WO2022216129A1 - 글루카곤 유도체를 포함하는 만성 신장 질환 예방 또는 치료용 약학 조성물 - Google Patents

글루카곤 유도체를 포함하는 만성 신장 질환 예방 또는 치료용 약학 조성물 Download PDF

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WO2022216129A1
WO2022216129A1 PCT/KR2022/005188 KR2022005188W WO2022216129A1 WO 2022216129 A1 WO2022216129 A1 WO 2022216129A1 KR 2022005188 W KR2022005188 W KR 2022005188W WO 2022216129 A1 WO2022216129 A1 WO 2022216129A1
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Prior art keywords
cysteine
composition
glp
exendin
conjugate
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PCT/KR2022/005188
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English (en)
French (fr)
Korean (ko)
Inventor
이선명
이종석
김정국
박은진
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Hanmi Pharmaceutical Co Ltd
Hanmi Pharmaceutical Industries Co Ltd
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Hanmi Pharmaceutical Co Ltd
Hanmi Pharmaceutical Industries Co Ltd
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Priority to US18/285,753 priority Critical patent/US20240197834A1/en
Priority to MX2023011759A priority patent/MX2023011759A/es
Priority to EP22785037.7A priority patent/EP4321170A4/en
Priority to CA3209923A priority patent/CA3209923A1/en
Priority to BR112023019917A priority patent/BR112023019917A2/pt
Priority to JP2023553081A priority patent/JP2024512895A/ja
Priority to IL305549A priority patent/IL305549A/en
Priority to AU2022254588A priority patent/AU2022254588A1/en
Priority to CN202280027283.1A priority patent/CN117157094A/zh
Publication of WO2022216129A1 publication Critical patent/WO2022216129A1/ko
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6847—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a hormone or a hormone-releasing or -inhibiting factor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • 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
    • C07K14/605—Glucagons

Definitions

  • the present invention relates to compositions comprising glucagon derivatives and uses thereof.
  • Glucagon is produced and secreted by the pancreas when blood sugar is lowered due to various causes, such as drug treatment, disease, or hormone or enzyme deficiency. It is known that secreted glucagon acts on the liver to break down glycogen to induce the release of glucose, and eventually plays a role in raising the blood sugar level to a normal level. In addition, it is known that it can lower blood lipid levels by inhibiting the synthesis of fat in the liver and promoting the burning of fatty acids. In addition, glucagon is known to show an effective weight loss effect by inducing fat burning and fat browning by acting directly or indirectly on white fat (Nat Rev Endocrinol. 11, 329-38 (2015); Physiol Rev. 97, 721-66) (2017)). Such glucagon exhibits activity by acting on the glucagon receptor.
  • Kidney (kidney, kidney) is located below the diaphragm and behind the stomach, it is an organ that removes wastes from the body, regulates the amount of water and salt, electrolyte and acid-base balance.
  • One of these diseases due to damage or dysfunction of the kidney, there is chronic kidney disease.
  • Chronic kidney disease also called chronic kidney disease, is a disease in which protein is continuously excreted in the urine (proteinuria) or the kidney function is reduced. have.
  • waste products accumulate in the body due to decreased kidney function, which can lead to increased blood pressure, anemia, weakened bones, nerve damage, and the like, and an increased risk of cardiovascular disease.
  • Treatment of chronic kidney disease is achieved through reduction of risk factors and maintenance or restoration of renal function.
  • high blood pressure is closely related to chronic kidney disease as it is known that hypertension is accompanied by 60% of patients with chronic kidney disease.
  • Inhibitors treatment methods using angiotensin receptor blockers, etc. are being studied.
  • side effects such as renal dysfunction, hyperkalemia, and ischemic kidney damage have also been reported when the known antihypertensive agents are administered.
  • One object of the present invention is to provide a pharmaceutical composition for preventing or treating chronic kidney disease comprising a glucagon derivative or a conjugate thereof.
  • Another object of the present invention is a glucagon derivative or a conjugate thereof; Or to provide a method for preventing or treating chronic kidney disease comprising administering a composition comprising the same.
  • Another object of the present invention is to provide a use for preventing or treating chronic kidney disease of a glucagon derivative or a conjugate thereof, or a composition comprising the same.
  • Another object of the present invention is to provide a use of a glucagon derivative or a conjugate thereof, or a composition comprising the same for the manufacture of a medicament for preventing or treating chronic kidney disease.
  • Another object of the present invention is a glucagon derivative or a conjugate thereof; And it relates to a pharmaceutical composition for preventing or treating chronic kidney disease comprising a GLP-1 receptor agonist or a conjugate thereof.
  • Another object of the present invention is a glucagon derivative or a conjugate thereof; and a GLP-1 receptor agonist or a combination thereof.
  • the glucagon derivative of the present invention exhibits an effect of lowering blood pressure and restoring renal function, it can be used as a therapeutic drug for patients with chronic kidney disease, and additional effects can be expected by using it in combination with a GLP-1 receptor agonist.
  • FIG. 1 is a diagram confirming the effect of reducing urine albumin output by the long-acting conjugate of the glucagon derivative of SEQ ID NO: 37 of the present invention in an essential hypertension (SHR) model.
  • FIG. 2 is a diagram confirming the systolic (left) and diastolic (right) blood pressure lowering effect of the long-acting conjugate of the glucagon derivative of SEQ ID NO: 37 of the present invention in an essential hypertension (SHR) model.
  • One aspect embodying the present invention is a composition comprising a glucagon derivative or a conjugate thereof.
  • the conjugate refers to a substance in which a peptide having activity against a glucagon derivative is covalently bonded to an immunoglobulin Fc region by a linker.
  • composition As a composition according to one embodiment, it is a pharmaceutical composition for preventing or treating chronic kidney disease including a glucagon derivative, or a conjugate thereof.
  • the glucagon derivative is a peptide comprising the amino acid sequence of the following general formula 1:
  • X1 is tyrosine (Y);
  • X2 is alpha-methyl-glutamic acid ( ⁇ -methyl-glutamic acid), Aib (aminoisobutyric acid), D-alanine, glycine (G), Sar (N-methylglycine), serine (S) or D-serine;
  • X7 is threonine (T), valine (V) or cysteine (C);
  • X10 is tyrosine (Y) or cysteine (C);
  • X12 is lysine (K) or cysteine (C);
  • X13 is tyrosine (Y) or cysteine (C);
  • X14 is leucine (L) or cysteine (C);
  • X15 is aspartic acid (D), glutamic acid (E) or cysteine (C);
  • X16 is glutamic acid (E), aspartic acid (D), serine (S), alpha-methyl-glutamic acid, or cysteine (C) or absent;
  • X17 is aspartic acid (D), glutamine (Q), glutamic acid (E), lysine (K), arginine (R), serine (S), cysteine (C), or valine (V) or absent;
  • X18 is alanine (A), aspartic acid (D), glutamic acid (E), arginine (R), valine (V), or cysteine (C) or absent;
  • X19 is alanine (A), arginine (R), serine (S), valine (V), or cysteine (C) or absent;
  • X20 is lysine (K), histidine (H), glutamine (Q), aspartic acid (D), arginine (R), alpha-methyl-glutamic acid, or cysteine (C) or absent;
  • X21 is aspartic acid (D), glutamic acid (E), leucine (L), valine (V), or cysteine (C) or absent;
  • X23 is isoleucine (I), valine (V), or arginine (R) or absent;
  • X24 is valine (V), arginine (R), alanine (A), cysteine (C), glutamic acid (E), lysine (K), glutamine (Q), alpha-methyl-glutamic acid, or leucine (L); absent;
  • X27 is isoleucine (I), valine (V), alanine (A), lysine (K), methionine (M), glutamine (Q), or arginine (R) or absent;
  • X28 is glutamine (Q), lysine (K), asparagine (N), or arginine (R) or absent;
  • X29 is threonine (T);
  • X30 is cysteine (C) or absent
  • the glucagon derivative is characterized in that it comprises any one amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 11, and 13 to 45.
  • X is a peptide comprising the amino acid sequence of Formula 1;
  • L is a linker containing an ethylene glycol repeating unit
  • F is an immunoglobulin Fc region
  • composition according to any one of the preceding embodiments comprising:
  • X2 is Aib (aminoisobutyric acid);
  • X7 is threonine (T), valine (V) or cysteine (C);
  • X10 is tyrosine (Y);
  • X12 is lysine (K);
  • X13 is tyrosine (Y);
  • X14 is leucine (L) or cysteine (C);
  • X15 is aspartic acid (D);
  • X16 is glutamic acid (E) or serine (S);
  • X17 is lysine (K), arginine (R), or cysteine (C);
  • X18 is arginine (R);
  • X19 is alanine (A), or cysteine (C);
  • X20 is glutamine (Q), or lysine (K);
  • X21 is aspartic acid (D) or glutamic acid (E);
  • X23 is valine (V);
  • X24 is glutamine (Q);
  • X27 is methionine (M).
  • X28 is asparagine (N);
  • X29 is threonine (T);
  • X30 is cysteine (C) or absent.
  • composition according to any one of the preceding embodiments, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 11, and 13 to 25, 27, 29, 31, 33, 35 to 45 .
  • composition according to any one of the preceding embodiments, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 22, 23, 27, 33, 35, 37, 38, 40, 41, 42, and 44 characterized in that
  • composition according to any one of the preceding embodiments, characterized in that the composition exhibits one or more of the following properties:
  • composition according to any one of the preceding embodiments, wherein the composition exhibits an effect of lowering blood pressure and/or restoring renal function of the subject when administered to the subject.
  • composition according to any one of the preceding embodiments, wherein the immunoglobulin Fc region is non-glycosylated.
  • the immunoglobulin Fc region comprises (a) a CH1 domain, a CH2 domain, a CH3 domain and a CH4 domain; (b) a CH1 domain and a CH2 domain; (c) a CH1 domain and a CH3 domain; (d) a CH2 domain and a CH3 domain; (e) a combination of one or two or more of the CH1 domain, CH2 domain, CH3 domain and CH4 domain with an immunoglobulin hinge region or portion of a hinge region; And (f) it is characterized in that it is selected from the group consisting of a dimer of each domain of the heavy chain constant region and the light chain constant region.
  • composition according to any one of the preceding embodiments, wherein, in the immunoglobulin Fc region, a site capable of forming a disulfide bond is removed, some amino acids at the N-terminus of the native Fc are removed, or the N-terminus of the native Fc It is characterized in that a methionine residue is added, a complement binding site is removed, or an antibody dependent cell mediated cytotoxicity (ADCC) site is removed.
  • ADCC antibody dependent cell mediated cytotoxicity
  • composition according to any one of the preceding embodiments, wherein the immunoglobulin Fc region is derived from IgG, IgA, IgD, IgE or IgM.
