aβ을 투과전자현미경으로 관찰하였고 인지질 이중막을 가진 구형의 나노입자 형태임을 확인하였다. Energy Dispersive X-ray Spectroscopy (EDS) 분석 결과 탄소, 산소, 아밀로이드 단백질에 함유된 질소, 지질에 함유된 인 등이 검출되었다. 본 발명의 LNP-R/Aβ은 232.7±84.0nm의 크기를 가진 나노입자이며, 그 크기는 7일간 관찰하였을 때 그 안정성을 유지하였다. 본 지질 나노입자의 주 구성 지질인 DOTAP에 의해 나노입자는 약간의 양전하를 가짐을 확인하였다(도 2a).LNP-R/Aβ prepared in Preparation Example 1 was observed under a transmission electron microscope and confirmed to be in the form of spherical nanoparticles with a phospholipid bilayer. As a result of Energy Dispersive X-ray Spectroscopy (EDS) analysis, carbon, oxygen, nitrogen contained in amyloid protein, and phosphorus contained in lipids were detected. LNP-R/Aβ of the present invention is a nanoparticle with a size of 232.7 ± 84.0 nm, and its size maintained its stability when observed for 7 days. It was confirmed that the nanoparticles had a slight positive charge due to DOTAP, the main lipid component of this lipid nanoparticle (Figure 2a).
31P-NMR 분석 결과 본 치료용 백신은 unilamellar 구조임을 확인하였고, HPLC 분석을 통해 LNP-R/Aβ내에 라파마이신이 함유되었음을 확인하였으며, BCA protein assay를 통해 함유된 항원(단백질)의 양을 정량하여 표 1에 나타내었다. 라파마이신이 LNP-R/Aβ와 단순히 혼합된 것이 아닌 봉입되어 있음을 확인하기 위해 DSC 분석을 통해 확인하였고 Free 라파마이신에서는 관찰된 피크가 LNP-R/Aβ에서는 관찰되지 않았음을 확인하였다. 아밀로이드 베타 단백질이 LNP-R/Aβ에 함유되어 있음을 확인하기 위해 FITC로 표지된 아밀로이드 베타 단백질을 이용하여 DiD 표지된 지질 나노입자를 제조하였고 공초점 형광현미경을 통해 관찰하여 두 형광이 동시에 검출됨을 확인하였다. 이를 유세포분석기를 통해 분석한 결과 약 91.7 %의 입자가 두 형광이 모두 검출되어 대부분의 지질 나노입자에 항원이 봉입되었음을 확인하였다. FT-IR을 통해 확인해본 결과 LNP-R/Aβ에서는 라파마이신, 아밀로이드 베타 단백질, 지질 나노입자에서 나타난 peak가 모두 나타남을 확인하였다(도 2b). LNP-R/Aβ의 세포 및 동물에서의 독성을 분석해본 결과 특별한 독성이나 염증 유발이 나타나지 않았다(도 2c).As a result of 31P-NMR analysis, it was confirmed that this therapeutic vaccine has a unilamellar structure, and through HPLC analysis, it was confirmed that rapamycin was contained in LNP-R/Aβ. The amount of antigen (protein) contained was quantified through BCA protein assay. It is shown in Table 1. To confirm that rapamycin was encapsulated and not simply mixed with LNP-R/Aβ, it was confirmed through DSC analysis, and it was confirmed that the peak observed in free rapamycin was not observed in LNP-R/Aβ. To confirm that amyloid beta protein is contained in LNP-R/Aβ, DiD-labeled lipid nanoparticles were prepared using FITC-labeled amyloid beta protein and observed through a confocal fluorescence microscope, confirming that both fluorescence were detected simultaneously. Confirmed. As a result of analyzing this using a flow cytometer, both fluorescence was detected in about 91.7% of the particles, confirming that antigens were encapsulated in most lipid nanoparticles. As a result of confirmation through FT-IR, it was confirmed that all peaks appearing in rapamycin, amyloid beta protein, and lipid nanoparticles were present in LNP-R/Aβ (Figure 2b). As a result of analyzing the toxicity of LNP-R/Aβ in cells and animals, no particular toxicity or inflammation was found (Figure 2c).
지질
나노입자lipids
nanoparticles 		라파마이신rapamycin 항원antigen
담지량(μg)Loading amount (μg) 담지 효율(%)Loading efficiency (%) 담지량(μg)Loading amount (μg) 담지 효율(%)Loading efficiency (%)
LNP-RLNP-R 18.36 ± 0.6018.36 ± 0.60 26.23 ± 0.8626.23 ± 0.86 N/AN/A N/AN/A
LNP-AβLNP-Aβ N/AN/A N/AN/A 106.18 ± 12.3106.18 ± 12.3 53.09 ± 6.1753.09 ± 6.17
LNP-R/AβLNP-R/Aβ 18.73 ± 0.8318.73 ± 0.83 26.75 ± 1.1926.75 ± 1.19 103.78 ± 9.25103.78 ± 9.25 51.89 ± 4.6351.89 ± 4.63
실시예 2. LNP-R/Aβ에 의한 Aβ 특이적인 항체 유도 및 알츠하이머병 동물 모델의 뇌에서 Aβ 응집체 집적에 미치는 효과 검증Example 2. Induction of Aβ-specific antibodies by LNP-R/Aβ and verification of effect on Aβ aggregate accumulation in the brain of an Alzheimer's disease animal model
