Classifications

• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • 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
  • 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
  • A61K47/56—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 organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
  • A61K47/59—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 organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
  • A61K47/60—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 organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• • 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
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Definitions

• the present invention relates to liver-targeted drugs and their therapeutic use for diseases requiring drug action in the liver.
• the present invention relates to a method of inducing an increase in distribution in liver tissue after targeting to liver tissue or intravitreal administration using a liver-targeting drug.
• the liver is one of the largest organs in our body and plays an important role in a variety of overall metabolic processes. Among the many vegetable or animal substances that we ingest and the metabolites of the body's functions, there are beneficial to the body, but there are also many harmful substances. The liver performs chemical processes that help the correct bioavailability of beneficial substances among these substances, and helps harmful substances to be safely excreted out of the body through urine or feces through chemical metabolism. The liver plays an essential role in biological functions by synthesizing and secreting various types of proteins, fats, and carbohydrates necessary for our body. Therefore, when liver function deteriorates, various functional abnormalities may appear. Insufficient production of coagulation factors involved in blood clotting leads to frequent bleeding from weak gums, etc.
• liver cirrhosis In the case of patients with liver cirrhosis, insulin decomposition does not work well and the liver's glycogen storage is insufficient, resulting in hypoglycemia due to fasting.
• the liver also plays a pivotal role in defense against bacterial invasion.
• Kupffer cells mainly perform a phagocytic action by consuming foreign substances or bacteria, and also play a role in inducing natural immunity in the body by exposing the virus entering the body to the immune system.
• about 800-1000 cc of bile a major substance synthesized and secreted by hepatocytes, is formed per day, and its main components are water, electrolytes, bile acids, cholesterol, phospholipids, and bilirubin.
• bile acids in bile play an important role in digesting and absorbing fat and fat-soluble vitamins in the small intestine, and bile itself plays a role in excreting many waste products produced in the body through feces. It is caused by various causes, and as a disease requiring drug action in the liver, there are various diseases such as liver disease, hypoglycemia, or congenital hyperinsulinism.
• Liver-targeting drugs can optimize drug treatment by minimizing the side effects of existing drugs and maximizing their efficacy and effectiveness so that the required amount of drug can be efficiently delivered. This increases bioavailability by increasing the amount of drug reaching the target site, enables more effective treatment, reduces side effects by preventing delivery to a place other than the target site, and improves patient compliance by improving the patient's response to the drug. play a major role in promoting
• liver-targeting drug In order to increase the therapeutic effect of diseases requiring drug action in the liver, it is required to develop an excellent liver-targeting drug that has a therapeutic effect and has a high degree of distribution of the drug in the liver among the body organs of the administered subject.
• One object of the present invention is to provide a pharmaceutical composition containing a liver-targeted drug, specifically, a pharmaceutical composition containing a liver-targeted drug and having a high distribution of the drug in the liver among internal organs of an administered subject.
• Another object of the present invention is to provide a pharmaceutical composition containing a liver-targeting drug, specifically, a pharmaceutical composition containing a liver-targeting drug for preventing or treating diseases requiring drug action in the liver.
• Another object of the present invention is to provide a method for preventing or treating diseases requiring drug action in the liver, comprising administering the pharmaceutical composition or a physiologically active substance targeted to liver tissue to a subject in need thereof. .
• Another object of the present invention is to provide a use of the liver-targeting drug, specifically, a bioactive substance targeted to liver tissue, for use in the preparation of a drug for preventing or treating a disease requiring drug action in the liver.
• Another object of the present invention is to provide a method for inducing liver targeting by administering the liver-targeting drug, specifically, a physiologically active substance targeted to liver tissue to a subject in need thereof.
• Another object of the present invention is to provide a method for inducing increased distribution of the bioactive substance in liver tissue by administering the liver-targeting drug, specifically, a bioactive substance targeted to liver tissue to a subject in need thereof.
• the drug of the present invention e.g., a physiologically active protein or peptide derivative
• a physiologically active substance that reaches liver tissue more effectively from blood vessels and is distributed in a relatively large amount among organs in the body after in vivo administration is a physiologically active substance that reaches liver tissue more effectively from blood vessels and is distributed in a relatively large amount among organs in the body after in vivo administration .
• glucagon receptor by having binding force with the glucagon receptor, it is not only provided as a bioactive substance that is relatively distributed in liver tissue, but also has a high distribution in the liver within about 7 days, reducing the patient's discomfort for medication, It can be applied to the treatment of diseases requiring drug action in the liver, including diseases, hypoglycemia, or congenital hyperinsulinism.
• One embodiment of the present invention is a pharmaceutical composition comprising a liver-targeting drug, specifically, a pharmaceutical composition in which the distribution of the drug in the liver is high among internal organs of an administered subject.
• the liver-targeting drug is characterized in that it is in the form of a peptide comprising the amino acid sequence of Formula 1 below or a long-acting conjugate comprising the same.
• the liver-targeting drug is characterized in that it is in the form of a peptide comprising the amino acid sequence of Formula 2 below or a long-acting conjugate comprising the same.
• the liver-targeting drug is characterized in that it is a peptide comprising the amino acid sequence of Formula 1 below:
• X1 is histidine (H), desamino-histidyl, dimethyl-histidyl, beta-hydroxy imidazopropionyl, 4-imidazoacetyl (4-imidazoacetyl), beta-carboxy imidazopropionyl, tryptophan (W), or tyrosine (Y), or absent;
• X2 is ⁇ -methyl-glutamic acid, aminoisobutyric acid (Aib), D-alanine, glycine (G), N-methylglycine (Sar), 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), glutamine (Q), 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), glutamic acid (E), 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 lysine (K), alanine (A), glycine (G), or threonine (T), or absent;
• X30 is cysteine (C) or absent.
• One embodiment of the present invention is a pharmaceutical composition comprising a liver-targeting drug, wherein the distribution of the drug in the liver is high among internal organs of an administered subject.
• the liver-targeting drug is characterized in that it is a peptide comprising the amino acid sequence of Formula 2 below:
• X7 is threonine (T), valine (V) or cysteine (C);
• X10 is tyrosine (Y) or cysteine (C);
• X12 is lysine (K) or cysteine (C);
• X15 is aspartic acid (D), or cysteine (C);
• X16 is glutamic acid (E) or serine (S);
• X17 is lysine (K) or arginine (R);
• X20 is glutamine (Q) or lysine (K);
• X21 is aspartic acid (D), or glutamic acid (E);
• X24 is valine (V) or glutamine (Q);
• X30 is cysteine (C) or absent
• amino acid sequence of Formula 2 is the same as SEQ ID NO: 12).
• the peptide is in the form of a long-acting conjugate
• the long-acting conjugate is a pharmaceutical composition represented by Formula 1 below:
• X is a peptide containing the amino acid sequence of Formula 2 above;
• L is a linker containing an ethylene glycol repeating unit
• F is an immunoglobulin Fc region
• the organ in the body is liver, heart, lung, large intestine, spleen, pancreas, adipose tissue, small intestine, stomach, muscle, kidney and brain, and among the above organs, the liver It is characterized by the highest degree of distribution.
• the composition is characterized in that it is for use in the prevention or treatment of diseases requiring drug action in the liver.
• liver-targeting drug has a tissue-to-serum ratio in the liver after administration of at least one selected from the following: (a) Administration After about 40 hours to about 50 hours, the T/S ratio is about 20% to about 40%; and (b) a T/S ratio of about 25% to about 40% from about 160 hours to about 180 hours after administration.
• liver-targeted drug has a T/S ratio in the liver after administration of at least one selected from the following: (a) T/S at about 2 days after administration at a ratio of about 25% to about 35%; and (c) a T/S ratio of about 27% to about 35% at about 7 days after administration.
• the distribution ratio of the liver-targeting drug in liver compared to lung tissue after administration is 1: about 2 to about 4.
• the distribution ratio of the liver-targeted drug to lung tissue after administration is 1: about 2.2 to about 3.2.
• the pharmaceutical composition according to any one of the preceding embodiments characterized in that the distribution ratio in liver compared to lung tissue after administration is the distribution ratio at about 40 hours to about 180 hours after administration.
• the pharmaceutical composition according to any one of the preceding embodiments characterized in that the distribution ratio in liver compared to lung tissue after administration is the distribution ratio at about 2 to about 7 days after administration.