  • composition according to any one of the preceding embodiments, wherein the immunoglobulin Fc region is a hybrid of domains having different origins derived from an immunoglobulin selected from the group consisting of IgG, IgA, IgD, IgE, and IgM.
  • composition according to any one of the preceding embodiments, wherein the immunoglobulin Fc region is in a dimeric form.
  • composition according to any one of the preceding embodiments, wherein the immunoglobulin Fc region is an IgG4 Fc region.
  • composition according to any one of the preceding embodiments, wherein the immunoglobulin Fc region is a non-glycosylated Fc region derived from human IgG4.
  • the conjugate is a covalent bond formed by reacting one end of L with an amine group or thiol group of F, and the other end of L with an amine group or thiol group of X, respectively, to F and X
  • F and X Each is characterized in that it is connected.
  • composition according to any one of the preceding embodiments, wherein L is polyethylene glycol.
  • composition according to any one of the preceding embodiments, wherein one end of the linker is linked to only one of the two Fc region chains of the dimeric immunoglobulin Fc region.
  • composition according to any one of the preceding embodiments, wherein the composition further comprises a GLP-1 receptor agonist (glucagon like peptide 1 receptor agonist).
  • GLP-1 receptor agonist glucagon like peptide 1 receptor agonist
  • composition according to any one of the preceding embodiments, wherein the GLP-1 receptor agonist is GLP-1, exendin-3, exendin-4, an agonist, derivative, or fragment thereof. , variants and combinations thereof.
  • CA exendin-4 imidazo-acetyl exendin-4
  • Another aspect embodying the present invention is a method for preventing or treating chronic kidney disease, comprising administering to an individual a glucagon derivative, a conjugate thereof, or a composition comprising the same.
  • Another aspect embodying the present invention is a glucagon derivative or a conjugate thereof, and a GLP-1 receptor agonist or a conjugate thereof; Or a method for preventing or treating chronic kidney disease comprising administering a composition comprising them to an individual.
  • Another aspect embodying the present invention is the use of a glucagon derivative or a conjugate thereof, or a composition comprising the same for the prevention or treatment of chronic kidney disease.
  • Another aspect embodying the present invention is a glucagon derivative or a conjugate thereof, and a GLP-1 receptor agonist or a conjugate thereof; Or the use of a composition comprising them for the prevention or treatment of chronic kidney disease.
  • Another aspect embodying the present invention is the use of a glucagon derivative or a conjugate thereof, or a composition comprising the same, for the manufacture of a medicament for preventing or treating chronic kidney disease.
  • Another aspect embodying the present invention is a glucagon derivative or a conjugate thereof, and a GLP-1 receptor agonist or a conjugate thereof; Or it is a use for the manufacture of a medicament having a preventive or therapeutic use of a composition comprising the same for chronic kidney disease.
  • Aib may be used interchangeably with “2-aminoisobutyric acid” or “aminoisobutyric acid”, and 2-aminoisobutyric acid and aminoiso Butyric acid (aminoisobutyric acid) may be used in combination.
  • compositions comprising a glucagon derivative or a conjugate thereof.
  • the composition is a pharmaceutical composition for preventing or treating chronic kidney disease including a glucagon derivative or a conjugate thereof.
  • the pharmaceutical composition for preventing or treating chronic kidney disease may be a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a glucagon derivative or a conjugate thereof in a pharmaceutically effective amount.
  • a composition comprising a glucagon derivative or a conjugate thereof according to the present invention as an active ingredient may have a preventive or therapeutic effect on chronic kidney disease by lowering blood pressure and restoring renal function through action on glucagon receptors.
  • composition of the present invention may exhibit an effect of preventing or treating chronic kidney disease.
  • chronic kidney disease may be used interchangeably with chronic kidney disease, and the glomerular filtration rate is 3 months or more regardless of whether there is a structural or functional abnormality of the kidney or kidney damage. It is an independent disease name defined as a decrease of less than 60 mL/min/1.73 m2 for more than one month. Chronic kidney disease is divided into five stages according to the progression and applied to clinical practice. Damage to the kidneys caused by high blood sugar or high blood pressure for a long time increases the risk of developing chronic kidney disease. It is known to be a cause of chronic kidney disease.
  • the chronic kidney disease is from the group consisting of nephrotic syndrome, renal tubular dysfunction, renal hypertension, uremia, renal failure, and chronic renal failure. It may include any one or more selected diseases, but is not limited thereto.
  • the glucagon derivative or conjugate thereof according to the present invention may have an effect of lowering blood pressure and/or restoring renal function.
  • the drop in blood pressure may mean a decrease in systolic blood pressure ( SBP) and diastolic blood pressure ( DBP), but is not limited thereto.
  • the kidney function recovery refers to the recovery of the kidney's intrinsic function of filtering out wastes in the blood, and can be measured in various ways.
  • albumin output As a non-limiting example of the measurement method, albumin output, glomerular filtration rate, proteinuria, creatine concentration, etc. may be measured, but the present invention is not limited thereto.
  • proteinuria refers to a symptom in which a protein (eg, albumin, globulin, etc.) higher than a normal value is included in the urine of a chronic kidney disease patient whose glomerular filtration rate is lowered.
  • Albumin is a protein that accounts for about 40% of urine protein, and the level of proteinuria can be confirmed by measuring the level of albumin in the urine.
  • the glucagon derivative or conjugate thereof according to the present invention, or a composition comprising the same may reduce the amount of protein (eg, albumin) excreted outside the body by restoring renal function. Therefore, in the present invention, proteinuria reduction is It can be used in the same sense as the restoration of kidney function.
  • the glucagon derivative or conjugate thereof according to the present invention, or a composition comprising the same may exhibit a blood pressure lowering effect on an individual when administered to an individual.
  • the glucagon derivative or conjugate thereof, or a composition comprising the same may be to restore the renal function of the subject when administered to the subject.
  • the lowering of blood pressure and restoration of renal function may occur independently of each other by the glucagon derivative or conjugate thereof according to the present invention, or a composition comprising the same, or may occur together.
  • subject may mean mammals including rats, livestock, etc. including humans with kidney damage, kidney dysfunction, and/or chronic kidney disease, and may reduce blood pressure and/or restore kidney function. Therefore, if a beneficial effect can be obtained, it is not particularly limited.
  • the essential hypertension model (spontaneously hypertensive rat, SHR) used in the examples of the present invention is known as a chronic kidney disease-related model because kidney damage due to congenital hypertension is induced, and is used to evaluate the efficacy of a blood pressure lowering agent.
  • SHR spontaneous hypertensive rat
  • the glucagon derivative or conjugate thereof according to the present invention may exhibit one or more of the following characteristics in the administered subject, but is not limited thereto:
  • the glucagon derivative or conjugate thereof according to the present invention has the above properties and may have a preventive or therapeutic effect on chronic kidney disease.
  • glucagon is a kind of hormone secreted by alpha cells of pancreas, and serves to increase the concentration of glucose by promoting the decomposition of glycogen, thereby raising blood sugar in the body.
  • Native glucagon may have the following amino acid sequence:
  • the glucagon derivative of the present invention is a peptide having one or more differences in amino acid sequence compared to native glucagon, a peptide modified through modification of the native glucagon sequence, and a glucagon receptor capable of activating the same as native glucagon. mimics of native glucagon.
  • Such a natural glucagon derivative may have an effect of lowering blood pressure and/or restoring renal function in vivo.
  • the glucagon derivative may be non-naturally occurring.
  • the glucagon derivative refers to a peptide capable of acting on the glucagon receptor at a significant level compared to native glucagon.
  • the glucagon derivative may be used in combination with “peptide having activity on the glucagon receptor”, “glucagon analog” or “peptide”.
  • the glucagon derivative of the present invention is a peptide having an activity on a glucagon receptor, wherein the activity on the receptor is 0.1% or more, 0.2% or more, 1% or more, 2% or more of the in vitro activity of the receptor compared to the native type. , 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60 % or more, 70% or more, 80% or more, 90% or more, 100% or more, or 200% or more. However, it is not limited thereto.
  • the glucagon derivative of the present invention has an in vitro activity for the glucagon receptor of about 0.001% or more, about 0.01% or more, about 0.1% or more, 0.2% or more, about 1% or more, compared to the natural ligand (native glucagon) of the receptor.
  • the peptide having an activity on the glucagon receptor of the present invention may have a relative activity on the glucagon receptor of 5% or more, 10% or more, 20% or more, or 30% or more compared to native glucagon, but is not limited thereto.
  • the term "about” includes all ranges including ⁇ 0.5, ⁇ 0.4, ⁇ 0.3, ⁇ 0.2, ⁇ 0.1, and the like, and includes all values in a range equal to or similar to the value following the term about, but not limited
  • the glucagon derivative is an agonist of the glucagon receptor, or an analog in which a mutation selected from the group consisting of substitution, addition, deletion, modification, and combinations thereof to one or more amino acids in native glucagon , or a fragment.
  • agonist of glucagon receptor has the same meaning as “agonist” or “agonist”, and refers to a substance that can function like glucagon in an actual living body by binding to the glucagon receptor.
  • glucagon analog means a non-natural glucagon different from natural glucagon.
  • Such glucagon analogs include analogs in which some amino acids of native glucagon are modified in the form of addition, deletion or substitution.
  • fragment refers to a form in which one or more amino acids are removed from the amino terminus or carboxy terminus of natural glucagon or a derivative of glucagon while having the effect of preventing or treating chronic kidney disease in the body.
  • the glucagon derivative of the present invention may be modified through any one of substitution, addition, deletion and modification of some amino acids in native glucagon or a combination of these methods. Substitution of such amino acids may generally occur based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of residues, and conservative substitutions with amino acids having such similar properties are It can be expected to exhibit the same or similar activity.
  • glucagon derivatives prepared by a combination of these methods include a peptide having an amino acid sequence different from that of native glucagon by one or more and deamination at the N-terminal amino acid residue and having an activating function for the glucagon receptor. , but is not limited thereto, and a natural glucagon derivative applied to the present invention can be prepared by a combination of various methods for preparing the derivative.
  • L- or D-form amino acids for the production of peptides, and / or modifications using non-natural amino acids; and/or by modifying the native sequence, e.g., modification of side chain functional groups, intramolecular covalent bonds, such as inter-side chain ring formation, methylation, acylation, ubiquitination, phosphorylation, aminohexylation, biotinylation, etc. includes all that Also, the above modifications include all substitutions with non-naturally occurring compounds.
  • amino acids to be substituted or added may be atypical or non-naturally occurring amino acids as well as the 20 amino acids commonly observed in human proteins.
  • Commercial sources of atypical amino acids include Sigma-Aldrich, ChemPep and Genzyme Pharmaceuticals. Peptides containing these amino acids and canonical peptide sequences can be synthesized and purchased from commercial peptide synthesis companies, for example, American peptide company or Bachem in the United States, or Anygen in Korea.
