[go: up one dir, main page]

WO2023106845A1 - Nouvel analogue et conjugué d'adiponectine - Google Patents

Nouvel analogue et conjugué d'adiponectine Download PDF

Info

Publication number
WO2023106845A1
WO2023106845A1 PCT/KR2022/019864 KR2022019864W WO2023106845A1 WO 2023106845 A1 WO2023106845 A1 WO 2023106845A1 KR 2022019864 W KR2022019864 W KR 2022019864W WO 2023106845 A1 WO2023106845 A1 WO 2023106845A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
analog
adiponectin
amino acid
formula
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/KR2022/019864
Other languages
English (en)
Korean (ko)
Inventor
박준섭
김진영
이종석
최재혁
신민경
이종민
오의림
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hanmi Pharmaceutical Co Ltd
Hanmi Pharmaceutical Industries Co Ltd
Original Assignee
Hanmi Pharmaceutical Co Ltd
Hanmi Pharmaceutical Industries Co Ltd
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 Hanmi Pharmaceutical Co Ltd, Hanmi Pharmaceutical Industries Co Ltd filed Critical Hanmi Pharmaceutical Co Ltd
Publication of WO2023106845A1 publication Critical patent/WO2023106845A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • the present invention relates to novel adiponectin analogs, conjugates thereof, and uses.
  • Adipose tissue is an endocrine organ that is involved in regulating systemic metabolism by secreting various substances with various biological activities. These substances are called adipokines, and include adiponectin, leptin, interleukin-6, and tumor necrosis factor alpha (TNF ⁇ ). Since these adipokines regulate lipid and sugar metabolism, insulin sensitivity, etc., they are known to be closely related to the occurrence of metabolic diseases related to obesity.
  • adiponectin is an adipokine that exists in a very high concentration (2-20 ⁇ g/mL) in the blood and accounts for about 0.01% of total plasma protein.
  • Adiponectin is a peptide consisting of 244 amino acids consisting of four domains: a short signal sequence targeting hormones for extracellular secretion, a short domain that varies between species, a 65 amino acid domain similar to collagen protein, and a globular domain. , and exists as a multimer in plasma.
  • Adiponectin is a protein that is expressed in adipose tissue and circulates in the blood when body fat increases. It is involved in the catabolism of fat. In muscle, adiponectin activates AMP activated protein kinase (AMPK) to promote ⁇ -oxidation of fatty acids. It is known to inhibit fat synthesis in tissues. Reduction of fat increases adiponectin production, and obesity reduces the amount of adiponectin in the blood.
  • AMPK AMP activated protein kinase
  • adiponectin increases insulin sensitivity
  • administration of adiponectin is known to restore insulin resistance.
  • the expression of adiponectin receptors AdipoR1 and AdipoR2 is decreased in obesity-related insulin resistance state, and it is known that AdipoR1 and AdipoR2 mediate the antidiabetic metabolic activity of adiponectin.
  • adiponectin is involved in various diseases, it is expected to be used as a treatment for various diseases, in particular, as a treatment for obesity-related diseases.
  • adiponectin is also used as an indicator for diagnosing the degree of fibrosis, and is known to have an anti-fibrotic effect by activating AMP activated protein kinase (AMPK).
  • AMPK AMP activated protein kinase
  • Adiponectin which has effects such as improvement of insulin sensitivity and anti-fibrosis, is expected to be a useful target for treating obesity-related diseases, and various studies are being conducted. In particular, studies are being conducted to develop useful therapeutic agents by increasing the activity of adiponectin.
  • One object of the present invention is to provide an adiponectin analog.
  • Another object of the present invention is to provide a long-acting conjugate containing the analog.
  • Another object of the present invention is to provide an acylated adiponectin analog.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating liver fibrosis, comprising the adiponectin analog, a conjugate thereof, or an acylated adiponectin analog.
  • Another object of the present invention is to provide a method for preventing or treating liver fibrosis, comprising administering the adiponectin analog, the conjugate thereof, the acylated adiponectin analog, or a composition containing the same to a subject in need thereof. .
  • Another object of the present invention is to provide a preventive or therapeutic use of the adiponectin analog, the conjugate thereof, the acylated adiponectin analog, or a composition containing the same for preventing or treating liver fibrosis.
  • Another object of the present invention is to provide a use of the adiponectin analog, the conjugate thereof, the acylated adiponectin analog, or a composition containing the same to provide a medicament for preventing or treating liver fibrosis.
  • One aspect of the present invention is a novel adiponectin analog.
  • the adiponectin analog may include a sequence in which one or more amino acids are mutated from a natural analog or a known sequence of adiponectin.
  • the adiponectin analog is an adiponectin analog comprising the amino acid sequence of Formula 1 below:
  • Ra is represented by Xa1-Xa2-F-Xa3-Xa4-Y, wherein
  • Xa1 is aspartic acid (D), glutamic acid (E), phenylalanine (F), histidine (H), threonine (T), or tyrosine (Y);
  • Xa2 is tyrosine (Y), glutamic acid (E), glycine (G), or phenylalanine (F);
  • Xa3 is glycine (G) or absent;
  • Xa4 is alanine (A), lysine (K), leucine (L), valine (V), or norleucine ( NL );
  • Rb is represented by Xb1-Xb2-Xb3-Xb4-Xb5-Xb6, where
  • Xb1 is glutamic acid (E), phenylalanine (F), histidine (H), serine (S), or leucine (L);
  • Xb2 is serine (S), proline (P), glutamic acid (E), glycine (G), arginine (R), aspartic acid (D), or hydroxyproline (HyP);
  • Xb3 is glycine (G), asparagine (N), glutamic acid (E), glutamine (Q), arginine (R), lysine (K), tyrosine (Y), or absent;
  • Xb4 is serine (S), aspartic acid (D), proline (P), asparagine (N), or absent;
  • Xb5 is glutamine (Q), hydroxyproline (HyP), or absent;
  • Xb6 is glutamic acid (E) or absent;
  • Rc is represented by Xc1-Xc2, where
  • Xc1 is proline (P) or hydroxy proline (HyP);
  • Xc2 is phenylalanine (F), norleucine ( NL ), norvaline ( NV ), glycine (G), or leucine (L);
  • Rd is represented by Xd1-Xd2-Xd3-Xd4-Xd5, where
  • Xd1 is phenylalanine (F), arginine (R), or tyrosine (Y);
  • Xd2 is glutamic acid (E), glycine (G), or tyrosine (Y);
  • Xd3 is phenylalanine (F) or tyrosine (Y);
  • Xd4 is alanine (A), phenylalanine (F), or absent;
  • Xd5 is alanine (A), cysteine (C), glutamic acid (E), serine (S), tyrosine (Y), or absent.
  • Ra is any one of DYFAY, EYFAY, FEF NL Y, FGF NL Y, FYF NL Y, FYFAY, FYFG NL Y, FYFKY, FYFLY, FYFVY, HYFAY, TFFAY, YFF NL Y, YYF NL Y, and YYFAY;
  • Rb is any one of ESGSQ, FPE, HHyPE, HDN, HEN, HGN, HP, HPD, HPE, HPK, HPKN, HPQ, HPQSQ, HRNPQ, HRRDHyPE, LDE, and SDYSQ;
  • Rc is any one of HyPF, P NL , P NV , PF, PG, and PL;
  • Rd is any one of FEFA, FGFA, FGYFA, FYF, FYFA, FYFAC, FYFAE, FYFAS, FYFAY, RYFA, and YYF; It may include an amino acid sequence of Formula 1, which is.
  • Ra is FYF NL Y, FYFAY, FYFG NL Y, or YYF NL Y;
  • Rb is HHyPE, HP, HPE, or HPQ
  • Rc is HyPF, P NV , PF, or PG
  • Rd is FGYFA, FYFA, FYFAE, or FYFAY; It may include an amino acid sequence of Formula 1, which is.
  • the adiponectin analog may further include one or more amino acids at the C-terminus.
  • the adiponectin analog comprising the amino acid sequence of Formula 1 may additionally bind any one of the following amino acid sequences to the N-terminus.
  • H HP, HDG, HHH, HHP, HEDP, HEHH, HEHP, HEHYFT NL IFYDEEDHEHH, HESG, HSHH, HSHP, HYDG, HAibEG, or HyPTSGH;
  • the adiponectin analog comprising the amino acid sequence of Formula 1 may additionally bind any one of the following amino acid sequences to the N-terminus.
  • H HP, or HEHH, HEHYFT NL IFYDEEDHEHH;
  • GGGGSFT NL IFYDEEDHEHH GGGGSGGGGSGGGGSHEHH.
  • the adiponectin analog comprising the amino acid sequence of Formula 1 may additionally bind any one of the following amino acid sequences to the C-terminus.
  • the adiponectin analog may be amidated at the C-terminus.
  • adiponectin analogs are SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42 to 63, and SEQ ID NOs: 65 to 93, characterized in that it comprises any one amino acid sequence selected from the group consisting of.
  • adiponectin analog is SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 31, SEQ ID NO: 40, SEQ ID NO: 42 to 44, SEQ ID NO: 48 to 50, SEQ ID NO: 57 to 63, sequence It is characterized by comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 66, SEQ ID NO: 73 to 75, SEQ ID NO: 81 to 85, and SEQ ID NO: 91.
  • the adiponectin analog is characterized in that the C-terminus is amidated.
  • the adiponectin analog is characterized in that it further comprises one or more amino acids at the C-terminus.
  • Another aspect of the present invention is an adiponectin analog comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 93.
  • SEQ ID NO: 31 SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 66, SEQ ID NO: 74, and sequences It is an adiponectin analog comprising any one amino acid sequence selected from the group consisting of number 75.
  • the adiponectin analog comprises any one amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6 to 28, SEQ ID NO: 30, SEQ ID NO: 32 to 36, SEQ ID NO: 38, and SEQ ID NO: 41 It is characterized by including.
  • the adiponectin analog is characterized by comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 93.
  • the adiponectin analog is characterized in that it comprises an amino acid sequence of Formula 2 below.
  • X1 is phenylalanine (F) or tyrosine (Y);
  • X2 is alanine (A), lysine (K), leucine (L), valine (V), or norleucine ( NL );
  • X3 is histidine (H) or leucine (L);
  • X4 is proline (P) or aspartic acid (D);
  • X5 is glutamic acid (E), glutamine (Q), or absent;
  • X6 is aspartic acid (D) or absent
  • X7 is phenylalanine (F), norleucine ( NL ), norvaline ( NV ), glycine (G), or leucine (L);
  • X8 is alanine (A) or absent.
  • the adiponectin analog is characterized in that it comprises an amino acid sequence of Formula 3 below.
  • X9 is phenylalanine (F) or tyrosine (Y);
  • X10 is glutamic acid (E) or absent.
  • the adiponectin analog is characterized in that it comprises an amino acid sequence of Formula 4 below.
  • X11 is phenylalanine (F) or tyrosine (Y);
  • X12 is tyrosine (Y) or phenylalanine (F);
  • X13 is glycine (G) or absent
  • X14 is proline (P), arginine (R), or hydroxyproline (HyP);
  • X15 is glutamic acid (E) or arginine (R);
  • X16 is aspartic acid (D) or absent
  • X17 is hydroxyproline (HyP) or absent
  • X18 is glutamic acid (E) or absent
  • X19 is proline (P) or hydroxy proline (HyP);
  • X20 is glycine (G) or tyrosine (Y);
  • X21 is phenylalanine (F) or tyrosine (Y);
  • X22 is alanine (A) or phenylalanine (F);
  • X23 is alanine (A) or absent.
  • Another aspect of the present invention is a long-acting conjugate comprising an adiponectin analog represented by Formula 1 below:
  • X is an adiponectin analog
  • L is a linker containing an ethylene glycol repeating unit
  • a is 0 or a natural number, provided that when a is 2 or more, each L is independent of each other;
  • F is an immunoglobulin Fc region in the form of a dimer
  • the adiponectin analog is characterized in that it comprises an amino acid sequence of Formula 1 below:
  • Ra is represented by Xa1-Xa2-F-Xa3-Xa4-Y, wherein
  • Xa1 is aspartic acid (D), glutamic acid (E), phenylalanine (F), histidine (H), threonine (T), or tyrosine (Y);
  • Xa2 is tyrosine (Y), glutamic acid (E), glycine (G), or phenylalanine (F);
  • Xa3 is glycine (G) or absent;
  • Xa4 is alanine (A), lysine (K), leucine (L), valine (V), or norleucine ( NL );
  • Rb is represented by Xb1-Xb2-Xb3-Xb4-Xb5-Xb6, where
  • Xb1 is glutamic acid (E), phenylalanine (F), histidine (H), serine (S), or leucine (L);
  • Xb2 is serine (S), proline (P), glutamic acid (E), glycine (G), arginine (R), aspartic acid (D), or hydroxyproline (HyP);
  • Xb3 is glycine (G), asparagine (N), glutamic acid (E), glutamine (Q), arginine (R), lysine (K), tyrosine (Y), or absent;
  • Xb4 is serine (S), aspartic acid (D), proline (P), asparagine (N), or absent;
  • Xb5 is glutamine (Q), hydroxyproline (HyP), or absent;
  • Xb6 is glutamic acid (E) or absent;
  • Rc is represented by Xc1-Xc2, where
  • Xc1 is proline (P) or hydroxy proline (HyP);
  • Xc2 is phenylalanine (F), norleucine ( NL ), norvaline ( NV ), glycine (G), or leucine (L);
  • Rd is represented by Xd1-Xd2-Xd3-Xd4-Xd5, where
  • Xd1 is phenylalanine (F), arginine (R), or tyrosine (Y);
  • Xd2 is glutamic acid (E), glycine (G), or tyrosine (Y);
  • Xd3 is phenylalanine (F) or tyrosine (Y);
  • Xd4 is alanine (A), phenylalanine (F), or absent;
  • Xd5 is alanine (A), cysteine (C), glutamic acid (E), serine (S), tyrosine (Y), or absent.
  • the adiponectin analog is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 42 to 63, And it is characterized by comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 65 to 93.
  • the adiponectin analog is SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 31, SEQ ID NO: 40, SEQ ID NO: 42 to 44, SEQ ID NO: 48 to 50, SEQ ID NO: 57 to 63, SEQ ID NO: 66, SEQ ID NO: 66 It is characterized in that it comprises any one amino acid sequence selected from the group consisting of SEQ ID NOs: 73 to 75, SEQ ID NOs: 81 to 85, and SEQ ID NO: 91.
  • adiponectin analog is SEQ ID NO: 31, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 62 It is characterized by comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 66, SEQ ID NO: 74, and SEQ ID NO: 75.
  • the adiponectin analog is any one amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6 to 28, SEQ ID NO: 30, SEQ ID NO: 32 to 36, SEQ ID NO: 38, and SEQ ID NO: 41 It is characterized in that it includes.
  • the adiponectin analogs are SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6 to 30, SEQ ID NO: 32 to 39, SEQ ID NO: 41, SEQ ID NO: 45 to 47, SEQ ID NO: 51 to 56, SEQ ID NO: 65 , SEQ ID NOs: 67 to 72, SEQ ID NOs: 76 to 80, SEQ ID NOs: 86 to 90, SEQ ID NO: 92, and SEQ ID NO: 93 characterized in that it comprises any one amino acid sequence selected from the group consisting of.
  • the adiponectin analog is characterized by comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 93.
