WO2014065370A1 - Pulmonary hypertension therapeutic agent - Google Patents

Pulmonary hypertension therapeutic agent Download PDF

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Publication number
WO2014065370A1
WO2014065370A1 PCT/JP2013/078828 JP2013078828W WO2014065370A1 WO 2014065370 A1 WO2014065370 A1 WO 2014065370A1 JP 2013078828 W JP2013078828 W JP 2013078828W WO 2014065370 A1 WO2014065370 A1 WO 2014065370A1
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pulmonary hypertension
alogliptin
dpp
inhibitor
acid
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PCT/JP2013/078828
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French (fr)
Japanese (ja)
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剛 原口
奨 細川
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国立大学法人 東京医科歯科大学
武田薬品工業株式会社
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Publication of WO2014065370A1 publication Critical patent/WO2014065370A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
    • 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
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives

Definitions

  • the present invention relates to a preventive or therapeutic agent for pulmonary hypertension and the like, which contains a dipeptidyl peptidase-IV (sometimes abbreviated as DPP-IV in this specification) inhibitor.
  • a dipeptidyl peptidase-IV sometimes abbreviated as DPP-IV in this specification
  • DPP-IV a kind of peptidase, specifically binds to a peptide containing proline (or alanine) second from the N-terminus, and produces a dipeptide by cleaving the C-terminal side of the proline (or alanine).
  • Serine protease DPP-IV has also been shown to be the same molecule as CD26, and has been reported to be related to the immune system. The role of DPP-IV in mammals has not been fully clarified, but it plays an important role in neuropeptide metabolism, T cell activation, cancer cell adhesion to endothelial cells, and HIV entry into cells.
  • DPP-IV is involved in inactivation of incretins GLP-1 (glucagon-like peptide-1) or GIP (Gastric inhibitory peptide / Glucose-dependent insulinotropic peptide).
  • GLP-1 has a short plasma half-life of 1-2 minutes
  • GLP-1 (9-36) amide a degradation product of DPP-IV, acts as an antagonist to the GLP-1 receptor. It is known that its physiological activity is significantly impaired by being decomposed into DPP-IV. From this, it is also known that suppressing the degradation of GLP-1 by inhibiting DPP-IV activity enhances the physiological activity of GLP-1 such as promoting insulin secretion in a glucose concentration-dependent manner. ing.
  • Pulmonary arterial hypertension is a serious disease that causes right heart failure and eventually death by increasing and sustaining pulmonary artery pressure.
  • the current main treatments are endothelin receptor antagonists, phosphodiesterase 5 inhibitors, and intravenous epoprostenol. Although these treatments improve the patient's subjective symptoms, the survival rate is It has not improved, and the fatality rate continues to be a high problem.
  • alogliptin benzoate can treat pulmonary hypertension for the first time, and have intensively studied to complete the present invention.
  • the present invention [1] A prophylactic or therapeutic agent for pulmonary hypertension comprising a dipeptidyl peptidase-IV inhibitor (preferably alogliptin or a salt thereof) (hereinafter sometimes abbreviated as the agent of the present invention); [2] The agent according to [1] above, wherein the dipeptidyl peptidase-IV inhibitor is alogliptin or a salt thereof; [3] The agent according to [1] above, wherein the dipeptidyl peptidase-IV inhibitor is alogliptin benzoate; [4] The agent according to [1], [2] or [3] above, wherein the pulmonary hypertension is pulmonary arterial pulmonary hypertension; [5] A method for preventing or treating pulmonary hypertension in a mammal, comprising administering an effective amount of a dipeptidyl peptidase-IV inhibitor (preferably alogliptin or a salt thereof) to the mammal; [6] Dipeptidyl peptidas
  • the agent of the present invention has an excellent DPP-IV inhibitory action and is useful as a preventive or therapeutic agent for pulmonary hypertension.
  • the agent of the present invention can be administered orally, it has few side effects such as infectious diseases and has an excellent therapeutic effect and prognosis improving effect on pulmonary hypertension.
  • FIG. 5 is a graph of a survival curve in Experimental Example 1.
  • 2 is a photomicrograph of immunohistochemical staining in Experimental Example 1.
  • HE shows a photomicrograph of immunohistochemical staining using hematoxylin and eosin staining, EVG using Elastica-Wangeson staining, SMa using smooth muscle ⁇ -actin antibody, and PCNA using nuclear proliferation antigen antibody.
  • 4 is a graph showing measurement results of mRNA expression of transforming growth factor (TGF) - ⁇ in Experimental Example 1.
  • 6 is a graph showing the results of cell proliferation assay in Experimental Example 1. It is a figure which shows the result of the western blotting in Experimental example 1.
  • the vertical axis shows the ratio of phosphorylated-nuclear factor-kappaB-p65 protein level to non-phosphorylated nuclear factor-kappaB-p65 protein level.
  • 4 is a photomicrograph of immunohistochemical staining in Experimental Example 2.
  • HE shows a photomicrograph of immunohistochemical staining using hematoxylin and eosin staining, EVG using Elastica-Wangeson staining, SMa using smooth muscle ⁇ -actin antibody, and PCNA using nuclear proliferation antigen antibody.
  • 4 is a photomicrograph of immunohistochemical staining using phosphorylated-nuclear factor-kappaBp65 antibody in Experimental Example 2.
  • Phosphorylated-nuclear factor-kappaB p65 positive cells are indicated by arrows.
  • 4 is a photomicrograph of immunohistochemical staining using CD8a antibody (CD8) and CD68 antibody (CD68) in Experimental Example 2.