  • Amino acid derivatives can also be obtained in the same manner, and 4-imidazoacetic acid can be used to name just a few examples.
  • glucagon derivative of the present invention may be prepared by using the above preparation methods independently or in combination.
  • the glucagon derivative may be a peptide comprising the amino acid sequence of the following general formula (1).
  • X1 is tyrosine
  • X2 is alpha-methyl-glutamic acid ( ⁇ -methyl-glutamic acid), Aib (aminoisobutyric acid), D-alanine, glycine, Sar (N-methylglycine), serine or D-serine;
  • X7 is threonine, valine or cysteine
  • X10 is tyrosine or cysteine
  • X12 is lysine or cysteine
  • X13 is tyrosine or cysteine
  • X14 is leucine or cysteine
  • X15 is aspartic acid, glutamic acid or cysteine
  • X16 is glutamic acid, aspartic acid, serine, alpha-methyl-glutamic acid, or cysteine or is absent;
  • X17 is aspartic acid, glutamine, glutamic acid, lysine, arginine, serine, cysteine, or valine or is absent;
  • X18 is alanine, aspartic acid, glutamic acid, arginine, valine, or cysteine or is absent;
  • X19 is alanine, arginine, serine, valine, or cysteine or is absent;
  • X20 is lysine, histidine, glutamine, aspartic acid, arginine, alpha-methyl-glutamic acid, or cysteine or is absent;
  • X21 is aspartic acid, glutamic acid, leucine, valine, or cysteine or is absent;
  • X23 is isoleucine, valine, or arginine or is absent;
  • X24 is valine, arginine, alanine, cysteine, glutamic acid, lysine, glutamine, alpha-methyl-glutamic acid, or leucine or is absent;
  • X27 is isoleucine, valine, alanine, lysine, methionine, glutamine, or arginine or is absent;
  • X28 is glutamine, lysine, asparagine, or arginine or is absent;
  • X29 is threonine (T);
  • X30 may be cysteine or absent
  • X2 is Aib (aminoisobutyric acid);
  • X7 is threonine (T), valine (V) or cysteine (C);
  • X10 is tyrosine (Y);
  • X12 is lysine (K);
  • X13 is tyrosine (Y);
  • X14 is leucine (L) or cysteine (C);
  • X15 is aspartic acid (D);
  • X16 is glutamic acid (E) or serine (S);
  • X17 is lysine (K), arginine (R), or cysteine (C);
  • X18 is arginine (R);
  • X19 is alanine (A), or cysteine (C);
  • X20 is glutamine (Q), or lysine (K);
  • X21 is aspartic acid (D) or glutamic acid (E);
  • X23 is valine (V);
  • X24 is glutamine (Q);
  • X27 is methionine (M).
  • X28 is asparagine (N);
  • X29 is threonine (T);
  • X30 may be cysteine (C) or absent.
  • the glucagon derivative comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 45, specifically, (essentially) consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 45 may be, but is not limited thereto.
  • the glucagon derivative comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 11, 13 to 45, specifically, an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 11, 13 to 45 (essentially ) may be configured, but is not limited thereto.
  • the glucagon derivative may include or (essentially) an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 11, and 13 to 25, 27, 29, 31, 33, 35 to 45. , but not limited thereto.
  • the glucagon derivative contains or (essentially) an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 22, 23, 27, 33, 35, 37, 38, 40, 41, 42, and 44 may, but is not limited thereto.
  • the glucagon derivative of the present invention has at least 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more sequence identity may be mentioned, and as long as it has a preventive or therapeutic effect on chronic kidney disease, it is not limited to a specific sequence.
  • the glucagon derivative of the present invention comprises the amino acid sequence of any one of SEQ ID NOs: 2 to 45, (essentially) consists of the amino acid sequence of any one of SEQ ID NOs: 2 to 45, or any one of SEQ ID NOs: 2 to 45 one amino acid sequence and at least 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, Or it may include a peptide having a sequence identity of 95% or more, and as long as it has a preventive or therapeutic effect on chronic kidney disease, it is not limited to a specific sequence.
  • the term 'homology' or 'identity' refers to the degree to which two given amino acid sequences or nucleotide sequences are related to each other and may be expressed as a percentage.
  • Whether any two peptide sequences have homology, similarity or identity can be determined, for example, by Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444, using a known computer algorithm such as the "FASTA” program. or, as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later), The Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) can be used to determine.
  • GAP program is defined as the total number of symbols in the shorter of two sequences divided by the number of similarly aligned symbols (ie, amino acids).
  • 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 above-described glucagon derivative of the present invention may include an intramolecular bridge (eg, a covalent bridge or a non-covalent bridge), and specifically may have a form including a ring between amino acid residues.
  • an intramolecular bridge eg, a covalent bridge or a non-covalent bridge
  • it may be in the form of a ring formed between amino acids 12 and 16 or 16 and 20 of the peptide, but is not particularly limited thereto.
  • Non-limiting examples of the ring may include a lactam bridge (or lactam ring).
  • the glucagon derivative includes all those modified to include a ring and an amino acid capable of forming a ring at a desired position.
  • Such a ring may be formed between the amino acid side chains in the glucagon derivative, for example, a lactam ring may be formed between the lysine side chain and the glutamic acid side chain, but is not particularly limited thereto.
  • the peptide comprising the amino acid sequence of the general formula 1 is the amino acid pair of X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24, and X24 and X28 of the general formula 1, each amino acid pair
  • Each of the amino acids may be substituted with glutamic acid or lysine, but is not limited thereto.
  • Xn (n is a natural number)
  • n represents an amino acid position from the N-terminus of the presented amino acid sequence.
  • the peptide comprising the amino acid sequence of Formula 1 is substituted with glutamic acid or lysine in which each amino acid of the amino acid pair of X12 and X16, the amino acid pair of X16 and X20, or the amino acid pair of X17 and X21 can form a ring may be, but is not limited thereto.
  • each amino acid of each amino acid pair in the above general formula 1 It may form a ring (eg, a lactam ring), but is not limited thereto.
  • X16 is glutamic acid
  • X20 is lysine
  • the side chains of X16 and X20 may form a lactam ring, but is not limited thereto.
  • the glucagon derivative of the present invention may have an unmodified N-terminus and/or C-terminus, but its N-terminus and/or C-terminus, etc.
  • This chemically modified form, protected by an organic group, or modified by adding an amino acid to the terminus of the glucagon derivative is also included in the scope of the glucagon derivative according to the present invention.
  • the terminal of the glucagon derivative according to the present invention has a carboxyl group, but is not particularly limited thereto.
  • the N-terminus is acetylated and/or the C-terminus is amidated to remove these charges. ), but is not particularly limited thereto.
  • the N-terminus or C-terminus of the glucagon derivative of the present invention may have an amine group (-NH2) or a carboxyl group (-COOH), but is not limited thereto.
  • the glucagon derivative may be in the form of a conjugate to which a biocompatible material portion capable of increasing its in vivo half-life is bound, but is not limited thereto.
  • the term "persistent conjugate” or “conjugate” has a structure in which the glucagon derivative and a biocompatible material are combined, and may exhibit increased potency persistence compared to a glucagon derivative to which the biocompatible material is not bound.
  • the biocompatible material may be covalently linked to a glucagon derivative, but is not particularly limited thereto.
  • the glucagon derivative which is a component of the conjugate, may be a peptide or fragment in which one or more amino acids are substituted, added, removed, modified, or a combination thereof is mutated in the native glucagon sequence. It may not lose activity by binding, but as long as it has a preventive or therapeutic effect on chronic kidney disease, it may be used as one component of the conjugate of the present invention without limitation.
  • the glucagon derivative contained in the conjugate may be a peptide comprising the amino acid sequence of General Formula 1 described above.
  • the glucagon derivative included in the conjugate may be a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 2 to 45.
  • the glucagon derivative included in the conjugate may be a peptide comprising any one of the amino acid sequences of SEQ ID NOs: 2 to 11, and 13 to 45, but is not limited thereto.
  • biocompatible material refers to a physiologically active material that is bound to the glucagon derivative of the present invention to increase the durability of the effect of the physiologically active material compared to the physiologically active material not bound to the biocompatible material part or carrier. refers to the substance.
  • the biocompatible material may be covalently linked to a physiologically active material, but is not particularly limited thereto.
  • the biocompatible material includes high molecular weight polymers, fatty acids, cholesterol, albumin and fragments thereof, albumin binding materials, polymers of repeat units of specific amino acid sequences, antibodies, antibody fragments, FcRn binding materials, in vivo connective tissue, nucleotides, fibronectin, It may be selected from the group consisting of transferrin, polysaccharide, heparin, and elastin, but is not particularly limited thereto.
  • the FcRn binding material may be an immunoglobulin Fc region, more specifically, an IgG Fc region, but is not particularly limited thereto.
  • One or more amino acid side chains in the peptides of the invention may be conjugated to such biocompatible materials to increase solubility and/or half-life and/or increase bioavailability in vivo. Such modifications may also reduce clearance of therapeutic proteins and peptides.
  • the biocompatible material described above may be water-soluble (amphiphilic or hydrophilic) and/or non-toxic and/or pharmaceutically acceptable.
  • the long-acting conjugate of the glucagon derivative of the present invention refers to a form in which the immunoglobulin Fc region is linked to the glucagon derivative.
  • the conjugate may be one in which the immunoglobulin Fc region is covalently linked to the glucagon derivative through a linker, but is not particularly limited thereto.
  • the long-acting conjugate of the glucagon derivative may exhibit increased potency persistence compared to the glucagon derivative to which the immunoglobulin Fc region is not bound. and "long-acting conjugate of glucagon derivative" may be used interchangeably.
  • the glucagon derivative or peptide is in the form of a long-acting conjugate
  • the long-acting conjugate may be a conjugate represented by Formula 1 below:
  • X is a peptide (glucagon derivative) comprising the amino acid sequence of Formula 1;
  • L is a linker containing an ethylene glycol repeating unit
  • F is an immunoglobulin Fc region
  • the long-acting conjugate of the present invention is characterized in that it contains a peptide (glucagon derivative) comprising the amino acid sequence of Formula 1 as a part of the conjugate.
  • the conjugate of the present invention can exhibit significant activity on the glucagon receptor like the glucagon derivative even in the form of the conjugate, and thus can exert the prevention or treatment efficacy of chronic kidney disease through lowering blood pressure and restoring renal function.
  • the conjugate of the present invention has about 0.01% or more, 0.1% or more, 0.2% or more, 0.5% or more, 0.7% or more, 1% or more, 2% or more, 3 % or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more , 70% or more, 80% or more, 90% or more, 100% or more, but is not limited thereto.