  • adiponectin analog further comprises one or more amino acids at the C-terminus.
  • immunoglobulin Fc region is derived from IgG, IgA, IgD, IgE, or IgM, a combination thereof, or a hybrid thereof.
  • the long-acting conjugate according to any one of the preceding embodiments characterized in that the immunoglobulin Fc region is an IgG4 Fc region.
  • the long-acting conjugate according to any one of the preceding embodiments, characterized in that the immunoglobulin Fc region is aglycosylated.
  • L contains an ethylene glycol repeating unit
  • the formula weight of the ethylene glycol repeating unit moiety is in the range of 1 to 100 kDa.
  • L is a polyethylene glycol linker, characterized in that a linker having a molecular weight of 2 kDa to 30 kDa.
  • a long-acting conjugate according to any one of the preceding embodiments wherein one end of L is an amine group or thiol group of F, and the other end of L is a covalent bond formed by reacting with an amine group or thiol group of X, respectively, to F and X characterized in that they are connected to each other.
  • Another aspect of the present invention is an acylated adiponectin analog.
  • the adiponectin analog is characterized in that it is acylated with a C 1 -C 30 straight-chain or branched-chain acyl group containing one or two carbolic acids.
  • the acyl group is a C 4 to C 30 fatty acid or dicarboxylic acid.
  • the adiponectin analog is acylated at an amino acid or lysine residue located at the N-terminus.
  • the acylated adiponectin analog is characterized in that it further comprises a linker.
  • the linker is characterized in that it contains an ethylene glycol repeating unit.
  • Another aspect of the present invention is the adiponectin analog; long-acting complex; or a pharmaceutical composition for preventing or treating liver fibrosis containing an acylated adiponectin analog.
  • the pharmaceutical composition comprises a pharmaceutically effective amount of an adiponectin analog; long-acting complex; or an acylated adiponectin analog and a pharmaceutically acceptable excipient.
  • composition according to any one of the preceding embodiments, wherein said pharmaceutical composition exhibits one or more of the following properties:
  • the pharmaceutical composition is intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, or It is characterized in that it is administered by the intrarectal route of administration.
  • Another aspect of the present invention is a method for preventing or treating liver fibrosis comprising administering the adiponectin analog, a conjugate thereof, an acylated adiponectin analog, or a composition containing the same to a subject in need thereof.
  • Another aspect of the present invention is the use of the adiponectin analog, a conjugate thereof, the acylated adiponectin analog, or a composition containing the same for preventing or treating liver fibrosis.
  • Another aspect of the present invention is the use of the adiponectin analog, the conjugate thereof, the acylated adiponectin analog, or a composition containing the same to provide a medicament for preventing or treating liver fibrosis.
  • the adiponectin analog according to the present invention has higher activity than the conventional adiponectin, while the acylated analog and the long-acting conjugate of the analog have excellent stability in the body and thus have an advantage as a therapeutic agent.
  • FIG. 1 is a diagram confirming the effect of improving liver fibrosis according to administration of an adiponectin analog of SEQ ID NO: 49 by H&E and Sirius red staining.
  • FIG. 2 is a diagram confirming the effect of improving liver fibrosis according to the administration of an adiponectin analog of SEQ ID NO: 49 through a decrease in the level of hydroxyproline.
  • FIG. 3 is a diagram confirming the effect of improving liver fibrosis according to administration of an adiponectin analog of SEQ ID NO: 49 through the reduction in expression levels of TGF ⁇ , Col1a1, and Col3a1 genes.
  • Aib may be used interchangeably with “2-aminoisobutyric acid” or “aminoisobutyric acid”, and 2-aminoisobutyric acid and aminoisobutyric acid Butyric acid (aminoisobutyric acid) may be used in combination.
  • the adiponectin analog means that one or more amino acids are mutated in the natural sequence, and in the present invention, a sequence modified by amino acid substitution, insertion, deletion, modification, or combination thereof in the natural sequence can mean In particular, insertion or substitution of hydrophobic amino acids to facilitate hydrophobic interactions and hydrogen bonds to enhance binding force with adiponectin receptors, substitution of sequences with amino acids known to form hydrogen bonds, or extension of sequences having hydrophobic properties and It may be prepared through insertion, partial sequence repetition of native adiponectin, extension of N-terminal and C-terminal sequences, insertion or substitution of negatively or positively charged amino acids, etc., but is not limited thereto as long as it exhibits excellent adiponectin activity. . Specifically, the adiponectin analogs of the present invention may be non-naturally occurring.
  • adiponectin is a type of adipokine, a substance secreted by adipose tissue, which is known to be closely related to the occurrence of metabolic diseases related to obesity by regulating lipid and sugar metabolism, insulin sensitivity, etc.
  • type 2 diabetes, insulin resistance, and obesity increase, the plasma adiponectin concentration is low, and its concentration is also reduced in hypertension, arteriosclerosis, and cardiovascular diseases, so it is expected to be a marker and therapeutic agent for these diseases.
  • Adiponectin is known to act on two types of receptors, AdipoR1 and AdipoR2.
  • the adiponectin of the present invention is human-derived adiponectin, and specifically, amino acids 149 to 166 of the active site. It may be a region including, and may include the amino acid sequence of SEQ ID NO: 1.
  • adiponectin analog means one or more amino acids mutated in the sequence of native adiponectin, and may specifically mean one or more amino acids mutated in the active site of native adiponectin.
  • the adiponectin analog of the present invention has increased binding ability to the AdipoR1 and/or AdipoR2 receptors than that of the native type, and has higher activity than known adiponectin, so it can be used as a treatment for various diseases. More specifically, the adiponectin analogs of the present invention can inhibit TGF ⁇ activity.
  • the adiponectin analog is a naturally-occurring adiponectin or an analog modified to have higher activity than BHD1028 (Encuragen), a known adiponectin analog.
  • BHD1028 Endagen
  • the adiponectin analog of the present invention has superior activity compared to BHD1028, a known adiponectin analog known to have higher activity than native adiponectin.
  • the adiponectin analog according to the present invention is about 100% or more, 200% or more, 300% or more, 400% or more, 500% or more, 700% or more, 1000% or more, 1500% or more, 2000% or more, compared to BHD1028. It may exhibit an activity of 2500% or more, 3000% or more, or 3500% or more.
  • the activity of such an adiponectin analog can be measured by a method known in the art, and is not limited to a specific method.
  • the activity of an adiponectin analog can be analyzed by measuring TGF ⁇ activity inhibited by the adiponectin analog.
  • BHD1028 is an adiponectin analog of Encuragen, and is designed to have a high affinity for AdipoR1, a receptor for adiponectin, and is being developed as a treatment for obesity. Specifically, it has the amino acid sequence of SEQ ID NO: 94.
  • the term "about” is a range including ⁇ 0.5, ⁇ 0.4, ⁇ 0.3, ⁇ 0.2, ⁇ 0.1, etc., and includes all numerical values in a range equivalent to or similar to the numerical value following the term about, but, therefore, Not limited.
  • the adiponectin analogs of the present invention have at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12 at the active site of the native sequence, particularly adiponectin. 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, or 20 or more amino acids may be substituted or added. The addition of the amino acid may be made at the N-terminus or C-terminus, or may be made inside the peptide.
  • amino acids when substituting or adding the above amino acids, not only 20 amino acids commonly observed in human proteins, but also atypical or non-naturally occurring amino acids, amino acid derivatives, and isomers thereof may be used.
  • Commercial sources of atypical amino acids include Sigma-Aldrich, ChemPep, Genzyme Pharmaceuticals, and others.
  • Peptides containing these amino acids and canonical peptide sequences can be synthesized and purchased through commercially available peptide synthesis companies, such as American Peptide Company or Bachem in the US or Anygen in Korea. Amino acid derivatives can be obtained in a similar manner.
  • amino acids to be substituted or inserted are desamino-histidine, beta-hydroxyimidazopropionic acid, 4-imidazoacetic acid, and beta-carboxy imidazopropionic acid.
  • Beta-carboxyimidazopropionic acid, Norleucine, Norvaline, Hydroxyproline, 2-aminoisobutyric acid (Aib), and the like can be used.
  • the adiponectin analog of the present invention may have some sequences deleted from the natural sequence. Specifically, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or more amino acid residues may be deleted, but are not limited thereto. In addition, 1, 2, 3, 4, 5 or more amino acid residues may be deleted from the C-terminus of the adiponectin active site including SEQ ID NO: 1, but is not limited thereto.
  • the adiponectin analog of the present invention may be one in which the terminal sequence is cleaved or another amino acid is substituted or inserted at the cleaved position, but is not limited thereto.
  • the cleaved position, N-terminus or C-terminus may be acetylated and/or amidated, but is not particularly limited thereto.
  • adiponectin analog of the present invention may be modified to have hydrogen instead of an amino group at the N-terminus, but is not limited thereto.
  • the adiponectin analog of the present invention may include 15 to 40 amino acid residues, 15 to 35 amino acid residues, or 15 to 30 amino acid residues, but is not limited thereto.
  • the adiponectin analogs are SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42 to 63, and SEQ ID NO: 65 to It may include, consist essentially of, or consist of any one amino acid sequence selected from the group consisting of 93, but is not limited thereto.
  • the adiponectin analogs are SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 31, SEQ ID NO: 40, SEQ ID NO: 42 to 44, SEQ ID NO: 48 to 50, SEQ ID NO: 57 to 63, SEQ ID NO: 66, SEQ ID NO: 73 to 75, SEQ ID NOs: 81 to 85, and SEQ ID NO: 91, may include, consist essentially of, or consist of any one amino acid sequence selected from the group consisting of, but is not limited thereto.
  • the adiponectin analog may include, consist essentially of, or consist of any one amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 44, and 49, but is not limited thereto.
  • the adiponectin analogs are SEQ ID NO: 31, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 66, SEQ ID NO: 74, and SEQ ID NO: 75 It may contain, consist essentially of, or consist of any one amino acid sequence selected from the group consisting of, but is not limited thereto.
  • the adiponectin analog comprises any one amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6 to 28, SEQ ID NO: 30, SEQ ID NO: 32 to 36, SEQ ID NO: 38, and SEQ ID NO: 41, It may be essentially configured or configured, but is not limited thereto.
  • the adiponectin analog may include, consist essentially of, or consist of any one amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 93, but is not limited thereto.
  • the adiponectin analogs of the present invention are 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83% of the amino acid sequences of SEQ ID NOs: 2 to 93. , 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or greater than 99% It may include amino acid sequences having homology or identity, but is not limited thereto as long as it acts on the adiponectin receptor and retains the activity of adiponectin.
  • the term 'homology' or 'identity' refers to the degree to which two given amino acid sequences or base sequences are related to each other and can be expressed as a percentage.
  • Sequence homology or identity of conserved polynucleotides or polypeptides can be determined by standard alignment algorithms, together with default gap penalties established by the program used. Substantially homologous or identical sequences are generally capable of hybridizing with all or part of the sequence under moderate or high stringent conditions. It is obvious that hybridization also includes hybridization with polynucleotides containing common codons or codons in consideration of codon degeneracy in polynucleotides.
  • GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215] : 403 (1990);Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO ETA/.] (1988) SIAM J Applied Math 48: 1073) Homology, similarity or identity can be determined using, for example, BLAST of the National Center for Biotechnology Information Database, or ClustalW.
  • GAP program defines the total number of symbols in the shorter of the two sequences divided by the number of similarly aligned symbols (i.e., nucleotides or amino acids).
  • the default parameters for the GAP program are (1) a binary comparison matrix (containing values of 1 for identity and 0 for non-identity) and Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp.
  • the preparation of the adiponectin analog of the present invention includes modification using L- or D-type amino acids and/or non-natural amino acids; and/or modification of the native sequence, e.g., modification of side chain functional groups, intramolecular covalent linkages such as inter-side chain ring formation, methylation, acylation, ubiquitination, phosphorylation, aminohexaylation, biotinylation, etc. everything to do is included
  • adiponectin analog of the present invention is chemically modified at its N-terminus and/or C-terminus, protected with an organic group, or amino acids at the end of a peptide in order to protect it from protein cleavage enzymes in vivo and increase stability. It may be added and modified form.
