WO2022030580A1 - 長時間作用型新規アドレノメデュリン誘導体、その製造方法及びその医薬用途 - Google Patents

長時間作用型新規アドレノメデュリン誘導体、その製造方法及びその医薬用途 Download PDF

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WO2022030580A1
WO2022030580A1 PCT/JP2021/029112 JP2021029112W WO2022030580A1 WO 2022030580 A1 WO2022030580 A1 WO 2022030580A1 JP 2021029112 W JP2021029112 W JP 2021029112W WO 2022030580 A1 WO2022030580 A1 WO 2022030580A1
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peptide
amino acid
group
acid sequence
seq
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和雄 北村
基生 山▲崎▼
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国立大学法人宮崎大学
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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
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • 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
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • 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
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • the present invention relates to a novel long-acting adrenomedullin derivative, a method for producing the same, and a pharmaceutical use thereof.
  • Adrenomedullin (hereinafter, also referred to as "AM”) is a bioactive peptide isolated and identified from brown cell tissue in 1993 (Non-Patent Document 1). Initially discovered, AM was found to exert a strong vasodilatory antihypertensive effect. For example, Patent Document 1 describes a peptide having a blood pressure lowering effect, which comprises an amino acid sequence of human AM.
  • AM exerts various pharmacological actions such as cardiovascular protective action, anti-inflammatory action, angiogenic action and tissue repair promoting action.
  • administration studies of AM to patients with various diseases have been conducted with the aim of applying the pharmacological action of AM to the treatment of diseases.
  • the usefulness of AM as a therapeutic agent for inflammatory bowel disease, pulmonary hypertension, peripheral vascular disease or acute myocardial infarction is expected.
  • Patent Document 2 describes an adrenomedullin or a derivative thereof having an activity of suppressing non-bacterial inflammation, or a salt thereof having an activity of suppressing non-bacterial inflammation as an active ingredient. Describes a prophylactic or therapeutic agent for non-bacterial inflammatory bowel disease contained as.
  • Patent Document 3 describes the method for preventing or treating an inflammatory bowel disease in a patient who requires the prevention or treatment of an inflammatory bowel disease in which the use of a steroid preparation, an immunosuppressive agent or a biological preparation is difficult or inadequate.
  • the present invention comprises administering to the patient an effective amount of adrenomedulin, a derivative thereof having an activity of suppressing inflammation, or a salt of the adrenomedulin or the derivative thereof having an activity of suppressing inflammation.
  • the prevention or treatment method is described.
  • AM is a peptide, it has a short half-life in vivo due to a metabolic reaction in vivo (for example, in blood). Therefore, when administering AM to a subject, it is necessary to select a continuous administration method such as continuous intravenous infusion.
  • AM has a strong vasodilatory action in addition to a pharmacological action such as a cardiovascular protective action, an anti-inflammatory action, an angiogenesis action and a tissue repair promoting action. Therefore, when AM is administered to a subject, it may cause unwanted side effects such as excessive blood pressure reduction due to its strong vasodilatory effect.
  • adrenomedullin derivatives have been developed that can substantially suppress unwanted side effects while maintaining the pharmacological action of adrenomedullin (Patent Documents 4 to 6).
  • Patent Documents 7 and 8 As a technique for improving pharmacokinetics, a conjugate of a drug and serum albumin is known (Patent Documents 7 and 8).
  • Japanese Patent No. 2774769 Japanese Patent No. 4830093 Japanese Patent No. 5954736 International Publication No. 2015/14 1919 International Publication No. 2017/047788 International Publication No. 2018/181638 Japanese Patent No. 5599822 Japanese Patent No. 5639039
  • Adrenomedullin a novel hypotensive peptide isolated from human pheochromocytoma. , Pp. 553-560
  • Adrenomedullin has a strong vasodilatory action in addition to pharmacological actions such as cardiovascular protective action, anti-inflammatory action, angiogenic action and tissue repair promoting action. Therefore, when adrenomedullin or a derivative thereof is administered to a subject, it is not desirable such as excessive decrease in blood pressure due to a strong vasodilatory effect, tachycardia and / or increase in renin activity associated with an increase in reflex sympathetic nerve activity.
  • a drug containing adrenomedullin or a derivative thereof as an active ingredient is administered to a subject by continuous intravenous infusion at a dose that substantially does not cause undesired side effects. Needed to be done. Such a method of administration may impose a burden on the subject.
  • the adrenomedulin derivative which maintains the pharmacological action of adrenomedullin and has improved persistence in vivo, exhibits the pharmacological effect of adrenomedullin even when administered once to a subject, without causing unwanted side effects.
  • the present inventors have examined various means for solving the above-mentioned problems.
  • the present inventors maintain the same biological activity as the parent compound adrenomedullin, and adrenomedullin.
  • the present inventors have completed the present invention based on the above findings.
  • the linking group L is a substituted or unsubstituted divalent hydrocarbon group (the divalent hydrocarbon group is one or more complex atoms, a heterocycle, an amide group (-CO-NH-), an ester.
  • the compound according to the above embodiment (1) or (2) or a salt thereof, which is a group (-CO-O-) or a urethane group (-O-CO-NH-) may be contained), or a salt thereof. Hydrocarbon.
  • the linking group L has an ethylene oxide unit having a number of repetitions in the range of 2 to 24, and has one or more complex atoms, a heterocycle, an amide group (-CO-NH-), and an ester group (-CO). -O-), or a divalent hydrocarbon group which may contain a urethane group (-O-CO-NH-), the compound according to embodiment (3) or a salt thereof, or a hydrate thereof. ..
  • Adrenomedullin or its modified product is as follows: (I) A peptide consisting of the amino acid sequence of adrenomedulin, (Ii) A peptide consisting of the amino acid sequence of adrenomedullin, in which two cysteine residues in the amino acid sequence form a disulfide bond. (Iii) In the peptide of (ii), the peptide in which the disulfide bond is substituted with an ethylene group, (Iv) A peptide in which 1 to 15 amino acid residues are deleted, substituted or added in any of the peptides of (i) to (iii).
  • Adrenomedullin or its modified product is as follows: (I) A peptide consisting of the amino acid sequence of adrenomedulin, (Ii) A peptide consisting of the amino acid sequence of adrenomedullin, in which two cysteine residues in the amino acid sequence form a disulfide bond. (V) In the peptide of (i) or (ii), the peptide in which the C-terminal is amidated, and in the peptide of (vi) (i) or (ii), a glycine residue is added to the C-terminal.
  • Adrenomedullin or its modified product is as follows: (Iv') In any of the peptides (i) to (iii), a peptide in which the amino acid residues at positions 1 to 15, 1 to 10 or 1 to 5 from the N-terminal side are deleted, (V) Select from the group consisting of the peptide in which the C-terminal is amidated in the peptide of (iv') and the peptide in which the glycine residue is added to the C-terminal in the peptide of (vi) (iv').
  • Adrenomedullin or a modified product thereof is as follows: (A) A peptide consisting of the amino acid sequence of SEQ ID NO: 1 or a peptide consisting of the amino acid sequence of SEQ ID NO: 1 and in which the cysteine residue at position 16 and the cysteine residue at position 21 form a disulfide bond; (B) A peptide consisting of the amino acid sequence of SEQ ID NO: 4, or a peptide consisting of the amino acid sequence of SEQ ID NO: 4 and in which the cysteine residue at position 16 and the cysteine residue at position 21 form a disulfide bond; (C) A peptide consisting of the amino acid sequence of SEQ ID NO: 6 or a peptide consisting of the amino acid sequence of SEQ ID NO: 6 in which the cysteine residue at position 16 and the cysteine residue at position 21 form a disulfide bond; (D) A peptide consisting of the amino acid sequence of SEQ ID NO: 1 or a peptide consisting
  • Adrenomedullin or its modified product is as follows: (A) A peptide consisting of the amino acid sequence of SEQ ID NO: 1 or a peptide consisting of the amino acid sequence of SEQ ID NO: 1 and in which the cysteine residue at position 16 and the cysteine residue at position 21 form a disulfide bond; (B) A peptide consisting of the amino acid sequence of SEQ ID NO: 4, or a peptide consisting of the amino acid sequence of SEQ ID NO: 4 and in which the cysteine residue at position 16 and the cysteine residue at position 21 form a disulfide bond; (C) A peptide consisting of the amino acid sequence of SEQ ID NO: 6 or a peptide consisting of the amino acid sequence of SEQ ID NO: 6 in which the cyste
  • Adrenomedullin or a modified product thereof is as follows: (H') In any of the peptides (a) to (d), the amino acid residues at positions 1 to 15, 1 to 10 or 1 to 5 from the N-terminal side are deleted, or ( In the peptide of e) or (f), the peptide in which the amino acid residues at positions 1 to 13, 1 to 8 or 1 to 5 are deleted from the N-terminal side; (I) In the peptide of (h'), the peptide in which the C-terminal is amidated; and in the peptide of (j) (h'), the peptide in which the glycine residue is added to the C-terminal; The compound according to the above-mentioned embodiment (8), a salt thereof, or a hydrate thereof, which is a
  • Heart failure acute myocardial infarction, arrhythmia, atrial fibrillation, pulmonary hypertension, peripheral vascular disease, cerebral infarction, dementia, inflammatory bowel disease, Crohn's disease, ulcerative colitis, intestinal Bechet's disease, diabetes, diabetic
  • Heart failure, acute containing the compound according to any one of the above embodiments (1) to (10), a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable hydrate thereof as an active ingredient.