LNP-R/Aβ는 쥐에 피내 주사한 후 수지상세포에 의해 uptake되어 림프절로 이동하여 작용할 것이라 기대되므로, 세포 수준에서 수지상세포에 LNP-R/Aβ을 처리하고 시간에 따라 관찰하였다. 시간에 따라 항원이 수지상세포에 의해 processing하여 표면에 제시됨을 확인하였다. 지질나노입자가 없는 라파마이신과 항원의 혼합물과 수지상세포의 항원 제시 능력을 비교해본 결과 지질나노입자를 이용하였을 때 그 효율이 월등히 높아짐을 확인하였다. MHC class II에 항원이 로딩된 복합체를 검출하기 위하여 이 때에는 아밀로이드 베타가 아닌 Eα 펩타이드를 항원으로 사용하였고(LNP-R/Eα), Eα-MHC class II를 검출하는 항체를 이용하였다. LNP-R/Aβ를 피내주사한 24 시간 후 IVIS를 통해 분석한 결과 림프절로 이동하였음을 확인하였다(도 3a). Since LNP-R/Aβ is expected to be uptaken by dendritic cells after intradermal injection into mice and move to the lymph nodes to act, dendritic cells were treated with LNP-R/Aβ at the cellular level and observed over time. It was confirmed that the antigen was processed by dendritic cells and presented on the surface over time. As a result of comparing the antigen presentation ability of dendritic cells with a mixture of rapamycin and antigen without lipid nanoparticles, it was confirmed that the efficiency was significantly increased when lipid nanoparticles were used. To detect complexes loaded with antigens on MHC class II, Eα peptide rather than amyloid beta was used as the antigen (LNP-R/Eα), and an antibody that detects Eα-MHC class II was used. Analysis using IVIS 24 hours after intradermal injection of LNP-R/Aβ confirmed that it had migrated to the lymph nodes (Figure 3a).
림프절을 동결시편제작하여 조직면역염색을 통해 확인해 본 결과 수지상세포에서 LNP-R/Aβ가 검출되었다. 체내에서 LNP-R/Aβ에 의해 항원 특이적 folicular helper T (Tfh) 세포가 유도됨을 확인하기 위해 항원으로 Ovalbumin을 이용하여 LNP-R/OVA를 제조하였고 이를 마우스에 1주일 간격으로 2회 피내주사하고 5일 후 림프절을 수확하여 OVA-specific Tfh 세포의 비율을 정량하였을 때, LNP-OVA, LNP-R/OVA 그룹에서 유사한 수준의 Tfh와 항원 특이적 Tfh가 유도됨을 확인하였다(도 3b).As a result of making frozen specimens of lymph nodes and confirming them through tissue immunostaining, LNP-R/Aβ was detected in dendritic cells. To confirm that antigen-specific folicular helper T (Tfh) cells are induced by LNP-R/Aβ in vivo, LNP-R/OVA was prepared using ovalbumin as an antigen and intradermally injected into mice twice at one-week intervals. When the lymph nodes were harvested 5 days later and the proportion of OVA-specific Tfh cells was quantified, it was confirmed that similar levels of Tfh and antigen-specific Tfh were induced in the LNP-OVA and LNP-R/OVA groups (Figure 3b).
알츠하이머병 마우스 모델인 5XFAD 마우스를 이용하여 LNP-R/Aβ의 체내 항체 유도 효과를 확인하였다. 5개월 령 5XFAD 마우스에 LNP-R/Aβ 또는 그 대조군들을 1주일 간격으로 5회 피내주사하고 2주 뒤 혈청과 뇌 조직을 얻었다 (도 3b). 그 결과 Aβ 항원이 포함되어 있는 LNP-Aβ와 LNP-R/Aβ 투여군의 혈청 및 뇌 조직에서 Aβ 특이적인 항체가 관측되었다. 두 투여군에서 생성된 항체들은 Aβ 항원의 N-말단의 8개 아미노산 서열 이내의 에피토프를 가지고 있는 것으로 확인되었다(도 3c). 5XFAD 마우스의 비장에서 Tfh 및 형질 세포의 비율을 조사해본 결과 LNP-Aβ, LNP-R/Aβ 그룹에서 유의미하게 증가하는 경향성을 보였다 (도 3d). 상기 투여 조건에서 알츠하이머병 마우스 모델의 뇌 내에서 Aβ 플라크의 집적을 면역조직염색법(immunohistochemistry)를 통해 형광현미경으로 관측하였다. 항 Aβ 항체인 6E10을 이용하여 뇌 조직 내의 Aβ 플라크의 양을 확인한 결과, LNP-Aβ와 LNP-R/Aβ 투여군에서 해마와 대뇌피질을 비롯한 전체 뇌 조직에 걸쳐 Aβ 플라크가 현저히 감소한 것을 확인하였다. 따라서 실시예 1과 2에서의 결과로부터 LNP-Aβ와 LNP-R/Aβ가 항 Aβ 항체의 유도를 통해 뇌 내 Aβ를 제거하였음을 유추할 수 있다 (도 3e, 3f).The antibody-inducing effect of LNP-R/Aβ in vivo was confirmed using 5XFAD mice, a mouse model of Alzheimer's disease. LNP-R/Aβ or its control group was injected intradermally five times at 1-week intervals into 5-month-old 5 As a result, Aβ-specific antibodies were observed in the serum and brain tissue of the LNP-Aβ and LNP-R/Aβ administration groups containing Aβ antigen. The antibodies produced in both administration groups were confirmed to have an epitope within the 8 amino acid sequence of the N-terminus of the Aβ antigen (Figure 3c). As a result of examining the ratio of Tfh and plasma cells in the spleen of 5XFAD mice, it showed a tendency to significantly increase in the LNP-Aβ and LNP-R/Aβ groups (Figure 3d). Under the above administration conditions, accumulation of Aβ plaques in the brain of an Alzheimer's disease mouse model was observed using a fluorescence microscope through immunohistochemistry. As a result of checking the amount of Aβ plaques in brain tissue using 6E10, an anti-Aβ antibody, it was confirmed that Aβ plaques were significantly reduced throughout the entire brain tissue, including the hippocampus and cerebral cortex, in the LNP-Aβ and LNP-R/Aβ administration groups. Therefore, from the results in Examples 1 and 2, it can be inferred that LNP-Aβ and LNP-R/Aβ removed Aβ in the brain through induction of anti-Aβ antibodies (FIGS. 3e, 3f).