  • the N-terminus is acetylated and/or the C-terminus is amidated to remove these charges. It may be, but is not particularly limited thereto.
  • the N-terminus or C-terminus of the adiponectin analog of the present invention may have an amine group (-NH 2 ) or a carboxyl group (-COOH), but is not limited thereto.
  • it may have hydrogen instead of the amide group at the N-terminus, but is not limited thereto.
  • the adiponectin analog of the present invention may further include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 amino acid residues at the C-terminus. However, it is not limited thereto.
  • the adiponectin analog of the present invention can be synthesized by a method well known in the art, for example, an automatic peptide synthesizer, or can be produced by genetic engineering technology.
  • adiponectin analogs of the present invention can be prepared by standard synthetic methods, recombinant expression systems, or any other method in the art.
  • adiponectin analogs according to the present invention can be synthesized in a number of ways, including, for example, methods comprising:
  • a method of obtaining a peptide fragment by any combination of (a), (b) and (c), then linking the fragments to obtain a peptide, and recovering the peptide.
  • the adiponectin analog according to the present invention includes a peptide itself, a salt thereof (eg, a pharmaceutically acceptable salt of the peptide), or a solvate thereof.
  • the peptide may be in any pharmaceutically acceptable form.
  • the type of salt is not particularly limited. However, it is preferably in a form that is safe and effective for an individual, such as a mammal, but is not particularly limited thereto.
  • pharmaceutically acceptable refers to a substance that can be effectively used for a desired purpose without causing excessive toxicity, irritation, or allergic reaction within the scope of medical or pharmaceutical judgment.
  • the term "pharmaceutically acceptable salt” includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases.
  • suitable acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid.
  • Salts derived from suitable bases may include alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium.
  • solvate used in the present invention refers to a compound in which the adiponectin analog or salt thereof according to the present invention forms a complex with solvent molecules.
  • the adiponectin analog of the present invention may have an increased in vivo half-life compared to native adiponectin or conventional adiponectin (eg, BHD1028), but is not particularly limited thereto.
  • a biocompatible material eg, an immunoglobulin Fc region
  • a linker e.g., an immunoglobulin Fc region
  • the long-acting conjugate according to the present invention not only includes an adiponectin analog with increased binding ability to the adiponectin receptor, but also increases the half-life of the adiponectin analog by binding to the immunoglobulin Fc region as a representative carrier for increasing its half-life, and also increases the half-life of the adiponectin analog and blood exposure. can be used as a useful therapeutic agent.
  • Another aspect for implementing the present invention provides a long-acting conjugate of an adiponectin analog.
  • the adiponectin analog long-acting conjugate may be a form in which the adiponectin analog according to the present invention is conjugated with a biocompatible material for increasing its in vivo half-life.
  • biocompatible material may be used interchangeably with the term carrier.
  • the long-acting conjugate has increased efficacy compared to the adiponectin analog to which the carrier is not bound, and in the present invention, such conjugate is referred to as a "long-acting conjugate” or "conjugate".
  • conjugates may be non-naturally occurring.
  • the long-acting conjugate is a long-acting conjugate represented by Formula 1 below:
  • X is an adiponectin analog
  • L is a linker containing an ethylene glycol repeating unit
  • a is 0 or a natural number, provided that when a is 2 or more, each L is independent of each other;
  • F is an immunoglobulin Fc region in the form of a dimer
  • X and L, and L and F may be linked to each other through a covalent bond, and in this case, the conjugate may be a conjugate in which X, L, and F are linked through a covalent bond, respectively, in the order of Formula 1. .
  • F may be directly linked to X (ie, a is 0 in Formula 1) or linked through a linker (L).
  • the term "long-acting conjugate” or “conjugate” of the present invention has a structure in which the adiponectin analog and a biocompatible material are combined, and can exhibit increased durability compared to an adiponectin analog to which the biocompatible material is not bound. .
  • the biocompatible material may be covalently linked to an adiponectin analog, but is not particularly limited thereto.
  • the adiponectin analog may correspond to one moiety constituting the conjugate. Specifically, it corresponds to X in Formula 1, and the adiponectin analog is as described above.
  • the adiponectin analog may be the adiponectin analog described above, specifically, a peptide or fragment comprising any one of the amino acid sequences of Formula 1 or SEQ ID NOs: 2 to 93, and the biocompatible material may increase the half-life of the adiponectin analog.
  • the biocompatible material may increase the half-life of the adiponectin analog.
  • a material that can be used it corresponds to one component (F) of the moiety constituting the conjugate of the present invention.
  • the adiponectin analog contained in the conjugate may be a peptide comprising or (essentially) consisting of the amino acid sequence of any one of Formula 1 or SEQ ID NOs: 2 to 93.
  • the adiponectin analog is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 42 to 63, and SEQ ID NO: 65 It may be a peptide comprising or (essentially) consisting of any one amino acid sequence selected from the group consisting of 93 to 93.
  • the adiponectin analogs are SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 31, SEQ ID NO: 40, SEQ ID NO: 42 to 44, SEQ ID NO: 48 to 50, SEQ ID NO: 57 to 63, SEQ ID NO: 66, SEQ ID NO: 73 to 75, SEQ ID NOs: 81 to 85, and SEQ ID NO: 91, and may be a peptide comprising or (essentially) consisting of any one amino acid sequence selected from the group consisting of.
  • the adiponectin analogs are SEQ ID NO: 31, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 66, SEQ ID NO: 74, and SEQ ID NO: 75 It may be a peptide comprising or (essentially) consisting of any one amino acid sequence selected from the group consisting of.
  • the adiponectin analog comprises any one amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6 to 28, SEQ ID NO: 30, SEQ ID NO: 32 to 36, SEQ ID NO: 38, and SEQ ID NO: 41, ( It may be a peptide consisting essentially of).
  • the adiponectin analog may be a peptide comprising or (essentially) consisting of any one amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 93.
  • the half-life may be increased without losing activity by binding to a biocompatible material, but is not limited thereto.
  • biocompatible material refers to a material that is bound to the peptide of the present invention, which is a physiologically active material, and can increase the durability of the effect of the physiologically active material compared to a physiologically active material that is not bound to a biocompatible material part or a carrier.
  • the biocompatible material may be covalently linked to a physiologically active material (eg, an adiponectin analog), but is not particularly limited thereto.
  • F is a substance capable of increasing the half-life of X, that is, an adiponectin analog, and corresponds to one component of the moiety constituting the conjugate of the present invention.
  • the F may be bonded to X and X through a covalent chemical bond, and F and X may be bonded to each other through L through a covalent chemical bond.
  • the F is an immunoglobulin Fc region
  • the immunoglobulin Fc region may be an IgG Fc region or a non-glycosylated IgG4 Fc region, but is not particularly limited thereto.
  • the biocompatible material may be an immunoglobulin Fc region, more specifically an IgG Fc region, but is not particularly limited thereto.
  • the F immunoglobulin Fc region
  • the F is a dimer composed of two polypeptide chains, and may have a structure in which one end of L is connected to only one polypeptide chain of the two polypeptide chains, but is not limited thereto.
  • the long-acting conjugate of the present invention may be one in which an adiponectin analog and an immunoglobulin Fc region are linked, but is not limited thereto.
  • One or more amino acid side chains within an adiponectin analog of the invention may be conjugated to such biocompatible materials to increase solubility and/or half-life in vivo and/or to increase bioavailability. Such modifications may also reduce clearance of adiponectin analogs.
  • the aforementioned biocompatible material may be water-soluble (amphiphilic or hydrophilic) and/or non-toxic and/or pharmaceutically acceptable.
  • the F may be directly linked to X (ie, a is 0 in Formula 1) or linked through a polyethylene glycol linker (L).
  • conjugates may be non-naturally occurring.
  • the adiponectin analog conjugate of the present invention has an activity of about 0.1% or more, 1% or more, 10% or more, 50% or more, 100% or more, 200% or more, 300% or more, 400% or more, 500% or more compared to BHD1028. It may be 700% or more, 1000% or more, 1500% or more, 2000% or more, 2500% or more, 3000% or more, or 3500% or more, but is not limited thereto.
  • the "immunoglobulin Fc region" which is one component of the long-acting conjugate, includes heavy chain constant region 2 (CH2) and/or heavy chain constant region 3 (CH3), excluding the heavy chain and light chain variable regions of immunoglobulin.
  • area that contains The immunoglobulin Fc region may be one constituent of the moiety of the conjugate of the present invention.
  • the Fc region refers to a natural sequence obtained from papain digestion of immunoglobulin as well as a derivative thereof, such as one or more amino acid residues in the natural sequence, which are transformed by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. It includes even the sequence that is different from the form.
  • the F is a structure in which two polypeptide chains are connected by a disulfide bond, and may be a structure in which only one chain of the two chains is connected through a nitrogen atom, but is not limited thereto.
  • the nitrogen atom may be connected to the epsilon amino atom or the N-terminal amino group of lysine through reductive amination.
  • the reductive amination reaction refers to a reaction in which an amine group or an amino group of a reactant reacts with an aldehyde (ie, a functional group capable of reductive amination) of another reactant to generate an amine, and then a reduction reaction forms an amine bond, It is an organic synthesis reaction widely known in the art.
  • the F may be connected through the nitrogen atom of the N-terminal proline, but is not limited thereto.
  • the immunoglobulin Fc region constitutes a moiety of the conjugate of Formula 1 of the present invention, and may specifically correspond to F in Formula 1 above.
  • Such an immunoglobulin Fc region may include a hinge portion in a heavy chain constant region, but is not limited thereto.
  • the immunoglobulin Fc region may include a specific hinge sequence at the N-terminus.
  • flankinge sequence refers to a region located in a heavy chain to form a dimer of an immunoglobulin Fc region through an inter disulfide bond.
  • the hinge sequence may be mutated to have only one cysteine residue by deleting a part of the hinge sequence having the following amino acid sequence, but is not limited thereto:
  • the hinge sequence may include only one cysteine residue by deletion of the 8th or 11th cysteine residue of the hinge sequence of SEQ ID NO: 97.
  • the hinge sequence of the present invention may consist of 3 to 12 amino acids including only one cysteine residue, but is not limited thereto.
  • the hinge sequence of the present invention may have the following sequence: Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 98), Glu-Ser- Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Pro (SEQ ID NO: 99), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 100), Glu- Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Pro (SEQ ID NO: 101), Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 102), Glu-Ser-Lys- Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 103), Glu-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 98
  • the hinge sequence may include an amino acid sequence of SEQ ID NO: 107 (Pro-Ser-Cys-Pro) or SEQ ID NO: 116 (Ser-Cys-Pro), but is not limited thereto.
  • the immunoglobulin Fc region of the present invention may be in the form of a dimer formed by two molecules of the immunoglobulin Fc chain due to the presence of a hinge sequence, and in the conjugate of Formula 1 of the present invention, one end of the linker is a dimer immunoglobulin It may be linked to one chain of the Fc region, but is not limited thereto.
  • N-terminus refers to the amino terminus of a protein or polypeptide, one, two, three, four, five, six, It may contain up to 7, 8, 9, or 10 or more amino acids.
  • the immunoglobulin Fc region of the present invention may include a hinge sequence at the N-terminus, but is not limited thereto.
  • the immunoglobulin Fc region of the present invention except for the heavy chain and light chain variable regions of the immunoglobulin, part or all of the heavy chain constant region 1 (CH1) and / or light chain constant region, as long as it has substantially equivalent or improved effects to the native type, 1 (CL1) may be an extended Fc region. In addition, it may be a region in which a part of a fairly long amino acid sequence corresponding to CH2 and/or CH3 is removed.
  • the immunoglobulin Fc region of the present invention includes 1) CH1 domain, CH2 domain, CH3 domain and CH4 domain, 2) CH1 domain and CH2 domain, 3) CH1 domain and CH3 domain, 4) CH2 domain and CH3 domain, 5) It may be a combination of one or two or more domains of the CH1 domain, CH2 domain, CH3 domain, and CH4 domain with an immunoglobulin hinge region (or part of the hinge region), 6) a dimer of each domain of the heavy chain constant region and the light chain constant region. .