  • One or more symptoms, diseases and / or disorders include heart failure, acute myocardial infarction, arrhythmia, atrial fibrillation, pulmonary hypertension, peripheral vascular disease, cerebral infarction, dementia, inflammatory bowel disease, Crohn's disease, ulcer.
  • the compound for use according to the embodiment (19) or pharmaceutical thereof which is sexual colitis, intestinal Bechette's disease, diabetes, diabetic nephropathy, diabetic retinopathy, pulmonary fibrosis, sepsis or septic shock. Acceptable salts, or their pharmaceutically acceptable arrhythmias.
  • (21) The compound according to any one of the above embodiments (1) to (10) or a pharmaceutically acceptable salt thereof in the manufacture of a pharmaceutical agent for the prevention or treatment of one or more symptoms, diseases and / or disorders. , Or the use of their pharmaceutically acceptable solvates.
  • Each aspect of the present invention makes it possible to provide a novel long-acting adrenomedullin derivative that exhibits high biological stability when administered to a subject while maintaining the pharmacological action of the parent compound adrenomedullin. ..
  • FIG. 1 shows the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting (WB) of the synthesized novel adrenomedullin derivative (hSA-AM) in Experiment I.
  • A shows the gel stained with Coomassie Brilliant Blue after SDS-PAGE
  • B shows the membrane obtained by WB.
  • M represents a molecular weight marker (Precision Plus Protein Dual Xtra Standards)
  • lane 1 represents 68 ng of hSA
  • lane 2 represents 180 ng of hSA-AM.
  • FIG. 1 shows the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting (WB) of the synthesized novel adrenomedullin derivative (hSA-AM) in Experiment I.
  • A shows the gel stained with Coomassie Brilliant Blue after SDS-PAGE
  • B shows the
  • FIG. 2 shows a UV chromatogram with a wavelength of 280 nm by gel filtration chromatography (GF) of the novel adrenomedullin derivative (hSA-AM) synthesized in Experiment I.
  • A is a chromatogram of 30 ⁇ g hSA-AM
  • B is a chromatogram of 30 ⁇ g hSA
  • C is a chromatogram of purified hSA-AM.
  • the horizontal axis is the retention time (minutes) of the reverse phase HPLC
  • the vertical axis is the UV absorption intensity (mV) at a wavelength of 280 nm.
  • FIG. 3 shows the dose-response curve of intracellular cAMP production in HEK293 cells to the added concentration of native adrenomedullin (natural AM) or novel adrenomedullin derivative (hSA-AM) in Experiment II-1.
  • the black circles ( ⁇ ) are the average values of the experiments to which natural AM was added
  • the white squares ( ⁇ ) are the average values of the experiments to which hSA-AM was added
  • the error bars represent SEM.
  • the horizontal axis is the logarithm (M) of the added concentration of natural AM or hSA-AM
  • the vertical axis is the intracellular cAMP production amount (fmol) in HEK293 cells that stably express the AM1 receptor.
  • FIG. 4 shows the time course of the amount of natural adrenomedullin (natural AM) or novel adrenomedullin derivative (hSA-AM) in rat plasma after subcutaneous administration in Experiment II-2.
  • A is the result of natural AM
  • B is the result of hSA-AM.
  • the horizontal axis is the elapsed time (A: time, B: day) after subcutaneous administration
  • the vertical axis is the plasma peptide concentration (pM) after administration of natural AM or hSA-AM. ..
  • A is the time course of systolic blood pressure (sBP)
  • B is the time course of diastolic blood pressure (dBP).
  • the horizontal axis is the elapsed time (days), and the vertical axis shows the difference in blood pressure (A: difference in sBP, B: difference in dBP) (mmHg) obtained by subtracting the blood pressure before administration from the blood pressure on each measurement day. ..
  • the values in the figure are the average values of each group, and the error bars represent SEM.
  • DAI disease activity index
  • Adrenomedullin (AM) is a bioactive peptide isolated and identified from human brown cell tissue in 1993 (SEQ ID NO: 1, Non-Patent Document 1). A peptide consisting of the amino acid sequence of SEQ ID NO: 1, the C-terminal is amidated, and the two cysteine residues at positions 16 and 21 in the amino acid sequence form a disulfide bond is a mature natural peptide. Corresponds to type human adrenomedulin (hereinafter, also referred to as "hAM (1-52)").
  • the peptide consisting of the amino acid sequence of SEQ ID NO: 1 corresponds to the pre-translational (ie immature) form of natural human adrenomedullin that has undergone post-translational modification of C-terminal amidation and disulfide of cysteine residues.
  • hAM (1-52) YRQSMNNFQGLRSFGCRFGTC-TVQKLAHQIYQFTDKDKDNVA-PRSKISPQGY-CONH 2 (SEQ ID NO: 1)
  • AM was found to exert a strong vasodilatory antihypertensive effect. Subsequent studies have revealed that AM exerts a variety of pharmacological actions such as cardiovascular protective action, anti-inflammatory action, angiogenic action and tissue repair promoting action. In addition, administration studies of AM to patients with various diseases have been conducted with the aim of applying the pharmacological action of AM to the treatment of diseases.
  • AM Since AM is a peptide, it has a short half-life in vivo due to a metabolic reaction in vivo (for example, in blood). In addition, AM has a strong vasodilatory action in addition to a pharmacological action such as a cardiovascular protective action, an anti-inflammatory action, an angiogenesis action and a tissue repair promoting action. Therefore, when AM is administered to subjects, it causes undesired side effects such as excessive decrease in blood pressure due to strong vasodilatory action, tachycardia associated with increased reflex sympathetic nerve activity, and / or increased renin activity. there is a possibility.
  • the prior art drug containing AM or its derivative as an active ingredient is administered to the subject by continuous intravenous infusion at a dose that substantially does not cause undesired side effects. Needed to be done. Such a method of administration may impose a burden on the subject.
  • the present inventors By linking the N-terminal ⁇ -amino group of AM and serum albumin via a divalent linking group, the present inventors maintain the same biological activity as the parent compound AM, and AM. We have found that they have significantly better pharmacokinetics, for example with respect to biological stability.
  • one aspect of the present invention is the formula (I) :.
  • ALB ALB
  • the present invention relates to a compound represented by the above, a salt thereof, or a hydrate thereof.
  • the compound represented by the formula (I) or a salt thereof, or a solvate thereof may be referred to as an "adrenomedullin derivative" or an "AM derivative".
  • AM is not only a human-derived peptide (SEQ ID NO: 1, Non-Patent Document 1), but also, for example, pig (SEQ ID NO: 4), dog (SEQ ID NO: 6), bovine (SEQ ID NO: 8), and the like. It may be a peptide (ortholog) derived from another non-human mammal (eg, warm-blooded animal) such as rat (SEQ ID NO: 10) or mouse (SEQ ID NO: 12). In vivo, these peptides have two cysteine residues in their amino acid sequence forming a disulfide bond and the C-terminus being amidated.
  • the peptide having a disulfide bond and a C-terminal amide group may be referred to as "natural adrenomedullin” or simply “adrenomedullin”.
  • Naturally adrenomedullin or simply "adrenomedullin”.
  • Each aspect of the present invention can be applied to any of the above peptides.
  • C-terminal amidation means an aspect of post-translational modification of a peptide in vivo, and specifically, the main chain carboxyl group of the C-terminal amino acid residue of the peptide is an amide group. It means a reaction that is transformed into a form.
  • formation of a disulfide bond of a cysteine residue or “disulfide formation of a cysteine residue” means one aspect of post-translational modification of a peptide in vivo, and specifically, the peptide. It means a reaction in which two cysteine residues in an amino acid sequence form a disulfide bond (-SS-).
  • bioactive peptides produced in vivo are initially biosynthesized as higher molecular weight precursor proteins, such as C-terminal amidation and / or cysteine residue disulfide during intracellular translocation. After translation, it undergoes a modification reaction to become a mature physiologically active peptide.
  • C-terminal amidation usually proceeds by the action of C-terminal amyloid enzyme on precursor protein.
  • a physiologically active peptide having a C-terminal amide group in the precursor protein, a glycine (Gly) residue is bound to the C-terminal carboxyl group to be amidated, and the Gly residue is a C-terminal amidating enzyme. Is converted to a C-terminal amide group.
  • the C-terminal propeptide of precursor protein contains a repeating sequence of a combination of basic amino acid residues such as Lys-Arg or Arg-Arg (Mizuno, Biochemistry Vol. 61, No. 12, No. 12). Pp. 1435-1461 (1989)).
  • Disulfide formation of cysteine residues can proceed under oxidative conditions. In vivo, disulfide formation of cysteine residues usually proceeds by the action of protein disulfide isomerase on the precursor protein.
  • B is a peptide moiety derived from adrenomedullin or a variant thereof.
  • Peptide moiety B preferably has adrenomedulin activity by itself. That is, the peptide moiety B is preferably an adrenomedullin or a peptide moiety derived from a modified form thereof having adrenomedulin activity.