실시예 3. LNP-R/Aβ에 의한 면역관용 수지상세포 및 조절 T세포 유도 확인Example 3. Confirmation of induction of immune tolerance dendritic cells and regulatory T cells by LNP-R/Aβ
LNP-R/Aβ에 의해 면역관용 수지상세포 및 조절 T세포가 유도됨을 확인하기 위하여 OVA를 항원으로 하여 LNP-R/OVA를 만들었다. 이를 먼저 야생형 C57BL/6 마우스 골수 유래 수지상세포에 처리하여 유세포 분석기와 qRT-PCR을 통해 분석하였고 면역관용 수지상세포의 특징으로 알려진 낮은 수준의 MHC class II와 공동자극분자(co-stimulatory molecule)의 발현과 높은 수준의 CCR7, TGF-beta1, IL-10, HO-1, IDO, PD-L1의 발현을 확인하였다. 이 수지상세포를 OT-2 마우스 유래 naive CD4 T cell과 공배양하였고, 그 결과 높은 수준의 조절 T 세포가 유도됨을 확인하였다. qRT-PCR 결과에서도 높은 수준의 Foxp3, IL-10, TGF-beta1의 발현, 낮은 수준의 IFN-gamma, TNF-alpha 발현을 확인하였다 (도 4a). To confirm that tolerant dendritic cells and regulatory T cells are induced by LNP-R/Aβ, LNP-R/OVA was created using OVA as an antigen. First, wild-type C57BL/6 mouse bone marrow-derived dendritic cells were treated and analyzed through flow cytometry and qRT-PCR, and low levels of MHC class II and co-stimulatory molecule expression, known as characteristics of immune-tolerant dendritic cells, were observed. and high levels of CCR7, TGF-beta1, IL-10, HO-1, IDO, and PD-L1 expression were confirmed. These dendritic cells were co-cultured with naïve CD4 T cells derived from OT-2 mice, and as a result, it was confirmed that high levels of regulatory T cells were induced. qRT-PCR results also confirmed high levels of Foxp3, IL-10, and TGF-beta1 expression, and low levels of IFN-gamma and TNF-alpha expression (Figure 4a).
항원만 봉입된 LNP-OVA와 라파마이신과 항원이 동시에 봉입된 LNP-R/OVA를 비교하고자 웨스턴 블랏을 실시하였고 LNP-R/OVA 그룹에서 더 높은 수준의 Foxp3, 더 낮은 수준의 T helper 1 세포의 마커인 T-bet이 발현되었다. 신경세포의 보호에 관여한다고 알려진 amphirgulin도 LNP-R/OVA에서 더 많이 발현함을 확인하였다. LNP가 아닌 라파마이신과 항원의 혼합물의 경우 LNP-R/OVA에 비해 더 낮은 수준의 조절 T 세포를 유도하였고 이를 통해 LNP의 필요성을 확인하였다(도 4b).Western blot was performed to compare LNP-OVA encapsulated with only the antigen and LNP-R/OVA encapsulated with rapamycin and antigen simultaneously. Higher levels of Foxp3 and lower levels of T helper 1 cells were observed in the LNP-R/OVA group. The marker T-bet was expressed. Amphirgulin, known to be involved in the protection of nerve cells, was also confirmed to be expressed more in LNP-R/OVA. In the case of a mixture of rapamycin and antigen rather than LNP, a lower level of regulatory T cells was induced compared to LNP-R/OVA, confirming the necessity of LNP (Figure 4b).
야생형 C57BL/6 마우스에 1 주일 간격으로 2 회 피내주사하고 5 일 후 비장과 림프절을 수확하여 조절 T 세포와 T helper 1 세포의 비율을 정량하였을 때 두 장기 모두에서 LNP-R/OVA는 높은 수준의 조절 T 세포을 유도하면서 T helper 1 세포는 증가시키지 않음을 확인하였다(도 4c). 상기 실시예 2에서의 5XFAD 알츠하이머병 마우스 모델에서도 뇌 조직 내에서 LNP-R/Aβ는 가장 높은 수준의 조절 T 세포를 유도함을 확인하였고(도 4c), 비장에서도 비슷한 경향성을 확인하였다 (도 4d).When wild-type C57BL/6 mice were injected intradermally twice at 1-week intervals and the spleen and lymph nodes were harvested 5 days later to quantify the ratio of regulatory T cells and T helper 1 cells, LNP-R/OVA was found to be at high levels in both organs. It was confirmed that while inducing regulatory T cells, T helper 1 cells were not increased (Figure 4c). It was confirmed that LNP-R/Aβ induced the highest level of regulatory T cells in brain tissue in the 5 .
실시예 4. LNP-R/Aβ에 의해 유도된 Aβ 특이적 조절 T세포의 Aβ 타겟 효율성 확인.Example 4. Confirmation of Aβ targeting efficiency of Aβ-specific regulatory T cells induced by LNP-R/Aβ.