• liver-targeted drug has (a) a distribution ratio in the liver to the heart at 40 to 50 hours after administration of 1: 1.6 to 3.0; and (b) at 160 to 180 hours after administration, the distribution ratio in the liver to the heart is 1:2.8 to 7.
• liver-targeting drug is in the form of a peptide or a long-acting conjugate containing the same that is distributed in a relatively large amount in the middle of the body after administration.
• the liver-targeting drug is characterized in that it has therapeutic activity for diseases requiring drug action in the liver.
• composition according to any one of the preceding embodiments, characterized in that the disease requiring drug action in the liver is liver disease, hypoglycemia or congenital hyperinsulinism.
• the hypoglycemia is characterized by acute hypoglycemia or chronic hypoglycemia.
• liver-targeting drug is a protein or peptide containing a peptide sequence having binding ability to the glucagon receptor.
• composition according to any one of the above embodiments, characterized in that a ring is formed between the amino acids X16 and X20 of Formula 2.
• the peptide comprising the amino acid sequence of Formula 2 is characterized in that it comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 33 and 36 to 44.
• the peptide comprising the amino acid sequence of Formula 1 is SEQ ID NO: 20, 22, 23, 27, 33, 35, 37, 38, 40, 41, 42, and It is characterized in that it comprises any one amino acid sequence of 44.
• the peptide comprising the amino acid sequence of Formula 1 is any one of the amino acid sequences of SEQ ID NOs: 20, 22, 23, 27, 33, 37, 38, and 44 It is characterized by including.
• composition according to any one of the preceding embodiments wherein at least one amino acid pair of amino acid pairs of X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24, and X24 and X28 in Formula 1 It is characterized in that each amino acid is substituted with glutamic acid or lysine capable of forming a ring.
• 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: 1 to 44.
• the peptide is characterized in that its C-terminus is amidated or has a free carboxyl group (-COOH).
• composition according to any one of the preceding embodiments, characterized in that the C-terminus of the peptide is amidated.
• composition according to any one of the preceding embodiments, wherein the C-terminus of the peptide is not modified.
• composition according to any one of the preceding embodiments characterized in that the formula weight of the ethylene glycol repeating unit portion in L is in the range of 1 to 100 kDa.
• X and F are as defined in Formula 1.
• the value of n is characterized in that the average molecular weight of the [OCH 2 CH 2 ]n site in the peptide conjugate, for example, the number average molecular weight, is determined to be 10 kDa.
• composition according to any one of the preceding embodiments, wherein X is linked through a sulfur atom of cysteine in the peptide.
• composition according to any one of the preceding embodiments, wherein the immunoglobulin Fc region is derived from IgG4.
• composition according to any one of the preceding embodiments, characterized in that the immunoglobulin Fc regions F and X are not glycosylated.
• Another aspect embodying the present invention is a bioactive material targeted to liver tissue, including a material having binding force with a receptor present in the liver, specifically, a bioactive material targeted to liver tissue.
• Another aspect embodying the present invention is a method for preventing or treating a disease requiring drug action in the liver, comprising administering the pharmaceutical composition or a physiologically active substance targeted to liver tissue to a subject in need thereof am.
• liver-targeting drug specifically a bioactive substance targeted to liver tissue
• a bioactive substance targeted to liver tissue for use in the preparation of a drug for preventing or treating a disease requiring drug action in the liver.
• Another embodiment of the present invention is a method of inducing liver targeting by administering the liver-targeting drug, specifically, a physiologically active substance targeted to liver tissue, to a subject in need thereof.
• Another aspect embodying the present invention is a method of inducing an increase in the distribution of the bioactive substance in liver tissue by administering the liver-targeting drug, specifically, a bioactive substance targeted to liver tissue to a subject in need thereof is to provide
• Aib may be used interchangeably with “2-aminoisobutyric acid” or “aminoisobutyric acid”, and 2-aminoisobutyric acid and aminoisobutyric acid Butyric acid (aminoisobutyric acid) may be used in combination.
• compositions such as a pharmaceutical composition, comprising a liver-targeting drug.
• a pharmaceutical composition comprising a liver-targeting drug, which has a high degree of distribution in the liver of the body organs of an administered subject.
• the high distribution in the liver among organs in the body means that among the body organs consisting of liver, heart, lungs, large intestine, spleen, pancreas, adipose tissue, small intestine, stomach, muscle, kidney and brain, after administration of a liver-targeted drug into the body, It may mean that the distribution of the drug is highest in the liver, but is not limited thereto.
• the pharmaceutical composition may be a pharmaceutical composition for use in preventing or treating diseases requiring drug action in the liver.
• liver-targeted drug refers to a drug that can be targeted to liver tissue. Targeting to liver tissue may mean that the distribution of the drug in the liver is higher than that of other organs after administration, but is not limited thereto.
• the liver-targeted drug may have therapeutic activity against a disease requiring drug action in the liver.
• the liver-targeted drug may have a T/S ratio in the liver after administration of one or more selected from the following, but is not limited thereto: (a) T at about 40 hours to about 50 hours after administration /S ratio of about 20% to about 40%, about 24% to about 40%, about 25% to about 40%, about 25% to about 38%, about 25% to about 35%, about 25% to about 34% %, about 25% to about 33%, about 26% to about 33%, about 26% to about 32%, about 27% to about 32%, about 28% to about 32%, about 29% to about 31%, or from about 29.5% to about 30.5%; and (b) a T/S ratio of about 25% to about 40%, about 26% to about 38%, about 27% to about 35%, about 27% to about 34% at about 160 hours to about 180 hours after administration.
• the feature may be one or more selected from (a) or (b), or both, but is not limited thereto.
• the liver-targeted drug may have a T/S ratio in the liver after administration of one or more selected from the following, but is not limited thereto: (a) a T/S ratio of about 25% to about 35% at about 2 days after administration; about 26% to about 34%, about 27% to about 33%, about 28% to about 32%, about 29% to about 31%, about 29.5% to about 30.5%, or about 29.8%; and (c) a T/S ratio of about 27% to about 35%, about 27% to about 34%, about 28% to about 33%, about 29% to about 32%, about 30% at about 7 days after administration. to about 31.5%, or about 31%.
• the feature may be at least one selected from (a) or (b), or both, but is not limited thereto.
• T/S ratio tissue-to-serum ratio
• tissue-to-serum ratio tissue-to-serum ratio
• T/S ratio tissue-to-serum ratio
• T/S ratio % is calculated as tissue concentration/serum concentration x 100.
• concentration of the substance is measured by an ELISA method after removing the organ.
• the higher the T/S ratio the higher the distribution of the administered substance in the tissue compared to the tissue having a low T/S ratio.
• the lung and heart are organs with a high distribution of the drug, so the distribution in the lung tissue and the heart tissue is compared with the liver tissue, and the distribution in the lung tissue and the heart tissue is compared with the liver tissue. If the distribution is large, it may mean that the liver-targeting drug of the present invention is effectively targeted to the liver tissue.
• tissues that can be compared with the T/S ratio of liver tissue by measuring the T/S ratio, which can confirm that the liver-targeting drug is effectively targeted to the liver tissue may be included without limitation.
• the liver-targeted drug has a distribution ratio of 1: about 2 to about 4.0, 1: about 2 to about 3.5, 1: about 2.1 to about 3.4, 1: about 2.2 to 3.2, 1: about 2.2 to about 3.3, 1: about 2.3 to about 3.2, 1: about 2.4 to about 3.2, 1: about 2.4 to about 3.1, 1: about 2.4 to about 3.0, 1: about 2.5 to about 2.95, 1: about 2.5 to 2.9 , 1:2.55 to about 2.87, 1: or 2.6 to 2.9, 1: about 2.5 to 3.9, but is not limited thereto.
• the distribution ratio can be measured based on T/S (%), and when the T/S (%) of the lung is set as 1, the T/S (%) in the liver can be confirmed as a multiple. .
• the distribution ratio in liver compared to lung tissue after administration may be about 40 hours to about 180 hours, about 45 hours to about 170 hours, or about 2 days to about 7 days after administration, but is not limited thereto.
• the distribution ratio in liver compared to lung tissue after administration is 1: about 2.4 to about 2.7, 1: about 2.4 to about 2.7, 1: about 2.5 to about 2.6, or 1: about 2.5 to 3.7 on about 2 days after administration; At about 7 days, it may be a distribution ratio of 1: about 2.5 to 3, 1: about 2.7 to about 3, 1: about 2.8 to about 3.0, 1: about 2.8 to about 2.9, or 1: about 2.8 to about 3.9. Not limited.