  • the "peptide moiety derived from adrenomedulin or a modification thereof” is one hydrogen atom from AM or a modification thereof (usually one hydrogen atom of an amino group, typically one hydrogen atom).
  • modified adrenomedullin means a peptide chemically modified from the native AM described above.
  • adrenomedullin activity means the biological activity of AM. Examples of the adrenomedullin activity include the following.
  • Cardiovascular system vasodilatory effect, blood pressure lowering effect, blood pressure increase inhibitory effect, heart rate increase / heart failure improving effect, pulmonary hypertension improving effect, angiogenesis effect, lymphangiogenesis effect, vascular endothelial function improving effect , Vascular permeability control, Endothelial cell adhesion control, Endothelial barrier protective action, Anti-arteriosclerotic action, Myocardial protective action (eg, Myocardial protective action in ischemia-reperfusion injury or inflammation), Remodeling inhibitory action after myocardial infarction , Cardiac hypertrophy inhibitory action, and andothetensin converting enzyme inhibitory action.
  • Myocardial protective action eg, Myocardial protective action in ischemia-reperfusion injury or inflammation
  • Remodeling inhibitory action after myocardial infarction Cardiac hypertrophy inhibitory action
  • Cardiac hypertrophy inhibitory action Cardiac hypertrophy inhibitory action
  • Kidney / water electrolyte system diuretic effect, natriuretic effect, antidiuretic hormone inhibitory effect, aldosterone lowering effect, renal protective effect (for example, myocardial protective effect in hypertension or ischemia-reperfusion injury), diabetic nephropathy suppression Action, C3 nephropathy suppressing action, drinking behavior suppressing action, and salt requirement suppressing action.
  • Brain / nervous system neuroprotective / cerebral disorder inhibitory action, anti-inflammatory action, apoptosis inhibitory action (for example, ischemia-reperfusion injury or apoptosis inhibitory action in inflammation), autoregulatory ability maintenance action, oxidative stress suppressive action, Dementia improving effect and sympathetic depressant effect.
  • Genitourinary system erection improving action, blood flow improving action, and implantation promoting action.
  • Digestive system anti-ulcer action, tissue repair action, mucosal neoplastic action, intestinal barrier protection action, blood flow improving action, anti-inflammatory action, and liver function improving action.
  • Orthopedic system Osteoblast stimulating action and arthritis improving action.
  • Endocrine metabolism system adipocyte differentiation effect, lipolysis control effect, insulin sensitivity improving effect, insulin secretion control effect, antidiuretic hormone secretion inhibitory effect, and aldosterone secretion inhibitory effect.
  • Respiratory system Bronchial dilation effect, lung protection effect, emphysema improving effect, pulmonary fibrosis suppression, pneumonia suppression, bronchitis suppression effect, and respiratory improvement effect.
  • Immune system C3b degradation promoting action.
  • Others Circulation improving action, anti-inflammatory action, cytokine control action, organ protection action, oxidative stress suppressing action, tissue repair action (for example, anti-decubitus action), sepsis improving action, septic shock improving action, many Suppressive action of organ failure, suppressive action of autoimmune disease, suppressive action of diabetic retinopathy, antibacterial action, hair growth action, and hair nourishing action.
  • the blood pressure lowering action is preferably a vasodilatory antihypertensive action.
  • the anti-inflammatory action in the digestive system is a preventive or therapeutic action for inflammatory bowel diseases such as steroid-resistant or steroid-dependent inflammatory bowel diseases (eg, ulcerative colitis, Crohn's disease or intestinal Behcet's disease). It is preferable to have.
  • the adrenomedulin activity exemplified above expressed by AM is usually expressed through an increase in the concentration of intracellular cAMP. Therefore, an increase in the concentration of intracellular cAMP can be used as an index of the adrenomedullin activity of the compound of this embodiment.
  • the action of increasing the intracellular cAMP concentration is, for example, by adding a target compound to a cultured cell line (HEK293 cell line) in which AM type 1 receptor (AM1 receptor) is stably expressed, and cells are used. It can be evaluated by measuring the production amount of intracellular cAMP.
  • the compound of this embodiment has an action of increasing the concentration of intracellular cAMP substantially equivalent to that of natural AM. Therefore, the compound of this embodiment can express biological activity (that is, adrenomedullin activity) substantially equivalent to that of natural AM through an increase in the concentration of intracellular cAMP.
  • Adrenomedullin or its modifications are listed below: (I) A peptide consisting of the amino acid sequence of adrenomedulin, (Ii) A peptide consisting of the amino acid sequence of adrenomedullin, in which two cysteine residues in the amino acid sequence form a disulfide bond. (Iii) In the peptide of (ii), the peptide in which the disulfide bond is substituted with an ethylene group, (Iv) A peptide in which 1 to 15 amino acid residues are deleted, substituted or added in any of the peptides of (i) to (iii).
  • the peptide in which the C-terminal is amidated and in any of the peptides (vi), (i) to (iv), the glycine residue at the C-terminal remains. It is preferably a peptide selected from the group consisting of peptides to which a group is added.
  • the adrenomedullin or a modification thereof is as follows: (I) A peptide consisting of the amino acid sequence of adrenomedulin, (Ii) A peptide consisting of the amino acid sequence of adrenomedullin, in which two cysteine residues in the amino acid sequence form a disulfide bond. (V) In the peptide of (i) or (ii), the peptide whose C-terminal is amidated, and in the peptide of (vi) (i) or (ii), a glycine residue is added to the C-terminal. It is more preferable that the peptide is selected from the group consisting of peptides.
  • the peptide consists of the amino acid sequence of AM contained in (v), the C-terminal is amidated, and the two cysteine residues in the amino acid sequence are disulfides.
  • the peptide forming the bond corresponds to mature native AM.
  • the peptide consisting of the amino acid sequence of AM in (i) corresponds to the pre-translational modification (ie, immature) form of native AM with C-terminal amidation and disulfide of cysteine residues.
  • the peptides other than the peptides described above correspond to modified versions of AM.
  • the peptide (ii) is formed by air-oxidizing the thiol groups of the two cysteine residues of the peptide (i) or by oxidizing them with an appropriate oxidizing agent to convert them into disulfide bonds. Can be made to.
  • the three-dimensional structure of the peptide portion B can be made to resemble the three-dimensional structure of the natural AM.
  • the adrenomedullin activity of the compound represented by the formula (I) can be made substantially equivalent to that of the natural AM.
  • the peptide of (iii) can be formed by converting the disulfide bond of the peptide of (ii) into an ethylene group. Substitution of disulfide bonds to ethylene groups can be performed by methods well known in the art (O. Keller et al., Helv. Chim. Acta, 1974, Vol. 57, p. 1253). By using the peptide of (iii) above, the three-dimensional structure of the peptide portion B can be stabilized. Thereby, the compound represented by the formula (I) can continuously express the adrenomedulin activity in the living body.
  • the number of amino acid residues deleted, substituted or added is preferably in the range of 1 to 15, more preferably in the range of 1 to 10, and 1 to 8.
  • the range of 1 is more preferable, the range of 1 to 5 is particularly preferable, and the range of 1 to 3 is most preferable.
  • Suitable peptides (iv) are 1 to 15 positions, 1 to 12 positions, 1 to 10 positions, 1 to 8 positions, and 1 to 5 positions from the N-terminal side in any of the peptides (i) to (iii).
  • a peptide in which the amino acid residue at the position or the 1st to 3rd position is deleted, and the more preferable peptide (iv) is the peptide of any of (i) to (iii), 1 to 1 to 1 from the N-terminal side. It is a peptide in which amino acid residues at positions 15, 1 to 10 or 1 to 5 are deleted. In the preferred peptide, one or more (eg, 1-5, 1-3, or 1 or 2) amino acid residues may be further deleted, substituted or added.
  • the adrenomedullin activity of the compound represented by the formula (I) can be made substantially equivalent to that of natural AM.
  • the compound represented by the formula (I) can continuously express adrenomedulin activity in vivo.
  • the peptide of (vi) can be converted into the peptide of (v) by converting the C-terminal glycine residue into a C-terminal amide group by the action of the C-terminal amidating enzyme. Therefore, by administering the peptide (vi) to a subject, a C-terminal amidated peptide can be formed in the living body of the subject after a lapse of a certain period of time. Thereby, the compound represented by the formula (I) can continuously express the adrenomedulin activity in the living body.