상기 실시예 3에서와 마찬가지로 야생형 C57BL/6 마우스에 1 주일 간격으로 2 회 피내주사하고 5 일 후 비장을 수확하여 추가로 3 일 간 OVA 항원에 의해 재자극을 실시하였다. 그 후 qRT-PCR, FACS, ELISA를 통해 분석하였고 이를 통해 항원 특이적 조절 T 세포가 유도됨을 확인하였다. 특히 OVA-specific TCR을 인식하는 tetramer를 이용한 tetramer staining에서 항원 특이적 조절 T 세포가 다른 그룹에 비해 현저히 높음을 확인하였다 (도 5a).As in Example 3, wild-type C57BL/6 mice were injected intradermally twice at one-week intervals, and 5 days later, the spleens were harvested and restimulated with OVA antigen for an additional 3 days. Afterwards, it was analyzed through qRT-PCR, FACS, and ELISA, and it was confirmed that antigen-specific regulatory T cells were induced. In particular, tetramer staining using a tetramer that recognizes OVA-specific TCR confirmed that the number of antigen-specific regulatory T cells was significantly higher than that of other groups (Figure 5a).
체내에서 LNP-R/Aβ에 의해 유도된 Aβ 특이적인 조절 T세포가 비특이적인 조절 T세포에 비해 효율적으로 뇌 내의 Aβ를 타겟하고 Aβ 플라크의 집적을 억제하는 것을 확인하기 위하여 adoptive Treg cell transfer를 진행하였다. 이는 LNP-R/Aβ의 주사에 의해 형성되는 항 Aβ 항체의 효과를 제외한 조절 T세포에 의한 효과만을 보기 위한 시험이다. 6 주령의 야생형 마우스 모델에 LNP와 LNP-R/Aβ를 1 주일 간격으로 2 회 투여 후, 1 주일 후 두 그룹에서 동일한 세포수의 조절 T세포를 분리하여 각각 5 개월령 5XFAD 마우스에 정맥 주사하고, 2 주 뒤에 그 효과를 면역조직염색법으로 확인하였다(도 5b). 이 때 transfer한 조절 T세포에 CFSE dye로 염색하여 뇌 내에서 transfer된 조절 T세포가 존재하는지 형광현미경을 통해 관측하였다. 또한 Aβ 플라크의 변화를 확인하기 위해 항 Aβ 항체를 통하여 면역조직염색하였다. 그 결과, LNP-R/Aβ에 의해 유도된 조절 T세포를 투여한 마우스의 뇌에서 Aβ 플라크가 대조군에 비해 유의하게 감소한 것을 확인하였으며, 대조군과 달리 Aβ 플라크 주변에서 transfer한 조절 T세포를 발견하였다. 따라서, LNP-R/Aβ에 의해 Aβ 특이적으로 작용하는 조절 T세포가 유도되고, Aβ 특이적이지 않은 조절 T세포에 비해 더욱 효율적인 Aβ 타겟팅 및 제거 작용을 보임을 확인하였다(도 5b). 또한 뇌 조직 내에 조절 T세포가 실험군에서 월등히 높음을 확인하였고, 염증성 미세아교세포(M1 microglia)는 감소하고, 항염증성 미세아교세포(M2 microglia)는 증가함을 확인하였다(도 5c). 이를 통해 LNP-R/Aβ가 유도한 항원 특이적 조절 T세포는 M1 미세아교세포를 M2 미세아교세포로 전환시키고 이 M2 미세아교세포로 인해 아밀로이드 베타 플라크가 감소함을 확인하였다.Adoptive Treg cell transfer was performed to confirm that Aβ-specific regulatory T cells induced by LNP-R/Aβ in the body target Aβ in the brain more efficiently than non-specific regulatory T cells and suppress the accumulation of Aβ plaques. did. This is a test to see only the effect of regulatory T cells, excluding the effect of anti-Aβ antibodies formed by injection of LNP-R/Aβ. LNP and LNP-R/Aβ were administered twice at 1-week intervals to a 6-week-old wild-type mouse model. After 1 week, the same number of regulatory T cells were isolated from both groups and intravenously injected into 5-month-old 5XFAD mice, respectively. Two weeks later, the effect was confirmed by immunohistostaining (Figure 5b). At this time, the transferred regulatory T cells were stained with CFSE dye and the presence of transferred regulatory T cells in the brain was observed using a fluorescence microscope. Additionally, to confirm changes in Aβ plaques, immunohistostaining was performed using an anti-Aβ antibody. As a result, it was confirmed that Aβ plaques were significantly reduced in the brains of mice administered regulatory T cells induced by LNP-R/Aβ compared to the control group. Unlike the control group, regulatory T cells transferred around the Aβ plaques were found. . Therefore, it was confirmed that Aβ-specific regulatory T cells were induced by LNP-R/Aβ and showed more efficient Aβ targeting and removal compared to non-Aβ-specific regulatory T cells (Figure 5b). In addition, it was confirmed that the number of regulatory T cells in brain tissue was significantly higher in the experimental group, and that inflammatory microglia (M1 microglia) decreased and anti-inflammatory microglia (M2 microglia) increased (Figure 5c). Through this, it was confirmed that antigen-specific regulatory T cells induced by LNP-R/Aβ convert M1 microglial cells into M2 microglial cells and that amyloid beta plaques are reduced due to these M2 microglial cells.
실시예 5. LNP-R/Aβ가 뇌에서 신경염증 억제에 미치는 치료 효과 확인.Example 5. Confirmation of the therapeutic effect of LNP-R/Aβ on suppressing neuroinflammation in the brain.