• the distribution ratio in liver to heart after the administration is (a) the distribution ratio in liver to heart is 1: about 1.6 to about 3.0 at about 40 hours to about 50 hours after administration; and (b) from about 160 hours to about 180 hours after administration, the distribution ratio in the liver to the heart may be 1: about 2.8 to about 7.0, but is not limited thereto.
• the distribution ratio in liver to heart after the administration is (a) at about 40 hours to about 50 hours, about 45 hours to about 50 hours, or about 2 days after administration, the distribution ratio in heart to liver is 1: about 1.6 to 3.0 , 1: about 1.6 to 2.5, 1: about 1.6 to about 2.2, 1: about 1.7 to about 2.1, 1: about 1.7 to about 2.0, 1: about 1.8 to 2.0, 1: about 1.8 to 2.0, 1: about 1.89 to 2.0, 1: from about 1.8 to 2.0, or 1: from about 1.8 to 2.9; and (b) from about 160 hours to about 180 hours, from about 160 hours to about 180 hours, or about 7 days after administration, a distribution ratio in liver to heart of 1 : about 2.8 to about 4, 1 : about 2.9 to about 3.8; 1: about 3.0 to about 3.8, 1: about 3.0 to about 3.6, 1: about 3.0 to about 3.5, 1: about 3.0 to about 3.4, 1: about 3.1 to about 3.4, 1: about 3.2 to about 3.4
• the distribution ratio can be measured based on T/S (%), and when the T/S (%) of the heart is set as 1, the T/S (%) of the liver can be confirmed as a multiple. .
• the term "about” is a range including ⁇ 0.5, ⁇ 0.4, ⁇ 0.3, ⁇ 0.2, ⁇ 0.1, ⁇ 0.05, etc., and includes all values in a range equivalent to or similar to the numerical value following the term about. However, it is not limited thereto.
• the liver-targeting drug may include a substance that can be targeted to liver tissue. Specifically, the liver-targeting drug can induce liver targeting.
• a peptide or protein containing glucagon or a derivative thereof which may include a peptide sequence having activity on the glucagon receptor.
• it may be a peptide comprising the amino acid sequence of Formula 1 or a long-acting conjugate comprising the same, or a peptide comprising the amino acid sequence of Formula 2 or a long-acting conjugate comprising the same, but is not limited thereto.
• the peptide is a peptide having activity on the glucagon receptor, and may specifically be a glucagon derivative, but is not limited thereto.
• glucagon or a derivative thereof which is a liver-targeting drug, means that when the drug is delivered into the blood of an animal, it retains binding ability to the glucagon receptor to such an extent that it can be targeted from the blood to the liver.
• the organ distribution ratio of the drug was confirmed at least 6 hours, 30 hours, at least 48 hours, or at least 168 hours after the drug was delivered into the animal's blood, the heart, lungs, large intestine, spleen, pancreas, About 5% or more, about 10% or more, about 15% or more, about 20% or more, about 30% or more distributed in the liver compared to at least one other organ, such as adipose tissue, small intestine, stomach, muscle, kidney, or brain It means higher than or higher than about 40%, but is not particularly limited thereto, as long as the distribution ratio is higher than that of the organs to be compared.
• the substance having activity on the glucagon receptor includes various substances having a significant level of activity on the glucagon receptor, such as compounds or peptide-type substances.
• substances having a significant level of activity for glucagon include not only native glucagon, but also those whose in vitro activity against the glucagon receptor is 0.1% or more compared to the native ligand (native glucagon) of the corresponding receptor, 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% It may represent 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more.
• Examples of the substance having activity for the glucagon receptor include native glucagon, an agonist thereof, and a derivative thereof, but may be specifically limited thereto.
• the glucagon derivatives according to the present invention are peptides having one or more differences in amino acid sequence compared to native glucagon, peptides modified through modification of the native glucagon sequence, and those capable of activating glucagon receptors like native glucagon. If it includes a mimetic of native glucagon, and the distribution of the peptide in the liver of the administered subject is high, it may fall within the scope of the present invention.
• glucagon derivative As the type of the glucagon derivative, the types described in International Publication Patent WO 2016/108586 and International Publication Patent WO 2017/003191 may be mentioned, and the entire specification of the International Publication Patent is incorporated herein by reference. In addition, the method for preparing the long-acting conjugate of the glucagon derivative peptide is described in WO 2017/003191, and it is clear that the entire specification of the international publication patent is also incorporated herein as a reference.
• Such glucagon derivatives may exhibit improved physical properties by having a changed pI with respect to native glucagon.
• the glucagon derivative may have improved solubility while retaining the activity of activating the glucagon receptor, but is not limited thereto.
• the glucagon derivative may be non-naturally occurring.
• native glucagon may have the following amino acid sequence:
• the term "isoelectric point" or "pI" refers to a pH value at which a polypeptide or molecule such as a peptide has no net charge (0).
• pI isoelectric point
• the sum of these charges at pi is zero.
• the net charge of the polypeptide will be negative, and at pH values lower than the pI, the net charge of the polypeptide will be positive.
• pi can be determined by isoelectric focusing on a fixed pH gradient gel composed of polyacrylamide, starch or agarose or by pI/MW tool (http://expasy.org/tools/pi_tool.html; Gasteiger et al., 2003) can be determined by estimating the pi from the amino acid sequence.
• altered pi means that a portion of the amino acid sequence of natural glucagon is substituted with negatively and positively charged amino acid residues to have a pI that is different from, that is, decreased or increased from, the pi of natural glucagon. Peptides with such altered pI may exhibit improved solubility and/or high stability at neutral pH as glucagon derivatives. However, it is not particularly limited thereto.
• the glucagon derivative may have a pI value other than the pI value of native glucagon (6.8), and may have a pI value of less than 6.8, less than 6.7, less than 6.5, greater than 6.8, greater than 7, and greater than 7.5, but is limited thereto. It is not, and if it has a pI value different from that of native glucagon, it is included in the scope of the present invention. In particular, if the degree of aggregation is low by showing improved solubility at neutral pH compared to native glucagon by having a pI value different from native glucagon, it may be included in the scope of the present invention, but is not limited thereto.
• it may have a pI value of 4 to 6.5 and/or 7 to 9.5, 7.5 to 9.5, and 8.0 to 9.3, but is not limited thereto.
• it since it has a higher or lower pI than native glucagon, it may exhibit improved solubility and higher stability compared to native glucagon at neutral pH.
• it is not limited thereto.
• some amino acids in native glucagon may be modified through any one method among substitution, addition, deletion, and modification, or a combination of these methods. there is.
• glucagon derivatives prepared by a combination of these methods include peptides having an amino acid sequence different from native glucagon by one or more, deaminated at the N-terminal amino acid residue, and having an activating function for the glucagon receptor. , It is not limited thereto, and a derivative of native glucagon applied to the present invention can be prepared by a combination of several methods for preparing the derivative.
• modifications for the production of derivatives of native glucagon include modifications using L- or D-type amino acids, and/or non-natural amino acids; and/or modification of the native sequence, e.g., modification of side chain functional groups, intramolecular covalent linkages such as inter-side chain ring formation, methylation, acylation, ubiquitination, phosphorylation, aminohexaylation, biotinylation, etc. including all that
• the above modifications include all substitutions with non-natural compounds.
• it includes all amino acids with one or more amino acids added to the amino and/or carboxy termini of native glucagon.
• the substituted or added amino acids may be 20 amino acids commonly observed in human proteins as well as atypical or non-naturally occurring amino acids.
• Commercial sources of atypical amino acids include Sigma-Aldrich, ChemPep and Genzyme pharmaceuticals. Peptides containing these amino acids and typical peptide sequences can be synthesized and purchased through commercialized peptide synthesis companies, such as American Peptide Company or Bachem in the US or Anygen in Korea.
• Amino acid derivatives can also be obtained in the same way, and examples thereof include 4-imidazoacetic acid and the like.