  • an immunoglobulin hinge region or part of the hinge region
  • the immunoglobulin Fc region F is a dimer (dimer dimer) composed of two polypeptide chains, wherein the Fc region dimer F and X are ethylene glycol They are covalently linked through one linker L containing repeating units.
  • X is covalently linked through a linker L to only one polypeptide chain of the two polypeptide chains of the Fc region dimer F.
  • only X of one molecule is covalently linked via L to one polypeptide chain to which X is linked among the two polypeptide chains of the Fc region dimer F.
  • the F is a homodimer.
  • the immunoglobulin Fc region F is a dimer composed of two polypeptide chains, and one end of L may be linked to only one polypeptide chain of the two polypeptide chains, but is not limited thereto.
  • the long-acting conjugate of the present invention it is also possible for two X molecules to bind symmetrically to one Fc region in the form of a dimer.
  • the immunoglobulins Fc and X may be connected to each other by a non-peptide linker.
  • a non-peptide linker it is not limited to the examples described above.
  • the immunoglobulin Fc region of the present invention is transformed by deletion, insertion, non-conservative or conservative substitution, or a combination thereof of one or more amino acid residues in the natural sequence as well as its derivatives, substitutions, etc., in addition to the natural amino acid sequence. It is included including variants such as sequences that are different from the above.
  • the above derivatives, substituents and variants are premised on having the ability to bind to FcRn.
  • amino acid residues from 214 to 238, from 297 to 299, from 318 to 322, or from 327 to 331 known to be important for binding in the case of IgG Fc can be used as suitable sites for modification.
  • various types of derivatives are possible, such as removal of a site capable of forming a disulfide bond, removal of some amino acids at the N-terminus of native Fc, or addition of a methionine residue to the N-terminus of native Fc. do.
  • a complement binding site eg, a C1q binding site
  • ADCC antibody dependent cell mediated cytotoxicity
  • Amino acid exchanges in proteins and peptides that do not entirely alter the activity of the molecule are known in the art (H. Neurath, R.L. Hill, The Proteins, Academic Press, New York, 1979).
  • the most commonly occurring exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/ Exchange between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly.
  • it is modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, and amidation. may be modified.
  • the above-described Fc derivative may exhibit biological activity equivalent to that of the Fc region of the present invention and may have increased structural stability of the Fc region against heat and pH.
  • an Fc region may be obtained from a natural form isolated in vivo from an animal such as human, cow, goat, pig, mouse, rabbit, hamster, rat or guinea pig, or obtained from transformed animal cells or microorganisms. It may be recombinant or a derivative thereof.
  • the method of obtaining from the natural form may be a method of obtaining whole immunoglobulin by isolating it from a human or animal living body and then treating it with a proteolytic enzyme. When treated with papain, it is cleaved into Fab and Fc, and when treated with pepsin, it is cleaved into pF'c and F(ab)2. Fc or pF'c can be separated from this using size-exclusion chromatography or the like.
  • the human-derived Fc region is a recombinant immunoglobulin Fc region obtained from a microorganism.
  • the immunoglobulin Fc region may be a native type sugar chain, an increased sugar chain compared to the native type, a reduced sugar chain compared to the natural type, or a form in which the sugar chain has been removed.
  • Conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms may be used to increase or decrease immunoglobulin Fc sugar chains.
  • the immunoglobulin Fc region in which sugar chains are removed from Fc has significantly reduced binding ability to complement (c1q), and antibody-dependent cytotoxicity or complement-dependent cytotoxicity is reduced or eliminated, so that unnecessary immune responses are not induced in vivo.
  • a form more suitable for its original purpose as a drug carrier will be referred to as an immunoglobulin Fc region in which sugar chains are removed or non-glycosylated.
  • deglycosylation refers to an Fc region from which sugars are removed by an enzyme
  • aglycosylation refers to a prokaryotic animal, in a more specific embodiment, refers to an Fc region that is not glycosylated produced by Escherichia coli. .
  • the immunoglobulin Fc region may be of human origin or animal origin such as cow, goat, pig, mouse, rabbit, hamster, rat, guinea pig, etc., and in a more specific embodiment, it is of human origin.
  • the immunoglobulin Fc region may be an Fc region derived from IgG, IgA, IgD, IgE, or IgM, a combination thereof, or a hybrid thereof. In a more specific embodiment, it is derived from IgG or IgM, which is most abundant in human blood, and in a more specific embodiment, it is derived from IgG known to improve the half-life of ligand binding proteins. In a more specific embodiment, the immunoglobulin Fc region is an IgG4 Fc region, and in the most specific embodiment, the immunoglobulin Fc region is a non-glycosylated Fc region derived from human IgG4, but is not limited thereto.
  • the immunoglobulin Fc fragment is a region of human IgG4 Fc, in which two monomers are linked through a disulfide bond (inter-chain form) between cysteine, which is the 3rd amino acid of each monomer. It may be in the form of a homodimer, wherein each monomer of the homodimer independently has an internal disulfide bond between cysteines 35 and 95 and an internal disulfide bond between cysteines 141 and 199, that is, two internal Has/can have disulfide bonds (intra-chain form).
  • the number of amino acids of each monomer may consist of 221 amino acids, and amino acids forming a homodimer may consist of a total of 442 amino acids, but are not limited thereto.
  • two monomers having the amino acid sequence of SEQ ID NO: 96 (consisting of 221 amino acids) form a homodimer through a disulfide bond between cysteine, the 3rd amino acid of each monomer, and the homodimer
  • the monomers of may each independently form an internal disulfide bond between cysteines at positions 35 and 95 and an internal disulfide bond between cysteines at positions 141 and 199, but are not limited thereto.
  • F in Formula 1 may include a monomer having an amino acid sequence of SEQ ID NO: 96, and F may be a homodimer of a monomer having an amino acid sequence of SEQ ID NO: 96, but is not limited thereto.
  • the immunoglobulin Fc fragment may be a homodimer including the amino acid sequence of SEQ ID NO: 96 (consisting of 442 amino acids), but is not limited thereto.
  • “combination" related to the immunoglobulin Fc region means that a polypeptide encoding a single-chain immunoglobulin Fc region of the same origin binds to a single-chain polypeptide of a different origin when forming a dimer or multimer.
  • a dimer or a multimer can be prepared from two or more regions selected from the group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc region.
  • hybrid is a term meaning that sequences corresponding to immunoglobulin Fc regions of two or more different origins exist in a single-chain immunoglobulin constant region.
  • various types of hybrids are possible. That is, a hybrid of one to four domains from the group consisting of CH1, CH2, CH3, and CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc, and IgD Fc is possible, and may include a hinge.
  • IgG can also be divided into subclasses of IgG1, IgG2, IgG3 and IgG4, and combinations thereof or hybridization thereof are also possible in the present invention. Specifically, it is an IgG2 and IgG4 subclass, and most specifically, it is an Fc region of IgG4 that has almost no effector function such as complement dependent cytotoxicity (CDC).
  • CDC complement dependent cytotoxicity
  • the above-described long-acting conjugate may have increased efficacy compared to native adiponectin or an existing adiponectin analog (eg, BHD1028) or to an F-unmodified X, and such a conjugate is described above. Not only forms, but also forms encapsulated in biodegradable nanoparticles, etc. are included, but are not limited thereto.
  • L may be a non-peptide linker, for example, a linker containing an ethylene glycol repeating unit.
  • the "non-peptide linker” includes a biocompatible polymer in which two or more repeating units are bonded.
  • the repeating units are connected to each other through any covalent bond other than a peptide bond.
  • the non-peptide linker may be one constituent of the moiety of the conjugate of the present invention, and corresponds to L in Formula 1 above.
  • Non-peptide linkers that can be used in the present invention can be used without limitation as long as they are polymers resistant to in vivo proteolytic enzymes.
  • the non-peptide linker may be used in combination with a non-peptide polymer.
  • the non-peptidyl linker includes a reactive group at its end, and may form a conjugate through reaction with other components constituting the conjugate.
  • a non-peptide linker having reactive functional groups at both ends binds to X and F of Formula 1 through each reactive group to form a conjugate
  • the non-peptide linker or non-peptide polymer is a non-peptide polymer linker moiety) or a non-peptide linker linkage.
  • the non-peptide linker may be a linker containing an ethylene glycol repeating unit, for example, polyethylene glycol, and derivatives thereof already known in the art and readily available at the level of skill in the art. Derivatives that can be prepared are also included in the scope of the present invention.
  • a specific example of the non-peptide linker may be a polyethylene glycol linker, but is not limited thereto.
  • the repeating unit of the non-peptide linker may be an ethylene glycol repeating unit, and specifically, the non-peptide linker may include an ethylene glycol repeating unit and include a functional group used for preparing a conjugate at a terminal thereof.
  • the long-acting conjugate according to the present invention may be a form in which X and F are linked through the functional group of the linker, but is not limited thereto.
  • the non-peptide linker may include two or three or more functional groups, and each functional group may be the same or different from each other, but is not limited thereto.
  • the linker L including the ethylene glycol repeating unit may include a functional group used for preparing the conjugate before being formed as a conjugate at its terminal.
  • the long-acting conjugate according to the present invention may be a form in which X and F are linked through the functional group, but is not limited thereto.
  • the linker may be polyethylene glycol (PEG) represented by Formula 2 below, but is not limited thereto:
  • the PEG moiety may include not only a -(CH 2 CH 2 O)n- structure, but also an oxygen atom intervening between the linking element and the -(CH 2 CH 2 O)n-. It is not limited.
  • the conjugate comprises an adiponectin analog (X) comprising the amino acid sequence of any one of Formula 1 or SEQ ID NOs: 2 to 93 and the immunoglobulin Fc region (F) containing an ethylene glycol repeating unit. It may be a structure linked by a covalent bond through a linker, but is not limited thereto.
  • the polyethylene glycol is a term encompassing all types of ethylene glycol homopolymers, PEG copolymers, and monomethyl-substituted PEG polymers (mPEG), but is not particularly limited thereto.
  • the ethylene glycol repeating unit may be represented by, for example, [OCH 2 CH 2 ]n, and the n value is a natural number, and the average molecular weight of the [OCH 2 CH 2 ]n site in the conjugate, such as the number average It can be set to have a molecular weight of greater than 0 to about 100 kDa, but is not limited thereto.
  • the n value is a natural number
  • the average molecular weight of the [OCH 2 CH 2 ]n site in the conjugate for example, the number average molecular weight is about 1 to about 100 kDa, about 1 to about 80 kDa, about 1 to about 50 kDa, about 1 to about 30 kDa, about 1 to about 25 kDa, about 1 to about 20 kDa, about 1 to about 15 kDa, about 1 to about 13 kDa, about 1 to about 11 kDa, about 1 to about 10 kDa , about 1 to about 8 kDa, about 1 to about 5 kDa, about 1 to about 3.4 kDa, about 3 to about 30 kDa, about 3 to about 27 kDa, about 3 to about 25 kDa, about 3 to about 22 kDa, About 3 to about 20 kDa, about 3 to about 18 kDa, about 3 to about 16 kDa, about 3 to about
  • L may be a polyethylene glycol linker having a molecular weight of 2 kDa to 30 kDa, but is not limited thereto.
  • both ends of the linker may bind to a thiol group, an amino group, a hydroxyl group of an immunoglobulin Fc region, and a thiol group, an amino group, an azide group, or a hydroxyl group of an adiponectin analog, but are not limited thereto.
  • the linker has a reactive group capable of binding to an immunoglobulin Fc region and an adiponectin analog at both ends, specifically, a thiol group of cysteine of an immunoglobulin Fc region; an amino group located at the N-terminus, lysine, arginine, glutamine and/or histidine; and/or a thiol group of cysteine of an adiponectin analog bonded to a hydroxyl group located at the C-terminus; amino groups at the N-terminus, lysine, arginine, glutamine and/or histidine; the azide group of azidoricin; And / or may include a reactive group capable of bonding with a hydroxyl group, but is not limited thereto.
  • the reactive group of the linker may be at least one selected from the group consisting of an aldehyde group, a maleimide group, and a succinimide derivative, but is not limited thereto.
  • examples of the aldehyde group include a propion aldehyde group or a butyl aldehyde group, but are not limited thereto.
  • both ends of the non-peptide linker may bind to an amine or thiol group of F, for example, an amine or thiol group of an immunoglobulin Fc region and an amine or thiol group of X, respectively.
  • the non-peptide linker is a reactive group capable of bonding to F (eg, immunoglobulin Fc region) and X at both ends, specifically, X, or the N-terminus of F (eg, immunoglobulin Fc region). or a reactive group capable of bonding with an amine group located on lysine or a thiol group of cysteine, but is not limited thereto.