LNP-R/Aβ가 뇌에서 신경염증을 완화할 수 있음을 확인하기 위하여, OT-2 마우스 유래 비장세포(splenocyte)에 LNP-R/OVA를 처리하고, 4 일간 배양하여 조절 T 세포를 유도하였다. 그 후, 각각 미세아교세포를 모사하는 LPS-treated Raw264.7 cell 혹은 교세포와 공배양하여 분석하였다. Raw264.7 세포의 qRT-PCR와 ELISA 분석 결과, M1 미세아교세포의 마커인 iNOS나 염증성 사이토카인 (TNF-alpha)는 LNP-R/OVA에 의해 감소하였고, M2 미세아교세포의 마커인 Arg-1나 항염증성 사이토카인 (IL-10)는 증가하였다. 이를 통해 M2 미세아교세포로의 polarization이 LNP-R/OVA에 의해 유도됨을 확인하였다(도 6a). 교세포와의 공배양에서도 LNP-R/OVA는 교세포의 성장인자 발현을 증가시켰고, 특히 LNP-OVA에 비해 LNP-R/OVA는 반응성 있는 성상세포(reactive asctrocyte)가 발현하는 것으로 알려진 사이토카인의 분비를 감소할 수 있음을 확인하였다. 또한 LNP-R/OVA가 유도하는 조절 T 세포는 그 자체로도 신경을 보호할 수 있음을 확인하였다(도 6b).To confirm that LNP-R/Aβ can alleviate neuroinflammation in the brain, splenocytes derived from OT-2 mice were treated with LNP-R/OVA and cultured for 4 days to induce regulatory T cells. . Afterwards, the cells were analyzed by co-culturing them with LPS-treated Raw264.7 cells or glial cells, respectively, which mimic microglial cells. As a result of qRT-PCR and ELISA analysis of Raw264.7 cells, iNOS and inflammatory cytokine (TNF-alpha), markers of M1 microglial cells, were decreased by LNP-R/OVA, and Arg-, a marker of M2 microglial cells, were decreased by LNP-R/OVA. 1I anti-inflammatory cytokine (IL-10) increased. Through this, it was confirmed that polarization into M2 microglial cells was induced by LNP-R/OVA (Figure 6a). Even in co-culture with glial cells, LNP-R/OVA increased the expression of growth factors in glial cells, and in particular, compared to LNP-OVA, LNP-R/OVA secreted cytokines known to be expressed by reactive astrocytes. It was confirmed that can be reduced. In addition, it was confirmed that regulatory T cells induced by LNP-R/OVA can protect nerves by themselves (Figure 6b).
상기 실시예의 5XFAD 마우스 실험에서도 유사하게 뇌 내에 M1 미세아교세포는 감소하고 M2 미세아교세포는 증가하는 경향성을 보였다(도 6b). 또한 LNP-R/Aβ는 기존 백신과 유사한 LNP-Aβ에 비해 염증성 사이토카인은 크게 증가하지 않은 반면, 항염증성 사이토카인은 증가하는 경향성을 보였다(도 6c). 특히 기존 백신의 부작용의 원인으로 지목된 T helper 1 세포를 뇌 및 비장에서 분석한 결과, 동일하게 항원을 주사해주었음에도 불구하고 본 실험군에서 LNP-Aβ에 비해 낮은 Th1 세포가 관찰되었다 (도 6c, 6d).Similarly, in the 5XFAD mouse experiment of the above example, M1 microglial cells tended to decrease and M2 microglial cells increased in the brain (FIG. 6b). In addition, LNP-R/Aβ did not significantly increase inflammatory cytokines compared to LNP-Aβ, which was similar to the existing vaccine, while anti-inflammatory cytokines tended to increase (Figure 6c). In particular, as a result of analyzing T helper 1 cells in the brain and spleen, which were identified as the cause of side effects of existing vaccines, a lower number of Th1 cells compared to LNP-Aβ was observed in this experimental group even though the same antigen was injected (Figure 6c, 6d).
알츠하이머병의 주요 병변 중 하나인 만성적인 신경염증에 LNP-R/Aβ이 미치는 영향을 확인하기 위하여, 상기 실시예 2의 5XFAD 마우스 뇌 내에서 염증반응의 주요 바이오마커인 활성화된 미세아교세포와 교세포(각 Iba1, GFAP)를 면역조직염색을 통하여 관찰하였다. 뇌 내에 집적된 Aβ의 양과 밀접한 상관관계를 보이는 미세아교세포는 LNP-Aβ와 LNP-R/Aβ 투여군에서 모두 상당히 감소하였으며, 교세포의 활성화와 같은 경우에는 LNP-Aβ 투여군에 비해 LNP-R/Aβ 투여군에서 현저히 감소하였다. 이를 통해 알츠하이머병 마우스 모델의 뇌 내에서 LNP-R/Aβ 투여군이 LNP-Aβ 투여군과 같이 항체 형성을 통해 뇌 내의 Aβ 집적을 줄여 만성적인 미세아교세포의 활성을 억제하며, 추가로 LNP-R/Aβ 투여군에서 Aβ 특이적인 조절 T세포를 유도하여 교세포의 활성화를 감소시키는 것을 확인하였다 (도 3f, 6d). 또한, LNP-R/Aβ는 5XFAD 마우스의 피질에서 시냅스의 기능 또한 회복시키는 것을 확인하였다 (도 6d).In order to confirm the effect of LNP-R/Aβ on chronic neuroinflammation, one of the major lesions of Alzheimer's disease, activated microglia and glial cells, which are major biomarkers of inflammatory response, were tested in the 5XFAD mouse brain of Example 2. (Iba1, GFAP) were observed through immunohistostaining. Microglial cells, which show a close correlation with the amount of Aβ accumulated in the brain, were significantly reduced in both the LNP-Aβ and LNP-R/Aβ administration groups, and in the case of glial cell activation, LNP-R/Aβ compared to the LNP-Aβ administration group. It decreased significantly in the administration group. Through this, in the brain of an Alzheimer's disease mouse model, the LNP-R/Aβ administration group, like the LNP-Aβ administration group, reduces Aβ accumulation in the brain through antibody formation, suppressing chronic microglial activity, and further LNP-R/Aβ administration group. In the Aβ administration group, it was confirmed that activation of glial cells was reduced by inducing Aβ-specific regulatory T cells (Figures 3f, 6d). In addition, it was confirmed that LNP-R/Aβ also restored synaptic function in the cortex of 5XFAD mice (Figure 6d).