  • Adrenomedullin or its modifications are listed below: (A) A peptide consisting of the amino acid sequence of SEQ ID NO: 1 or a peptide consisting of the amino acid sequence of SEQ ID NO: 1 and in which the cysteine residue at position 16 and the cysteine residue at position 21 form a disulfide bond; (B) A peptide consisting of the amino acid sequence of SEQ ID NO: 4, or a peptide consisting of the amino acid sequence of SEQ ID NO: 4 and in which the cysteine residue at position 16 and the cysteine residue at position 21 form a disulfide bond; (C) A peptide consisting of the amino acid sequence of SEQ ID NO: 6 or a peptide consisting of the amino acid sequence of SEQ ID NO: 6 in which the cysteine residue at position 16 and the cysteine residue at position 21 form a disulfide bond; (D) A peptide consisting of the amino acid sequence of SEQ ID NO: 8 or a peptide consisting of the amino
  • Adrenomedullin or its modifications are listed below: (A) A peptide consisting of the amino acid sequence of SEQ ID NO: 1 or a peptide consisting of the amino acid sequence of SEQ ID NO: 1 and in which the cysteine residue at position 16 and the cysteine residue at position 21 form a disulfide bond; (B) A peptide consisting of the amino acid sequence of SEQ ID NO: 4, or a peptide consisting of the amino acid sequence of SEQ ID NO: 4 and in which the cysteine residue at position 16 and the cysteine residue at position 21 form a disulfide bond; (C) A peptide consisting of the amino acid sequence of SEQ ID NO: 6 or a peptide consisting of the amino acid sequence of SEQ ID NO: 6 in which the cysteine residue at position 16 and the cysteine residue at position 21 form a disulfide bond; (D) A peptide consisting of the amino acid sequence of SEQ ID NO: 8 or a peptide consisting of the amino
  • the adrenomedullin or a modification thereof is as follows: (A) A peptide consisting of the amino acid sequence of SEQ ID NO: 1 or a peptide consisting of the amino acid sequence of SEQ ID NO: 1 and in which the cysteine residue at position 16 and the cysteine residue at position 21 form a disulfide bond; (I) In the peptide of (a), the peptide in which the C-terminal is amidated; and in the peptide of (j) (a), the peptide in which the glycine residue is added to the C-terminal; It is preferably a peptide selected from the group consisting of.
  • the number of amino acid residues deleted, substituted or added is preferably in the range of 1 to 12, more preferably in the range of 1 to 10, and is preferably in the range of 1 to 8.
  • the range of 1 is more preferable, the range of 1 to 5 is particularly preferable, and the range of 1 to 3 is most preferable.
  • Suitable peptides (h) are 1 to 15 positions, 1 to 12 positions, 1 to 10 positions, 1 to 8 positions, and 1 to 5 positions from the N-terminal side in any of the peptides (a) to (g).
  • one or more amino acids eg, 1-5, 1-3, or 1 or 2 may be further deleted, substituted or added.
  • the adrenomedullin activity of the compound represented by the formula (I) can be made substantially equivalent to that of natural AM. Further, by using the peptide of the above (h), the compound represented by the formula (I) can continuously express the adrenomedullin activity in the living body.
  • peptides exemplified above as adrenomedulin or a modified form thereof usually have adrenomedulin activity.
  • the peptide having the above-mentioned characteristics and adrenomedullin activity is, for example, biological as compared with AM while maintaining substantially the same pharmacological action as the parent compound AM. It can have significantly better pharmacokinetics with respect to stability.
  • A is a modifying group that is serum albumin.
  • Serum albumin is the major protein in mammalian blood. Serum albumin is involved in the transport and metabolism of various substances in the living body. For example, serum albumin recognizes and binds to endothelial cell gp60. As a result, serum albumin is known to have excellent migration activity on endothelial cells. Therefore, the conjugate of the drug and serum albumin is known as a technique for improving pharmacokinetics (for example, Japanese Patent No. 5959822 (Patent Document 7) and Japanese Patent No. 5639039 (Patent Document 8)). Therefore, the compound represented by the formula (I) of this embodiment having the modifying group A which is serum albumin has significantly superior pharmacokinetics, for example, in terms of biological stability as compared with the native AM. be able to.
  • the mammal from which the serum albumin used as the modifying group A is derived is a drug containing the compound represented by the formula (I) of one aspect of the present invention as an active ingredient, which is described below. It can be selected as appropriate based on the target to be applied.
  • Modifying group A is serum albumin from human or non-human mammals (eg, warm-blooded animals such as pigs, dogs, cows, rats, mice, guinea pigs, rabbits, chickens, sheep, cats, monkeys, hamadryas baboons or chimpanzees).
  • the serum albumin is derived from the same human or non-human mammal as the subject to which the medicine of one aspect of the present invention is applied.
  • the modifying group A is preferably human serum albumin consisting of the amino acid sequence of SEQ ID NO: 14.
  • Human serum albumin (hereinafter, also referred to as “hSA”) consisting of the amino acid sequence of SEQ ID NO: 14 is a protein consisting of 585 amino acid residues having a molecular weight of about 66,000 Da.
  • L is a divalent linking group.
  • the linking group L is preferably a substituted or unsubstituted divalent hydrocarbon group, a substituted or unsubstituted divalent aliphatic hydrocarbon group, a substituted or unsubstituted divalent alicyclic group or a substituent. Alternatively, it is more preferably an unsubstituted divalent aromatic group, and further preferably a substituted or unsubstituted C 1 to C 20 alkylene group, a C 2 to C 20 alkenylene group or a C 2 to C 20 alkynylene group. preferable.
  • the group may be one or more complex atoms (eg, O or S), a heterocycle, an amide group (-CO-NH-), an ester group (-CO-O-), or a urethane group (-O-CO).
  • -NH- is preferably included.
  • the substituent may include halogen (eg, F, Cl, Br or I), cyano, nitro and C 1 to C 5 alkoxy.
  • the linking group L has an ethylene oxide unit having a repeat number in the range of 2 to 24, particularly 4 to 12, and one or more complex atoms (eg, O or S), a heterocycle, -CO-NH-,.
  • the compound represented by the formula (I) of this embodiment can be compared with, for example, biologically, while maintaining the pharmacological action of the native AM. It can have significantly better pharmacokinetics with respect to stability.
  • the linking group L has a carboxyl group at at least one end.
  • the linking group L preferably has a functional group such as a carboxyl group, an amino group or a succinimide group at the other end.
  • the linking group L preferably has a carboxyl group at one end and a succinimide group at the other end.
  • the linking group L forms an amide bond with the ⁇ -amino group at the N-terminal of the peptide moiety B via the carboxyl group, and the cysteine residue of the modifying group A via the succinimide group.
  • a thioether bond with the thiol group, it is linked to the modifying group A and the peptide moiety B.
  • the peptide moiety B has an N-terminal ⁇ -amino group, specifically, an ⁇ -amino group of an N-terminal amino acid residue forming an amide bond with a carboxyl group at the end of the linking group L. It is connected to the rest.
  • the modifying group A which is serum albumin
  • the modifying group A is such that any functional group contained in the constituent amino acid residues of serum albumin forms a covalent bond with the functional group exemplified above at the terminal of the linking group L. Is linked to the rest by.
  • the functional group forming the linkage with the linking group L include an ⁇ -amino group at the N-terminal of serum albumin, a carboxyl group at the C-terminal, a thiol group (sulfhydryl group) at the cysteine residue, and a lysine residue.
  • Examples thereof include the ⁇ amino group of the group, the imidazole ring of the histidine residue, the guanidino group of the arginine residue, the ⁇ carboxyl group of the glutamate residue, or the ⁇ carboxyl group of the aspartic acid residue.
  • the linking group L is a substituted or unsubstituted divalent hydrocarbon group having a carboxyl group at at least one end and a functional group exemplified above at the other end (the group is one or more complex atoms (the group is one or more complex atoms). For example, it is preferably O or S), a heterocycle, -CO-NH-, -CO-O- or -O-CO-NH-), with a carboxyl group at at least one end and the other.
  • the group may be one or more complex atoms (eg, O or S), a heterocycle, -CO-NH-, -CO-O- or -O-CO-NH-). More preferably, substituted or unsubstituted C 1 to C 20 alkylene groups, C 2 to C 20 alkenylene groups or C 2 to C 20 having a carboxyl group at at least one end and the functional group exemplified above at the other end.
  • Alquinylene group (the group may include one or more complex atoms (eg, O or S), a heterocycle, -CO-NH-, -CO-O- or -O-CO-NH-). It is more preferable to have.
  • the linking group L has a carboxyl group at at least one end, a functional group exemplified above at the other end, and an ethylene oxide unit having a repetition number in the range of 2 to 24, particularly 4 to 12.
  • a divalent hydrocarbon group which may contain one or more complex atoms (eg, O or S), a heterocycle, -CO-NH-, -CO-O- or -O-CO-NH-.
  • the ethylene oxide unit has a carboxyl group at at least one end, the functional group exemplified above at the other end, and has a repetition number in the range of 2 to 24, particularly 4 to 12, and 1 More preferably, it is a divalent hydrocarbon group that may contain more than one O or -CO-NH-.
  • the linking group L is, for example, the following formula: (In the formula, # represents the linking position with the modifying group A, and * represents the linking position with the peptide moiety B.).
  • a suitable compound represented by the formula (I) is Modification group A is human serum albumin consisting of the amino acid sequence of SEQ ID NO: 14.
  • Peptide part B is as follows: (A) A peptide consisting of the amino acid sequence of SEQ ID NO: 1 or a peptide consisting of the amino acid sequence of SEQ ID NO: 1 and in which the cysteine residue at position 16 and the cysteine residue at position 21 form a disulfide bond; (B) A peptide consisting of the amino acid sequence of SEQ ID NO: 4, or a peptide consisting of the amino acid sequence of SEQ ID NO: 4 and in which the cysteine residue at position 16 and the cysteine residue at position 21 form a disulfide bond; (C) A peptide consisting of the amino acid sequence of SEQ ID NO: 6 or a peptide consisting of the amino acid sequence of SEQ ID NO: 6 in which the cysteine residue at position 16 and the cysteine residue at position 21 form a disulfide bond;
  • the linking group L is a substituted or unsubstituted divalent hydrocarbon group having a carboxyl group at at least one end and a functional group such as a carboxyl group, an amino group or a succinimide group at the other end (the above group is 1). More than one complex atom (eg, O or S), heterocycle, -CO-NH-, -CO-O- or -O-CO-NH- may be included).