  • the reactive group of the non-peptidic linker which can bind to F, such as an immunoglobulin Fc region and X, may be selected from the group consisting of an aldehyde group, a maleimide group, and a succinimide derivative, but is not limited thereto.
  • examples of the aldehyde group include a propion aldehyde group or a butyl aldehyde group, but are not limited thereto.
  • succinimide derivatives include succinimidyl valerate, succinimidyl methylbutanoate, succinimidyl methylpropionate, succinimidyl butanoate, succinimidyl propionate, N-hydroxysuccini Mead, hydroxy succinimidyl, succinimidyl carboxymethyl or succinimidyl carbonate may be used, but is not limited thereto.
  • the non-peptide linker may be connected to X and F through such a reactive group and converted into a non-peptide linker linkage, but is not particularly limited thereto.
  • the end product produced by reductive amination by aldehyde linkage is much more stable than that linked by amide linkage.
  • the aldehyde reactive group reacts selectively at the N-terminus at low pH and can form a covalent bond with a lysine residue at high pH, for example, pH 9.0.
  • the reactive groups at both ends of the non-peptide linker may be the same or different from each other, for example, having an aldehyde group at both ends, or a maleimide group at one end and an aldehyde group at the other end, It may have a propionic aldehyde group or a butyl aldehyde group.
  • F specifically, an immunoglobulin Fc region and X can be coupled to each end of the non-peptide linker, it is not particularly limited thereto.
  • a maleimide group as a reactive group may be included at one end of the non-peptide linker, and an aldehyde group, propion aldehyde group, or butyl aldehyde group may be included at the other end.
  • the hydroxyl group can be activated with various reactive groups by a known chemical reaction, or polyethylene glycol having a commercially available modified reactive group can be used.
  • the long-acting conjugate of the invention can be prepared.
  • the non-peptidic linker may be linked to a cysteine residue of X, more specifically to a -SH group of cysteine, but is not limited thereto.
  • a reactive group of a non-peptide linker may be connected to the -SH group of the cysteine residue, and all of the above descriptions apply to the reactive group.
  • maleimide-PEG-aldehyde is used, the maleimide group is connected to the -SH group of X through a thioether bond, and the aldehyde group is subjected to a reductive amination reaction with F, specifically -NH 2 group of immunoglobulin Fc It can be connected through, but is not limited thereto, and this corresponds to one example.
  • the N-terminal amino group of the immunoglobulin Fc region is linked to an oxygen atom located at one end of PEG, a non-peptide linker, through a linker functional group having a structure of -CH 2 CH 2 CH 2 - , -PEG-O-CH 2 CH 2 CH 2 NH- can form a structure such as immunoglobulin Fc, and one end of PEG is linked to the sulfur atom located at the cysteine of the adiponectin analog through a thioether bond.
  • the above-mentioned thioether bond is may contain the structure of
  • the non-peptide polymer may be linked to a lysine residue of X, more specifically, to an amine group of lysine, but is not limited thereto.
  • the reactive group of the non-peptide linker may be linked to -NH 2 located at the N-terminus of the immunoglobulin Fc region, but this corresponds to one example.
  • the adiponectin analog may be connected to a linker having a reactive group through the C-terminus, but this corresponds to one example.
  • C-terminus refers to the carboxy terminal of an adiponectin analog, and refers to a position capable of binding to a linker for the purpose of the present invention.
  • it may include not only the most terminal amino acid residue at the C-terminus but also all of the amino acid residues around the C-terminus, and specifically include the first to twentieth amino acid residues from the terminal end. It may, but is not limited thereto.
  • Such a salt form may be, for example, a form using any pharmaceutically acceptable salt.
  • the type of salt is not particularly limited. However, it is preferably in a form that is safe and effective for an individual, such as a mammal, but is not particularly limited thereto.
  • the type of salt is not particularly limited. However, it is preferably in a form that is safe and effective for an individual, such as a mammal, but is not particularly limited thereto.
  • pharmaceutically acceptable refers to a substance that can be effectively used for a desired purpose without causing excessive toxicity, irritation, or allergic reaction within the scope of medical or pharmaceutical judgment.
  • the term "pharmaceutically acceptable salt” includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases.
  • suitable acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid.
  • Salts derived from suitable bases may include alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium.
  • solvate used in the present invention refers to a compound in which the adiponectin analog or salt thereof according to the present invention forms a complex with solvent molecules.
  • the long-acting conjugate of the present invention may be prepared by covalently linking the above-described adiponectin analog to a biocompatible material, but is not limited thereto.
  • the medium used for culturing the transformants must meet the requirements of host cell culture in an appropriate manner.
  • the carbon source that can be included in the medium for the growth of the host cell can be appropriately selected by the judgment of a person skilled in the art according to the type of the prepared transformant, and appropriate culture conditions can be adopted to control the culture time and amount. .
  • Sugar sources that can be used include sugars and carbohydrates such as glucose, saccharose, lactose, fructose, maltose, starch and cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, palmitic acid, stearic acid, These include fatty acids such as linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid. These materials may be used individually or as a mixture.
  • sugars and carbohydrates such as glucose, saccharose, lactose, fructose, maltose, starch and cellulose
  • oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, palmitic acid, stearic acid, These include fatty acids such as linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid. These materials may be used individually or as a mixture.
  • Nitrogen sources that can be used include peptone, yeast extract, broth, malt extract, corn steep liquor, soybean meal and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. Nitrogen sources may also be used individually or as a mixture.
  • the culture medium may contain metal salts such as magnesium sulfate or iron sulfate necessary for growth.
  • essential growth substances such as amino acids and vitamins may be used in addition to the above substances.
  • precursors suitable for the culture medium may be used.
  • the above-mentioned raw materials may be added in a batchwise or continuous manner during cultivation by a method suitable for the culture.
  • Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or acid compounds such as phosphoric acid or sulfuric acid can be used in an appropriate manner to adjust the pH of the culture.
  • antifoaming agents such as fatty acid polyglycol esters may be used to suppress foam formation. Oxygen or an oxygen-containing gas (eg air) is injected into the culture to maintain aerobic conditions.
  • Cultivation of the transformant according to the present invention is usually carried out at a temperature of 20 ° C to 45 ° C, specifically 25 ° C to 40 ° C.
  • the culture is continued until the maximum amount of production of the desired adiponectin analog is obtained, and for this purpose, the culture can usually be continued for 10 to 160 hours.
  • the transformant according to the present invention produces an adiponectin analog, and the adiponectin analog produced according to the composition of the vector and the characteristics of the host cell is It can be secreted into the periplasmic space or extracellularly.
  • Proteins expressed in or outside the host cell can be purified in a conventional manner.
  • purification methods include salting out (eg, ammonium sulfate precipitation, sodium phosphate precipitation, etc.), solvent precipitation (eg, protein fraction precipitation using acetone, ethanol, etc.), dialysis, gel filtration, ion exchange, reverse phase column chromatography, and the like. Techniques such as chromatography and ultrafiltration may be applied alone or in combination.
  • an adiponectin analog prepared by peptide synthesis may be linked to a biocompatible material (eg, an immunoglobulin Fc region) through a non-peptide polymer.
  • a biocompatible material eg, an immunoglobulin Fc region
  • Such peptide synthesis can be prepared without limitation using a known peptide synthesis method as the adiponectin analog sequence of the present invention is provided.
  • Adiponectin analogs are as described above.
  • Another aspect for implementing the present invention provides acylated adiponectin analogs.
  • the adiponectin analog is as described above. Specifically, the adiponectin analog may include, consist essentially of, or consist of the amino acid sequence of any one of Formula 1 or SEQ ID NOs: 2 to 93, but is not limited thereto.
  • Acylation is known as a method for improving the pharmacokinetic and pharmacodynamic properties of peptides and drugs.
  • peptide drugs there is a problem in that it is difficult to exert drug effects due to enzymatic degradation in the body. Accordingly, a technique for increasing the stability and half-life of a peptide drug by blocking an enzyme action site through acylation of a peptide that attaches a fatty acid to a peptide is being studied (PLoS One 2014 Oct 8;9(10):e109939; Tetrahedron , 49(20), 4141-4146).
  • adiponectin analogs of the present invention may be in an acylated form to increase half-life.
  • the acyl group used for acylation may be a carbon chain having an arbitrary length, and may be linear or branched.
  • the chain may include a linear aliphatic chain, a branched aliphatic chain, a chain containing a cyclic alkyl moiety, a hydrophobic natural product such as a steroid, an aralkyl chain, or an alkyl chain containing an acyl moiety. there is.
  • One specific example may be an acylated adiponectin analog in which an acyl group is attached to the adiponectin analog.
  • the acyl group is N-terminus of the amino acid sequence of the adiponectin analog; or a side chain amine of a lysine residue present in an adiponectin analog amino acid sequence; it may be attached to.
  • the adiponectin analog is acylated with a C 1 -C 40 straight-chain or branched-chain acyl group containing one or more, two or more, specifically one or two carbolic acids or carboxylic acid derivatives.
  • the acyl group may be a fatty acid or dicarboxylic acid, or a derivative compound thereof, such as an ester compound of a fatty acid or dicarboxylic acid, specifically a C 4 to C 40 fatty acid or dicarboxylic acid, or an ester compound thereof. Not limited.
  • C 18 , C 20 , C 22 , C 24 , C 26 , C 28 , C 30 , C 30 It may be a C 35 , or C 40 fatty acid or dicarboxylic acid, or an ester compound thereof.
  • Other exemplary acyl groups include cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, taurocholic acid, glycocholic acid, bile acids such as cholesterol acid, succinic acid or succinic acid derivatives, maleic acid, or maleic acid derivatives. , but not limited thereto.
  • acyl group may be one or more selected from the group consisting of an aldehyde group, a maleimide group, and a succinimid derivative, but are not limited thereto.
  • the reactive group of the acyl group may be carboxylic acid, succinic acid ester, succinimide, or hydroxysuccinimide.
  • an acyl group may be attached to 1 to 5, such as 1 to 3, or a side chain amine of one lysine residue present in an adiponectin analog amino acid sequence.
  • the acylated adiponectin analog may be acylated with a C 1 -C 40 straight-chain or branched-chain acyl group containing one or two carbolic acids.
  • the acyl group may be a C 4 to C 40 fatty acid or dicarboxylic acid.
  • the acylated adiponectin analog of the present invention may be one in which an acyl group is attached to the adiponectin analog by a method known in the art.
  • an acyl group may be attached to a desired position using a protecting group and/or a linker, and the reaction may be carried out in the presence of an organic solvent or aqueous solution.
  • the adiponectin analog may be in the form of an acyl group attached directly to its amino acid residue.
  • an acyl group may be attached through an ester, thioester, imide, or amide bond, but is not limited thereto.
  • the adiponectin analog may be acylated at an amino acid residue having an amine, hydroxyl, or thiol group.
  • the adiponectin analog may be acylated at an amino acid or lysine residue located at the N-terminus, but is not limited thereto.
  • the acylation of the lysine residue may be by attaching an acyl group to the side chain amine (epsilon amino atom of lysine) of the lysine residue.
  • the acylated adiponectin analog of the present invention may further include a linker.
  • the acylated adiponectin analog according to the present invention may be acylated after connecting a linker, but is not limited thereto.
  • the linker may be one or more amino acids having an amine, hydroxyl, or thiol group, a linker containing an ethylene glycol repeating unit, or a linker in which one or more amino acids and a linker containing an ethylene glycol repeating unit are connected. .
  • the linker may be polyethylene glycol (PEG) having a structure of [-OCH 2 CH 2 -]n.
  • the n may be a natural number of 1 to 10, but is not limited thereto.
  • the linker may include an amine, hydroxyl, thiol, and/or carboxyl group, aldehyde, maleimide, succinimide, etc. as a reactive group, but is not limited thereto.