  • the peptide (glucagon derivative) or a conjugate thereof has an activity of about 0.01% or more, about 0.1% or more, 1% or more, 2% or more, 3% or more, 4% or more on the glucagon receptor compared to native glucagon. or more, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 200% or more
  • the present invention is not limited thereto.
  • composition of the present invention may include (i) a glucagon derivative having activity on the glucagon receptor or (ii) a long-acting conjugate of the glucagon derivative, and the long-acting conjugate is based on increased persistence in the body, excellent chronic It can show the effect of preventing or treating kidney disease.
  • F is a biocompatible substance that increases the half-life, specifically X, that is, a substance capable of increasing the half-life of the glucagon derivative of the present invention, and corresponds to one configuration of the moiety constituting the conjugate of the present invention. .
  • the F may be bonded to each other by a covalent chemical bond or a non-covalent chemical bond with X, and F and X may be bonded to each other through L through a covalent chemical bond, a non-covalent chemical bond, or a combination thereof.
  • X and L, and L and F may be connected to each other by a covalent bond, and in this case, the conjugate is a compound in which X, L, and F are each connected via a covalent bond in the order of Formula 1 .
  • F may be an immunoglobulin Fc region.
  • immunoglobulin Fc region refers to a region including heavy chain constant region 2 (CH2) and/or heavy chain constant region 3 (CH3) portions, excluding the heavy and light chain variable regions of immunoglobulin.
  • the immunoglobulin Fc region may be one component constituting a moiety of the conjugate of the present invention. Specifically, it corresponds to F in Formula 1 above.
  • F may be a human immunoglobulin region, but is not limited thereto.
  • F is a structure in which two polypeptide chains are connected by a disulfide bond, and is connected only through a nitrogen atom of one of the two chains. It may have a structure that is, but is not limited thereto. The linkage via the nitrogen atom may be linked via reductive amination to the epsilon amino atom or the N-terminal amino group of lysine.
  • Reductive amination reaction means a reaction in which an amine group or an amino group of a reactant reacts with an aldehyde (i.e., a functional group capable of reductive amination) of another reactant to form an amine, and then forms an amine bond by a reduction reaction, It is an organic synthesis reaction well known in the art.
  • aldehyde i.e., a functional group capable of reductive amination
  • the F may be connected through the nitrogen atom of the N-terminal proline, but is not limited thereto.
  • the immunoglobulin Fc region is one component constituting a moiety of the conjugate of Formula 1 of the present invention, and specifically, may correspond to F in Formula 1 above.
  • the immunoglobulin Fc region may include a hinge region in the heavy chain constant region, but is not limited thereto.
  • flankinge sequence refers to a region that is located on a heavy chain and forms 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 portion 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 deleting the 8th or 11th cysteine residue in the hinge sequence of SEQ ID NO:47.
  • the hinge sequence of the present invention may be composed 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: 48), Glu-Ser- Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Pro (SEQ ID NO: 49), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 50), Glu- Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Pro (SEQ ID NO: 51), Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 52), Glu-Ser-Lys- Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 53), Glu-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 54),
  • the hinge sequence may include the amino acid sequence of SEQ ID NO: 57 (Pro-Ser-Cys-Pro) or SEQ ID NO: 66 (Ser-Cys-Pro), but is not limited thereto.
  • the immunoglobulin Fc region of the present invention may be in a form in which two immunoglobulin Fc chain molecules form a dimer due to the presence of a hinge sequence.
  • one end of the linker is a dimer immunoglobulin It may be in a form 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, and 1, 2, 3, 4, 5, 6, It may include 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.
  • part or all of the heavy chain constant region 1 (CH1) and/or light chain constant region except for only the heavy and light chain variable regions of immunoglobulin 1 (CL1) may be an extended Fc region. It may also be a region in which some fairly long amino acid sequences corresponding to CH2 and/or CH3 have been removed.
  • the immunoglobulin Fc region of the present invention comprises 1) a CH1 domain, a CH2 domain, a CH3 domain and a CH4 domain, 2) a CH1 domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, 5) a combination of one or two or more of the CH1 domain, the CH2 domain, the CH3 domain and the CH4 domain with an immunoglobulin hinge region (or a part of the hinge region), 6) heavy chain constant region
  • Each domain may be a dimer of each domain and the light chain constant region . However, it is not limited thereto.
  • the immunoglobulin Fc region may be in the form of a dimer or multimer composed of single-chain immunoglobulins composed of domains of the same origin, but is not limited thereto.
  • the immunoglobulin Fc region F is a dimer (dimer) consisting of two polypeptide chains, wherein the Fc region dimer F and X are shared through one and the same linker L containing an ethylene glycol repeating unit. are associatively connected.
  • X is covalently linked via a linker L to only one of the two polypeptide chains of this Fc region dimer F.
  • only one molecule of X is covalently linked via L to one of the two polypeptide chains of the Fc region dimer F to which X is linked.
  • F is a homodimer.
  • the immunoglobulin Fc region F is a dimer consisting of two polypeptide chains, and one end of L may be connected to only one of the two polypeptide chains, but is not limited thereto. .
  • the long-acting conjugate of the present invention it is also possible for two molecules of X to bind symmetrically to one Fc region in a dimeric form.
  • the immunoglobulin Fc region and X may be linked 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 includes a native amino acid sequence as well as a sequence derivative thereof.
  • An amino acid sequence derivative means that one or more amino acid residues in a natural amino acid sequence have a different sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof.
  • amino acid residues 214 to 238, 297 to 299, 318 to 322, or 327 to 331 known to be important for binding may be used as suitable sites for modification.
  • various types of derivatives are possible, such as a site capable of forming a disulfide bond is removed, some amino acids at the N-terminus of the native Fc are removed, or a methionine residue may be added to the N-terminus of the native Fc do.
  • the complement binding site eg, the C1q binding site
  • the ADCC antibody dependent cell mediated cytotoxicity
  • 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 against heat, pH, etc. of the Fc region.
  • the Fc region may be obtained from a native type isolated in vivo from animals such as humans, cows, goats, pigs, mice, rabbits, hamsters, rats or guinea pigs, or obtained from transformed animal cells or microorganisms. It may be recombinant or a derivative thereof.
  • the method of obtaining from the native type may be a method of obtaining whole immunoglobulin by isolating it from a living body of a human or animal, 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 may be separated using size-exclusion chromatography or the like.
  • it is a recombinant immunoglobulin Fc region obtained by obtaining a human-derived Fc region from a microorganism.
  • the immunoglobulin Fc region may have a native sugar chain, an increased sugar chain compared to the native type, a decreased sugar chain compared to the native type, or a form in which the sugar chain is removed.
  • Conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms may be used for the increase or decrease or removal of such immunoglobulin Fc sugar chains.
  • the immunoglobulin Fc region from which the sugar chain is removed from the Fc has significantly reduced binding to complement (c1q) and reduced or eliminated antibody-dependent cytotoxicity or complement-dependent cytotoxicity, so that unnecessary immune responses in vivo are not induced. does not In this respect, a form more suitable for the original purpose as a drug carrier will be an immunoglobulin Fc region in which sugar chains are removed or non-glycosylated.
  • deglycosylation refers to an Fc region from which sugars are removed with an enzyme
  • aglycosylation refers to an Fc region that is not glycosylated by production in prokaryotes, in a more specific embodiment, in E. coli. .
  • the immunoglobulin Fc region may be of human or animal origin such as cattle, goats, pigs, mice, rabbits, hamsters, rats, and guinea pigs, 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, or a combination or 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, which is known to enhance the half-life of ligand binding proteins. In an even 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 region is a region of human IgG4 Fc, and is a homologous region in which two monomers are linked through a disulfide bond (inter-chain form) between cysteine, amino acid 3 of each monomer. It may be in the form of a dimer, where each monomer of the homodimer is independently an internal disulfide bond between cysteines at positions 35 and 95 and an internal disulfide bond between cysteines at positions 141 and 199, that is, two internal and/or may have disulfide bonds (intra-chain form).
  • the number of amino acids of each monomer may consist of 221 amino acids, and the amino acids forming the homodimer may consist of a total of 442 amino acids, but is not limited thereto.
  • two monomers having the amino acid sequence of SEQ ID NO: 67 (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 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 is not limited thereto.
  • F of Formula 1 may include a monomer having an amino acid sequence of SEQ ID NO: 67, and F may be a homodimer of a monomer having an amino acid sequence of SEQ ID NO: 67, but is not limited thereto.
  • the immunoglobulin Fc region may be a homodimer comprising the amino acid sequence of SEQ ID NO: 68 (consisting of 442 amino acids), but is not limited thereto.
  • the immunoglobulin Fc region and X may not be glycosylated, but is not limited thereto.
  • “combination” means that when a dimer or a multimer is formed, a polypeptide encoding a single-chain immunoglobulin Fc region of the same origin forms a bond with a single-chain polypeptide of a different origin. That is, it is possible to prepare a dimer or multimer 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 that means 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 domains consisting of 1 to 4 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 in the present invention, a combination thereof or hybridization thereof is also possible. Specifically, they are subclasses of IgG2 and IgG4, and most specifically, the Fc region of IgG4, which has little effector function such as complement dependent cytotoxicity (CDC).
  • CDC complement dependent cytotoxicity
  • the above-described conjugate may have an increased duration of effect compared to native glucagon, or compared to unmodified X, such a conjugate is not only in the above-described form, but also in the form encapsulated in biodegradable nanoparticles.
  • L may be a non-peptidyl linker, for example, a linker containing an ethylene glycol repeating unit.
  • non-peptidyl linker includes a biocompatible polymer in which two or more repeating units are bonded. The repeating units are linked to each other through any covalent bond other than a peptide bond.
  • the non-peptidyl linker may be one component constituting the moiety of the conjugate of the present invention, and corresponds to L in Formula 1 above.
  • the non-peptidyl linker that can be used in the present invention may be used without limitation as long as it is a polymer resistant to proteolytic enzymes in vivo.
  • the non-peptidyl linker may be used in combination with a non-peptidyl polymer.
  • the non-peptidyl linker may include a reactive group at the end thereof to form a conjugate through reaction with other components constituting the conjugate.
  • a non-peptidyl 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 (linker). moiety) or a non-peptidyl linker linkage.
  • non-peptidyl linker may be a linker containing an ethylene glycol repeating unit, for example, polyethylene glycol, and also derivatives thereof known in the art and easily at the level of skill in the art. Derivatives that can be prepared are also included in the scope of the present invention.
  • the repeating unit of the non-peptidyl linker may be an ethylene glycol repeating unit, and specifically, the non-peptidyl linker includes an ethylene glycol repeating unit, and before being formed into a conjugate, a functional group used in the preparation of the conjugate is at the terminal end. may include.
  • the long-acting conjugate according to the present invention may be in a form in which X and F are linked through the functional group, but is not limited thereto.