  • the peptide moiety B is linked to the rest by the N-terminal ⁇ -amino group forming an amide bond with the carboxyl group at the end of the linking group L.
  • a more suitable compound represented by the formula (I) is Modification group A is human serum albumin consisting of the amino acid sequence of SEQ ID NO: 14.
  • Peptide part B is as follows: (A) A peptide consisting of the amino acid sequence of SEQ ID NO: 1 or a peptide consisting of the amino acid sequence of SEQ ID NO: 1 and in which the cysteine residue at position 16 and the cysteine residue at position 21 form a disulfide bond; (I) In the peptide of (a), the peptide in which the C-terminal is amidated; and in the peptide of (j) (a), the peptide in which the glycine residue is added to the C-terminal; A peptide moiety derived from adrenomedullin or a modification thereof, which is a peptide selected from the group consisting of
  • the linking group L is a substituted or unsubstituted divalent hydrocarbon group having a carboxyl group at at least one end and a functional group
  • More than one complex atom eg, O or S
  • heterocycle e.g., -CO-NH-, -CO-O- or -O-CO-NH- may be included).
  • the peptide moiety B is linked to the rest by the N-terminal ⁇ -amino group forming an amide bond with the carboxyl group at the end of the linking group L.
  • a more suitable compound represented by the formula (I) is Modification group A is human serum albumin consisting of the amino acid sequence of SEQ ID NO: 14.
  • Peptide part B is as follows: (A) A peptide consisting of the amino acid sequence of SEQ ID NO: 1 or a peptide consisting of the amino acid sequence of SEQ ID NO: 1 and in which the cysteine residue at position 16 and the cysteine residue at position 21 form a disulfide bond; (I) In the peptide of (a), the peptide in which the C-terminal is amidated; and in the peptide of (j) (a), the peptide in which the glycine residue is added to the C-terminal; A peptide moiety derived from adrenomedullin or a modification thereof, which is a peptide selected from the group consisting of
  • the linking group L has a carboxyl group at at least one end and a functional group such as a carboxyl group, an amino group or a succin
  • the peptide moiety B is linked to the rest by the N-terminal ⁇ -amino group forming an amide bond with the carboxyl group at the end of the linking group L.
  • the compound represented by the formula (I) of this embodiment having the above-mentioned characteristics is a drug that is significantly superior in, for example, to biological stability, as compared with the natural AM, while maintaining the pharmacological action of the natural AM.
  • the compound of this aspect includes not only the compound itself but also a salt thereof.
  • the compound of this embodiment is in the form of a salt, it is preferably a pharmaceutically acceptable salt.
  • the counter ion of the salt of the compound of this embodiment is not limited, but is, for example, a cation such as a sodium ion, a potassium ion, a calcium ion, a magnesium ion, or a substituted or unsubstituted ammonium ion, or a chloride ion.
  • the compound of this aspect includes not only the compound itself but also a solvate of the compound or a salt thereof.
  • the compound of this embodiment or a salt thereof is in the form of a solvate, it is preferably a pharmaceutically acceptable solvate.
  • the solvent that can form a solvent with the compound or a salt thereof is not limited, but is, for example, water, or methanol, ethanol, 2-propanol (isopropyl alcohol), dimethyl sulfoxide (DMSO), acetic acid, ethanol, and the like.
  • Organic solvents such as amine, acetonitrile or ethyl acetate are preferred.
  • the compound of this aspect includes not only the compound itself described above or below, but also a protected form thereof.
  • protected form means a form in which a protecting group is introduced into one or more functional groups (eg, side chain amino groups of lysine residues).
  • the "protecting group” is a group introduced into a specific functional group in order to prevent the undesired progress of the reaction, and is quantitatively removed under the specific reaction conditions, and the same. It means a group that is substantially stable under the reaction conditions other than the above, that is, the reaction is inactive.
  • Protecting groups that can form the protected form of the compound are, but are not limited to, for example, t-butoxycarbonyl (Boc), 2-bromobenzyloxycarbonyl (BrZ), 9-fluorenylmethoxycarbonyl (Fmoc).
  • the adrenomedullin activity of the compound may be substantially equivalent to that of native AM.
  • the compound of this aspect also includes individual enantiomers and diastereomers of the compound, as well as mixtures of stereoisomers of the compound, such as racemates.
  • the compound of this embodiment is remarkable in terms of, for example, biological stability as compared with the natural AM, while maintaining substantially the same pharmacological action as the parent compound, the natural AM.
  • adrenomedullin derivative ⁇ 2.
  • the compound of one aspect of the present invention can have significantly superior pharmacokinetics as compared with AM while maintaining a pharmacological action substantially equivalent to that of the parent compound AM. Therefore, another aspect of the present invention relates to a medicine containing the compound of one aspect of the present invention as an active ingredient.
  • the compound of one aspect of the present invention When the compound of one aspect of the present invention is applied to pharmaceutical use, the compound may be used alone or in combination with one or more pharmaceutically acceptable ingredients.
  • the pharmaceutical of this embodiment can be formulated into various dosage forms commonly used in the art, depending on the desired method of administration. Therefore, the pharmaceutical of this embodiment also comprises a compound of one aspect of the invention or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, and one or more pharmaceutically acceptable carriers.
  • the pharmaceutical composition is, in addition to the above-mentioned components, one or more pharmaceutically acceptable media (for example, a solvent such as sterile water or a solution such as physiological saline), an excipient, and the like.
  • Binders such as agents, antioxidants, sweeteners and flavoring agents may be included.
  • the dosage form of the drug of this embodiment is not particularly limited and may be a preparation for use in parenteral administration, and may be transmucosal (for example, nasal, sublingual or oral cavity mucosa, etc.), transdermal, transdermal. It may be a preparation for use in administration of an anal (enema), transvaginal or the like, or a preparation for use in oral administration.
  • the dosage form of the pharmaceutical product of this embodiment may be a unit-dose form or a plurality of dosage forms.
  • Formulations for use in parenteral administration include, for example, injections such as sterile solutions or suspensions with water or other pharmaceutically acceptable liquids.
  • Additives that can be mixed with the injection are, but are not limited to, isotonic containing, for example, physiological saline, glucose or other adjuvants (eg, D-sorbitol, D-mannitol or sodium chloride).
  • Vehicles such as liquids, solubilizers such as alcohols (eg ethanol or benzyl alcohol), esters (eg benzyl benzoate), polyalcohols (eg propylene glycol or polyethylene glycol), polysorbate 80 or polyoxyethylene hydrogenated castor oil.
  • Nonionic surfactants such as oily liquids such as sesame oil or soybean oil, buffers such as phosphate buffers or sodium acetate buffers, soothing agents such as benzalconium chloride or prokine hydrochloride, humans.
  • Stabilizers such as serum albumin or polyethylene glycol, preservatives, antioxidants and the like can be mentioned.
  • the prepared injection is usually filled in a suitable container (eg, vial or ampoule) and stored in a suitable environment until use.
  • Additives contained in the preparation for use in transmucosal administration include, for example, vehicles, emulsifiers, suspensions, antibacterial agents (eg, chlorobutanol), isotonic agents (eg, sodium chloride), pH regulators and Penetrants can be mentioned.
  • Additives contained in the pharmaceutical product for use in transdermal administration include, for example, a vehicle, an antipruritic agent, an antifoaming agent, a palliative agent, a surfactant, an emulsifier, a thickener, a suspending agent, a buffer, and a viscosity. Examples thereof include excipients, moisturizers, antioxidants, chemical stabilizers, colorants and decolorizers.
  • additive contained in the preparation for use in transanal administration examples include a medium, an emulsifier and a solid fat base.
  • Additives contained in the formulation for use in vaginal administration include, for example, vehicles, buffers, oily liquids, suspensions, wetting agents, surfactants, antioxidants, antibacterial agents and isotonic agents. be able to.
  • the preparation for use in oral administration examples include tablets, pills, powders, capsules, soft capsules, microcapsules, elixirs, liquids, syrups, slurrys and suspensions. ..
  • the tablet may be formulated as a sugar-coated or soluble-coated sugar-coated tablet, gelatin-encapsulated tablet, enteric-coated tablet, orally disintegrating tablet (OD tablet) or film-coated tablet, or two. It may be formulated as a dosage form of a heavy tablet or a multi-layer tablet.
  • Additives that can be mixed with tablets, capsules, etc. are not limited, but are, for example, water, ethanol, propanol, simple syrup, glucose solution, carboxymethyl cellulose, cellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone.
  • Excipients such as starch, lactose, kaolin, bentonite or colloidal silicic acid;
  • Lubricants such as purified talc, stearate (eg magnesium stearate), powder borate or polyethylene glycol;
  • Sucrose lactose
  • a sweetener such as saccharin
  • a flavoring agent such as peppermint, red mono oil or cherry can be mentioned.
  • the pharmaceutical product is a capsule, it may further contain a liquid carrier such as fat or oil.
  • the pharmaceutical product of this embodiment can also be formulated as a depot preparation.