  • the acylation of the present invention may be attaching one reactive group of the linker to an acyl group after coupling the linker, or, when two or more carboxylic acids are included, all but one are protected by a protecting group such as an ester. It may be by attaching an oily moiety, but as long as an acyl group can be introduced into the adiponectin analog according to the present invention, the introduction method of the acyl group is not limited to a specific method.
  • the acylated adiponectin analog of the present invention may have an acyl group attached to the N-terminus, C-terminus, or an internal residue of the peptide.
  • an acyl group may be attached to the N-terminus, C-terminus, or side chain of a tyrosine, phenylalanine, or lysine residue inside the peptide, but is not limited thereto.
  • an acyl group may be attached to an amine group of tyrosine, phenylalanine, or lysine, a hydroxyl group, or a thiol group of cysteine, but is not limited thereto.
  • the acyl group may be represented by Formula 3-1 below.
  • an acyl group is attached to the side chain amine (lysine side chain) of a lysine residue present in the N-terminal acylation of the amino acid sequence of the adiponectin analog or the adiponectin analog amino acid sequence to form an acyl group. It may be in a realized form, but is not limited thereto. Specifically, it may have structural formulas of Formulas 3-2 and 3-3 below, but is not limited thereto.
  • R represents an adiponectin analog.
  • an acyl group may be attached to the N-terminus of the adiponectin analog.
  • an acyl group may be attached to the side chain amine of a lysine residue present in the adiponectin analog amino acid sequence.
  • Chemical Formula 3-3 represents a lysine residue having an acyl group of Chemical Formula 3-1 attached to a side chain amine.
  • the acylated adiponectin analog of the present invention described above has an activity of about 0.1% or more, 1% or more, 10% or more, 50% or more, 100% or more, 200% or more, 300% or more, 400% or more, 500% or more compared to BHD1028. % or more, 700% or more, 1000% or more, 1500% or more, 2000% or more, 2500% or more, 3000% or more, 3500% or more, but is not limited thereto.
  • the acylated adiponectin analog of the present invention may have an adiponectin receptor activity and thus perform the same function as non-acylated adiponectin or an adiponectin analog, but may have a high activity level, stability, and increased half-life.
  • composition according to the present invention may be a pharmaceutical composition, and in a specific embodiment may be a pharmaceutical composition having a purpose for preventing or treating liver fibrosis.
  • composition according to the present invention comprises an adiponectin analog; Sustainable conjugates containing them; or an acylated adiponectin analog, specifically, a pharmacologically effective amount of an adiponectin analog; Sustainable conjugates containing them; or an acylated adiponectin analog.
  • a pharmaceutically acceptable carrier may be further included.
  • composition according to the present invention include an adiponectin analog comprising the amino acid sequence of any one of Formula 1 or SEQ ID NOs: 2 to 93; Sustainable conjugates containing them; or an acylated adiponectin analog, more specifically, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, and an adiponectin analog comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 42 to 63 and SEQ ID NOs: 65 to 93; Any one amino acid selected from the group consisting of SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 66, SEQ ID NO: 74, and SEQ ID NO: 75 an adiponectin analog
  • composition according to the present invention may exhibit one or more of the following properties, but is not limited thereto as long as it exhibits activity on adiponectin receptors to have anti-inflammatory activity and/or to improve fibrosis:
  • Adiponectin is an adipokine that acts on AdipoR1 and AdipoR2 receptors, and is involved in the development of metabolic diseases by regulating lipid and sugar metabolism and insulin sensitivity. Preventive and therapeutic effects on metabolic diseases, particularly liver fibrosis, can be obtained through the adiponectin analog of the present invention modified to have high activity.
  • the adiponectin analog of the present invention is about 0.01-fold or more, about 0.1-fold or more, about 1-fold or more, about 3-fold or more, or about 5-fold or more than native adiponectin or an existing known adiponectin analog (eg, BHD1028). , about 10 times more, about 15 times more, about 20 times more, about 25 times more, or about 30 times more active, but the number is not limited and has a preventive and therapeutic effect on liver fibrosis. fall within the scope of the present invention.
  • liver fibrosis means the formation of excessive fibrous connective tissue in an organ or tissue in a reparative or reactive process as a result of a wound healing process for repetitive liver damage. Unlike liver cirrhosis, it is reversible, composed of thin fibrils, and is known to have no nodule formation. When the cause of liver damage is eliminated, normal recovery may be possible, but this liver fibrosis process repetitively If it persists, crosslinking between ECM (extra cellular matrix) increases, leading to irreversible liver cirrhosis with nodules.
  • the composition according to the present invention may exhibit preventive or therapeutic effects on hepatic fibrosis.
  • the composition according to the present invention can (i) improve liver fibrosis in the subject to whom it is administered; and/or (ii) reducing the expression of TGF ⁇ , Col1a1, or Col3a1 to prevent or treat liver fibrosis, but is not limited thereto.
  • an adiponectin analog according to the present invention may exhibit an effect on liver fibrosis.
  • prevention refers to the adiponectin analog; Sustainable conjugates containing them; acylated adiponectin analogs; Or any action that inhibits or delays liver fibrosis by administration of a composition containing the same.
  • treatment refers to the adiponectin analog; Sustainable conjugates containing them; acylated adiponectin analogs; Or any action that improves or benefits the symptoms of liver fibrosis by administering a composition containing the same.
  • acylated adiponectin analogs of the present invention can improve the quality of life of patients by reducing the number of administrations to chronic patients who need to be administered daily due to a dramatic increase in blood exposure, half-life in blood, and sustained effect in vivo.
  • the pharmaceutical composition of the present invention may contain the adiponectin analog, the long-acting conjugate thereof, or the acylated adiponectin analog in a pharmaceutically effective amount.
  • a pharmacologically effective amount means the degree to which the desired pharmacological activity (eg, prevention, improvement or treatment of liver fibrosis) can be obtained due to an adiponectin analog, a long-acting conjugate thereof, or an acylated adiponectin analog.
  • it may also mean a pharmaceutically acceptable level at an insignificant level that does not cause toxicity or side effects in the administered subject, but is not limited thereto.
  • Such a pharmaceutically effective amount may be determined by comprehensively considering the number of administrations, patients, dosage forms, and the like.
  • the pharmaceutical composition of the present invention may contain the ingredient (active ingredient) in an amount of 0.01 to 99% by weight by volume.
  • the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier or diluent.
  • a pharmaceutically acceptable carrier or diluent may be non-naturally occurring.
  • the term "pharmaceutically acceptable” means an amount sufficient to exhibit a therapeutic effect and not causing side effects, and the type of disease, age, weight, health, sex, and sensitivity of the patient to the drug , It can be easily determined by a person skilled in the art according to factors well known in the medical field, such as an administration route, an administration method, the number of administrations, a treatment period, combination or drugs used simultaneously.
  • the pharmaceutical composition comprising the acylated adiponectin analog may include a pharmaceutically acceptable excipient.
  • the excipient is not particularly limited thereto, but for oral administration, a binder, a lubricant, a disintegrant, a solubilizer, a dispersant, a stabilizer, a suspending agent, a colorant, a flavoring agent, etc. may be used.
  • a buffer, a preservative, A pain reliever, solubilizer, tonicity agent, stabilizer, etc. may be mixed and used, and in the case of topical administration, a base, excipient, lubricant, preservative, etc. may be used.
  • the dosage form of the composition of the present invention may be variously prepared by mixing with the pharmaceutically acceptable excipients as described above.
  • it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, it can be prepared in the form of unit dose ampoules or multiple doses.
  • it may be formulated into solutions, suspensions, tablets, pills, capsules, sustained-release preparations, and the like.
  • examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil and the like can be used.
  • fillers, anti-coagulants, lubricants, wetting agents, flavoring agents, preservatives, and the like may be further included.
  • the pharmaceutical composition of the present invention is any one selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, freeze-dried preparations and suppositories. may have a form.
  • composition is formulated into a unit dosage form suitable for administration into the body of a patient according to a conventional method in the pharmaceutical field, specifically, a formulation useful for the administration of a protein drug, an administration method commonly used in the art.
  • Oral, dermal, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intragastric, topical, sublingual, vaginal or rectal It may be administered by a parenteral administration route including, but not limited to, these routes.
  • the conjugate may be mixed with various pharmaceutically acceptable carriers such as physiological saline or organic solvents, and carbohydrates such as glucose, sucrose or dextran, ascorbic acid (ascorbic acid) may be used to increase stability or absorption. acid) or glutathione, antioxidants, chelating agents, low-molecular-weight proteins or other stabilizers may be used as drugs.
  • pharmaceutically acceptable carriers such as physiological saline or organic solvents, and carbohydrates such as glucose, sucrose or dextran, ascorbic acid (ascorbic acid) may be used to increase stability or absorption. acid) or glutathione, antioxidants, chelating agents, low-molecular-weight proteins or other stabilizers may be used as drugs.
  • Another aspect of the present invention is an adiponectin analog; Sustainable conjugates containing them; or an acylated adiponectin analog; Or it provides a method for preventing or treating liver fibrosis comprising administering a pharmaceutical composition containing the same to a subject in need thereof.
  • adiponectin analog The adiponectin analog, the long-acting conjugate thereof, the acylated adiponectin analog, the composition containing the same, and the prevention and treatment of liver fibrosis are as described above.
  • the subject is an individual who has or is suspected of having liver fibrosis, and refers to mammals including humans, rats, livestock, etc., but the adiponectin analog of the present invention, a long-acting conjugate thereof, an acylated adiponectin analog, Or a subject that can be treated with the composition comprising the same is included without limitation.
  • the term "administration” means introducing a predetermined substance (eg, an adiponectin analog, a long-acting conjugate thereof, or an acylated adiponectin analog) to a patient by any suitable method, and the route of administration is not particularly limited thereto. However, it can be administered through any general route that can reach the target in vivo, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration. administration, intrapulmonary administration, or intrarectal administration, and the like.
  • a predetermined substance eg, an adiponectin analog, a long-acting conjugate thereof, or an acylated adiponectin analog
  • the method of the present invention may include administering the adiponectin analog, the long-acting conjugate thereof, the acylated adiponectin analog, or a pharmaceutical composition containing the same in a pharmaceutically effective amount.
  • An appropriate total daily amount can be determined by a treating physician within the scope of sound medical judgment, and can be administered once or divided into several times.
  • a specific therapeutically effective amount for a particular patient is determined by the type and extent of the response to be achieved, the specific composition, including whether other agents are used as the case may be, the patient's age, weight, general state of health, It is preferable to apply differently according to various factors including gender and diet, administration time, administration route and secretion rate of the composition, treatment period, drugs used together with or concurrently used with a specific composition, and similar factors well known in the medical field.
  • the dosage and frequency of administration are determined according to the type of drug as the active ingredient, as well as various related factors such as the disease to be treated, the route of administration, the age, sex, and weight of the patient, and the severity of the disease.
  • the composition of the present invention may contain the adiponectin analog, the long-acting conjugate thereof, or the acylated adiponectin analog in a pharmaceutically effective amount, but is not limited thereto.
  • adiponectin analog, its long-acting conjugate, or acylated adiponectin analog in a pharmaceutically effective amount means the degree to which the desired pharmacological activity can be obtained due to the adiponectin analog, its long-acting conjugate, or acylated adiponectin analog, , It may also mean a pharmaceutically acceptable level at which toxicity or side effects do not occur or are insignificant in the subject being administered, but is not limited thereto. Such a pharmaceutically effective amount may be determined by comprehensively considering the number of administrations, patients, dosage forms, and the like.
  • the pharmaceutical composition of the present invention may contain the ingredient (active ingredient) in an amount of 0.01 to 99% by weight by volume.