  • HE shows a photomicrograph of hematoxylin and eosin staining. The numbers of CD8-positive and CD68-positive cells per field obtained from the photomicrograph of immunohistochemical staining in Experimental Example 2 (strong enlargement; 400 times) are shown.
  • the DPP-IV inhibitor means a compound that inhibits the enzyme activity of DPP-IV [Classification by International Union of Biochemistry (IUBMB): EC 3.4.14.5].
  • the compound may be either peptidic or non-peptidic, but is preferably non-peptidic.
  • the form of the DPP-IV inhibitor may be different before and after administration to the living body as long as the DPP-IV inhibitory activity is retained. That is, the DPP-IV inhibitor may be an “active metabolite” having DPP-IV inhibitory activity after becoming a structural change body after undergoing metabolism in vivo.
  • the DPP-IV inhibitor may be a “prodrug” that is converted into an active form by a reaction with an enzyme, gastric acid or the like under physiological conditions in vivo.
  • the DPP-IV inhibitory activity can be confirmed, for example, by a method utilizing “Method of Raymond et al. (Diabetes, 47, 1253-1258, 1998)”.
  • DPP-IV inhibitors include nitrogen-containing heterocyclic compounds, specifically the following compounds (1) to (7).
  • alogliptin or a salt thereof is preferable.
  • alogliptin benzoate is particularly preferable.
  • Examples of the salt of the above compound include pharmacologically acceptable salts such as salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like.
  • Preferable examples of the salt with inorganic acid include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like.
  • Preferable examples of salts with organic acids include benzoic acid, formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, and benzenesulfonic acid.
  • salts with p-toluenesulfonic acid and the like Preferable examples of the salt with basic amino acid include salts with arginine, lysine, ornithine and the like, and preferable examples of the salt with acidic amino acid include salts with aspartic acid, glutamic acid and the like.
  • alogliptin salt examples include salts with benzoic acid, trifluoroacetic acid, p-toluenesulfonic acid, hydrochloric acid and the like, and among them, the salt with benzoic acid is preferable.
  • the salt of trelagliptin examples include salts with trifluoroacetic acid, succinic acid, hydrochloric acid and the like, and among them, the salt with succinic acid is preferable.
  • the DPP-IV inhibitor can be produced according to the method described in US Patent Application Publication No. 2005/0261271, or a method analogous thereto.
  • the agent of the present invention has low toxicity (eg, acute toxicity, chronic toxicity, genotoxicity, reproductive toxicity, cardiotoxicity, drug interaction, carcinogenicity), and the DPP-IV inhibitor is used as it is or pharmacologically acceptable.
  • toxicity eg, acute toxicity, chronic toxicity, genotoxicity, reproductive toxicity, cardiotoxicity, drug interaction, carcinogenicity
  • the DPP-IV inhibitor is used as it is or pharmacologically acceptable.
  • Prevention of pulmonary hypertension for mammals eg, humans, mice, rats, rabbits, dogs, cats, cows, horses, pigs, monkeys
  • the content of the DPP-IV inhibitor in the agent of the present invention varies depending on the type of DPP-IV inhibitor, the size of the preparation, etc., but is, for example, 1 to 90% by weight, preferably 5 to 80% by weight.
  • pharmacologically acceptable carrier various organic or inorganic carrier substances commonly used as pharmaceutical materials are used, and excipients, lubricants, binders, disintegrants in solid preparations; solvents in liquid preparations, It is blended as a solubilizing agent, suspending agent, isotonic agent, buffering agent, soothing agent and the like. If necessary, preparation additives such as preservatives, antioxidants, colorants, sweeteners and the like can also be used.
  • excipients include lactose, sucrose, D-mannitol, D-sorbitol, starch, pregelatinized starch, dextrin, crystalline cellulose, low-substituted hydroxypropylcellulose, sodium carboxymethylcellulose, gum arabic, pullulan, light Anhydrous silicic acid, synthetic aluminum silicate, magnesium magnesium metasilicate, etc. are mentioned.
  • lubricant examples include magnesium stearate, calcium stearate, talc, colloidal silica and the like.
  • Preferred examples of the binder include pregelatinized starch, sucrose, gelatin, gum arabic, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, crystalline cellulose, sucrose, D-mannitol, trehalose, dextrin, pullulan, hydroxypropylcellulose, hydroxy Examples thereof include propylmethylcellulose and polyvinylpyrrolidone.
  • disintegrant examples include lactose, sucrose, starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, croscarmellose sodium, carboxymethyl starch sodium, light anhydrous silicic acid, low substituted hydroxypropyl cellulose and the like.
  • the solvent include water for injection, physiological saline, Ringer's solution, alcohol, propylene glycol, polyethylene glycol, sesame oil, corn oil, olive oil, cottonseed oil and the like.
  • solubilizer examples include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, sodium acetate. Etc.
  • suspending agent examples include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate; polyvinyl alcohol, polyvinylpyrrolidone And hydrophilic polymers such as sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose; polysorbates, polyoxyethylene hydrogenated castor oil, and the like.
  • surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate
  • polyvinyl alcohol polyvinylpyrrolidone
  • hydrophilic polymers such as sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethyl
  • the isotonic agent include sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose and the like.
  • buffer solutions such as phosphate, acetate, carbonate and citrate.
  • Benzyl alcohol etc. are mentioned as a suitable example of a soothing agent.
  • preservative examples include p-hydroxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.