  • the non-peptidyl 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 may be polyethylene glycol (PEG) represented by the following formula (2), but is not limited thereto:
  • the PEG moiety in the long-acting conjugate may include, but is not limited to, the -(CH2CH2O)n-structure as well as an oxygen atom intervening between the linking element and the -(CH2CH2O)n-.
  • the ethylene glycol repeating unit may be represented by, for example, [OCH 2 CH 2 ]n, and the value of n is a natural number, the average molecular weight of the [OCH 2 CH 2 ]n site in the long-acting conjugate,
  • the number average molecular weight may be set to be greater than 0 to about 100 kDa, but is not limited thereto.
  • the n value is a natural number and the average molecular weight of the [OCH 2 CH 2 ]n site in the long-acting conjugate, for example, has a number average molecular weight of 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,
  • the conjugate may have a structure in which the glucagon derivative (X) and the immunoglobulin Fc region (F) are covalently linked through a linker containing an ethylene glycol repeating unit, but is not limited thereto.
  • L in the long-acting conjugate, L may be a linker containing an ethylene glycol repeating unit, and F may be a dimer-type immunoglobulin Fc region. More specifically, one molecule of X to one Fc region of the dimer-type immunoglobulin Fc region may be covalently linked to each other through a linker containing the ethylene glycol repeating unit, but is not limited thereto. In addition, in another specific embodiment, one end of the linker containing the ethylene glycol repeating unit may be linked to only one of the two Fc region chains of the dimer immunoglobulin Fc region, but is not limited thereto. .
  • the polyethylene glycol is a term encompassing all forms of ethylene glycol homopolymer, PEG copolymer, or monomethyl-substituted PEG polymer (mPEG), but is not particularly limited thereto.
  • the formula weight of the ethylene glycol repeating unit moiety in L may be in the range of 1 to 100 kDa, but is not limited thereto.
  • the conjugate is shared between F and X through a non-peptide linker comprising a reactive group capable of binding to F, specifically an immunoglobulin Fc region, and X, specifically, a glucagon derivative at both ends. It may be conjointly linked.
  • the non-peptidyl linker that can be used in the present invention may be used without limitation as long as it is a polymer including an ethylene glycol repeating unit that is resistant to proteolytic enzymes in vivo.
  • the molecular weight of the non-peptidyl polymer is, but is not limited to, greater than 0 in the range of about 100 kDa, in the range of about 1 to about 100 kDa, specifically in the range of about 1 to about 20 kDa, or in the range of about 1 to about 10 kDa.
  • the non-peptidyl linker of the present invention coupled to the polypeptide corresponding to F not only one type of polymer but also a combination of different types of polymers may be used.
  • the term "about” includes all ranges including ⁇ 0.5, ⁇ 0.4, ⁇ 0.3, ⁇ 0.2, ⁇ 0.1, and the like, and includes all values in a range equal to or similar to the value following the term about, but not limited
  • the non-peptidyl linker may have reactive groups at both ends in a state in which it is not bound to F and X, and may bind to F and X through the reactive groups.
  • both ends of the linker may bind to a thiol group, an amino group, a hydroxyl group of the immunoglobulin Fc region, and a thiol group, an amino group, an azide group, or a hydroxyl group of the glucagon derivative (X), It is not limited thereto.
  • the linker comprises a reactive group capable of binding to the immunoglobulin Fc region and the glucagon derivative (X) at both ends, specifically, a thiol group of the cysteine of the immunoglobulin Fc region; an amino group located at the N-terminus, lysine, arginine, glutamine and/or histidine; and/or a thiol group of the cysteine of the glucagon derivative (X) bonded to a hydroxyl group located at the C-terminus; amino groups of lysine, arginine, glutamine and/or histidine; azide group of azidolysin; and/or a reactive group capable of bonding to 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.
  • the aldehyde group may be exemplified by a propion aldehyde group or a butyl aldehyde group, but is not limited thereto.
  • succinimidyl valerate succinimidyl methylbutanoate, succinimidyl methylpropionate, succinimidyl butanoate, succinimidyl propionate, N-hydroxysuccini Mead, hydroxy succinimidyl, succinimidyl carboxymethyl or succinimidyl carbonate may be used, but not limited thereto.
  • the linker may be connected to F, which is an immunoglobulin Fc region, and X, which is a glucagon derivative, through a reactive group as described above, and may be converted into a linker linking portion.
  • the final product resulting from reductive amination by aldehyde bonds is much more stable than those linked by amide bonds.
  • the aldehyde reactive group selectively reacts with the N-terminus at a low pH, and can form a covalent bond with a lysine residue at a high pH, for example, pH 9.0.
  • the reactive groups at both ends of the non-peptidyl 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 propion aldehyde group, or a butyl aldehyde group.
  • F specifically, an immunoglobulin Fc region and X can be bound to each end of the non-peptide linker, it is not particularly limited thereto.
  • one end of the non-peptidyl linker may include a maleimide group as a reactive group, and an aldehyde group, a propionaldehyde group, or a butyl aldehyde group at the other end thereof.
  • the hydroxyl group can be activated into the various reactive groups by a known chemical reaction, or a commercially available polyethylene glycol having a modified reactive group is used.
  • the long-acting protein conjugate of the invention can be prepared.
  • the non-peptidyl polymer may be linked to a cysteine residue of X, more specifically, a -SH group of cysteine, but is not limited thereto.
  • cysteine residue 7 cysteine 10
  • cysteine 12 cysteine 13
  • cysteine 14 cysteine 15
  • cysteine 16 cysteine 17, 18
  • the non-peptidyl polymer may be linked to cysteine residue No. 19, cysteine residue 19, cysteine 20, cysteine 21, or cysteine 30, but is not particularly limited thereto.
  • a reactive group of the non-peptidyl polymer may be linked 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 by a thioether bond, and the aldehyde group is F, specifically, the -NH 2 group of immunoglobulin Fc by reductive amination reaction may be connected through, but is not limited thereto, and this corresponds to one example.
  • the N-terminal amino group of the immunoglobulin Fc fragment is linked to an oxygen atom located at one end of PEG 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 the same structure as immunoglobulin Fc, and through a thioether bond, one end of PEG can form a structure linked to a sulfur atom located at the cysteine of the peptide of Formula 1 .
  • the above-mentioned thioether bond is may contain the structure of
  • the non-peptidyl polymer may be linked to a lysine residue of X, more specifically, an amino group of lysine, but is not limited thereto.
  • the reactive group of the non-peptidyl polymer may be linked to -NH 2 located at the N-terminus of the immunoglobulin Fc region, but this corresponds to one example.
  • the glucagon derivative according to the present invention may be linked to a linker having a reactive group through the C-terminus, but this corresponds to one example.
  • C-terminal refers to the carboxy terminus of a peptide, and refers to a position capable of binding to a linker for the purpose of the present invention.
  • it may include all amino acid residues around the C-terminus as well as the most terminal amino acid residue at the C-terminus, and specifically includes the first to 20th amino acid residues from the most terminal. can, but is not limited thereto.
  • the conjugate of Formula 1 may have a structure of Formula 3 below.
  • X is a peptide of Formula 1 described above;
  • F is a human immunoglobulin Fc region
  • n may be a natural number. In this case, the description of n is the same as described above.
  • the long-acting conjugate of Formula 3 is a structure in which the peptide X of Formula 1 of SEQ ID NO: 46 and the human immunoglobulin Fc domain F are covalently linked through an ethylene glycol repeat, each X is Formula 3 In the succinimide ring of , F may be connected to the oxypropylene group of Formula 3.
  • n may be such that the average molecular weight of the [OCH 2 CH 2 ]n site in the peptide conjugate is 1 to 100 kDa, or 1 to 20 kDa or 10 kDa. However, it is not limited thereto.
  • X of the conjugate may be a peptide having activity on the glucagon receptor.
  • the site at which X is connected to the succinimide ring of Formula 3 may be the sulfur atom of the C-terminal cysteine of X.
  • F is a human immunoglobulin Fc region, and a site connected to the oxypropylene group in F is not particularly limited.
  • the moiety of F connected to the oxypropylene group may be an N-terminal nitrogen or a nitrogen atom of an F internal moiety (eg, epsilon nitrogen of lysine).
  • the site where F is connected to the oxypropylene group of Formula 3 may be the N-terminal proline of F, but is not limited thereto.
  • the F may be connected through the nitrogen atom of the N-terminal proline, but is not limited thereto.
  • glucagon derivative As the kind of the glucagon derivative, the kind described in International Patent Application Publication No. WO 2016/108586 and International Patent Publication WO 2017/003191 may be mentioned, and the entire specification of the International Patent Publication is incorporated herein by reference.
  • the method for producing a long-acting conjugate of the glucagon derivative is described in WO 2017/003191, and it is clear that the entire specification of the International Patent Publication is also incorporated herein as a reference.
  • the above-mentioned binder may have increased durability of effect compared to X in which F is not modified.
  • the detailed description of the specification or the description of the claims of the "peptide” or the “conjugate” in which such a peptide is covalently linked to a biocompatible material according to the present invention, as well as the corresponding peptide or conjugate It also applies to the category including all forms of a salt of a peptide or a conjugate (eg, a pharmaceutically acceptable salt of the peptide), or a solvate thereof. Accordingly, even if only a “peptide” or “conjugate” is described in the specification, the description also applies to the specific salt, the specific solvate, and the specific solvate of the specific salt.
  • Such salt form may be, for example, a form using any pharmaceutically acceptable salt.
  • the type of the 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 means a substance that can be effectively used for a desired purpose without causing excessive toxicity, irritation, or allergic reaction within the scope of medical judgment.
  • salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases 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 , benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like.
  • Salts derived from suitable bases may include alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium.
  • solvate refers to a compound in which the peptide, conjugate, or salt thereof according to the present invention forms a complex with a solvent molecule.
  • the length of the peptide of the present invention can be synthesized by a method well known in the art, for example, an automatic peptide synthesizer, or it can be produced by a genetic engineering technique.
  • glucacon derivatives according to the present invention can be synthesized by a number of methods including, for example, methods comprising:
  • a method for obtaining a fragment of a peptide by any combination of (a), (b) and (c), and then ligating the fragments to obtain a peptide, and recovering the peptide.
  • the composition according to the present invention may include a glucagon derivative or a conjugate thereof, and specifically, may include a pharmacologically effective amount of a glucagon derivative or a conjugate thereof. In addition, it may further include a pharmaceutically acceptable carrier.
  • the composition of the present invention may have a use for preventing or treating chronic kidney disease, and may have an effect of lowering blood pressure and/or restoring renal function, but is not limited thereto.
  • the term "pharmaceutically acceptable” means a sufficient amount to exhibit a therapeutic effect and does not cause side effects, and the type of disease, the patient's age, weight, health, sex, and the patient's sensitivity to the drug , administration route, administration method, frequency of administration, treatment period, combination or drugs used at the same time can be easily determined by those skilled in the art according to factors well known in the medical field.
  • Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, dyes and fragrances, etc. for oral administration, and in the case of injections, buffers, preservatives, pain relief
  • a topical agent, solubilizer, isotonic agent, and stabilizer may be mixed and used, and in the case of topical administration, a base, excipient, lubricant, and preservative may be used.
  • the dosage form of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above.
  • oral administration in the case of oral administration, it may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups and wafers, and in the case of injections, it may be prepared in the form of unit dose ampoules or multiple doses.
  • it can be formulated into solutions, suspensions, tablets, pills, capsules, sustained-release preparations, and the like.
  • suitable carriers, excipients and diluents 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 may be used.
  • fillers, anti-agglomeration agents, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be further included.
  • composition of the present invention is any one formulation selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, freeze-dried preparations and suppositories can have
  • the conjugate can be used by mixing with various pharmaceutically acceptable carriers such as physiological saline or organic solvents, and carbohydrates such as glucose, sucrose or dextran, ascorbic acid (ascorbic acid) to increase stability or absorption. acid) or antioxidants such as glutathione, chelating agents, low molecular weight proteins or other stabilizers, etc. may be used as agents.
  • pharmaceutically acceptable carriers such as physiological saline or organic solvents, and carbohydrates such as glucose, sucrose or dextran, ascorbic acid (ascorbic acid) to increase stability or absorption. acid) or antioxidants such as glutathione, chelating agents, low molecular weight proteins or other stabilizers, etc.
  • composition of the present invention is determined according to the type of drug as the active ingredient, along with 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 include the glucagon derivative or a conjugate thereof in a pharmaceutically effective amount, but is not limited thereto.
  • the inclusion of the glucagon derivative or its conjugate in a pharmaceutically effective amount means the degree to which the desired pharmacological activity (eg, prevention or treatment of chronic kidney disease) can be obtained due to the glucagon derivative or its conjugate, and is also administered It may mean a pharmaceutically acceptable level as a level that does not cause toxicity or side effects in an individual or is insignificant, but is not limited thereto. Such a pharmaceutically effective amount may be determined by comprehensively considering the number of administration, patient, formulation, and the like.
  • the pharmaceutical composition of the present invention may contain the component (active ingredient) in an amount of 0.01 to 99% by weight to volume.
  • the total effective amount of the composition of the present invention may be administered to a patient as a single dose, or may be administered by a fractionated treatment protocol in which multiple doses are administered for a long period of time.
  • the composition of the present invention may vary the content of the active ingredient depending on the severity of the disease.
  • the preferred total dose of the conjugate of the present invention may be about 0.0001 mg to 500 mg per kg of body weight of the patient per day.
  • the dosage of the conjugate is determined by considering various factors such as the age, weight, health status, sex, severity of disease, diet and excretion rate of the patient, as well as the administration route and number of treatments of the pharmaceutical composition, and the effective dosage for the patient is determined.
  • the pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route and administration method as long as the effect of the present invention is exhibited.
  • composition of the present invention may be administered once a week, once every 2 weeks, once every 4 weeks, or once a month, but is not limited thereto.
  • composition according to the present invention may further include a GLP-1 receptor agonist (glucagon like peptide 1 receptor agonist), but is not limited thereto.
  • GLP-1 receptor agonist glutagon like peptide 1 receptor agonist
  • the composition of the present invention may be a pharmaceutical composition for preventing or treating chronic kidney disease comprising a glucagon derivative and a GLP-1 receptor agonist, more specifically, a glucagon derivative, a GLP-1 receptor agonist, and a pharmaceutical It may be a pharmaceutical composition for preventing or treating chronic kidney disease, including an excipient that is acceptable, but is not limited thereto.
  • glucagon derivative of the present invention (i) a glucagon derivative of the present invention; and (ii) a pharmaceutical composition comprising a GLP-1 receptor agonist.
  • a glucagon derivative (i) a glucagon derivative; and (ii) the GLP-1 receptor agonist may be administered in an isolated form, but is not limited thereto.
  • the glucagon derivative and the GLP-1 receptor agonist may be formulated as one formulation or separately formulated. glucagon derivatives; And when the GLP-1 receptor agonist is in an isolated form, the glucagon derivative and the GLP-1 receptor agonist may be formulated as separate formulations and administered simultaneously, separately, sequentially, or in reverse order.
  • concurrent administration does not only mean simultaneous administration, but also allows the glucagon derivative and the GLP-1 receptor agonist to act together on an individual so that each substance can perform at a level equal to or higher than its original function. It should be understood as a dosage form. Accordingly, as used herein, the term “combination” should be understood to denote simultaneous, separate, sequential, or reverse administration of a glucagon derivative and a GLP-1 receptor agonist. When the administration is sequential, reversed or separate, the order of administration is not particularly limited, provided that the interval between administration of the second component is such that the beneficial effect of the combination is not lost.
  • kits may include a composition according to the present invention for co-administration of a glucagon derivative and a GLP-1 receptor agonist.
  • the kit according to the present invention may include separate preparations of a glucagon derivative and a GLP-1 receptor agonist, or a glucagon derivative and a GLP-1 receptor agonist formulated as one formulation, and concurrent administration of the two substances It may additionally include a material required for, but is not limited thereto.
  • the glucagon derivative and the GLP-1 receptor agonist may be in the form of a long-acting conjugate in which an immunoglobulin Fc region is linked, but is not limited thereto.
  • GLP-1 glucagon-like peptide-1
  • GLP-1 is a hormone secreted from the small intestine and is known to promote intracellular glucose uptake. In the small intestine, the glucagon precursor is broken down into three peptides: glucagon, GLP-1, and GLP-2.
  • GLP-1 refers to GLP-1(1-37), which is a form without insulin secretion function, and is processed into GLP-1(7-37) form to become active GLP-1(7-37).
  • the GLP-1 (7-37) amino acid sequence is as follows:
  • the GLP-1 receptor agonist refers to an agent capable of acting on the GLP-1 receptor, and may include a variety of substances, such as compounds or peptides, but is not limited thereto.
  • the GLP-1 receptor agonist has an in vitro activity of not only native GLP-1 but also GLP-1 receptor of 0.1% or more, 1% or more, or 2% of the corresponding receptor's native ligand (native GLP-1). or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more may be a substance (eg, a peptide) having a significant level of activity for the GLP-1 receptor, but is not limited thereto .
  • a substance eg, a peptide
  • a method for measuring the in vitro activity of such a GLP-1 receptor agonist can be achieved using various in vitro activity measurements known in the art.
  • the GLP-1 receptor agonist according to the present invention has a significant level of activity on the GLP-1 receptor, and compares the native GLP-1 with a peptide having at least one difference in the amino acid sequence and the native GLP-1 sequence. It may include, but is not limited to, peptides modified through modification, or mimics of native GLP-1.
  • the GLP-1 receptor agonist is GLP-1, exendin-3, exendin-4, agonists, derivatives, fragments, variants, and agonists thereof. It may include, but is not limited to, those selected from the group consisting of combinations thereof.
  • agonist has the same meaning as “agonist” or “agonist”, and refers to a substance capable of performing the same function as a natural ligand in an actual living body by binding to a receptor.
  • a "derivative" has a significant level of activity against the GLP-1 receptor, some sequences of native GLP-1 are modified, and some groups of amino acid residues are chemically substituted (eg, alpha-methylation, alpha -hydroxylation), removed (eg, deamination) or modified (eg, N-methylation) may be in the form, but is not limited thereto.
  • the GLP-1 receptor agonist of the present invention is a derivative in which the N-terminal amino group is removed (Desamino-histidyl derivative), a derivative in which an amino group is substituted with a hydroxyl group (beta-hydroxy imidazopropionyl-derivative), and two amino groups are added to the amino group.
  • Derivatives modified with a methyl (methyl) residue Dimethyl-histidyl-derivatives
  • derivatives in which the amino group at the amino terminus is substituted with a carboxyl group beta-carboxyimidazopropionyl-derivatives
  • Derivatives from which the positive charge of the amino group is removed by leaving only the (imidazoacetyl) group Imidazoacetyl-derivatives
  • other types of amino-terminal mutated derivatives are included in the scope of the present invention.
  • the GLP-1 receptor agonist is a derivative of chemically mutating the N-terminal amino group or amino acid residue of exendin-4, and more specifically, the first amino acid at the amino terminus of exendin-4.
  • exendin-4 derivative in which the alpha amino group or alpha carbon present in the alpha carbon of the histidine residue is substituted or removed, and more specifically, desamino-histidyl-exendin-4 (Desamino- histidylexendin-4, DA-exendin-4), beta-hydroxy imidazopropionyl-exendin-4 substituted with a hydroxyl group or a carboxyl group (beta-hydroxy imidazopropionyl-exendin-4, HY-exendin-4) ), beta-carboxyimidazopropyl-exendin-4 (beta-carboxyimidazopropionyl-exendin-4, CX-exendin-4), dimethyl-histidyl-exendin-4 modified with two methyl residues (Dimethyl-histidyl -exendin-4, DM-exendin-4), or imidazoacetyl-exendin-4 (Imidazoacetyl-
  • GLP-1 receptor agonists of the present invention include, wherein the N-terminal histidine residue is des-amino-histidyl, dimethyl-histidyl, beta-hydroxy imidazopropionyl, 4-imidazoacetyl and beta and a GLP-1 receptor agonist derivative substituted with a substance selected from the group consisting of -carboxy imidazopropionyl.
  • GLP-1 fragment refers to a form in which one or more amino acids are added or deleted at the N-terminus or C-terminus of native GLP-1 while having a significant level of activity against the GLP-1 receptor. and the added amino acid may be an amino acid that does not exist in nature (eg, D-type amino acid).
  • GLP-1 variant refers to a peptide having a significant level of activity against the GLP-1 receptor and having at least one amino acid sequence different from that of native GLP-1, and may be used interchangeably with “analog”. have.
  • the GLP-1 mutant is modified through any one of substitution, addition, deletion and modification of some amino acids in native GLP-1 or a combination of these methods. it could be
  • the amino acid sequence is different from that of native GLP-1 by at least one, and the activation function for the GLP-1 receptor is deamination at the N-terminal amino acid residue. and peptides possessed, but is not limited thereto.
  • amino acids are added to the amino and/or carboxy terminus of native GLP-.
  • the amino acids to be substituted or added may be atypical or non-naturally occurring amino acids as well as the 20 amino acids commonly observed in human proteins.
  • Commercial sources of atypical amino acids include Sigma-Aldrich, ChemPep and Genzyme Pharmaceuticals. Peptides containing these amino acids and canonical peptide sequences can be synthesized and purchased from commercial peptide synthesis companies, for example, American peptide company or Bachem in the United States, or Anygen in Korea.