  • the total effective amount of the composition of the present invention can be administered to the patient in a single dose or by a fractionated treatment protocol in which multiple doses are administered over a long period of time.
  • the pharmaceutical composition of the present invention may vary the content of the active ingredient according to the severity of the disease.
  • a preferred total dose of the adiponectin analog of the present invention, the long-acting conjugate thereof, or the acylated adiponectin analog may be about 0.0001 mg to 500 mg/kg of the patient's body weight per day.
  • the dose of the adiponectin analog, its long-acting conjugate, or acylated adiponectin analog varies widely, such as the route of administration of the pharmaceutical composition and the number of treatments, as well as the patient's age, weight, health condition, sex, severity of disease, diet and excretion rate. Since the effective dosage for the patient is determined in consideration of factors, those skilled in the art will be able to determine an appropriate effective dosage according to the specific use of the composition of the present invention considering these points.
  • the pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route and administration method as long as it exhibits the effects of the present invention.
  • the pharmaceutical composition of the present invention has excellent in vivo persistence and potency, and thus the number and frequency of administration of the pharmaceutical preparation of the present invention can be significantly reduced.
  • the pharmaceutical composition may be administered by intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, or intrarectal administration route, but It is not limited to a specific route of administration as long as the desired pharmacological effect can be obtained.
  • the pharmaceutical composition of the present invention may be administered once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, or once a month, or once a week to 1 It may be administered once or multiple times at time intervals in the range of months, but is not limited thereto.
  • Another aspect embodying the present invention is the use of the adiponectin analog, a long-acting conjugate thereof, an acylated adiponectin analog, or a composition containing the same in the preparation of a drug for preventing or treating liver fibrosis.
  • adiponectin analog the long-acting conjugate thereof, the acylated adiponectin analog, or the composition containing the same, and the prevention, treatment, route of administration, and frequency of administration of liver fibrosis are as described above.
  • Another aspect embodying the present invention is to provide an adiponectin analog, a long-acting conjugate thereof, an acylated adiponectin analog, or a composition containing the same for preventing or treating liver fibrosis.
  • adiponectin analog the long-acting conjugate thereof, the acylated adiponectin analog, or the composition containing the same, and the prevention, treatment, route of administration, and frequency of administration of liver fibrosis are as described above.
  • adiponectin analog with high therapeutic efficacy compared to natural and previously known adiponectin analogs (BHD1028, Encuragen; SEQ ID NO: 94).
  • Amino acid substitution, sequence extension, insertion, and partial sequence repetition were performed in the active sequence of native adiponectin (SEQ ID NO: 1; KFHCNIPGLYYFAYHITV) to prepare an adiponectin analog having higher activity than native and previously known adiponectin analogs. .
  • the sequence of the adiponectin analog prepared through this process is as follows.
  • Aib 2-Aminoisobutyric acid
  • NL Norleucine
  • HyP Hydroxyproline
  • NV Norvaline
  • Example 1 The synthesis of the adiponectin analog and comparative material (SEQ ID NO: 94, BHD1028, Encuragen) prepared in Example 1 was performed using an automatic peptide synthesizer (Symphony X, Gyros Protein Tech.) using a solid phase synthesis method. In order to maintain the carboxyl group at the C-terminus, A-wang resin was used for analogues of SEQ ID NOs: 3, 4 and 5, and F-wang resin was used for other analogues. Each amino acid was synthesized in the order from C-terminus to N-terminus.
  • Reverse phase chromatography was used to purify the synthesized adiponectin analog and comparative material.
  • the purity of the synthesized peptide was confirmed using an analytical liquid chromatogram (RP-HPLC), and if it was 90% or higher, it was judged to be usable for the experiment.
  • RP-HPLC analytical liquid chromatogram
  • the molecular weight and information of the peptides were confirmed using liquid chromatogram/mass spectrometry (LC/MS).
  • the synthesized peptides were stored at -20 °C until used in experiments.
  • Example 2 The in vitro activity of the novel adiponectin analogs obtained in Example 2 against the BHD1028 comparative material was evaluated by bioluminescence assay to determine the degree of inhibition of the fibrotic response induced by treatment with TGF ⁇ in human hepatocytes.
  • adiponectin analog test substance or comparative substance diluted by a 2-fold serial dilution method in SMAD/HepG2 (Signosis, USA), a human hepatocyte cell line stably expressing the luciferase reporter gene under the SMAD 2/3 response element, was administered at 250 It was treated in the range of mM to 0.49 mM. After 3 hours, each well was additionally treated with human TGF ⁇ at a concentration of 5 ng/mL and reacted for 16 hours, and then the level of luminescence of the expressed luciferase was measured using the Dual-Luciferase ® Reporter Assay System (E1910, Promega, USA). was measured according to the manufacturer's protocol.
  • IC 50 half inhibitory concentration
  • the novel adiponectin analogs showed about 2 to 15 times higher in vitro activity than the comparative material used as a control, and through this, the novel adiponectin analogs have the potential to improve the TGF ⁇ -mediated fibrosis response. confirmed.
  • the adiponectin analogs (SEQ ID NOs: 31, 44, and 49) prepared in the above example were acylated in the following two forms. The first is an N-terminal acylation form, and the second is an acylation form of a lysine side chain (a side chain amine of a lysine residue).
  • the adiponectin analog was synthesized according to the method of Example 2, and during synthesis, Fmoc (9H-fluoren-9-ylmethoxycarbonyl) protected amino acid, 4 equivalents compared to peptide-resin), HOBt (1-hydroxybenzotriazole, 8 equivalents compared to peptide-resin) and DIC (diisopropylcarbodiimide, 8 equivalents compared to peptide-resin) were used, and Fmoc was protected with 20% piperidine/DMF. It was deprotected.
  • the Fmoc protecting group was removed by adding 8 mL of 20% piperidine/DMF to the reaction vessel containing the resin in an automatic synthesizer for 5 minutes, and this process was repeated once more. In order to remove remaining impurities, after each process, it was washed 6 times for 10 seconds with 12 mL of DMF.
  • R represents an adiponectin analog. Specifically, an acyl group is attached to the N-terminus of the adiponectin analog.
  • Fmoc-L-Lys[Oct-(otBu) instead of Fmoc-L-Lys(Boc)-OH used in the synthesis of lysine residues of adiponectin analogues for side chain amine acylation of lysine residues of adiponectin analogues with the structure shown in Chemical Formula 3-3 It was synthesized using -Glu-(otBu)-AEEA-AEEA]-OH (4 equivalents compared to peptide-resin), and the subsequent process was reacted in the same way as the above acylation process.
  • the PEG linker As the PEG linker, 3.4K ALD-PEG-ALD (Korea Hanmi Fine Chemical Co., Ltd.), a modified PEG in which the hydroxy hydrogens at both ends are modified with propylaldehyde groups, and the formula weight of the ethylene glycol repeating unit is 3.4 kDa, or both ends 3.4K ALD-PEG-DEP, a modified PEG in which one of the hydroxyl hydrogens is modified with propylaldehyde and the other with 3-diethoxypropyl, and the formula weight of the ethylene glycol repeating unit is 3.4 kDa (Hanmi Fine Chemical Co., Ltd., Korea) was used for the preparation of the linkages.
  • the molar ratio of the analog and PEG linker is 1:5 to 1:30 and the concentration of the analog is 1 to 5 mg/mL at room temperature or 2 to 10 ° C. Reacted for 3 hours. At this time, the reaction is 0 to 60% isopropanol or 0 to 60% ethanol or 10 to 60% dimethyl sulfoxide (Dimethyl sulfoxide, below) in the presence of 10 to 50 mM sodium cyanoborohydride (SCB) as a reducing agent.
  • SBCB sodium cyanoborohydride
  • Example 5-2 Preparation of adiponectin analog-PEG-immunoglobulin Fc conjugate
  • the mono-PEGylated adiponectin analog obtained in Example 5-1 was mixed with a modified immunoglobulin Fc fragment (prepared according to Korean Patent Registration No. 10-725315, SEQ ID NO: 96) in the form of a dimer and 1:2 to 1:
  • the mixture was mixed at a molar ratio of 10, and the total peptide concentration was 1 to 20 mg/mL and reacted at room temperature or 2 to 10 ° C. for 12 to 16 hours.
  • the reaction was carried out in 100 mM K-P pH 6.0-8.0 or 50 mM Bis-Tris pH 6.0-8.0, and the reaction was performed by adding 0-20% IPA or 0-20% ethanol and 10-50 mM SCB as a reducing agent.
  • the reaction solution under the following conditions is a Source 15Q purification column (Cytiva), Sephaedex 75 or 200 purification column (Cytiva), Source 15 Phe purification column (Cytiva), Butyl-FF, Butyl-HP purification column (Cytiva) ) was purified using. From this, an adiponectin-3.4K PEG-immunoglobulin Fc conjugate was obtained.
  • Example 6 Effect of adiponectin analogs on improving liver fibrosis in TAA-induced liver fibrosis model mice
  • adiponectin analog SEQ ID NO: 49
  • TAA Thioacetamide
  • mice 7-week-old normal male C57BL/6 mice (Daehan Biolink, Korea) were intraperitoneally administered with TAA at a dose of 50 to 400 mg/kg three times a week for a total of 12 weeks to create a TAA-induced liver fibrosis model.
  • the administration of the drug started 2 weeks after the start of TAA administration, 7 mice per group, control G1 (normal mice administered only with vehicle without TAA), group G2 administered only with TAA (administered with TAA and vehicle), TAA and adiponectin analog (SEQ ID NO: 49 ) It was divided into 3 groups, administration group G3 (TAA administration and analogue administration 4823 mg/kg twice a day).
  • test substance was administered subcutaneously in distilled water containing 2% DMSO. After a total of 10 weeks of administration, blood was collected and autopsied. The left lateral lobe of the obtained liver tissue was fixed with formalin for histopathological analysis. The rest of the liver tissue was weighed, crushed in distilled water, and stored at -80 °C.
  • the collected blood was separated from serum in a serum separation tube (BD bioscience, USA) and stored at -80 °C until the time of analysis.
  • liver tissue samples fixed in formalin were sectioned in paraffin and stained with hematoxylin and eosin (H&E) and Sirius red (NovaUltra, IHC World, USA). Sirius Red Stain Kit) staining was analyzed by proceeding according to a standard protocol.
  • Sirius red staining which reflects the degree of fibrosis in liver tissue
  • the experimental group administered with the adiponectin analog significantly improved liver fibrosis index (area positive for Sirius red staining) compared to the control group (see FIG. 1 ).
  • hydroxyproline (Hydroxyproline) in the liver, which is an indicator of liver fibrosis, was measured according to the manufacturer's protocol by hydrolyzing the liver tissue lysate with hydrochloric acid and using a Hydroxyproline assay kit (Netherlands Quickzyme Biosciences, catalog number QZBHYPRO5).
  • the adiponectin analog administration group showed significantly improved liver fibrosis index compared to the control group (FIG. 2).
  • liver fibrosis marker genes the expression of TGF ⁇ , Col1a1, and Col3a1 genes was measured through quantitative real-time PCR.
  • mRNA was extracted from liver tissue using the RNeasy mini kit (Qiagen, 74106) and synthesized into cDNA using the iScript cDNA synthesis kit (Bio-rad, 1708891). cDNA reaction conditions were used the same as the manufacturer's test protocol. After diluting the synthesized cDNA, it was analyzed with a QuantStudio 6 FLEX (Thermofisher, USA) instrument using 2X Power SYBRTM Green PCR Master Mix (Applied biosystems, 4367659). The sequence of the primer used in the reaction is as follows.
  • TGF ⁇ -forward (SEQ ID NO: 117): 5'- ACT GCT TCC CGA ATG TCT GAC GTA - 3'
  • TGF ⁇ -reverse SEQ ID NO: 118: 5'- TAA AGA GGT CAC CCG CGT GCT AAT - 3'
  • Col1a1-forward (SEQ ID NO: 119): 5'-AGA GCA TGA CCG ATG GAT TC-3'
  • Col1a1-reverse SEQ ID NO: 120: 5'-CCT TCT TGA GGT TGC CAG TC-3'
  • Col3a1-forward (SEQ ID NO: 121): 5'-ACT GGG GAA ACA TGC ATA AA-3'
  • Col3a1-reverse (SEQ ID NO: 122): 5'- GGC CAT AGC TGA ACT GAA AA - 3'
  • GAPDH-forward (SEQ ID NO: 123): 5'- CAA CAG CAA CTC CCA CTC TT-3'
  • GAPDH-reverse (SEQ ID NO: 124): 5'-TGT TGC TGT AGC CGT ATT CA-3'
  • the expression level of the G1 group was converted to 100% to show the relative expression level.
  • the novel adiponectin analog according to the present invention exhibits superior activity compared to the conventionally used adiponectin comparative material. It was confirmed to have high in vitro activity.
  • the adiponectin analog according to the present invention has the effect of significantly improving liver fibrosis in liver fibrosis model mice. , confirmed through histopathological analysis and fibrosis index and gene expression analysis in liver tissue. This suggests that adiponectin analogs are effective in improving liver fibrosis.
  • the adiponectin analog according to the present invention has a higher activity than native adiponectin or previously known adiponectin, and has advantages as a therapeutic agent. In particular, it can be used as an excellent therapeutic agent for liver fibrosis. It suggests possibility.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Endocrinology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne un nouvel analogue d'adiponectine à activité améliorée.
PCT/KR2022/019864 2021-12-08 2022-12-08 Nouvel analogue et conjugué d'adiponectine Ceased WO2023106845A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2021-0175141 2021-12-08
KR1020210175141A KR20230086480A (ko) 2021-12-08 2021-12-08 신규한 아디포넥틴 아날로그 및 결합체