  • antioxidant examples include sulfite and ascorbate.
  • the colorant examples include water-soluble edible tar dyes (eg, edible dyes such as edible red Nos. 2 and 3, edible yellows Nos. 4 and 5, edible blue Nos. 1 and 2, etc.), water-insoluble lake dyes (Eg, the aluminum salt of the water-soluble edible tar dye), natural dyes (eg, ⁇ -carotene, chlorophyll, bengara) and the like.
  • water-soluble edible tar dyes eg, edible dyes such as edible red Nos. 2 and 3, edible yellows Nos. 4 and 5, edible blue Nos. 1 and 2, etc.
  • water-insoluble lake dyes Eg, the aluminum salt of the water-soluble edible tar dye
  • natural dyes eg, ⁇ -carotene, chlorophyll, bengara
  • Suitable examples of the sweetening agent include saccharin sodium, dipotassium glycyrrhizinate, aspartame, stevia and the like.
  • the agent of the present invention is produced by mixing a DPP-IV inhibitor with a pharmacologically acceptable carrier according to a method known per se as a method for producing a pharmaceutical preparation (eg, a method described in the Japanese Pharmacopoeia, etc.).
  • a method for producing a pharmaceutical preparation eg, a method described in the Japanese Pharmacopoeia, etc.
  • the Examples of the dosage form of the agent of the present invention include tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, orally disintegrating tablets, buccal tablets, etc.), pills, powders, granules, capsules (soft capsules, microcapsules).
  • These preparations may be controlled-release preparations (eg, sustained-release microcapsules)
  • oral preparations contain active ingredients such as excipients (eg, lactose, sucrose, starch, D-mannitol, etc.), disintegrants (eg, carboxymethylcellulose calcium, etc.), binders (eg, pregelatinized starch, Arabic Rubber, carboxymethyl cellulose, hydroxypropyl cellulose, polyvinyl pyrrolidone, etc.) or lubricant (eg, talc, magnesium stearate, polyethylene glycol 6000, etc.), etc., and compression molded, and if necessary, taste masking, enteric
  • active ingredients such as excipients (eg, lactose, sucrose, starch, D-mannitol, etc.), disintegrants (eg, carboxymethylcellulose calcium, etc.), binders (eg, pregelatinized starch, Arabic Rubber, carboxymethyl cellulose, hydroxypropyl cellulose, polyvinyl pyrrolidone, etc.) or lubricant (eg, talc
  • the coating base examples include sugar coating base, water-soluble film coating base, enteric film coating base, sustained-release film coating base and the like.
  • sugar coating base sucrose is used, and one or more selected from talc, precipitated calcium carbonate, gelatin, gum arabic, pullulan, carnauba wax and the like may be used in combination.
  • water-soluble film coating base examples include cellulose polymers such as hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, and methylhydroxyethylcellulose; polyvinyl acetal diethylaminoacetate, aminoalkyl methacrylate copolymer E [Eudragit E (trade name), Rohm Pharma Co., Ltd.], synthetic polymers such as polyvinylpyrrolidone; polysaccharides such as pullulan.
  • cellulose polymers such as hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, and methylhydroxyethylcellulose
  • polyvinyl acetal diethylaminoacetate, aminoalkyl methacrylate copolymer E [Eudragit E (trade name), Rohm Pharma Co., Ltd.]
  • synthetic polymers such as polyvinylpyrrolidone
  • polysaccharides such as pullulan.
  • enteric film coating bases include cellulose-based polymers such as hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, carboxymethylethylcellulose, and cellulose acetate phthalate; methacrylic acid copolymer L [Eudragit L (trade name), Acrylic polymers such as Rohm Pharma Co., Ltd., methacrylic acid copolymer LD [Eudragit L-30D55 (trade name), Rohm Pharma Co., Ltd.], and methacrylic acid copolymer S [Eudragit S (trade name), Rohm Pharma Co., Ltd.]; Examples include natural products such as shellac.
  • sustained-release film coating bases examples include cellulose polymers such as ethyl cellulose; aminoalkyl methacrylate copolymer RS [Eudragit RS (trade name), Rohm Pharma Co., Ltd.], ethyl acrylate / methyl methacrylate copolymer suspension Acrylic polymers such as suspensions (Eudragit NE (trade name), Rohm Pharma) are listed. Two or more kinds of the above-described coating bases may be mixed and used at an appropriate ratio. Moreover, you may use light-shielding agents, such as a titanium oxide, ferric oxide, etc. in the case of coating.
  • Injections contain active ingredients as dispersants (eg, polysorbate 80, polyoxyethylene hydrogenated castor oil 60, polyethylene glycol, carboxymethyl cellulose, sodium alginate, etc.), preservatives (eg, methyl paraben, propyl paraben, benzyl alcohol, chlorobutanol, Phenol), isotonic agents (eg, sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose, etc.), etc., and aqueous solvents (eg, distilled water, physiological saline, Ringer's solution, etc.) or oily solvents (eg, , Olive oil, sesame oil, cottonseed oil, vegetable oils such as corn oil, propylene glycol, etc.) and the like.
  • dispersants eg, polysorbate 80, polyoxyethylene hydrogenated castor oil 60, polyethylene glycol, carboxymethyl cellulose, sodium alginate, etc.
  • preservatives eg,
  • additives such as a solubilizer (eg, sodium salicylate, sodium acetate, etc.), a stabilizer (eg, human serum albumin, etc.), a soothing agent (eg, benzyl alcohol, etc.) may be used as desired.