실시예 6. LNP-R/Aβ가 알츠하이머병 마우스 모델의 인지능력 향상에 미치는 영향 확인.Example 6. Confirmation of the effect of LNP-R/Aβ on improving cognitive ability in an Alzheimer's disease mouse model.
공간 학습 및 기억 능력을 평가하는 모리스 수중 미로 실험을 통해 LNP-R/Aβ가 알츠하이머병 마우스 모델에 미치는 영향을 평가하였다. 5XFAD 마우스 모델의 모리스 수중 미로에서 뚜렷한 인지 기능 저하가 나오는 연령대에서 실험하기 위하여 7 개월령 5XFAD 마우스 모델에 1 주일 간격으로 5 회 LNP-R/Aβ와 대조군들을 피내주사하였다. 주사 후 2 주일 후부터 5 일의 학습 기간을 가진 후 6 일째에 학습 및 기억 능력을 평가하였다. 5 일의 학습기간 동안 쥐는 네 개의 사분면을 큐(Que)를 이용하여 표시한 직경 1 m, 수심 60 cm의 수조에서 보이지 않는 플랫폼을 찾을 수 있도록 훈련하였다. 물에는 불투명한 흰색 페인트를 풀어 플랫폼이 보이지 않도록 하였으며, 물의 온도는 21~22 ℃로 유지하였다. 마우스들은 하루에 각 사분면에서 출발하는 네 번의 시행을 5일의 학습기간 동안 반복하였으며, 각 시행에서 60 초 동안 자유롭게 수영하여 숨겨진 플랫폼을 찾도록 하였다. 플랫폼을 제한 시간 내에 찾지 못했을 경우 플랫폼 위로 실험자가 유도 후 15 초 동안 주변 큐를 통해 공간을 학습시켰다. 만일 제한 시간 내에 플랫폼을 찾은 경우 6 초 이상 플랫폼에 머무른 경우 마우스를 꺼내주고, 플랫폼 도달 시간을 측정하였다. 마지막 평가일에는 플랫폼을 없앤 후 하나의 사분면에서 출발하여 60 초간 자유롭게 수영한 후 꺼내주었으며, 플랫폼의 위치(타겟)에 도달하는데 걸린 시간, 타겟에 도달할 때까지 수영한 거리와 수영 궤적을 기록하였다. 5 일간의 학습기간 동안 LNP-R/Aβ 투여군의 마우스들이 LNP 투여군에 비해 플랫폼 도달 시간이 유의하게 빨라졌으며, 평가일에 더 빠르고 짧은 거리 내에 플랫폼의 위치를 찾아갔으며 야생형 대조군과 비슷한 수준의 수행능력을 보여주었다. 따라서 LNP-R/Aβ 투여에 의해 마우스들의 공간 학습 및 기억 능력이 야생형 수준으로 회복된 것을 확인하였다(도 7).The effect of LNP-R/Aβ on an Alzheimer's disease mouse model was evaluated through a Morris water maze experiment that evaluates spatial learning and memory abilities. In order to test the 5XFAD mouse model in an age group in which significant cognitive decline occurs in the Morris water maze, LNP-R/Aβ and the control group were intradermally injected into a 7-month-old 5XFAD mouse model 5 times at weekly intervals. Learning and memory abilities were assessed starting 2 weeks after injection and on day 6 following a 5-day learning period. During a 5-day learning period, rats were trained to find an invisible platform in a water tank with a diameter of 1 m and a water depth of 60 cm, with four quadrants marked using cues. Opaque white paint was added to the water to make the platform invisible, and the temperature of the water was maintained at 21-22°C. Mice repeated four trials starting from each quadrant per day over a learning period of 5 days, and were allowed to swim freely for 60 seconds in each trial to find the hidden platform. If the platform was not found within the time limit, the space was learned through peripheral cues for 15 seconds after the experimenter guided the participant onto the platform. If the mouse found the platform within the time limit and stayed on the platform for more than 6 seconds, the mouse was taken out and the time to reach the platform was measured. On the last evaluation day, the platform was removed and the animals were taken out after swimming freely for 60 seconds starting from one quadrant. The time taken to reach the position of the platform (target), the distance swam until reaching the target, and the swimming trajectory were recorded. . During the 5-day learning period, mice in the LNP-R/Aβ administration group reached the platform significantly faster than those in the LNP administration group, found the platform location faster and within a shorter distance on the evaluation day, and performed at a level similar to that of the wild-type control group. showed. Therefore, it was confirmed that the spatial learning and memory abilities of mice were restored to wild-type levels by administration of LNP-R/Aβ (Figure 7).