Publications (1)

Publication Number Publication Date
WO2023106845A1 true WO2023106845A1 (fr) 2023-06-15

Family

ID=86730989

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2022/019864 Ceased WO2023106845A1 (fr) 2021-12-08 2022-12-08 Nouvel analogue et conjugué d'adiponectine

Country Status (2)

Country Link
KR (1) KR20230086480A (fr)
WO (1) WO2023106845A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117330659A (zh) * 2023-09-13 2024-01-02 南京汉欣医药科技有限公司 一种检测四肽非活化酯异构体的高效液相色谱分析方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012142142A2 (fr) * 2011-04-12 2012-10-18 Temple University - Of The Commonwealth System Higher Education Agonistes des récepteurs de l'adiponectine et procédés d'utilisation
KR20150032401A (ko) * 2013-09-17 2015-03-26 강원대학교산학협력단 아디포넥틴으로부터 유래한 펩타이드 및 이를 포함하는 조성물
KR101838622B1 (ko) * 2016-09-20 2018-03-14 김브라이언 아디포넥틴 수용체에 대한 작용제 펩타이드
KR20210124071A (ko) * 2020-04-03 2021-10-14 한미약품 주식회사 Glp-2 유도체 또는 이의 지속형 결합체를 포함하는 방사선요법, 화학요법, 또는 이들의 조합으로 유발된 점막염의 예방 또는 치료용 약학적 조성물

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012142142A2 (fr) * 2011-04-12 2012-10-18 Temple University - Of The Commonwealth System Higher Education Agonistes des récepteurs de l'adiponectine et procédés d'utilisation
KR20150032401A (ko) * 2013-09-17 2015-03-26 강원대학교산학협력단 아디포넥틴으로부터 유래한 펩타이드 및 이를 포함하는 조성물
KR101838622B1 (ko) * 2016-09-20 2018-03-14 김브라이언 아디포넥틴 수용체에 대한 작용제 펩타이드
KR20210124071A (ko) * 2020-04-03 2021-10-14 한미약품 주식회사 Glp-2 유도체 또는 이의 지속형 결합체를 포함하는 방사선요법, 화학요법, 또는 이들의 조합으로 유발된 점막염의 예방 또는 치료용 약학적 조성물

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SUNGHWAN KIM, YOUNHO LEE, JUN WOO KIM, YOUNG-JIN SON, MIN JUNG MA, JEE-HYUN UM, NAM DOO KIM, SANG HYUN MIN, DONG IL KIM, BRIAN B. : "Discovery of a novel potent peptide agonist to adiponectin receptor 1", PLOS ONE, vol. 13, no. 6, 18 June 2018 (2018-06-18), pages e0199256, XP055587574, DOI: 10.1371/journal.pone.0199256 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117330659A (zh) * 2023-09-13 2024-01-02 南京汉欣医药科技有限公司 一种检测四肽非活化酯异构体的高效液相色谱分析方法
CN117330659B (zh) * 2023-09-13 2024-05-31 南京汉欣医药科技有限公司 一种检测四肽非活化酯异构体的高效液相色谱分析方法

Also Published As

Publication number Publication date
KR20230086480A (ko) 2023-06-15

Similar Documents

Publication Publication Date Title
WO2017116205A1 (fr) Conjugué persistant d'un triple activateur activant le récepteur du glucagon, du glp-1 et du gip
WO2017003191A1 (fr) Dérivé de glucagon et composition comprenant un conjugué à action prolongée de celui-ci
WO2012173422A9 (fr) Conjugué comprenant de l'oxyntomoduline et un fragment d'immunoglobuline, et son utilisation
WO2021066600A1 (fr) Glucagon, composition comprenant un agoniste double du récepteur de glp-1 et du récepteur de gip et utilisation thérapeutique associée
WO2020130749A1 (fr) Composition pharmaceutique comprenant de l'insuline et un triple agoniste ayant une activité par rapport à l'ensemble des récepteurs glp1, glucagon et gip
WO2022211537A1 (fr) Nouveau conjugué immunoactif d'analogue d'interleukine 2 et son procédé de préparation
WO2019066586A1 (fr) Conjugué à action prolongée du dérivé de peptide-2 apparenté au glucagon (glp-2)
WO2020071865A1 (fr) Utilisations thérapeutiques du glucagon et produit combiné comprenant celui-ci
WO2017116207A1 (fr) Analogue de fgf21, conjugué de fgf21 et leur utilisation
WO2022035302A1 (fr) Composition pharmaceutique à effet hypotenseur comprenant un activateur triple présentant une activité pour tous les récepteurs du glucagon, de la glp-1, et de la gip
WO2020263063A1 (fr) Utilisation thérapeutique, pour les maladies hépathiques, d'un triple agoniste ayant une activité par rapport à tous les récepteurs du glucagon, de glp-1 et de gip, ou conjugué de ceux-ci
WO2021235913A1 (fr) Formulation liquide de conjugué à action prolongée de glp-2
WO2023106845A1 (fr) Nouvel analogue et conjugué d'adiponectine
WO2018174668A2 (fr) Complexe d'analogue à l'insuline à affinité réduite pour le récepteur de l'insuline et son utilisation
WO2022015082A1 (fr) Utilisation thérapeutique d'un dérivé du glucagon ou d'un conjugué de celui-ci pour une maladie hépatique
WO2021010532A1 (fr) Procédé de préparation d'un conjugué de médicament à action prolongée par préparation d'un nouvel intermédiaire
WO2022035271A1 (fr) Composition pharmaceutique comprenant un conjugué persistant de triple activateur utilisé comme principe actif
WO2021235907A1 (fr) Formulation liquide d'un conjugué à action prolongée d'un dérivé du glucagon
WO2024136597A1 (fr) Composition pharmaceutique pour prévenir ou traiter une maladie du greffon contre l'hôte contenant un dérivé de glp-2 ou un conjugué de celui-ci à action prolongée
WO2023277620A1 (fr) Utilisation thérapeutique d'une combinaison comprenant un triple activateur présentant une activité sur l'ensemble des récepteurs suivants : au glucagon, glp-1 et gip
WO2022216129A1 (fr) Composition pharmaceutique pour prévenir ou traiter une maladie rénale chronique contenant un dérivé de glucagon
WO2020130751A1 (fr) Composition pharmaceutique contenant de l'insuline et du glucagon
WO2020214013A1 (fr) Utilisation thérapeutique, pour l'hyperlipidémie, d'un triple agoniste ayant une activité par rapport à tous les récepteurs du glucagon, glp -1 et gip, ou conjugué de ceux-ci
WO2022139538A1 (fr) Nouveau triple activateur activant tous les récepteurs du glucagon, du glp-1 et du gip, et son utilisation
WO2022065897A1 (fr) Usage thérapeutique, pour des maladies neurodégénératives, d'un triple agoniste ayant une activité par rapport à tous les récepteurs du glucagon, de glp-1 et de gip, ou conjugué associés

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: 22904674

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22904674

Country of ref document: EP

Kind code of ref document: A1