  • a solubilizer eg, sodium salicylate, sodium acetate, etc.
  • a stabilizer eg, human serum albumin, etc.
  • a soothing agent eg, benzyl alcohol, etc.
  • the agent of the present invention is useful for prevention or treatment of pulmonary hypertension.
  • the agent of the present invention is more preferably useful for the prevention or treatment of pulmonary arterial pulmonary hypertension.
  • prevention or treatment of pulmonary hypertension includes suppression of the progression of pulmonary hypertension.
  • the preventive or therapeutic effect of pulmonary hypertension for example, in the administration subject of the agent of the present invention, is improved survival rate, decreased medial thickness of pulmonary arteriole, decreased mRNA expression of transforming growth factor (TGF) - ⁇ Decreased expression of TGF- ⁇ protein, decreased expression of basic fibroblast growth factor (FGF2) mRNA or protein, suppressed proliferation of pulmonary artery smooth muscle cells, suppressed phosphorylated-nuclear factor-kappaB p65 (p-p65) protein level , Suppression of p-p65 nuclear translocation, suppression of infiltration of immune / inflammatory cells (eg, macrophages, T cells, etc.) into lung lesions, cell surface receptors involved in immune / inflammatory reactions or ligands thereof (eg, CTLA-4, CD86, LIGHT / TNFSF14, HVEM, etc.) are confirmed by evaluating the mRNA expression.
  • the average pulmonary artery pressure in resting position exceeds 25 mmHg, or in lung disease, sleep apnea syndrome, and alveolar hypoventilation syndrome Diagnosed as pulmonary hypertension if it exceeds 20 mmHg.
  • pulmonary hypertension includes various forms of pulmonary hypertension. Examples include idiopathic pulmonary arterial pulmonary hypertension; familial pulmonary arterial pulmonary hypertension; collagen vascular disease, congenital systemic or pulmonary shunt, portal hypertension, HIV infection, drugs or toxicants Pulmonary arterial hypertension; Thyroid disease, Glycogenosis, Goshe disease, Hereditary hemorrhagic telangiectasia, Abnormal hemoglobinosis, Myeloproliferative disease or Spleen removal surgery, Pulmonary hypertension; Pulmonary arterial hypertension; persistent neonatal pulmonary hypertension; chronic obstructive pulmonary disease, interstitial lung disease, alveolar hypoventilation caused by hypoxia, sleep disordered breathing caused by hypoxia or Pulmonary hypertension associated with chronic exposure to high altitude; pulmonary hypertension associated with abnormal occurrence; and pulmonary hypertension due to embolic obstruction of the distal pulmonary artery.
  • the dose of the agent of the present invention varies depending on the administration subject, administration route, target disease, and the like.
  • the active ingredient DPP-IV inhibitor when orally administered to an adult pulmonary hypertension patient, is usually administered in a single dose. About 0.01 to 100 mg / kg body weight, preferably 0.05 to 30 mg / kg body weight, more preferably 0.1 to 10 mg / kg body weight, and this amount can be administered once or twice a day. desirable.
  • the DPP-IV inhibitor is alogliptin or a salt thereof, 0.1 to 1 mg / kg body weight in terms of alogliptin free body is preferable, and it is desirable to administer this amount once a day.
  • the agent of the present invention can be used for the prevention or treatment of other pulmonary hypertension (eg, prostaglandin preparations, anticoagulants such as warfarin and aspirin, diuretics such as furosemide and spironolactone, cardiotonic drugs such as dopamine and digoxin, and ambri.
  • Endothelin antagonists such as sentan, bosentan, and macitentan, PDE5 inhibitors such as sildenafil and tadalafil, prostacyclin preparations such as epoprostenol, PDGF receptor kinase inhibitors such as imatinib, etc.
  • concomitant drugs may be low molecular weight compounds, and may be macromolecular proteins, polypeptides, antibodies, nucleic acids (including antisense nucleic acids, siRNA, shRNA), vaccines, and the like.
  • the administration timing of the DPP-IV inhibitor and the concomitant drug is not limited, and these may be administered simultaneously to the administration subject or may be administered with a time difference.
  • dosage forms include (1) administration of a single preparation obtained by simultaneously formulating a DPP-IV inhibitor and a concomitant drug, and (2) formulating the DPP-IV inhibitor and the concomitant drug separately. Simultaneous administration of the two preparations obtained by the same administration route, and (3) a time difference in the same administration route of the two preparations obtained by separately formulating the DPP-IV inhibitor and the concomitant drug.
  • the dose of the concomitant drug can be appropriately selected based on the clinically used dose.
  • the mixing ratio of the DPP-IV inhibitor and the concomitant drug can be appropriately selected depending on the administration subject, administration route, target disease, symptom, combination and the like. For example, when the administration subject is a human, 0.01 to 100 parts by weight of the concomitant drug may be used per 1 part by weight of the DPP-IV inhibitor.
  • the above concomitant drugs may be used in combination of two or more at an appropriate ratio.
  • the amount of each agent can be reduced within a safe range considering the opposite effects of those agents. Thus, the adverse effects that would be caused by these agents can be safely prevented.
  • the present invention further relates to “a method for preventing or treating pulmonary hypertension in a mammal, which comprises administering an effective amount of a dipeptidyl peptidase-IV inhibitor to the mammal”.
  • a method for preventing or treating pulmonary hypertension in a mammal which comprises administering an effective amount of a dipeptidyl peptidase-IV inhibitor to the mammal.
  • examples of the pulmonary hypertension and dipeptidyl peptidase-IV inhibitor include those described above.