  • Amino acid derivatives can also be obtained in the same manner, and 4-imidazoacetic acid can be used to name just a few examples.
  • modifications for the preparation of derivatives or variants include modifications with L-form or D-form amino acids, and/or non-natural amino acids; and/or by modifying the native sequence, e.g., modification of side chain functional groups, intramolecular covalent bonds, such as inter-side chain ring formation, methylation, acylation, ubiquitination, phosphorylation, aminohexylation, biotinylation, etc. includes all that Also, the above modifications include all substitutions with non-naturally occurring compounds.
  • Exendin-3 and exendin-4 of the present invention are GLP-1 receptor agonists consisting of 39 amino acids that show 53% amino acid sequence similarity to GLP-1, and amino acids of exendin-3 and exendin-4 The sequence is as follows.
  • HSDGT FTSDL SKQME EEAVR LFIEW LKNGG PSSGA PPPS (SEQ ID NO: 70)
  • HGEGT FTSDL SKQME EEAVR LFIEW LKNGG PSSGA PPPS (SEQ ID NO: 71)
  • exendin agonist refers to a substance that binds to the exendin receptor in vivo regardless of the structure of exendin and exhibits the same biological activity as exendin. It shows homology in the amino acid sequence, and some groups of amino acid residues may be chemically substituted (eg, alpha-methylation, alpha-hydroxylation), removed (eg, deamination) or modified (eg, N-methylation). It is not limited thereto.
  • Exendin derivatives include a derivative in which the N-terminal amino group is removed (Desamino-histidyl derivative), a derivative in which an amino group is substituted with a hydroxyl group (beta-hydroxy imidazopropionyl-derivative), and two methyl (methyl) residues in the amino group. (Dimethyl-histidyl-derivative), a derivative in which the amino group at the amino terminal is substituted with a carboxyl group (beta-carboxyimidazopropionyl-derivative), or an imidazoacetyl group by deleting the alpha carbon of the histidine residue at the amino terminal. It may include derivatives (Imidazoacetyl-derivatives) from which the positive charge of the amino group has been removed, and the like, and other types of amino-terminal mutated derivatives also fall within the scope of the present invention.
  • the exendin derivative is a derivative in which the N-terminal amino group or amino acid residue of exendin-4 is chemically mutated, more specifically, the alpha of the histidine residue, which is the first amino acid at the amino terminus of exendin-4.
  • exendin-4 derivative in which the alpha amino group or alpha carbon present in the carbon is substituted or removed, more specifically, desamino-histidyl-exendin-4 (Desamino-histidylexendin-4, DA) in which the N-terminal amino group is removed -Exendin-4), beta-hydroxy imidazopropionyl-exendin-4 substituted with a hydroxyl group or a carboxyl group (beta-hydroxy imidazopropionyl-exendin-4, HY-exendin-4), beta-carboxy imidazopropyl-exendin-4 (beta-carboxyimidazopropionyl-exendin-4, CX-exendin-4), dimethyl-histidyl-exendin-4 modified with two methyl residues (Dimethyl-histidyl-exendin-4, DM-exendin-4) or imidazoacetyl-exendin-4 (Imidazoacetyl
  • Non-limiting examples of the exendin derivative of the present invention include an exendin-4 derivative in which the N-terminal amine group of exendin-4 is removed, and exendin in which the N-terminal amine group of exendin-4 is substituted with a hydroxyl group.
  • exendin fragment refers to a form in which one or more amino acids are added or deleted at the N-terminus or C-terminus of the native exendin, and amino acids that do not exist in nature (eg, D-type amino acids) can also be added. do.
  • An exendin mutant is a peptide having at least one amino acid sequence different from that of the native exendin. Specifically, some amino acids in the native exendin are substitution, addition, deletion, and modification. It may be modified through any one method or a combination of these methods.
  • insulinotropic peptides differing in one or more amino acid sequences and deamination to the N-terminal amino acid residue are also included.
  • GLP-1 receptor agonist of the present invention [(1H-Imidazol-4-yl)-acetyl1]GEGTFTSDL SKQMEEEAVR LFIEWLKNGGPSSGAPPPS (SEQ ID NO: 72) (Bachem) or HGEGTFTSDV SSYLEEQAAK EFIAWLVKG (SEQ ID NO: 73) (Bachem) ), but is not limited thereto.
  • the amino acid sequence is described in the direction from N-terminus to C-terminus.
  • the GLP-1 receptor agonist may be in the form of a long-acting conjugate in which the GLP-1 receptor agonist is linked to an immunoglobulin Fc region, but is not limited thereto.
  • the GLP-1 receptor agonist may be one in which imidazo-acetyl exendin-4 (CA exendin-4) is linked to the immunoglobulin Fc region through a linker containing an ethylene glycol repeating unit, more specifically is a GLP-1 receptor agonist linking one end of a polyethylene glycol linker to lysine (Lys) of the GLP-1 receptor agonist of SEQ ID NO: 72 and linking an immunoglobulin Fc region to the other end of the polyethylene glycol linker It may be a long-acting combination of nist, but is not limited thereto.
  • the GLP-1 receptor agonist may be a GLP-1 receptor agonist efpegleantide, but is not limited thereto.
  • composition of the present invention may include any one of the following (i) to (iv):
  • the pharmaceutical composition of the present invention may contain the glucagon derivative or its conjugate, and the GLP-1 receptor agonist or its conjugate in an amount of 0.01 to 99% by weight to volume.
  • the composition of the present invention contains the glucagon derivative at 0.01 to 5 nmol/kg, 0.05 to 4 nmol/kg, 0.05 to 3.5 nmol/kg, or 0.09 to 2.99 nmol/kg, and the GLP-1 receptor agonist at 0.01 to 5 nmol/kg, 0.05 to 4 nmol/kg, 0.05 to 3.5 nmol/kg, or 0.09 to 2.99 nmol/kg may be included, but is not limited thereto.
  • the composition of the present invention may include a glucagon derivative: a GLP-1 receptor agonist in a molar ratio of 0.01 to 50, 0.02 to 40, or 0.03 to 33.22, but is not limited thereto.
  • the pharmaceutical composition of the present invention may be administered to an individual in an amount of 0.1 to 20 mg, or 0.5 to 16 mg, respectively, of a glucagon derivative or a conjugate thereof, and a GLP-1 receptor agonist or a conjugate thereof to the individual, but is not limited thereto. does not
  • Another aspect embodying the present invention is a method for preventing or treating chronic kidney disease, comprising administering to an individual a glucagon derivative, a conjugate thereof, or a composition comprising the same.
  • the method for preventing or treating chronic kidney disease according to the present invention may further comprise administering to the subject a GLP-1 receptor agonist, a conjugate thereof, or a composition comprising the same.
  • Another aspect embodying the present invention is a glucagon derivative, a conjugate thereof; and a method for preventing or treating chronic kidney disease, comprising co-administering a GLP-1 receptor agonist or a conjugate thereof to a subject.
  • the method comprises administering to a subject a composition comprising a glucagon derivative or a conjugate thereof, and a GLP-1 receptor agonist or a conjugate thereof, or a glucagon derivative or a conjugate thereof; and a GLP-1 receptor agonist or a combination thereof as a separate formulation, but is not limited thereto.
  • the method of the present invention may include, but is not limited to, administering (i) a glucagon derivative and (ii) a GLP-1 receptor agonist as one agent, or administering the separate agents simultaneously, separately, sequentially or in reverse order. .
  • the glucagon derivative, GLP-1 receptor agonist, conjugate, composition, blood pressure lowering, renal function recovery, chronic kidney disease, prevention, and treatment are the same as described above.
  • the subject refers to mammals including mice, livestock, etc., including humans, and may be an individual having chronic kidney disease or a risk of developing, but is not limited thereto.
  • the term "administration" refers to introducing a predetermined substance to a patient (individual) by any suitable method, and the route of administration of the glucagon derivative, GLP-1 receptor agonist, conjugate thereof, or composition is specifically selected therefor.
  • the glucagon derivative, GLP-1 receptor agonist, conjugate or composition thereof may be administered through any general route capable of reaching an in vivo target, for example, intraperitoneal administration, intravenous administration, intramuscular administration
  • Intradermal administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration and the like may be used.
  • the glucagon derivative, GLP-1 receptor agonist, conjugate or composition thereof is a preparation in a unit dosage form suitable for administration in a patient's body according to a conventional method in the pharmaceutical field, specifically, a preparation useful for administration of a protein drug Formulated in the form of oral or skin, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, It may be administered by parenteral routes of administration including, but not limited to, intranasal, intragastrointestinal, topical, sublingual, vaginal or rectal routes.
  • the method of the present invention may include administering a composition comprising the glucagon derivative, the GLP-1 receptor agonist, or a conjugate thereof in a pharmaceutically effective amount.
  • a suitable total daily amount may be determined by a treating physician within the scope of sound medical judgment, and may be administered once or divided into several doses.
  • a specific therapeutically effective amount for a particular patient depends on the type and extent of the response to be achieved, the specific composition, including whether other agents are used, if necessary, the specific composition, the patient's age, weight, general health, It is preferable to apply differently depending on various factors including sex and diet, administration time, administration route and secretion rate of the composition, treatment period, drugs used together with or concurrently with a specific composition, and similar factors well known in the pharmaceutical field.
  • the glucagon derivative, GLP-1 receptor agonist, conjugate thereof, or a composition comprising the same of the present invention may be administered once a week, once every 2 weeks, once every 4 weeks, or once a month. It may be administered once, but is not limited thereto.
  • the glucagon derivative and the GLP-1 receptor agonist when administered, may be administered at about 1 to 200 ⁇ g, 3 to 180 ⁇ g, or 5 to 160 ⁇ g per 1 kg of the patient's body weight, and when the two substances are used in combination, the total amount About 2 to 400 ⁇ g, about 6 to 360 ⁇ g, or 10 to 320 ⁇ g per 1 kg of the patient's body weight may be administered, but is not limited thereto.
  • the glucagon derivative and the GLP-1 receptor agonist administered in combination may be administered in a molar ratio of 0.01 to 50, 0.02 to 40, or 0.03 to 33.22, but is not limited thereto.
  • the glucagon derivative is administered at 0.09 to 2.99 nmol/kg and the GLP-1 receptor agonist at 0.09 to 2.99 nmol/kg, or the glucagon derivative: 0.01 to 50, 0.02 to 40 of the GLP-1 receptor agonist. , or may be administered at a molar ratio of 0.03 to 33.22, but is not limited thereto.
  • Another aspect embodying the present invention is the use of a glucagon derivative or a conjugate thereof, and a composition comprising the same for preventing or treating chronic kidney disease.
  • Another aspect embodying the present invention is the use of a composition comprising a glucagon derivative or a conjugate thereof and a GLP-1 receptor agonist or a conjugate thereof for preventing or treating chronic kidney disease.