Claims (7)

  1. 아밀로이드 베타 펩타이드 또는 이를 코딩하는 핵산; 및 라파마이신 또는 그 유도체;를 포함하는 알츠하이머병 예방 또는 치료용 약학 조성물.amyloid beta peptide or nucleic acid encoding it; and rapamycin or a derivative thereof; a pharmaceutical composition for preventing or treating Alzheimer's disease.
  2. 청구항 1에 있어서, 상기 아밀로이드 베타 펩타이드는 서열번호 1의 아미노산 서열의 적어도 일부를 포함하는 서열로 이루어진 것인 알츠하이머병 예방 또는 치료용 약학 조성물.The pharmaceutical composition for preventing or treating Alzheimer's disease according to claim 1, wherein the amyloid beta peptide consists of a sequence including at least part of the amino acid sequence of SEQ ID NO: 1.
  3. 청구항 1에 있어서, 상기 아밀로이드 베타 펩타이드는 서열번호 2 내지 서열번호 8의 아미노산 서열로 이루어진 군에서 선택되는 서열로 이루어진 것인 알츠하이머병 예방 또는 치료용 약학 조성물.The pharmaceutical composition for preventing or treating Alzheimer's disease according to claim 1, wherein the amyloid beta peptide consists of a sequence selected from the group consisting of amino acid sequences of SEQ ID NO: 2 to SEQ ID NO: 8.
  4. 청구항 1에 있어서, 상기 아밀로이드 베타 펩타이드 또는 이를 코딩하는 핵산; 및 라파마이신 또는 그 유도체;는 전달체에 담지된 것인 알츠하이머병 예방 또는 치료용 약학 조성물.The method according to claim 1, wherein the amyloid beta peptide or a nucleic acid encoding the same; and rapamycin or a derivative thereof; a pharmaceutical composition for preventing or treating Alzheimer's disease carried in a carrier.
  5. 청구항 4에 있어서, 상기 전달체는 바이러스 전달체, 바이러스 유사 입자(virus-like particle, VLP), 양전하성 폴리머, 리포좀, 지질나노입자(lipid nanoparticle), 금 또는 반도체 나노결정 입자(quantum dot)를 포함하는 군에서 선택되는 것인 알츠하이머병 예방 또는 치료용 약학 조성물.The method of claim 4, wherein the carrier includes a viral carrier, a virus-like particle (VLP), a positively charged polymer, a liposome, a lipid nanoparticle, gold, or a semiconductor nanocrystal particle (quantum dot). A pharmaceutical composition for preventing or treating Alzheimer's disease, which is selected from the group.
  6. 청구항 4에 있어서, 상기 전달체는 1,2-dioleoyl-3-trimethylammonium-propane(DOTAP), dioleoylphosphatidylethanolamine(DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](PEG2000 PE) 및 콜레스테롤을 포함하는 지질나노입자인 알츠하이머병 예방 또는 치료용 약학 조성물.The method of claim 4, wherein the carrier is 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioleoylphosphatidylethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol) -2000] (PEG2000 PE) and a pharmaceutical composition for preventing or treating Alzheimer's disease, which are lipid nanoparticles containing cholesterol.
  7. 청구항 4에 있어서, 상기 전달체의 직경은 50 nm 내지 600 nm인 알츠하이머병 예방 또는 치료용 약학 조성물.The pharmaceutical composition for preventing or treating Alzheimer's disease according to claim 4, wherein the diameter of the carrier is 50 nm to 600 nm.
PCT/KR2022/016873 2022-09-30 2022-11-01 Pharmaceutical composition for prevention or treatment of alzheimer's disease WO2024071509A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2022-0125296 2022-09-30
KR1020220125296A KR102593753B1 (en) 2022-09-30 2022-09-30 Pharmaceutical composition for treating or prventing alzheimer