  • the “effective amount” refers to a therapeutically effective amount of a dipeptidyl peptidase-IV inhibitor for the prevention or treatment of pulmonary hypertension.
  • the present invention further relates to a “dipeptidyl peptidase-IV inhibitor for use as a preventive or therapeutic agent for pulmonary hypertension”.
  • examples of the pulmonary hypertension and dipeptidyl peptidase-IV inhibitor include those described above.
  • alogliptin described in the following experimental examples means alogliptin benzoate.
  • dose the dose and concentration of alogliptin are shown in terms of free form.
  • MCT monocrotaline
  • TGF transforming growth factor
  • TGF- ⁇ Altered bone morphogenetic protein and transforming growth factor-beta signaling in rat.
  • the sequence of TGF- ⁇ is Forward; TGGCGTTACCTTGGTAACC (SEQ ID NO: 1), Reverse; GGTGTTGAGCCCTTTCCAG (SEQ ID NO: 2).
  • the housekeeping gene ⁇ -actin was used as a control. The results are shown in FIG. In the MCT + alogliptin group, TGF- ⁇ mRNA expression decreased.
  • PASMC pulmonary artery smooth muscle cells
  • TGF- ⁇ 1 protein (Abcam, Cambridge, UK) (final concentration 10 ng / ml) was administered to each well (37 ° C., 24 hours). Thereafter, WST-8 (0.5 mg / ml), which is a formazan salt, was added to each well and incubated at 37 ° C. for 30 to 240 minutes. Absorbance at 450 nm was measured using a Bio-Rad automated EIA analyzer (Bio-Rad Laboratories, Hercules, CA, US). The results are shown in FIG. Alogliptin inhibited the proliferation of pulmonary artery smooth muscle cells induced by TGF- ⁇ in a dose-dependent manner.
  • Samples are diluted with SDS-PAGE sample buffer (62.5 mM Tris-HCl pH 6.8, 25% glycerol, 2% SDS, 0.5% beta-mercaptoethanol, 0.01% bromophenol blue), heated at 95 ° C for 10 minutes, and then And separated on a 10% SDS polyacrylamide gel. Samples were separated by electrophoresis and transferred to a 0.45 ⁇ m nitrocellulose membrane (Bio-Rad). After the transfer, this membrane was blocked with 5% skim milk using Tris-Buffered Saline and Tween 20 (TTBS) for 2 hours at room temperature.
  • SDS-PAGE sample buffer 62.5 mM Tris-HCl pH 6.8, 25% glycerol, 2% SDS, 0.5% beta-mercaptoethanol, 0.01% bromophenol blue
  • the primary antibody phosphorylated NF-kappaB p65 polyclonal antibody (Cell Signaling, Danvers, US) was diluted 1000 times with TTBS, placed on the membrane, and shaken at 4 ° C. overnight. The next day, the membrane was washed with TTBS, the secondary antibody HRP-linked anti-rabbit IgG was diluted 5000 times with TTBS, placed on the membrane, and waited at room temperature for 1 hour. PCNA was used as positive control. Protein quantification was performed using LAS-1000 (GE Healthcare, UK), and the intensity of the obtained band was analyzed using Image J software. As a result, alogliptin suppressed the phosphorylated-nuclear factor-kappaB p65 protein level induced by TGF- ⁇ (FIG. 5).
  • DPP-IV is thought to be involved in the progression of pulmonary hypertension by causing TGF- ⁇ -induced inflammation.
  • DPP-IV inhibitors such as alogliptin or a salt thereof are effective as a novel preventive or therapeutic agent for pulmonary hypertension.
  • Experimental example 2 In the present experimental example (and experimental example 3), the following experiment was performed using lung tissue collected from the rat sacrificed in experimental example 1 (2).
  • the same amount of physiological saline was subcutaneously injected instead of MCT, and distilled water was orally administered daily for 2 weeks. It was.
  • (1) Immunohistochemical staining Paraffin continuous sections (1 section 3 ⁇ m) prepared from lung tissues collected from each group of rats were pretreated in the same manner as in Experimental Example 1 (3), and immunohistochemical staining was performed.
  • ⁇ -SMa antibody and PCNA antibody in addition to the ⁇ -SMa antibody and PCNA antibody described above, phosphorylated NF-kappaB p65 polyclonal antibody (Cell Signaling, Danvers, US), CD8a antibody (BD Biosciences, San Jose, US), CD68 antibody (Santa Cruz, Dallas, US) was used.
  • secondary antibody simple stain rat MAX PO (Nichireihist Fine, Tokyo, Japan) was used.
  • ⁇ -SMa and PCNA were also subjected to hematoxylin and eosin staining (HE) and elastica-Wangeson staining (EVG), and CD8 and CD68 were also subjected to hematoxylin and eosin staining (HE).
  • alogliptin decreased the medial thickness of the pulmonary arteriole as in Experimental Example 1 (3) (FIG. 6).
  • alogliptin suppresses phosphorylated NF-kappaB p65 levels (thus, NF ⁇ B translocation into the nucleus) (Fig. 7), and CTL (CD8 positive) and macrophages (CD68 positive) around the blood vessels of small pulmonary arteries was suppressed (FIGS. 8 and 9).
  • Experimental Example 4 According to the method of Experimental Example 1 (1), 5 new MCT + Alogliptin groups were prepared, and the survival of the rats up to 30 days after MCT administration was examined, and combined with the results of Experimental Example 1 (2) (that is, MCT + Alogliptin) The survival rate of each group was shown by the Kaplan-Meier curve (FIG. 14). The survival rate at 30 days after MCT administration was 5% (1/20) in the MCT group, compared with 44% (11/25) in the MCT + alogliptin group.