  • the drug of this embodiment in the dosage form of the depot preparation can be administered, for example, subcutaneously or intramuscularly, or by intramuscular injection.
  • the adrenomedullin activity of the compound of one embodiment of the present invention can be continuously expressed over a long period of time.
  • the compound of one embodiment of the present invention is significantly superior to AM, for example, in terms of biological stability, while maintaining substantially the same pharmacological action as the parent compound AM.
  • the pharmaceutical product of this embodiment is preferably formulated as a single-dose formulation, and more preferably formulated as a single-subcutaneous dosage form.
  • the drug of this embodiment can also be used in combination with one or more other drugs useful as a drug.
  • the pharmaceutical product of this embodiment is a single compound containing the compound of one embodiment of the present invention or a pharmaceutically acceptable salt thereof, or a solvate thereof and one or more other agents. It may be provided in the form of a pharmaceutical, and the compound of one aspect of the present invention or a pharmaceutically acceptable salt thereof, or a solvate thereof and one or more other agents are separately prepared. It may be provided in the form of a pharmaceutical combination or a kit containing multiple formulations. In the case of a pharmaceutical combination or in the form of a kit, the respective formulations can be administered simultaneously or separately (eg, sequentially).
  • the compound of one aspect of the present invention is not only the compound itself, but also a pharmaceutically acceptable salt of the compound and a pharmaceutically acceptable solvent thereof. Also includes Japanese products.
  • the pharmaceutically acceptable salt of the compound of one aspect of the present invention and the pharmaceutically acceptable solvate thereof are not limited, but for example, the salt or solvate exemplified above is preferable.
  • the compound of one aspect of the present invention is in the form of the salt or solvate, the compound can be applied to a desired pharmaceutical use.
  • the pharmaceutical product of this embodiment containing the compound of one aspect of the present invention, a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient has various symptoms prevented or treated by AM. Diseases and / or disorders can be prevented or treated as well. The symptoms, diseases and / or disorders include, but are not limited to, the following.
  • Cardiovascular disease heart failure, pulmonary hypertension, arteriosclerosis obliterans, Buerger's disease, myocardial infarction, lymphedema, Kawasaki disease, myocarditis, arrhythmia (for example, arrhythmia after catheter ablation surgery), atrial fibrillation, Aortitis, pulmonary hypertension, hypertension, organ damage due to hypertension, peripheral vascular disease, and arteriosclerosis.
  • Kidney / water electrolyte system diseases renal failure and nephritis.
  • Brain and neurological diseases cerebral infarction, dementia, cerebrovascular dementia, Alzheimer's disease, and encephalitis.
  • Genitourinary disease erectile dysfunction (ED).
  • Gastrointestinal diseases inflammatory diseases (eg, inflammatory bowel disease or Crohn's disease), ulcerative diseases (eg, ulcerative colitis), intestinal Behcet's disease, hepatitis, liver fibrosis, cirrhosis, and liver failure.
  • Orthopedic disease arthritis.
  • Endocrine and metabolic disorders diabetes and diabetic organ disorders (eg, diabetic nephropathy or diabetic retinopathy), and primary aldosteronism.
  • Respiratory disorders bronchial asthma, emphysema, pulmonary fibrosis, pneumonia, acute bronchitis, chronic bronchitis, and acute respiratory distress syndrome (ARDS).
  • ARDS acute respiratory distress syndrome
  • Immune disorders Diseases associated with the complement system (eg, C3 nephropathy).
  • Other diseases sepsis, septic shock, autoimmune diseases, multiple organ failure, pressure ulcers, wound healing, and alopecia.
  • Cardiovascular diseases prevented or treated by the pharmaceuticals of this embodiment are, in particular, heart failure, myocardial infarction (eg, acute myocardial infarction), arrhythmia (eg, arrhythmia after catheter ablation surgery), atrial fibrillation, pulmonary hypertension or peripheral. It is a vascular disease.
  • Brain / neurological disorders prevented or treated by the pharmaceuticals of this embodiment are, in particular, cerebral infarction or dementia.
  • the gastrointestinal diseases prevented or treated by the pharmaceuticals of this embodiment are, in particular, inflammatory diseases (eg, inflammatory bowel disease or Crohn's disease), ulcerative diseases (eg, ulcerative colitis) or intestinal Bechet's disease.
  • the endocrine and metabolic disorders prevented or treated by the pharmaceuticals of this embodiment are, in particular, diabetes and diabetic organ disorders (eg, diabetic nephropathy or diabetic retinopathy).
  • Respiratory disorders that are prevented or treated by the pharmaceuticals of this embodiment are, in particular, pulmonary fibrosis.
  • Other diseases prevented or treated by the pharmaceuticals of this embodiment are, in particular, sepsis or septic shock.
  • the pharmaceutical of this embodiment is preferably a pharmaceutical for use in the prevention or treatment of the symptoms, diseases and / or disorders described above (for example, cardiovascular disease, brain / neurological disease or digestive system disease), and heart failure. , Acute myocardial infarction, arrhythmia, atrial fibrillation, pulmonary hypertension, peripheral vascular disease, cerebral infarction, dementia, inflammatory bowel disease, Crohn's disease, ulcerative colitis, intestinal Bechet's disease, diabetes, diabetic nephropathy, diabetes More preferably, it is a drug for use in the prevention or treatment of sexual retinopathy, pulmonary fibrosis, sepsis or septic shock.
  • the drug of this embodiment for the prevention or treatment of the symptoms, diseases and / or disorders described above, the pharmacokinetics is significantly superior to that of AM, and the preventive or therapeutic effect is substantially equivalent to that of AM. Can be expressed.
  • prevention means substantially preventing the occurrence (onset or manifestation) of symptoms, diseases and / or disorders.
  • treatment means suppressing (eg, suppressing progression), ameliorating, repairing and / or curing symptoms, diseases and / or disorders that have occurred (onset or manifestation).
  • the compound of one aspect of the present invention has a structure in which AM, which is a natural bioactive peptide, and serum albumin are linked via a linking group L. Therefore, the compound of one aspect of the present invention is safe and has low toxicity. Therefore, the pharmaceutical product of this embodiment containing the compound of one aspect of the present invention, a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient has the above-mentioned symptoms, diseases and / or disorders. It can be applied to various subjects in need of prevention or treatment.
  • the subject is a subject or patient of a human or non-human mammal (eg, a warm-blooded animal such as a pig, dog, cow, rat, mouse, guinea pig, rabbit, chicken, sheep, cat, monkey, hamadryas baboon or chimpanzee). It is preferably present, and more preferably a human patient.
  • a human or non-human mammal eg, a warm-blooded animal such as a pig, dog, cow, rat, mouse, guinea pig, rabbit, chicken, sheep, cat, monkey, hamadryas baboon or chimpanzee.
  • the exact dosage and administration will be the age, gender, sign of the subject to be prevented or treated, the disease and / or the exact condition of the disorder (eg, severity). ), And many factors such as the route of administration, the attending physician should finally determine the therapeutically effective dosage and administration. Therefore, in the pharmaceutical of this embodiment, the compound of one aspect of the present invention which is an active ingredient, a pharmaceutically acceptable salt thereof, or a solvate thereof which is pharmaceutically acceptable thereof is used as a therapeutically effective dosage and administration ( For example, the dose, the number of doses, and the route of administration) are administered to the subject.
  • the dose of the compound of one aspect of the present invention used as an active ingredient, a pharmaceutically acceptable salt thereof, or a solvate thereof is pharmaceutically acceptable. Is usually in the range of 0.01 to 1000 ⁇ g / kg body weight / day, for example, in the range of 0.5 to 200 ⁇ g / kg body weight / day.
  • the drug of this embodiment may be administered at any number of times and by a route of administration.
  • the compound of one embodiment of the present invention is significantly superior to AM, for example, in terms of biological stability, while maintaining substantially the same pharmacological action as the parent compound AM.
  • the drug of this embodiment is preferably administered in a single dose.
  • the drug of this embodiment is preferably administered by a parenteral route such as intravenous administration, enema administration, subcutaneous administration, intramuscular administration or intraperitoneal administration, and more preferably subcutaneous administration.
  • the above-mentioned symptoms, diseases and / or disorders in a subject are prevented by using the drug of this embodiment containing AM or an adrenomedullin derivative as an active ingredient in the above-mentioned dosage and administration (for example, dose, frequency of administration and route of administration). Or it can be treated.
  • the compound of one aspect of the invention can similarly prevent or treat the symptoms, diseases and / or disorders described above that are prevented or treated by AM. Therefore, another aspect of the present invention has been described above, comprising the compound of one aspect of the invention or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof as an active ingredient. Concerning preventive or therapeutic agents for symptoms, diseases and / or disorders.
  • the prophylactic or therapeutic agent of one aspect of the present invention has the same characteristics as the pharmaceutical of the present aspect described above.
  • the prophylactic or therapeutic agent of this embodiment can be used in the same dosage and administration for the same symptoms, diseases and / or disorders as the medicine of this embodiment described above.
  • the compound of one aspect of the present invention can be used for the prevention or treatment of the symptom, the disease and / or the disorder in the subject having the symptom, the disease and / or the disorder described above. Therefore, another aspect of the invention is pharmaceutically acceptable in an effective amount of the compound of one aspect of the invention or pharmaceutically acceptable thereof in a subject requiring the prevention or treatment of the symptoms, diseases and / or disorders described above.