• Glucagon has a pI of about 7, so it is insoluble in solution at physiological pH (pH 4-8) and tends to precipitate at neutral pH. In aqueous solutions below pH 3, glucagon is initially soluble but precipitates due to gel formation within 1 hour. Gelated glucagon is mainly composed of ⁇ -sheet fibrils, and glucagon precipitated in this way is not suitable for use as an injectable agent because it clogs blood vessels when the gel is administered with an injection needle or intravenously. To retard the precipitation process, it is common to use acidic (pH 2-4) formulations, which allow glucagon to remain relatively unaggregated for a short period of time. However, since fibril formation of glucagon occurs very rapidly at low pH, these acidic formulations must be injected immediately after preparation.
• the glucagon derivatives of the present invention include those developed to have an extended action profile by changing the pI of natural glucagon by substitution of negatively and positively charged amino acid residues, and such derivatives have a changed pI compared to natural glucagon, resulting in neutral It is characterized by being capable of exhibiting improved solubility and/or high stability at pH.
• the glucagon or glucagon derivative may be a peptide comprising the amino acid sequence of Formula 1 below.
• X1 is histidine, desamino-histidyl, dimethyl-histidyl, beta-hydroxy imidazopropionyl, 4-imidazoacetyl (4- imidazoacetyl), beta-carboxy imidazopropionyl, tryptophan, or tyrosine, or absent;
• X2 is ⁇ -methyl-glutamic acid, aminoisobutyric acid (Aib), D-alanine, glycine, N-methylglycine (Sar), 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 absent;
• X17 is aspartic acid, glutamine, glutamic acid, lysine, arginine, serine, cysteine, or valine, or absent;
• X18 is alanine, aspartic acid, glutamine, glutamic acid, arginine, valine, or cysteine, or absent;
• X19 is alanine, arginine, serine, valine, or cysteine, or absent;
• X20 is lysine, histidine, glutamic acid, glutamine, aspartic acid, arginine, alpha-methyl-glutamic acid, or cysteine, or absent;
• X21 is aspartic acid, glutamic acid, leucine, valine, or cysteine, or absent;
• X23 is isoleucine, valine, or arginine, or absent;
• X24 is valine, arginine, alanine, cysteine, glutamic acid, lysine, glutamine, alpha-methyl-glutamic acid, or leucine, or absent;
• X27 is isoleucine, valine, alanine, lysine, methionine, glutamine, or arginine, or absent;
• X28 is glutamine, lysine, asparagine, or arginine, or absent;
• X29 is lysine, alanine, glycine, or threonine, or absent;
• X30 may be cysteine or may be absent.
• X1 is histidine, tryptophan, or tyrosine, or absent;
• X2 is serine or aminoisobutyric acid (Aib);
• 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, or cysteine
• X16 is glutamic acid, serine or cysteine
• X17 is aspartic acid, glutamic acid, lysine, arginine, serine, cysteine, or valine;
• X18 is aspartic acid, glutamic acid, arginine, or cysteine;
• X19 is alanine, or cysteine
• X20 is glutamine, aspartic acid, lysine, or cysteine
• X21 is aspartic acid, glutamic acid, leucine, valine, or cysteine;
• X23 is isoleucine, valine or arginine
• X24 is valine, arginine, alanine, glutamic acid, lysine, glutamine, or leucine;
• X27 is isoleucine, valine, alanine, methionine, glutamine or arginine;
• X29 is threonine
• X30 may be cysteine or may be absent.
• the peptide may contain an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 44, specifically SEQ ID NO: 1 to 44, but may (essentially) consist of an amino acid sequence selected from the group consisting of, Not limited to this.
• the peptide of the present invention may be one comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 45, or (essentially) composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 45 , but not limited thereto.
• 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]: can be determined using known computer algorithms such as the “FASTA” program using default parameters as in 2444. 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), It can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453).
• GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215] : 403 (1990);Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO ETA/.] (1988) SIAM J Applied Math 48: 1073) Homology, similarity or identity can be determined using, for example, BLAST of the National Center for Biotechnology Information Database, or ClustalW.
• the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 14, 17, 19 to 25, 27, 29, 33, 35 to 38, 40 to 42, and 44, specifically SEQ ID NO: 12, 14, 17, 19 to 25, 27, 29, 33, 35 to 38, and 40 to 42, may be (essentially) composed of an amino acid sequence selected from the group consisting of, but limited thereto It doesn't work.
• the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 13, 15, 17, 20 to 24, 26 to 30, and 32 to 44, specifically SEQ ID NOs: 2 to 13, It may be (essentially) composed of an amino acid sequence selected from the group consisting of 15, 17, 20 to 24, 26 to 30, and 32 to 44, but is not limited thereto.
• the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 22, 23, 27, 33, 37, 38, and 44, specifically SEQ ID NOs: 20, 22, 23, 27, It may be (essentially) composed of an amino acid sequence selected from the group consisting of 33, 37, 38, and 44, but is not limited thereto.
• the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 16, 18, 19, 25, and 31, specifically SEQ ID NOs: 14, 16, 18, 19, 25, And it may be (essentially) composed of an amino acid sequence selected from the group consisting of 31, but is not limited thereto.
• X7 is threonine (T), valine (V) or cysteine (C);
• X10 is tyrosine (Y) or cysteine (C);
• X12 is lysine (K) or cysteine (C);
• X15 is aspartic acid (D), or cysteine (C);
• X16 is glutamic acid (E) or serine (S);
• X21 is aspartic acid (D), or glutamic acid (E);
• X30 is cysteine (C) or absent
• amino acid sequence of Formula 2 is the same as SEQ ID NO: 12).
• X10 is tyrosine or cysteine
• X17 is lysine or arginine
• X21 is aspartic acid or glutamic acid
• X24 is glutamine
• X30 may be cysteine or absent, but is not particularly limited thereto.
• the peptide is another example, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 33 and 36 to 44, specifically selected from the group consisting of SEQ ID NO: 33 and 36 to 44 It may be (essentially) composed of an amino acid sequence that is, but is not limited thereto.
• Non-limiting examples of the ring may include a lactam bridge (or lactam ring).
• glucagon derivatives include those modified to include an amino acid capable of forming a ring at a desired position to include a ring.
• the peptide comprising the amino acid sequence of Formula 1 or Formula 2 is X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24, and X24 and X28 of Formula 1 or Formula 2
• each amino acid of each amino acid pair 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 given amino acid sequence.
• the peptide comprising the amino acid sequence of Formula 1 or Formula 2 is a glutamic acid in which each amino acid of an amino acid pair of X12 and X16, an amino acid pair of X16 and X20, or an amino acid pair of X17 and X21 can form a ring Or it may be substituted with lysine, but is not limited thereto.
• X16 is glutamic acid
• X20 is lysine
• side chains of X16 and X20 may form a lactam ring, but is not limited thereto.
• the peptide may have an increased in vivo half-life compared to native glucagon, but is not particularly limited thereto.
• the peptide according to the present invention may have an unmodified N-terminus and/or C-terminus, but in order to protect from protein cleavage enzymes in vivo and to increase stability, its N-terminus and/or C-terminus, etc.
• Forms chemically modified, protected with organic groups, or modified by adding amino acids to the ends of the peptides are also included in the scope of the peptides according to the present invention.
• the terminal of the peptide according to the present invention has a carboxyl group, but is not particularly limited thereto.
• the N-terminus may be acetylated and/or the C-terminus may be amidated to remove these charges. It may be, but is not particularly limited thereto.
• the term "pharmaceutically acceptable salt” includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases.
• suitable acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid.
• Salts derived from suitable bases may include alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium.
• solvate used in the present invention refers to a complex formed by a peptide, conjugate, or salt thereof according to the present invention with a solvent molecule.
• the C-terminus of the peptide according to the present invention may include a peptide having an amidated or free carboxyl group (-COOH), or a peptide having an unmodified C-terminus, but is not limited thereto.
• the peptide may be one in which the C-terminus is amidated, but is not limited thereto.
• the peptide may be aglycosylated, but is not limited thereto.
• the peptides of the present invention can be synthesized through solid phase synthesis, can be produced by recombinant methods, and can be produced by requesting commercially, but are not limited thereto.
• the peptide of the present invention can be synthesized according to its length by a method well known in the art, for example, an automatic peptide synthesizer, or can be produced by genetic engineering technology.
• a method of obtaining a peptide fragment by any combination of (a), (b) and (c), then linking the fragments to obtain a peptide, and recovering the peptide.
• the peptide or glucagon derivative comprising the amino acid sequence of Formula 1 or Formula 2 may be in the form of a long-acting conjugate coupled to a biocompatible material moiety that increases its half-life in vivo, but is not limited thereto.