  • the agent of the present invention contains a dipeptidyl peptidase-IV inhibitor and is useful as a preventive or therapeutic agent for pulmonary hypertension and the like.

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Abstract

The present invention provides a pulmonary hypertension preventive or therapeutic agent which exhibits excellent drug efficacy. The present invention pertains to a pulmonary hypertension preventive or therapeutic agent which contains a dipeptidyl peptidase IV inhibitor, and specifically, contains alogliptin or a salt thereof.

Description

肺高血圧症治療剤Pulmonary hypertension treatment
　本発明は、ジペプチジルペプチダーゼ－IV(本明細書中、ＤＰＰ－ＩＶと略記することがある)阻害薬を含有する、肺高血圧症などの予防または治療剤に関する。 The present invention relates to a preventive or therapeutic agent for pulmonary hypertension and the like, which contains a dipeptidyl peptidase-IV (sometimes abbreviated as DPP-IV in this specification) inhibitor.
（発明の背景）
　ペプチダーゼは、様々な疾患に関連していることが知られている。ペプチダーゼの１種であるＤＰＰ－ＩＶは、Ｎ末端から２番目にプロリン(あるいはアラニン)を含むペプチドに特異的に結合し、そのプロリン(あるいはアラニン)のＣ末端側を切断してジペプチドを産生するセリンプロテアーゼである。また、ＤＰＰ－ＩＶは、ＣＤ２６と同一分子であることも示されており、免疫系にも関係があることが報告されている。
　哺乳類におけるＤＰＰ－ＩＶの役割は完全には明らかになっていないが、神経ペプチドの代謝、Ｔ細胞の活性化、ガン細胞の内皮細胞への接着やＨＩＶの細胞内への侵入等において重要な役割を演じていると考えられている。特に糖代謝の面では、ＤＰＰ－ＩＶは、インクレチンであるＧＬＰ－１(glucagon-like peptide-1)あるいはＧＩＰ(Gastric inhibitory peptide/Glucose-dependent insulinotropic peptide)の不活性化に関与する。ＧＬＰ－１は、血漿中の半減期が１～２分と短い上、ＤＰＰ－ＩＶによる分解産物であるＧＬＰ－１(９－３６)アミドが、ＧＬＰ－１受容体に対してアンタゴニストとして働くなど、ＤＰＰ－ＩＶに分解されることによりその生理活性が著しく損なわれることが知られている。
　このことから、ＤＰＰ－ＩＶ活性を阻害することによりＧＬＰ－１の分解を抑制すれば、グルコース濃度依存的にインスリン分泌を促進するなど、ＧＬＰ－１の有する生理活性が増強されることも知られている。 (Background of the Invention)
Peptidases are known to be associated with various diseases. DPP-IV, a kind of peptidase, specifically binds to a peptide containing proline (or alanine) second from the N-terminus, and produces a dipeptide by cleaving the C-terminal side of the proline (or alanine). Serine protease. DPP-IV has also been shown to be the same molecule as CD26, and has been reported to be related to the immune system.
The role of DPP-IV in mammals has not been fully clarified, but it plays an important role in neuropeptide metabolism, T cell activation, cancer cell adhesion to endothelial cells, and HIV entry into cells. It is thought that he plays. Particularly in terms of sugar metabolism, DPP-IV is involved in inactivation of incretins GLP-1 (glucagon-like peptide-1) or GIP (Gastric inhibitory peptide / Glucose-dependent insulinotropic peptide). GLP-1 has a short plasma half-life of 1-2 minutes, and GLP-1 (9-36) amide, a degradation product of DPP-IV, acts as an antagonist to the GLP-1 receptor. It is known that its physiological activity is significantly impaired by being decomposed into DPP-IV.
From this, it is also known that suppressing the degradation of GLP-1 by inhibiting DPP-IV activity enhances the physiological activity of GLP-1 such as promoting insulin secretion in a glucose concentration-dependent manner. ing.
　ＤＰＰ－ＩＶ阻害薬としては、アログリプチンなどが報告されている（特許文献１）。 Alogliptin and the like have been reported as DPP-IV inhibitors (Patent Document 1).
　肺動脈性肺高血圧症（ＰＡＨ）は、肺動脈圧が上昇、持続することにより、右心不全を引き起こし、最終的には死に至る重大な疾患である。現行の主な治療は、エンドセリン受容体拮抗剤、ホスホジエステラーゼ５阻害剤、経静脈的エポプロステノールの３種類であるが、これらの治療を行うと、患者の自覚症状は改善するものの、生存率は改善せず、高い致死率が続いており、現在も大きな問題となっている。 Pulmonary arterial hypertension (PAH) is a serious disease that causes right heart failure and eventually death by increasing and sustaining pulmonary artery pressure. The current main treatments are endothelin receptor antagonists, phosphodiesterase 5 inhibitors, and intravenous epoprostenol. Although these treatments improve the patient's subjective symptoms, the survival rate is It has not improved, and the fatality rate continues to be a high problem.
米国特許出願公開第２００５／０２６１２７１号明細書US Patent Application Publication No. 2005/0261271
　肺高血圧症において疾病負荷を改善する医学的必要性は依然として高く、薬効、特異性、低毒性の点で優れた性質を有する、肺高血圧症の予防または治療剤の開発が望まれている。 There is still a high medical need for improving the disease burden in pulmonary hypertension, and it is desired to develop a preventive or therapeutic agent for pulmonary hypertension having excellent properties in terms of drug efficacy, specificity and low toxicity.