  • a method for preventing or treating the above-mentioned symptoms, diseases and / or disorders which comprises administering a salt or a solvate thereof which is pharmaceutically acceptable.
  • the compound or the like of one aspect of the present invention can be administered to a subject at the same dosage and administration as the pharmaceutical of this aspect described above.
  • the symptoms, diseases and / or disorders are preferably cardiovascular disease, brain / neurological disease, digestive system disease, endocrine and metabolic disease, respiratory system disease or other disease, such as heart failure, acute myocardial infarction, arrhythmia, and atriosphere. Fibrillation, pulmonary hypertension, peripheral vascular disease, cerebral infarction, dementia, inflammatory bowel disease, Crohn's disease, ulcerative colitis, intestinal Bechette's disease, diabetes, diabetic nephropathy, diabetic retinopathy, pulmonary fibrosis, More preferably, it is septicemia or septic shock.
  • An effective amount of a compound of one aspect of the present invention, a pharmaceutically acceptable salt thereof, or a solvate thereof is provided to a subject in need of prevention or treatment of the above-mentioned symptoms, diseases and / or disorders.
  • a pharmaceutically acceptable salt thereof, or a solvate thereof is provided to a subject in need of prevention or treatment of the above-mentioned symptoms, diseases and / or disorders.
  • the symptoms, diseases and / or disorders can be prevented or treated.
  • the compound of one aspect of the present invention and the like can be used for administration to a subject at the same dosage and administration as the pharmaceutical of this aspect described above.
  • the symptoms, diseases and / or disorders are preferably cardiovascular disease, brain / neurological disease, digestive system disease, endocrine and metabolic disease, respiratory system disease or other disease, such as heart failure, acute myocardial infarction, arrhythmia, and atriosphere.
  • Fibrillation Fibrillation, pulmonary hypertension, peripheral vascular disease, cerebral infarction, dementia, inflammatory bowel disease, Crohn's disease, ulcerative colitis, intestinal Bechette's disease, diabetes, diabetic nephropathy, diabetic retinopathy, pulmonary fibrosis, More preferably, it is septicemia or septic shock.
  • a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof for the prevention or treatment of the symptoms, diseases and / or disorders described above. Symptoms, diseases and / or disorders can be prevented or treated.
  • Yet another aspect of the present invention relates to a method for producing a compound according to one aspect of the present invention.
  • the method of this embodiment includes a connecting step.
  • the method of this embodiment may include a precursor preparation step.
  • Precursor preparation process The method of this embodiment prepares at least one of a precursor of peptide moiety B derived from AM or a variant thereof, a precursor of modifying group A which is serum albumin, and a precursor of divalent linking group L. , Precursor preparation steps may be included.
  • "precursor of modifying group A which is serum albumin” means serum albumin itself, or in the linking step described below, modifying group A and linking group L are fused with each other. Means those derivatives that have been appropriately modified or activated to be linked.
  • the precursor of modifying group A is preferably serum albumin itself or a protected form thereof.
  • the precursor of modifying group A may be prepared by means such as a transformation system usually used in the art, or may be prepared by purchasing pre-produced serum albumin or the like. good. In either case, it is included in the embodiment of this step.
  • precursor of peptide moiety B derived from adrenomedullin or a variant thereof means AM or the variant thereof itself, or in the linking step described below, peptide moiety B and It means their derivatives that have been appropriately modified or activated so that the linking groups L are linked to each other by a condensation reaction.
  • the precursor of peptide moiety B is preferably AM or its modifications themselves, or a protected form thereof.
  • the precursor of peptide moiety B can be formed by means commonly used in the art.
  • the precursor of peptide portion B is AM or a variant thereof itself
  • solid phase or liquid phase peptide synthesis methods may be used, and human or non-human mammalian tissues capable of producing AM.
  • a method of purifying a natural peptide from cells may be used.
  • a transformation system such as Escherichia coli or Saccharomyces cerevisiae using DNA encoding AM in human or non-human mammals capable of producing AM (eg, SEQ ID NO: 2, 5, 7, 9, 11 or 13). You may use the method of expressing a large amount of recombinant protein in.
  • a peptide prepared in advance may be purchased and used. In either case, it is included in the embodiment of this step.
  • the two cysteine residues in the amino acid sequence are disulfide-bonded.
  • the disulfide bond formed between the two cysteine residues in the amino acid sequence is replaced with an ethylene group, whereby the disulfide bond becomes ethylene.
  • a precursor substituted with a group can be obtained.
  • the disulfide reaction and the substitution reaction with an ethylene group can be carried out under the conditions usually used in the art.
  • the disulfide reaction and the substitution reaction with an ethylene group may be carried out in this step or may be carried out in the linking step described below. Both cases are included in the embodiments of the method of the present invention.
  • the "precursor of the divalent linking group L" is the linking group between the modifying group A and the linking group L and / or the peptide moiety B and the linking group L in the linking step described below. It means a compound in which at least one end, particularly both ends, is activated so that the spaces are linked together by a condensation reaction.
  • the linking group L has a carboxyl group at at least one end, and the carboxyl group forms an amide bond with the N-terminal ⁇ -amino group of the peptide moiety B. Therefore, in the precursor of the linking group L, the terminal linked to the peptide portion B is a carboxyl group or an activated form thereof.
  • the activated form of the carboxyl group examples include N-hydroxysuccinimide ester and p-nitrophenyl ester.
  • the linking group L has the functional group exemplified above at the other end, and the functional group forms a covalent bond with any functional group contained in the constituent amino acid residue of the modifying group A. do. Therefore, in the precursor of the linking group L, the terminal linked to the modifying group A is the functional group exemplified above or an activated form thereof.
  • the activated form of the functional group exemplified above examples include N-hydroxysuccinimide ester, p-nitrophenyl ester and maleimide.
  • the precursor of the linking group L may be prepared by using a chemical synthesis means usually used in the art, or may be prepared by purchasing the precursor prepared in advance. .. In either case, it is included in the embodiment of this step.
  • a precursor containing a modifying group A and a linking group L in which a modifying group A which is serum albumin and a divalent linking group L are linked may be prepared.
  • the precursor containing the modifying group A and the linking group L the precursor of the modifying group A and the precursor of the divalent linking group L are prepared by the procedure described above, respectively, and then the modifying group is prepared. It may be prepared by linking the precursor of A and the precursor of the divalent linking group L, or the precursor prepared in advance may be purchased or prepared. In either case, it is included in the embodiment of this step.
  • a precursor containing a peptide moiety B and a linking group L in which a precursor of the peptide moiety B derived from AM or a modified product thereof and a divalent linking group L are linked may be prepared. ..
  • the precursor of the peptide portion B and the precursor of the divalent linking group L are prepared by the procedure described above, respectively, and the precursor of the peptide portion B is prepared. It may be prepared by linking the precursor and the precursor of the divalent linking group L, or the precursor prepared in advance may be purchased or prepared. In either case, it is included in the embodiment of this step.
  • At least one of the precursor of modifying group A, the precursor of linking group L and the precursor of peptide moiety B is a protected form thereof, in this step, optionally, the precursor of modifying group A, the precursor of linking group L
  • a deprotection step may be performed to deprotect at least one or more protecting groups of at least one of the protective forms of the body.
  • the protection and deprotection steps can be carried out by the protection and deprotection reactions commonly used in the art.
  • the protection step and the deprotection step may be carried out in this step, or may be carried out in the connection step described below. Both cases are included in the embodiment of the method of this embodiment.
  • the method of this embodiment is between the precursor of modifying group A, which is serum albumin, and the precursor of divalent linking group L, and with the precursor of peptide moiety B derived from adrenomedullin or a modification thereof. Containing a linking step, linking at least one of the precursors of the linking group L to give the compound of formula (I).
  • the means for linking the precursor of the modifying group A and the precursor of the linking group L and the precursor of the peptide portion B and the precursor of the linking group L are not particularly limited. Based on the terminal structure, activation form, etc. of each precursor, a bond-forming reaction usually used in the art may be appropriately applied.
  • MAL-AM MAL-dPEG4-CO-NH-human AM
  • MAL-AM has a maleimide group at one end and a carboxyl group at the other end.
  • * indicates the connection position with the remaining part.
  • It consists of a group (MAL-dPEG4 group) having an ethylene oxide unit having 4 repetition numbers represented by, and an amino acid sequence of SEQ ID NO: 1, with the C-terminal amidated and a cysteine residue at position 16.
  • the mature natural human adrenomedulin (hAM (1-52)), which is a peptide in which the cysteine residue at position 21 forms a disulfide bond, is the carboxyl group of MAL-dPEG4 and the carboxyl group of hAM (1-52). It has a structure linked by forming an amide bond with the ⁇ -amino group of the N-terminal tyrosine residue.
  • hSA Human serum albumin
  • MAL-AM 1.1 mol
  • hSA 1.0 mol
  • PBS pH 7.2
  • N, N-dimethylformamide a novel human serum albumin conjugated adrenomedullin
  • the maleimide group at the end of MAL-AM can undergo a Michael addition reaction with the thiol group of the cysteine residue of hSA. By this reaction, the maleimide group is converted to a succinimide group, and a thioether bond is formed between the succinimide group and the thiol group, whereby MAL-AM and hSA are linked.