• the biocompatible material part may be used interchangeably with a carrier.
• the long-acting conjugate form of the peptide containing the amino acid sequence of Formula 1 or Formula 2 is a liver-targeting drug, administered It is an active ingredient of a pharmaceutical composition with a high degree of distribution in the liver of the body organs of the subject.
• the term "long-acting conjugate” is a form in which a biocompatible material moiety or a carrier is bound to a physiologically active substance (eg, glucagon derivative). It includes a biocompatible material portion, and the peptide portion may be the same as or including the amino acid sequence of Formula 1 or Formula 2 or SEQ ID NOs: 2 to 11 and 13 to 45.
• the biocompatible material unit or carrier may be covalently linked to the bioactive material, but is not particularly limited thereto.
• the peptide conjugate may exhibit increased efficacy persistence and/or increased blood half-life compared to the peptide to which the carrier is not bound, and in the present invention, such conjugate is referred to as a "long-acting conjugate".
• conjugates may be non-naturally occurring.
• the long-acting conjugate refers to a form in which an immunoglobulin Fc region is linked to a glucagon derivative.
• the conjugate may be one in which an immunoglobulin Fc region is covalently linked to a glucagon derivative through a linker, but is not particularly limited thereto.
• the immunoglobulin Fc region and X may not be glycosylated, but is not limited thereto.
• the long-acting conjugate is a conjugate represented by Formula 1 below:
• X is the above peptide
• L is a linker containing an ethylene glycol repeating unit
• F is an immunoglobulin Fc region
• X of the long-acting conjugate of Formula 1 may be the glucagon derivative described above, but is not limited thereto.
• X of the long-acting conjugate of Formula 1 may be a peptide including the amino acid sequence of Formula 2.
• the X may be a peptide comprising the amino acid sequence of any one SEQ ID NO: 13, 15, 19, 33, and 36 to 45 selected from the group consisting of, or SEQ ID NO: 33 and 36 to 44 It may be a peptide comprising an amino acid sequence of any one sequence number selected from the group consisting of, but is not limited thereto.
• the X may be a peptide comprising the amino acid sequence of Formula 1, or, the X may be a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 2 to 45, or SEQ ID NOs: 2 to 11 and It may be a peptide comprising any one of amino acid sequences from 13 to 45, or X is SEQ ID NO: 20, 22, 23, 27, 33, 35, 37, 38, 40, 41, 42, and 44 It may be a peptide comprising an amino acid sequence selected from the group, but is not limited thereto.
• F is X, that is, a liver target drug, specifically a peptide having activity against glucagon receptors, and corresponds to one component of the moiety constituting the conjugate of the present invention. All of the above-described liver-targeting drugs are applied.
• the F may be bonded to X through a covalent chemical bond or a non-covalent chemical bond, 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.
• the term "long-acting conjugate of Formula 1" refers to a form in which a glucagon derivative and an immunoglobulin Fc region are connected to each other by a linker, and the conjugate exhibits increased durability compared to a glucagon derivative to which the immunoglobulin Fc region is not coupled.
• F is a substance capable of increasing the half-life of X, that is, a glucagon derivative, and corresponds to one component of the moiety constituting the conjugate of the present invention.
• connection between the peptide X, which is a glucagon derivative, and the immunoglobulin Fc region may be a physical or chemical bond, or a non-covalent or covalent bond, and specifically, may be a covalent bond, but is not limited thereto.
• the method of linking the immunoglobulin Fc region with the glucagon derivative X of the long-acting conjugate of Formula 1 is not particularly limited, but the glucagon derivative and the immunoglobulin Fc region may be connected to each other through a linker.
• X and L and L and F may be linked to each other through a covalent bond, and in this case, the conjugate may be a conjugate in which X, L, and F are linked through a covalent bond, respectively, in the order of Formula 1.
• the X and F may be connected through a linker (L).
• L may be a non-peptide linker, for example, a linker containing an ethylene glycol repeating unit.
• the "non-peptide linker” includes a biocompatible polymer in which two or more repeating units are bonded. The repeating units are connected to each other through any covalent bond other than a peptide bond.
• the non-peptide linker may be one constituent of the moiety of the conjugate of the present invention, and corresponds to L in Formula 1 above.
• Non-peptide linkers that can be used in the present invention can be used without limitation as long as they are polymers resistant to in vivo proteolytic enzymes.
• the non-peptide linker may be used in combination with a non-peptide polymer.
• non-peptide linker may be a linker containing an ethylene glycol repeating unit, for example, polyethylene glycol, and derivatives thereof already known in the art and readily available at the level of skill in the art. Derivatives that can be prepared are also included in the scope of the present invention.
• the repeating unit of the non-peptide linker may be an ethylene glycol repeating unit.
• the non-peptide 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 attached to the terminal.
• the long-acting conjugate according to the present invention may be a form in which X and F are linked through the functional group, but is not limited thereto.
• the non-peptide linker may include two or three or more functional groups, and each functional group may be the same or different from each other, but is not limited thereto.
• the linker may be polyethylene glycol (PEG) represented by Formula 2 below, but is not limited thereto:
• the PEG moiety may include not only a -(CH 2 CH 2 O) n - structure but also an oxygen atom intervening between the linking element and the -(CH 2 CH 2 O) n - , but accordingly It is not limited.
• the ethylene glycol repeating unit may be represented by, for example, [OCH 2 CH 2 ]n, where n is a natural number and is the average molecular weight of the [OCH 2 CH 2 ]n site in the peptide conjugate, such as number It can be set to have an average molecular weight of greater than 0 to about 100 kDa, but is not limited thereto.
• the long-acting conjugate of Formula 1 is a covalent bond between the peptide (X) containing the amino acid sequence of Formula 2 and the immunoglobulin region (F) via a linker containing an ethylene glycol repeating unit. It may be a connected structure, 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 molecular weight of the non-peptide polymer ranges from 1 to 100 kDa, specifically from 1 to 20 kDa, or from 1 to 10 kDa, but is not limited thereto.
• the non-peptide 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 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.
• aldehyde group examples include a propion aldehyde group or a butyl aldehyde group, but are not limited thereto.
• the linker may be connected to F, an immunoglobulin Fc region, and X, a peptide (glucagon derivative), through the reactive group as described above, and converted into a linker linkage.
• aldehyde reactive group reacts selectively at the N-terminus at low pH, and may form a covalent bond with a lysine residue at high pH, for example, pH 9.0, but is not limited thereto.
• it may have a maleimide group at one end and an aldehyde group, propionic aldehyde group, or butyl aldehyde group at the other end.
• it may have a succinimidyl group at one end and a propion aldehyde group or a butyl aldehyde group at the other end.
• the hydroxyl group can be activated into the various reactive groups by a known chemical reaction, or commercially available polyethylene glycol having a modified reactive group can be used.
• the conjugate of the invention can be prepared.
• the reactive group of the linker may be connected to a cysteine residue of peptide (X), more specifically, a -SH group of cysteine, but is not limited thereto.
• the reactive group of the linker may be linked to -NH 2 located at the N-terminus of the immunoglobulin Fc region, but this corresponds to one example.
• the peptide 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-terminus refers to the carboxy terminal of a peptide, and refers to a position capable of binding to a linker for the purpose of the present invention.
• it may include not only the most terminal amino acid residue at the C-terminus but also all of the amino acid residues around the C-terminus, and specifically include the first to twentieth amino acid residues from the terminal end. It may, but is not limited thereto.
• the conjugate of Formula 1 may have a structure of Formula 3 below.
• X is the above-described peptide (glucagon derivative);
• n may be a natural number. At this time, the description of n is as described above.
• the long-acting conjugate of Formula 3 is a structure in which peptide X and human immunoglobulin Fc region F are covalently linked via ethylene glycol repeating units, each X being a succinimide ring of Formula 3, F may be linked to the oxypropylene group of Formula 3.
• the value of n may be determined so that the average molecular weight of the [OCH 2 CH 2 ]n site in the peptide conjugate, for example, the number average molecular weight is 1 to 100 kDa, or 1 to 20 kDa or 10 kDa However, it is not limited thereto.
• F in Formula 1 is an immunoglobulin Fc region.
• the immunoglobulin Fc region may be derived from IgG, but is not particularly limited thereto.