　本発明者らは、アログリプチン安息香酸塩が、肺高血圧症を治療し得ることを初めて見出し、鋭意研究を行い、本発明を完成するに至った。 The present inventors have found that alogliptin benzoate can treat pulmonary hypertension for the first time, and have intensively studied to complete the present invention.
　即ち、本発明は、
［１］　ジペプチジルペプチダーゼ－IV阻害薬（好ましくは、アログリプチンまたはその塩）を含有してなる、肺高血圧症の予防または治療剤（以下、本発明の剤と略記することがある。）；
［２］　ジペプチジルペプチダーゼ－IV阻害薬が、アログリプチンまたはその塩である、上記［１］記載の剤；
［３］　ジペプチジルペプチダーゼ－IV阻害薬が、アログリプチン安息香酸塩である、上記［１］記載の剤；
［４］　肺高血圧症が、肺動脈性肺高血圧症である上記［１］、［２］または［３］記載の剤；
［５］　哺乳動物に対して、有効量のジペプチジルペプチダーゼ－IV阻害薬（好ましくは、アログリプチンまたはその塩）を投与することを特徴とする、該哺乳動物における肺高血圧症の予防または治療方法；
［６］　肺高血圧症の予防または治療剤として使用するための、ジペプチジルペプチダーゼ－IV阻害薬（好ましくは、アログリプチンまたはその塩）；
［７］　肺高血圧症の予防または治療剤を製造するための、ジペプチジルペプチダーゼ－IV阻害薬（好ましくは、アログリプチンまたはその塩）の使用；に関する。 That is, the present invention
[1] A prophylactic or therapeutic agent for pulmonary hypertension comprising a dipeptidyl peptidase-IV inhibitor (preferably alogliptin or a salt thereof) (hereinafter sometimes abbreviated as the agent of the present invention);
[2] The agent according to [1] above, wherein the dipeptidyl peptidase-IV inhibitor is alogliptin or a salt thereof;
[3] The agent according to [1] above, wherein the dipeptidyl peptidase-IV inhibitor is alogliptin benzoate;
[4] The agent according to [1], [2] or [3] above, wherein the pulmonary hypertension is pulmonary arterial pulmonary hypertension;
[5] A method for preventing or treating pulmonary hypertension in a mammal, comprising administering an effective amount of a dipeptidyl peptidase-IV inhibitor (preferably alogliptin or a salt thereof) to the mammal;
[6] Dipeptidyl peptidase-IV inhibitor (preferably alogliptin or a salt thereof) for use as a preventive or therapeutic agent for pulmonary hypertension;
[7] Use of a dipeptidyl peptidase-IV inhibitor (preferably alogliptin or a salt thereof) for producing a prophylactic or therapeutic agent for pulmonary hypertension.
　本発明の剤は、優れたＤＰＰ－ＩＶ阻害作用を有し、肺高血圧症の予防または治療剤として有用である。
　また、本発明の剤は、経口投与が可能であることから、感染症などの副作用が少なく、肺高血圧症に対して優れた治療効果および予後改善効果を有する。 The agent of the present invention has an excellent DPP-IV inhibitory action and is useful as a preventive or therapeutic agent for pulmonary hypertension.
In addition, since the agent of the present invention can be administered orally, it has few side effects such as infectious diseases and has an excellent therapeutic effect and prognosis improving effect on pulmonary hypertension.
実験例１における生存曲線のグラフである。5 is a graph of a survival curve in Experimental Example 1. 実験例１における免疫組織化学染色の顕微鏡写真である。HEはヘマトキシリン・エオジン染色、EVGはエラスチカ・ワンギーソン染色、SMaは平滑筋αアクチン抗体、PCNAは核内増殖抗原抗体を使用した免疫組織化学染色の顕微鏡写真を示す。2 is a photomicrograph of immunohistochemical staining in Experimental Example 1. HE shows a photomicrograph of immunohistochemical staining using hematoxylin and eosin staining, EVG using Elastica-Wangeson staining, SMa using smooth muscle α-actin antibody, and PCNA using nuclear proliferation antigen antibody. 実験例１におけるトランスフォーミング増殖因子(TGF)-βのmRNA発現の測定結果を示すグラフである。4 is a graph showing measurement results of mRNA expression of transforming growth factor (TGF) -β in Experimental Example 1. 実験例１における細胞増殖アッセイの結果を示すグラフである。6 is a graph showing the results of cell proliferation assay in Experimental Example 1. 実験例１におけるウェスタンブロッティングの結果を示す図である。縦軸は、非リン酸化nuclear factor-kappaB p65 proteinレベルに対するphosphorylated-nuclear factor-kappaB p65 proteinレベルの比を示す。It is a figure which shows the result of the western blotting in Experimental example 1. The vertical axis shows the ratio of phosphorylated-nuclear factor-kappaB-p65 protein level to non-phosphorylated nuclear factor-kappaB-p65 protein level. 実験例２における免疫組織化学染色の顕微鏡写真である。HEはヘマトキシリン・エオジン染色、EVGはエラスチカ・ワンギーソン染色、SMaは平滑筋αアクチン抗体、PCNAは核内増殖抗原抗体を使用した免疫組織化学染色の顕微鏡写真を示す。