  • PVDF polyvinylidene difluoride
  • the PVDF membrane after protein transcription was blocked with 5% skim milk (room temperature, 60 minutes) and immersed in a primary antibody (mouse anti-adrenomedullin C-terminal sequence antibody, Shionogi Pharmaceutical Co., Ltd., Japan) (4 ° C, overnight). .. This primary antibody binds to the C-terminal portion of AM.
  • the treated membrane was rinsed with phosphate buffered saline and Tween-20 (PBST). Subsequently, this membrane was immersed in a solution of a secondary antibody (anti-mouse IgG antibody, Immuno-Star goat anti-mouse HRP Conjugate®; # 170-5047, 1: 2000, Bio-Rad, USA). (Room temperature, 60 minutes). The treated membrane was rinsed again with PBST. Finally, the membrane was immersed in Clarity western ECL substance® (# 1705060, Bio-Rad, USA) and allowed to stand in the dark for 5 minutes. The membrane was then imaged using Omega Lum TM G.
  • FIG. 1 shows the results of SDS-PAGE and WB.
  • A shows the gel stained with Coomassie Brilliant Blue after SDS-PAGE
  • B shows the membrane obtained by WB.
  • M represents a molecular weight marker (Precision Plus Protein Dual Xtra Standards)
  • lane 1 represents 68 ng of hSA
  • lane 2 represents 180 ng of hSA-AM.
  • both hSA and hSA-AM showed a single band at approximately the same position below the 75 kDa marker (lanes 1 and 2).
  • Fig. 1B the presence of the C-terminal part of AM was suggested in hSA-AM (lane 2).
  • hSA-AM and hSA were subjected to gel filtration chromatography using a Superdex TM 200 Increase 10/300 GL column (GE Healthcare UK, UK) in 20 mM citrate buffer (pH 7.0) containing 100 mM NaCl. Eluted.
  • a molecular weight marker Gel Filtration Calibration Kit LMW (GE Healthcare UK, UK) was used.
  • FIGS. 2A and 2B show the results of GF.
  • A is a chromatogram of 30 ⁇ g hSA-AM
  • B is a chromatogram of 30 ⁇ g hSA
  • C is a chromatogram of purified hSA-AM.
  • the horizontal axis is the retention time (minutes) of the reverse phase HPLC
  • the vertical axis is the UV absorption intensity (mV) at a wavelength of 280 nm.
  • hSA-AM and hSA showed similar peak profiles.
  • 3 fractions of eluate containing peaks with retention times (RT) of 15 minutes, 18 minutes and 25 minutes were separated.
  • the fractions were analyzed by enzyme immunoassay (EIA) using mouse anti-adrenomedulin C-terminal sequence antibody and anti-mouse IgG antibody. Fractions containing a 25-minute peak were negative (data not shown). Subsequently, the fraction containing the peak at RT 18 minutes, that is, the purified hSA-AM was subjected to gel filtration chromatography under the same conditions again. As a result, the peak seen at RT 15 minutes disappeared (Fig. 2C). The enzyme immunoassay (EIA) using mouse anti-adrenomedulin C-terminal sequence antibody and anti-mouse IgG antibody. Fractions containing a 25-minute peak were negative (data not shown). Subsequently, the fraction containing the peak at RT 18 minutes, that is, the purified hSA-AM was subjected to gel filtration chromatography under the same conditions again. As a result, the peak seen at RT 15 minutes disappeared (Fig. 2C). The
  • Example of using a novel adrenomedulin derivative> [Experiment II-1: Intracellular cAMP concentration increasing effect by adrenomedulin derivative]
  • the physiological effects of AM are known to be expressed through an increase in the concentration of intracellular cAMP (Kitamura K et al., Biochem Biophys Res Commun, April 30, 1993, Vol. 192 (2), pp. See 553-560). Therefore, an AM derivative was added to a cultured cell line (HEK293 cell line) in which the AM type 1 receptor (AM1 receptor) was stably expressed, and the amount of intracellular cAMP produced was measured.
  • HEK293 cell line HEK293 cell line
  • HEK293 cells were placed in a 24-well plate (Thermo Fisher Scientific Co., Ltd.) coated with fibronectin in Dalvecco-modified Eagle's medium (10% bovine fetal serum, 100 U / mL penicillin G, 100 ⁇ g / mL streptomycin, 0.25 ⁇ g /). Cultivated in mL amhotericin B, 100 ⁇ g / ml hyglomycin B and 250 ⁇ g / ml genetesin added (37 ° C, humidified, 5% CO 2 conditions). After culturing for 3 days, 90% confluent cells stimulated to accumulate intracellular cAMP were subjected to the experiment.
  • Dalvecco-modified Eagle's medium 10% bovine fetal serum, 100 U / mL penicillin G, 100 ⁇ g / mL streptomycin, 0.25 ⁇ g /.
  • the medium was replaced with a Hanks equilibrium salt solution containing 20 mM 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid (HEPES) and 0.1% bovine serum albumin in the presence of 0.5 mM isobutylmethylxanthine (IBMX).
  • HPES 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid
  • IBMX isobutylmethylxanthine
  • the cells were added with a given concentration of native AM or AM derivative (hSA-AM) and incubated for 15 minutes under 37 ° C., humidified, 5% CO 2 conditions. The reaction was stopped by adding cytolysis buffer. Then, using an enzyme immunoassay (EIA) kit for measuring cAMP (GE Healthcare UK, UK), the amount of intracellular cAMP produced in HEK293 cells in each well was measured. The measurement was performed in 4 iterations, and the mean value and standard error (SEM) were calculated
  • Figure 3 shows the dose-response curve of intracellular cAMP production in HEK293 cells with respect to the added concentration of natural AM or AM derivative (hSA-AM).
  • the black circles ( ⁇ ) are the average values of the experiments to which natural AM was added
  • the white squares ( ⁇ ) are the average values of the experiments to which hSA-AM was added
  • the error bars represent SEM.
  • the horizontal axis is the logarithm (M) of the added concentration of the natural AM or AM derivative (hSA-AM)
  • the vertical axis is the intracellular cAMP in HEK293 cells that stably express the AM1 receptor. Production amount (fmol / well). As shown in FIG.
  • hSA-AM increased the intracellular cAMP concentration in a dose-dependent manner, similar to that of natural AM. From the dose-response curve, the pEC50 of natural AM was calculated to be 8.660, and the pEC50 of hSA-AM was calculated to be 7.208.
  • Peripheral blood samples were taken for a given time (natural human AM: 0, 0.25, 1, 2 and 3 hours after administration, hSA-AM: 0, 1, 3, 6, 12, 24, 48, 72, 120 and after administration. It was collected from the tail vein into a test tube (Easy tube (registered trademark), containing heparin Na, Eiken, Tochigi Prefecture, Japan) at 168 hours. Plasma was obtained by centrifuging the test tube at 2,000 xg. Plasma was collected from the test tube and transferred to a test tube supplemented with 21 ⁇ g aprotinin and 0.3 g Na 2 -EDTA for storage. Human AM concentration in rat plasma was measured using specific fluorescence immunoassay (Toso Corporation) using antibodies with different recognition sites.
  • the first antibody binds to a cyclic structure in which the cysteine residue at position 16 and the cysteine residue at position 21 of hAM (1-52) form a disulfide bond, and the second antibody is the C-terminal portion of AM. Combine to.
  • full-length AM (tAM) of 52 amino acid residues and mature AM (mAM) capable of expressing activity can be distinguished and quantitatively analyzed (Ohta H et al., One-step direct). assay for mature-type adrenomedullin with monoclonal antibodies.
  • tAM full-length AM
  • mAM mature AM
  • FIGS. 4A and 4B show the change over time in the amount of natural AM or AM derivative (hSA-AM) in rat plasma after subcutaneous administration.
  • A is the result of natural AM
  • B is the result of AM derivative (hSA-AM).
  • the horizontal axis is the elapsed time (A: time, B: day) after subcutaneous administration
  • the vertical axis is the plasma peptide concentration after administration of natural AM or AM derivative (hSA-AM) ( pM). Plasma peptide concentrations are shown as tAM (black circles or squares) and mAMs (white circles or squares), respectively.
  • the residual blood duration of hSA-AM was significantly longer than that of AM.
  • hSA-AM which is a novel AM derivative, has significantly superior pharmacokinetics in terms of biological stability in a single dose as compared with the natural form.
  • Figure 5 shows the effect of a single subcutaneous dose of hSA-AM on SHR in suppressing the increase in blood pressure.
  • A is the time course of systolic blood pressure (sBP)
  • B is the time course of diastolic blood pressure (dBP).
  • the horizontal axis is the elapsed time (days), and the vertical axis shows the difference in blood pressure (A: difference in sBP, B: difference in dBP) (mmHg) obtained by subtracting the blood pressure before administration from the blood pressure on each measurement day. ..
  • the values in the figure are the average values of each group, and the error bars represent SEM.
  • the DAI score was significantly reduced in the hSA-AM-administered treatment group as compared with the control group.
  • the present invention is not limited to the above-described embodiment, but includes various modifications.
  • the above-described embodiment has been described in detail in order to explain the present invention in an easy-to-understand manner, and is not necessarily limited to the one including all the configurations described.
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