• the Fc region refers to a natural sequence obtained from papain digestion of immunoglobulin as well as a derivative thereof, such as one or more amino acid residues in the natural sequence, which are transformed by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. It includes even variants such as sequences that are different from the form.
• the above derivatives, substituents and variants are premised on having the ability to bind to FcRn.
• F may be a human immunoglobulin region, but is not limited thereto.
• the F is a structure in which two polypeptide chains are connected by a disulfide bond, and may be a structure in which only one chain of the two chains is connected through a nitrogen atom, but is not limited thereto.
• the nitrogen atom may be connected to the epsilon amino atom or the N-terminal amino group of lysine through reductive amination.
• the reductive amination reaction refers to a reaction in which an amine group or an amino group of a reactant reacts with an aldehyde (ie, a functional group capable of reductive amination) of another reactant to generate an amine, and then a reduction reaction forms an amine bond, It is an organic synthesis reaction widely known in the art.
• flankinge sequence refers to a region located in a heavy chain and forming a dimer of an immunoglobulin Fc region through an inter disulfide bond.
• the hinge sequence may be mutated to have only one cysteine residue by deleting a part of the hinge sequence having the following amino acid sequence, but is not limited thereto:
• the hinge sequence may include only one cysteine residue by deletion of the 8th or 11th cysteine residue of the hinge sequence of SEQ ID NO: 50.
• the hinge sequence of the present invention may consist of 3 to 12 amino acids including only one cysteine residue, but is not limited thereto.
• the hinge sequence of the present invention may have the following sequence: Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 51), Glu-Ser- Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Pro (SEQ ID NO: 52), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 53), Glu- Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Pro (SEQ ID NO: 54), Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 55), Glu-Ser-Lys- Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 56), Glu-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 57),
• the hinge sequence may include an amino acid sequence of SEQ ID NO: 60 (Pro-Ser-Cys-Pro) or SEQ ID NO: 69 (Ser-Cys-Pro), but is not limited thereto.
• the immunoglobulin Fc region of the present invention may be in the form of a dimer formed by two molecules of the immunoglobulin Fc chain due to the presence of a hinge sequence, and in the conjugate of Formula 1 of the present invention, one end of the linker is a dimer immunoglobulin It may be linked to one chain of the Fc region, but is not limited thereto.
• N-terminus refers to the amino terminus of a protein or polypeptide, and includes one, two, three, four, five, six, It may contain up to 7, 8, 9, or 10 or more amino acids.
• the immunoglobulin Fc region of the present invention may include a hinge sequence at the N-terminus, but is not limited thereto.
• the immunoglobulin Fc region of the present invention includes 1) CH1 domain, CH2 domain, CH3 domain and CH4 domain, 2) CH1 domain and CH2 domain, 3) CH1 domain and CH3 domain, 4) CH2 domain and CH3 domain, 5) Combination of one or more of the CH1 domain, CH2 domain, CH3 domain and CH4 domain with an immunoglobulin hinge region (or part of a hinge region) (e.g., a combination of a CH2 domain and a CH3 domain with a hinge region or a portion thereof) , and two dimer forms of a polypeptide having the above combination), 6) may be a dimer of each domain of the heavy chain constant region and the light chain constant region, but is not limited thereto.
• the immunoglobulin Fc region is composed of single-chain immunoglobulins composed of domains of the same origin, and may be in the form of a dimer or a multimer, but is not limited thereto.
• the immunoglobulin Fc region may be in a dimeric form, and one molecule of X may be covalently linked to one Fc region of the dimer form, in which case the immunoglobulin Fc and X may be linked to each other by a non-peptide polymer. Meanwhile, it is also possible for two X molecules to bind symmetrically to one Fc region in the form of a dimer. In this case, the immunoglobulins Fc and X may be connected to each other by a non-peptide linker. However, 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 exchanges in proteins and peptides that do not entirely alter the activity of the molecule are known in the art (H. Neurath, R.L. Hill, The Proteins, Academic Press, New York, 1979).
• the most commonly occurring exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/ Exchange between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly.
• it is modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, and amidation. may be modified.
• the above-described Fc derivative may exhibit biological activity equivalent to that of the Fc region of the present invention and may have increased structural stability of the Fc region against heat and pH.
• an Fc region may be obtained from a natural form isolated in vivo from an animal such as human, cow, goat, pig, mouse, rabbit, hamster, rat or guinea pig, or obtained from transformed animal cells or microorganisms. It may be recombinant or a derivative thereof.
• the method of obtaining from the natural form may be a method of obtaining whole immunoglobulin by isolating it from a human or animal living body and then treating it with a proteolytic enzyme. When treated with papain, it is cleaved into Fab and Fc, and when treated with pepsin, it is cleaved into pF'c and F(ab) 2 . Fc or pF'c can be separated from this using size-exclusion chromatography or the like.
• the human-derived Fc region is a recombinant immunoglobulin Fc region obtained from a microorganism.
• the immunoglobulin Fc region may be a native type sugar chain, an increased sugar chain compared to the native type, a reduced sugar chain compared to the natural type, or a form in which the sugar chain has been removed.
• Conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms may be used to increase or decrease immunoglobulin Fc sugar chains.
• the immunoglobulin Fc region in which sugar chains are removed from Fc has significantly reduced binding ability to complement (c1q), and antibody-dependent cytotoxicity or complement-dependent cytotoxicity is reduced or eliminated, so that unnecessary immune responses are not induced in vivo.
• a form more suitable for its original purpose as a drug carrier will be referred to as an immunoglobulin Fc region in which sugar chains are removed or non-glycosylated.
• deglycosylation refers to an Fc region from which sugars are removed by an enzyme
• aglycosylation refers to a prokaryotic animal, in a more specific embodiment, refers to an Fc region that is not glycosylated produced by Escherichia coli. .
• the immunoglobulin Fc region may be of human origin or animal origin such as cow, goat, pig, mouse, rabbit, hamster, rat, guinea pig, etc., and in a more specific embodiment, it is of human origin.
• the immunoglobulin Fc region may be an Fc region derived from IgG, IgA, IgD, IgE, or IgM, a combination thereof, or a hybrid thereof. In a more specific embodiment, it is derived from IgG or IgM, which is most abundant in human blood, and in a more specific embodiment, it is derived from IgG known to improve the half-life of ligand binding proteins. In a more specific embodiment, the immunoglobulin Fc region is an IgG4 Fc region, and in the most specific embodiment, the immunoglobulin Fc region is a non-glycosylated Fc region derived from human IgG4, but is not limited thereto.
• the immunoglobulin Fc region is a region of human IgG4 Fc, in which two monomers are linked through a disulfide bond (inter-chain form) between cysteine, which is amino acid number 3 of each monomer. It may be in the form of a homodimer, wherein each monomer of the homodimer independently has an internal disulfide bond between cysteines 35 and 95 and an internal disulfide bond between cysteines 141 and 199, that is, two internal Has/can have disulfide bonds (intra-chain form).
• the number of amino acids of each monomer may consist of 221 amino acids, and amino acids forming a homodimer may consist of a total of 442 amino acids, but are not limited thereto.
• two monomers having the amino acid sequence of SEQ ID NO: 70 (consisting of 221 amino acids) form a homodimer through a disulfide bond between cysteine, the 3rd amino acid of each monomer, and the homodimer
• the monomers of may each independently form an internal disulfide bond between cysteines at positions 35 and 95 and an internal disulfide bond between cysteines at positions 141 and 199, but are not limited thereto.
• F in Formula 1 may include a monomer having an amino acid sequence of SEQ ID NO: 70, and F may be a homodimer of a monomer having an amino acid sequence of SEQ ID NO: 70, but is not limited thereto.
• the immunoglobulin Fc region may be a homodimer including the amino acid sequence of SEQ ID NO: 71 (consisting of 442 amino acids), but is not limited thereto.
• dimers or multimers can be prepared from two or more fragments selected from the group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc and IgE Fc fragments.
• conjugates may have increased efficacy compared to native glucagon or compared to X where F is not modified, and such conjugates may have the above-described form, as well as a form encapsulated in biodegradable nanoparticles, etc. All inclusive.
• a composition containing the liver-targeting drug such as a composition containing the peptide (eg, the peptide itself or in the form of a long-acting conjugate in which an immunoglobulin Fc region is conjugated thereto) can be used to prevent or treat diseases requiring drug action in the liver.