Publications (1)

Publication Number Publication Date
WO2024071509A1 true WO2024071509A1 (en) 2024-04-04

Family

ID=88508821

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2022/016873 WO2024071509A1 (en) 2022-09-30 2022-11-01 Pharmaceutical composition for prevention or treatment of alzheimer's disease

Country Status (2)

Country Link
KR (1) KR102593753B1 (en)
WO (1) WO2024071509A1 (en)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004014367A2 (en) * 2002-08-12 2004-02-19 Pharmacia Corporation Use of an amyloid beta vaccination in combination with a selective cox-2 inhibitor for the treatment of alzheimer's disease

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102317080B1 (en) 2018-05-09 2021-10-25 한국과학기술연구원 Composition for preventing or treating tau related disease

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004014367A2 (en) * 2002-08-12 2004-02-19 Pharmacia Corporation Use of an amyloid beta vaccination in combination with a selective cox-2 inhibitor for the treatment of alzheimer's disease

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ARTI TYAGI: "Integrated Pathways of COX-2 and mTOR: Roles in Cell Sensing and Alzheimer’s Disease", FRONTIERS IN NEUROSCIENCE, FRONTIERS RESEARCH FOUNDATION, CH, vol. 14, 9 July 2020 (2020-07-09), CH , XP093153990, ISSN: 1662-453X, DOI: 10.3389/fnins.2020.00693 *
DATABASE Protein 18 February 1994 (1994-02-18), ANONYMOUS: "beta-amyloid-(1-42), A beta-(1-42)=amyloid major component {N-terminal} [human, leptomeningeal blood vessels, Peptide Partial, 19 aa]", XP093153996, Database accession no. AAB29001.1 *
DATABASE Protein 5 August 2021 (2021-08-05), ANONYMOUS: "Amyloid-beta A4 protein [Galemys pyrenaicus]", XP093153994, Database accession no. KAG8518514.1 *
SUNG PIL KWON: "Nanoparticle‐Mediated Blocking of Excessive Inflammation for Prevention of Heart Failure Following Myocardial Infarction", SMALL, WILEY, HOBOKEN, USA, vol. 17, no. 32, 1 August 2021 (2021-08-01), Hoboken, USA, XP093153998, ISSN: 1613-6810, DOI: 10.1002/smll.202101207 *

Also Published As

Publication number Publication date
KR102593753B1 (en) 2023-10-26

Similar Documents

Publication Publication Date Title
US11660329B2 (en) Materials and methods useful for treating glioblastoma
US7871765B2 (en) Composition having antitumor effect
CN110520438A (en) Oncolytic viral therapy
WO1999029343A1 (en) Method of treating bladder cancer with wild type vaccinia virus
WO2012165815A2 (en) Nano-vehicle derived from tumor tissue, and cancer vaccine using same
KR20190093141A (en) Nanovesicles from Adult Stem Cells and its use for targeted therapy
JP2015529685A (en) Compositions and methods for the treatment of amyotrophic lateral sclerosis
CN112755180B (en) Preparation method and application of universal bacterial vaccine
Qi et al. A (H1N1) vaccination recruits T lymphocytes to the choroid plexus for the promotion of hippocampal neurogenesis and working memory in pregnant mice
US20190307794A1 (en) Method for inducing transdifferentiation of immune cells based on exosomes
JP5412280B2 (en) Treatment of age-related macular degeneration
KR102162727B1 (en) Novel cell therapeutics composition by simultaneous administration of human cell-derived extracellular vesicles and cells
CN113750236A (en) Application of VEGFR inhibitor in preparation of anti-Alzheimer's disease drugs
US20240131119A1 (en) Use of pertussis toxin as a therapeutic agent
WO2024071509A1 (en) Pharmaceutical composition for prevention or treatment of alzheimer's disease
JP6474082B2 (en) Human monocyte subpopulations for the treatment of eye diseases and disorders
KR20220041397A (en) Composition for Preventing or Treating Dmentia Comprising Peptide Nucleic Acid Complex
Wang et al. Neuroprotective effect of vaccination with autoantigen-pulsed dendritic cells after spinal cord injury
WO2023008815A1 (en) Her2 vaccine composition
KR102122802B1 (en) Vaccine for treating and preventing tuberculosis comprising B cell loading α-galactosylceramide and ESAT6
Ha et al. Inhibitory Effects of the Attenuated Salmonella typhimurium Containing the IL‐2 Gene on Hepatic Tumors in Mice
WO2021031409A1 (en) Application of pseudomonas aeruginosa vaccine in respiratory disease
Alghonemy et al. Systemic immune response development in Albino rats after retrograde instillation of COVID-19 vaccine to submandibular salivary gland: An experimental study
US20220202955A1 (en) Composition and method for inhibiting tau protein accumulation, aggregation, and tangle formation
CN117500530A (en) Composition for inhibiting alpha-synuclein and aggregation inhibition method

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22961108

Country of ref document: EP

Kind code of ref document: A1