  • Another embodiment embodying the present invention is the combined use of a glucagon derivative or a conjugate thereof and a GLP-1 receptor agonist or a conjugate thereof for the prevention or treatment of chronic kidney disease.
  • the glucagon derivative, GLP-1 receptor agonist, conjugate, composition, blood pressure lowering, renal function recovery, chronic kidney disease, prevention, and treatment are the same as described above.
  • Another aspect embodying the present invention is the use of a glucagon derivative or a conjugate thereof, and a composition comprising the same for the preparation of a medicament for preventing or treating chronic kidney disease.
  • Another aspect embodying the present invention is the use of a composition comprising a glucagon derivative or a conjugate thereof and a GLP-1 receptor agonist or a conjugate thereof for the manufacture of a medicament for preventing or treating chronic kidney disease.
  • the glucagon derivative, GLP-1 receptor agonist, conjugate, composition, blood pressure lowering, renal function recovery, chronic kidney disease, prevention, and treatment are the same as described above.
  • another aspect of the present invention is the use of a glucagon derivative or a conjugate thereof, and a composition comprising the same for lowering blood pressure and/or restoring renal function.
  • Another aspect embodying the present invention is the use of a composition comprising a glucagon derivative or a conjugate thereof and a GLP-1 receptor agonist or a conjugate thereof to lower blood pressure and/or restore renal function.
  • Another aspect embodying the present invention is the combined use of a glucagon derivative or a conjugate thereof and a GLP-1 receptor agonist or a conjugate thereof for lowering blood pressure and/or restoring renal function.
  • the glucagon derivative, GLP-1 receptor agonist, conjugate, composition, blood pressure lowering, renal function recovery, chronic kidney disease, prevention, and treatment are the same as described above.
  • Another aspect embodying the present invention is the use of a glucagon derivative or a conjugate thereof, and a composition comprising the same for the manufacture of a medicament having the use of lowering blood pressure and/or restoring renal function.
  • Another embodiment embodying the present invention is the use of a composition comprising a glucagon derivative or a conjugate thereof and a GLP-1 receptor agonist or a conjugate thereof to lower blood pressure and/or restore renal function for the manufacture of a medicament.
  • the glucagon derivative, GLP-1 receptor agonist, conjugate, composition, blood pressure lowering, renal function recovery, chronic kidney disease, prevention, and treatment are the same as described above.
  • the present inventors tried to confirm whether the prevention or treatment of chronic kidney disease can be obtained by lowering blood pressure and restoring renal function through activity on the glucagon receptor.
  • Glucagon Natural Form and Glucagon Derivative Amino Acid Sequences SEQ ID NO: peptide sequence Whether a ring is formed pI In vitro activity (relative activity to SEQ ID NO: 1, %) SEQ ID NO: 1 HSQGTFTSDYSKYLDSRRAQDFVQWLMNT - 6.8 100 SEQ ID NO: 2 HSQGTFTSDYSKYLDCDRAQDFVQWLMNT - 4.56 0.6 SEQ ID NO: 3 HSQGTFTSDYSKYLDCERAQDFVQWLMNT - 4.66 6.1 SEQ ID NO: 4 HSQGTFTSDYSKYLDSCDAQDFVQWLMNT - 4.13 ⁇ 0.1 SEQ ID NO: 5 HSQGTFTSDYSKYLDSCEAQDFVQWLMNT - 4.22 0.3 SEQ ID NO: 6 HSQGTFTSDYSKYLDSCEADDFVQWLMNT - 4.03 ⁇ 0.1 SEQ ID NO: 7
  • the amino acid denoted by X is the non-natural amino acid aminoisobutyric acid (Aib)
  • the underline under the amino acid symbol indicates the formation of a lactam ring between the side chains of the corresponding amino acid pair indicated in underline and bold, and "-" indicates that there is no amino acid residue at that position.
  • "-" indicates that a ring is not formed in the corresponding sequence.
  • the glucagon derivative of SEQ ID NO: 37 prepared in the above Example and the immunoglobulin Fc region were combined using polyethylene glycol as a linker to prepare a glucagon derivative long-acting conjugate.
  • Maleimide-PEG-aldehyde which is a linearly modified polyethylene glycol with a molecular weight of 10 kDa, in which the hydrogens at both ends are substituted with 3-(3-maleimidopropionamido)propyl groups and 3-oxopropyl groups (propionaldehyde groups), respectively (Japan NOF g) was reacted with the glucagon derivative of SEQ ID NO: 37 to pegylate the cysteine residue of the glucagon derivative peptide to the maleimide end of maleimide-PEG-aldehyde.
  • the molar ratio of the glucagon derivative peptide of SEQ ID NO: 37 to maleimide-PEG-aldehyde was 1:1 to 5, and the protein concentration was 3 to 10 mg/ml, and the reaction was carried out at low temperature for 1 to 3 hours. At this time, the reaction was performed in an environment in which 20-60% isopropanol was added to 50 mM Tris buffer (pH 7.5). After the reaction was completed, the reaction solution was applied to SP sepharose HP (GE healthcare, USA) to purify the glucagon derivative mono-pegylated to cysteine.
  • SP sepharose HP GE healthcare, USA
  • the immunoglobulin Fc region is an immunoglobulin Fc region (49.8 kDa, two chains of SEQ ID NO: 67) having a hinge region of the Pro-Ser-Cys-Pro sequence (SEQ ID NO: 57) at the N-terminus is allogeneic linked by a disulfide bond. mer) was prepared by the method described in International Patent Publication WO2007/021129.
  • the purified mono-pegylated glucagon derivative and the immunoglobulin Fc region are reacted at a molar ratio of 1:2-10 and a protein concentration of 10-50 mg/ml at 4-8°C for 12-18 hours. did it
  • the reaction solution was carried out in an environment in which 10-50 mM sodium cyanoborohydride and 10-20% isopropanol as reducing agents were added to 100 mM potassium phosphate buffer (pH 6.0). After the reaction was completed, the reaction solution was applied to the Butyl sepharose FF purification column (GE healthcare, USA) and the Source ISO purification column (GE healthcare, USA),
  • a long-acting conjugate of the glucagon derivative peptide in which the aldehyde side polyethylene glycol terminus of the mono-pegylated glucagon derivative peptide is linked to the N-terminal proline nitrogen of one of the two chains of the immunoglobulin Fc homodimer was purified.
  • the conjugate is a structure in which a glucagon derivative, a polyethylene glycol (PEG) linker and the immunoglobulin Fc region of the dimer are covalently linked in a molar ratio of 1:1:1, and the PEG linker is one of the two polypeptide chains of the immunoglobulin Fc region. It is linked to the N-terminal proline nitrogen of one chain.
  • two monomers having the amino acid sequence of SEQ ID NO: 67 (consisting of 221 amino acids) form a homodimer through a disulfide bond between cysteine, which is amino acid 3 of each monomer, and the homodimer
  • the monomers 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.
  • the conjugate in which the glucagon derivative peptide and the immunoglobulin Fc region are linked via PEG was named 'persistent conjugate of glucagon derivative'.
  • Example 3 Effect of improving renal function in a rat model of chronic kidney disease
  • SHR spontaneous hypertensive rat
  • the 16-week-old SHR which is known to cause renal dysfunction due to the rise in blood pressure, was divided into an excipient control group and a long-acting conjugate of glucagon derivative of SEQ ID NO: 37 (3.9 nmol/kg, Q3D, subcutaneous) administration group, and repeated administration for 6 weeks did.
  • As a negative control group normal rats and a vehicle control group were used.
  • urine albumin excretion was measured at day 0, day 13, day 28, and day 46.
  • One-way ANOVA was used for statistical treatment (* ⁇ ***p ⁇ 0.05 ⁇ 0.001 vs. SHR vehicle control).
  • Example 4 Blood pressure lowering effect in a rat model of chronic kidney disease
  • SHR spontaneous hypertensive rat
  • a telemetry device was inserted into the aorta through surgery at a 15-week-old SHR whose blood pressure had risen to the level of hypertension, followed by an excipient control group and a long-acting conjugate of a glucagon derivative (2 nmol/kg, Q3D, subcutaneous) administration group. divided, and repeated administration was performed for 1 week. Changes in systolic (SBP) and diastolic (DBP) blood pressure during repeated dosing for 1 week were monitored every 2 hours through a telemetry device inserted into the aorta.
  • SBP systolic
  • DBP diastolic
  • the long-acting conjugate of the glucagon derivative of the present invention has therapeutic efficacy in lowering blood pressure in chronic kidney disease (CKD).
  • Example 5 Confirmation of renal function improvement effect by co-administration with GLP-1 receptor agonist
  • Imidazo-acetyl exendin-4 (CA exendin-4)-immunoglobulin in which the N-terminus of the immunoglobulin Fc region and specifically monopegylated imidazo-acetyl exendin-4 (CA exendin-4) are linked
  • the reaction was performed at pH 5.0-8.2.
  • a two-step purification method using a hydrophobic column and an anion exchange column was used, and finally the site-specifically bound GLP-1 receptor agonist-immunoglobulin Fc region conjugate was purified.
  • GLP-1 receptor agonist a conjugate in which the GLP-1 receptor agonist of SEQ ID NO: 72 (imidazo-acetyl exendin-4 (CA exendin-4)) and the immunoglobulin Fc region are linked through PEG is referred to as ‘GLP-1 receptor agonist’ It was named as 'long-acting conjugate' or 'long-acting conjugate of CA-Exd4 derivative'.
  • the immunoglobulin Fc region used for the preparation of the long-acting conjugate of the GLP-1 receptor agonist is the same as the Fc region used in Example 2.
  • Example 5-2 Confirmation of effect of co-administration with GLP-1 receptor agonist
  • 16-week-old SHR which is known to cause renal dysfunction due to increase in blood pressure, was treated with excipient control group, long-acting conjugate of glucagon derivative (3.0 nmol/kg, Q3D, subcutaneous) administration group, and long-acting conjugate of glucagon derivative (3.0 nmol/kg) , Q3D, subcutaneous) and a long-acting conjugate of a GLP-1 receptor agonist (6.9 nmol/kg, Q3D, subcutaneous) were divided into co-administration groups, and repeated administration was performed for 10 weeks.
  • As a negative control group normal rats and a vehicle control group were used.
  • ACR albumin to creatinine ratio
  • CKD chronic kidney disease
  • glucagon derivative of the present invention has an effect of lowering blood pressure and restoring renal function, so it has an effective effect in the treatment of chronic kidney disease, and additional therapeutic effect is expected by co-administration with a GLP-1 receptor agonist. suggests that it can

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EP22785037.7A EP4321170A4 (en) 2021-04-09 2022-04-11 PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING CHRONIC KIDNEY DISEASE CONTAINING A GLUCAGON DERIVATIVE
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