• a composition containing the peptide eg, the peptide itself or in the form of a long-acting conjugate in which an immunoglobulin Fc region is conjugated thereto
• prevention refers to any action that suppresses or delays the onset of a disease requiring drug action in the liver by administering a composition containing the liver-targeted drug
• treatment includes the liver-targeted drug. It means any action that improves or benefits the symptoms of a disease requiring a drug action in the liver by administration of a composition.
• the term "administration” means introducing a predetermined substance into a patient by any appropriate method, and the route of administration of the composition is not particularly limited thereto, but any general route by which the composition can reach the target in vivo It can be administered through, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, or intrarectal administration.
• intraperitoneal administration intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, or intrarectal administration.
• the term "disease requiring drug action in the liver” refers to a disease in which the liver-targeted drug according to the present invention can exhibit therapeutic activity, and the distribution of the drug administered to the liver tissue increases, thereby achieving a preventive or therapeutic effect.
• disease that can manifest may be liver disease, hypoglycemia or congenital hyperinsulinism, but is not particularly limited thereto, and diseases that can be treated by targeting the liver-targeted drug of the present invention are included without limitation.
• peptides having activity on glucagon receptors or long-acting conjugates thereof can be used to treat metabolic liver disease, simple steatosis, non-alcoholic fatty liver, liver inflammation, non-alcoholic steatohepatitis, cholestatic liver disease, liver It is effective for liver diseases such as fibrosis, liver cirrhosis, liver failure and liver cancer. Therefore, the peptide or long-acting conjugate thereof, which is a liver-targeted drug with excellent targeting to liver tissue of the present invention, has a preventive or therapeutic effect on the liver disease, and can be effectively provided as a liver disease treatment agent. there is.
• liver disease means a disease that develops in the liver, and includes nonalcoholic fatty liver disease (NAFLD), liver fibrosis, liver inflammation, liver cirrhosis ), liver decompensation, hepatocellular carcinoma, and cholestasis liver disease.
• NAFLD nonalcoholic fatty liver disease
• liver fibrosis liver inflammation
• liver cirrhosis liver cirrhosis
• liver decompensation hepatocellular carcinoma
• cholestasis liver disease cholestasis liver disease.
• hypoglycemia refers to a state in which the level of sugar in the blood is lower than that of a normal person. It usually refers to when blood sugar is 50 mg/dl or less, but is not particularly limited thereto.
• a common cause of hypoglycemia is when a person using oral hypoglycemic drugs or insulin consumes less food than usual or does excessive activity or exercise.
• drinking alcohol or use of some blood sugar lowering drugs serious physical illness, hormone deficiency such as adrenal cortical hormone or glucagon, insulin-producing pancreatic tumor, autoimmune disease against insulin, gastrectomy patients, hereditary carbohydrate metabolizing enzymes Hypoglycaemia can also occur due to abnormal diseases.
• hypoglycemia Symptoms of hypoglycemia include weakness, tremors, paleness, cold sweats, dizziness, excitement, anxiety, palpitations, hunger, headaches, and fatigue. Prolonged hypoglycemia may result in convulsions or seizures, and may result in shock and loss of consciousness.
• hypoglycemia may be caused by persistent hyperinsulinemia due to genetic defects.
• Hyperinsulinism caused by genetic defects is caused by mutations in the SUR or Kir6.2 gene on chromosome 11p15.1, GK (glucokinase) gene mutation on chromosome 7p15-p13, which increases GK activity, and GDH (glutamate dehydrogenase). It is known that GDH is activated by gene mutation, and ATP in beta islet cells increases due to this.
• congenital hyperinsulnism is one of the causes of severe and persistent hypoglycemia occurring in newborns and children. It can be caused by a temporary increase in insulin secretion in low birth weight babies or babies born to diabetic mothers, abnormal function of pancreatic cells due to gene mutation, etc.
• the liver can produce glucose through glycogenolysis and gluconeogenesis. Therefore, when hypoglycemia occurs, if a pharmaceutical composition that can induce glycogen breakdown and gluconeogenesis in the liver, such as glucagon and conjugates containing the same, can be targeted to the liver, more effective improvement of hypoglycemia and normalization of blood sugar can be expected. .
• the term "pharmaceutically acceptable” means an amount sufficient to exhibit a therapeutic effect and not causing side effects, and the type of disease, age, weight, health, sex, and sensitivity of the patient to the drug , It can be easily determined by a person skilled in the art according to factors well known in the medical field, such as an administration route, an administration method, the number of administrations, a treatment period, combination or drugs used simultaneously.
• the pharmaceutical composition including the peptide of the present invention may further include a pharmaceutically acceptable carrier.
• the carrier is not particularly limited thereto, but for oral administration, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavors, etc. may be used, and in the case of injections, buffers, Preservatives, analgesic agents, solubilizers, tonicity agents, stabilizers, etc. may be mixed and used, and in the case of topical administration, bases, excipients, lubricants, preservatives, etc. may be used.
• the dosage form of the composition of the present invention may be variously prepared by mixing with the pharmaceutically acceptable carrier as described above.
• the pharmaceutically acceptable carrier for example, for oral administration, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, it can be prepared in the form of unit dose ampoules or multiple doses.
• it may be formulated into solutions, suspensions, tablets, pills, capsules, sustained-release preparations, and the like.
• examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil and the like can be used.
• fillers, anti-coagulants, lubricants, wetting agents, flavoring agents, preservatives, and the like may be further included.
• the pharmaceutical composition of the present invention is any one selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, freeze-dried preparations, and suppositories.
• composition is formulated into a unit dosage form suitable for administration into the body of a patient according to a conventional method in the pharmaceutical field, specifically, a formulation useful for the administration of a protein drug, and is commonly used in the field of administration.
• the conjugate may be mixed with various pharmaceutically acceptable carriers such as physiological saline or organic solvents, and carbohydrates such as glucose, sucrose or dextran, ascorbic acid (ascorbic acid) may be used to increase stability or absorption. acid) or glutathione, antioxidants, chelating agents, low-molecular-weight proteins or other stabilizers may be used as drugs.
• pharmaceutically acceptable carriers such as physiological saline or organic solvents, and carbohydrates such as glucose, sucrose or dextran, ascorbic acid (ascorbic acid) may be used to increase stability or absorption. acid) or glutathione, antioxidants, chelating agents, low-molecular-weight proteins or other stabilizers may be used as drugs.
• the dosage and number of doses of the pharmaceutical composition of the present invention are determined according to the type of drug as an active ingredient, together with various related factors such as the disease to be treated, the route of administration, the patient's age, sex and weight, and the severity of the disease.
• the total effective amount of the composition of the present invention can be administered to the patient in a single dose or by a fractionated treatment protocol in which multiple doses are administered over a long period of time.
• the pharmaceutical composition of the present invention may vary the content of the active ingredient according to the severity of the disease.
• a preferred total dose of the conjugate of the present invention may be about 0.0001 mg to 500 mg/kg of the patient's body weight per day.
• the dose of the conjugate is determined by considering various factors such as the patient's age, weight, health condition, sex, severity of disease, diet and excretion rate, as well as the route of administration of the pharmaceutical composition and the number of treatments.
• composition according to the present invention is not particularly limited in its formulation, administration route and administration method as long as it exhibits the effects of the present invention.
• the pharmaceutical composition of the present invention has excellent in vivo persistence and potency, and thus the number and frequency of administration of the pharmaceutical preparation of the present invention can be significantly reduced.
• Another embodiment of the present invention provides a bioactive material targeted to liver tissue, including a material having binding force with a glucagon receptor, specifically, a bioactive material targeted to liver tissue.
• Another aspect embodying the present invention is a method for preventing or treating a disease requiring drug action in the liver, comprising administering the pharmaceutical composition or a physiologically active substance targeted to liver tissue to a subject in need thereof provides
• the subject is suspected of having a disease requiring drug action in the liver, and the subject suspected of having a disease requiring liver drug action includes mice, livestock, etc., including humans who have or may develop the disease. It means a mammal, but subjects that can be treated with the composition containing the liver-targeting drug of the present invention are included without limitation.
• the method of the present invention may include administering a pharmaceutical composition containing the liver-targeting drug in a pharmaceutically effective amount.
• An appropriate total daily amount can be determined by a treating physician within the scope of sound medical judgment, and can be administered once or divided into several times.
• a specific therapeutically effective amount for a particular patient is determined by the type and extent of the response to be achieved, the specific composition, including whether other agents are used as the case may be, the patient's age, weight, general state of health, It is preferable to apply differently according to various factors including gender and diet, administration time, administration route and secretion rate of the composition, treatment period, drugs used together with or concurrently used with a specific composition, and similar factors well known in the medical field.
• liver-targeting drug specifically a bioactive substance targeted to liver tissue
• a bioactive substance targeted to liver tissue for use in the preparation of a drug for preventing or treating a disease requiring drug action in the liver is to provide
• Another embodiment of the present invention is to provide a method for inducing liver targeting of the liver-targeting drug by administering the liver-targeting drug, specifically, a physiologically active substance targeted to liver tissue to a subject in need thereof.
• liver-targeting drug the bioactive substance targeted to the liver tissue, and the subject.
• Another aspect embodying the present invention is a method of inducing an increase in the distribution of the bioactive substance in liver tissue by administering the liver-targeting drug, specifically, a bioactive substance targeted to liver tissue to a subject in need thereof is to provide
• liver-targeting drug the bioactive substance targeted to the liver tissue, and the subject.
• the method of inducing liver targeting may be administered through an appropriate route of administration so that the substance can be targeted to the liver, such as subcutaneously (s.c.), but is not particularly limited thereto.
• an appropriate route of administration so that the substance can be targeted to the liver, such as subcutaneously (s.c.), but is not particularly limited thereto.
• Peptides of glucagon and derivatives thereof exhibiting activity at the glucagon receptor were prepared, and their sequences are shown in Table 1 below. Specifically, the amino acid sequence of natural glucagon of SEQ ID NO: 1 was substituted with negatively and positively charged amino acid residues to synthesize glucagon derivative peptides as shown in Table 1 below. The relative in vitro activity described herein was measured by the method described in Experimental Example 1 below.
• the amino acid represented by X represents aminoisobutyric acid (Aib), a non-natural amino acid, and the underline under the amino acid symbol indicates the formation of a lactam ring between the side chains of the underlined amino acid pair, and "-" indicates that there is no amino acid residue at that position.
• "-" in the column for whether or not a ring is formed indicates that a ring is not formed in the corresponding sequence.
• the glucagon derivative of SEQ ID NO: 37 was selected among the derivative peptides of Table 1 prepared in Example 1 above, and a conjugate was prepared by the following method.
• Maleimide-PEG-aldehyde Japanese NOF, a linear modified polyethylene glycol with a molecular weight of 10 kDa in which hydrogens at both ends are substituted with 3-(3-maleimidopropionamido)propyl group and 3-oxopropyl group (propionaldehyde group), respectively.
• G was reacted with the glucagon derivative peptide derivative of SEQ ID NO: 37 having cysteine, and the cysteine residue of the glucagon derivative peptide was PEGylated at the maleimide end of maleimide-PEG-aldehyde.
• the glucagon derivative peptide of SEQ ID NO: 37 and maleimide-PEG-aldehyde were reacted at a molar ratio of 1:1 to 5 and at a protein concentration of 3 to 10 mg/ml at a low temperature for 1 to 3 hours.
• the reaction was performed in an environment in which 20 to 60% isopropanol was added to 50 mM Tris buffer (pH 7.5).
• the reaction solution was applied to SP sepharose HP (GE healthcare, USA) to purify glucagon derivatives mono-pegylated to cysteine.
• the immunoglobulin Fc fragment is an immunoglobulin Fc fragment (49.8 kDa, two monomeric chains of SEQ ID NO: 70 linked by a disulfide bond) having a hinge region of Pro-Ser-Cys-Pro sequence (SEQ ID NO: 60) at the N-terminus. mer) was prepared by the method described in International Publication Patent WO2007/021129.
• the purified mono-PEGylated glucagon derivative peptide and immunoglobulin Fc fragment were incubated at a molar ratio of 1:2 to 10 and a protein concentration of 10 to 50 mg/mL at 4 to 8° C. for 12 to 18 hours. reacted
• the reaction solution was performed in an environment in which 10 to 50 mM sodium cyanoborohydride and 10 to 20% isopropanol as reducing agents were added to 100 mM potassium phosphate buffer (pH 6.0).
• reaction solution was applied to a Butyl sepharose FF purification column (GE healthcare, USA) and a Source ISO purification column (GE healthcare, USA) to obtain polyethylene glycol on the aldehyde side of the mono-pegylated glucagon derivative peptide.
• GE healthcare, USA a Butyl sepharose FF purification column
• Source ISO purification column GE healthcare, USA
• a conjugate in which a glucagon derivative peptide and an immunoglobulin Fc fragment are linked through PEG is referred to as a 'long-acting conjugate of glucagon derivative peptide',
• Each of the above cell lines was transformed to express a human GCG receptor gene in Chinese hamster ovary (CHO), and is suitable for measuring GCG activity. Therefore, activity on GCG receptors was measured using each transformed cell line.
• SEQ ID NOs: 48 and 49 are made using the part corresponding to the ORF in the cDNA of the human glucagon receptor gene (OriGene Technologies, Inc. USA) as a template and contain the EcoRI cleavage site and the XhoI cleavage site, respectively. PCR was performed using primers.
• the PCR reaction was performed 30 times, including denaturation at 95°C for 60 seconds, annealing at 55°C for 60 seconds, and elongation at 68°C for 30 seconds.
• the PCR product amplified therefrom was electrophoresed on a 1.0% agarose gel, and then a band having a size of 450 bp was eluted.
• the PCR product was cloned into a known animal cell expression vector x0GC/dhfr to prepare a recombinant vector x0GC/GCGR.
• the activity of the glucagon derivative synthesized in Example 1 was measured in the cell line. Specifically, the transformed cell line was subcultured 3 or 4 times a week, and then 6X10 3 subcultured cell lines were dispensed per well in a 384-well plate and cultured for 24 hours.
• Hank's Balanced HBSS HBSS containing 0.5 mM IBMX (3-isobutyl-1-methylxanthine), 0.1% BSA (Bovine serum albumin), and 5 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) in the cultured cells.
• the above-prepared glucagon derivative has high activity on the glucagon receptor, so targeting to hepatocytes is increased, and can also be used as a therapeutic substance for target diseases in the liver by activating the glucagon receptor.
• natural glucagon of SEQ ID NO: 1 has a pI of 6.8, while some glucagon derivatives according to the present invention have a pI ranging from about 4 to 6. Since these glucagon derivatives have a pI lower or higher than that of natural glucagon, they may exhibit improved solubility and higher stability than native glucagon at neutral pH.
• glucagon derivative according to the present invention When the glucagon derivative according to the present invention is used as a therapeutic agent for a target disease in the liver, patient compliance can be increased.
• tissue and organ distribution of the long-acting conjugate of SEQ ID NO: 37 which was selected as a representative example of the long-acting conjugate of glucagon derivative, was compared in 3 SD rats.
• tissue concentration / serum concentration * 100 For example, the 48-hour data in Table 3 below is serum: 15500.8 ⁇ 2686.9, liver: 4625.4 ⁇ 1216.9, and the T / S ratio (% ) was calculated as about 29.8% as 4625.4/15500.8*100. Concentration was measured by ELISA method after organ extraction.
• the tissue distribution of the long-acting glucagon derivative conjugate was most strongly detected at 48 hours after administration, and showed a high distribution ratio in the liver in particular.
• Tissue distribution was highest in the order of liver, heart, lung, colon, spleen, small intestine, muscle, stomach, pancreas, adipose tissue, and kidney. Even 168 hours after administration, it was still detected at the highest rate in the liver, and it was found that it was present in the liver at a high rate even on the 7th day after administration. In addition, at 168 hours after administration, it was present in the highest proportion in the liver, and the distribution ratio was higher in the lung than in the heart compared to 48 hours.
• Table 2 summarizes the results of the serum-to-tissue distribution ratio of the long-acting conjugate of SEQ ID NO: 37 confirmed in the above example.
• ng/ml is serum, ng/g is tissue concentration
• long-acting conjugate of the glucagon derivative of the present invention has excellent tissue distribution in the liver compared to other tissues, and can be used as a therapeutic substance for target diseases. Therefore, long-acting conjugates of glucagon derivatives can be used for new uses that can optimize drug treatment by inducing targeting to liver tissue to efficiently deliver a required amount of drug.

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