4 is a photomicrograph of immunohistochemical staining in Experimental Example 2. HE shows a photomicrograph of immunohistochemical staining using hematoxylin and eosin staining, EVG using Elastica-Wangeson staining, SMa using smooth muscle α-actin antibody, and PCNA using nuclear proliferation antigen antibody. 実験例２におけるphosphorylated-nuclear factor-kappaB p65抗体を使用した免疫組織化学染色の顕微鏡写真である。phosphorylated-nuclear factor-kappaB p65陽性細胞を矢印で示す。4 is a photomicrograph of immunohistochemical staining using phosphorylated-nuclear factor-kappaBp65 antibody in Experimental Example 2. Phosphorylated-nuclear factor-kappaB p65 positive cells are indicated by arrows. 実験例２におけるCD8a抗体（CD8）、CD68抗体（CD68）を使用した免疫組織化学染色の顕微鏡写真である。HEはヘマトキシリン・エオジン染色の顕微鏡写真を示す。4 is a photomicrograph of immunohistochemical staining using CD8a antibody (CD8) and CD68 antibody (CD68) in Experimental Example 2. HE shows a photomicrograph of hematoxylin and eosin staining. 実験例２における免疫組織化学染色の顕微鏡写真（強拡大; 400倍）から得られた、視野あたりのCD8陽性、CD68陽性細胞数を示す。The numbers of CD8-positive and CD68-positive cells per field obtained from the photomicrograph of immunohistochemical staining in Experimental Example 2 (strong enlargement; 400 times) are shown. 実験例１におけるトランスフォーミング増殖因子(TGF)-βのmRNA発現（左パネル）および実験例２における塩基性線維芽細胞増殖因子(FGF2)のmRNA発現（右パネル）の測定結果を示すグラフである。It is a graph which shows the measurement result of the mRNA expression (left panel) of the transforming growth factor (TGF) -β in Experimental Example 1 and the mRNA expression (right panel) of the basic fibroblast growth factor (FGF2) in Experimental Example 2. . 実験例１（左パネル）および実験例２（右パネル）における細胞増殖アッセイの結果を示すグラフである。It is a graph which shows the result of the cell proliferation assay in Experimental example 1 (left panel) and Experimental example 2 (right panel). 実験例２におけるウェスタンブロッティングの結果を示す図である。縦軸は、非リン酸化nuclear factor-kappaB p65 proteinレベルに対するphosphorylated-nuclear factor-kappaB p65 proteinレベルの比を示す。It is a figure which shows the result of the western blotting in Experimental example 2. The vertical axis shows the ratio of phosphorylated-nuclear factor-kappaB-p65 protein level to non-phosphorylated nuclear factor-kappaB-p65 protein level. 実験例３におけるCTLA-4、CD86およびLIGHT mRNAの発現の測定結果を示すグラフである。6 is a graph showing measurement results of expression of CTLA-4, CD86 and LIGHT mRNA in Experimental Example 3. 実験例４における生存曲線のグラフである。It is a graph of the survival curve in Experimental example 4.
（発明の詳細な説明）
　本明細書中、ＤＰＰ－ＩＶ阻害薬は、ＤＰＰ－ＩＶ［生化学国際連合命名委員会（ＩＵＢＭＢ）による分類：EC3.4.14.5］の酵素活性を阻害する化合物を意味する。該化合物は、ペプチド性または非ペプチド性のいずれであってもよいが、非ペプチド性のものが好ましい。
　また、ＤＰＰ－ＩＶ阻害薬は、ＤＰＰ－ＩＶ阻害活性が保持されている限り、その形態が生体内への投与前後で異なっていてもよい。すなわち、ＤＰＰ－ＩＶ阻害薬は、生体内での代謝を受けて構造変化体となった後にＤＰＰ－ＩＶ阻害活性を有する「活性代謝物」であってもよい。さらに、ＤＰＰ－ＩＶ阻害薬は、生体内における生理条件下で酵素や胃酸等による反応により活性体に変化する「プロドラッグ」であってもよい。
　なお、ＤＰＰ－ＩＶ阻害活性は、例えば「レイモンド（Raymond）らの方法（ダイアビーティーズ（Diabetes）、４７巻、１２５３－１２５８頁、１９９８年）」を利用した方法により確認することができる。 (Detailed description of the invention)
In the present specification, the DPP-IV inhibitor means a compound that inhibits the enzyme activity of DPP-IV [Classification by International Union of Biochemistry (IUBMB): EC 3.4.14.5]. The compound may be either peptidic or non-peptidic, but is preferably non-peptidic.
In addition, the form of the DPP-IV inhibitor may be different before and after administration to the living body as long as the DPP-IV inhibitory activity is retained. That is, the DPP-IV inhibitor may be an “active metabolite” having DPP-IV inhibitory activity after becoming a structural change body after undergoing metabolism in vivo. Furthermore, the DPP-IV inhibitor may be a “prodrug” that is converted into an active form by a reaction with an enzyme, gastric acid or the like under physiological conditions in vivo.
The DPP-IV inhibitory activity can be confirmed, for example, by a method utilizing “Method of Raymond et al. (Diabetes, 47, 1253-1258, 1998)”.
　ＤＰＰ－ＩＶ阻害薬としては、例えば含窒素複素環化合物、具体的には以下の化合物（１）～（７）等が挙げられる。
（１）アログリプチンまたはその塩。
（２）トレラグリプチンまたはその塩。
（３）シタグリプチンまたはその塩。
（４）ビルダグリプチンまたはその塩。
（５）サクサグリプチンまたはその塩。
（６）リナグリプチンまたはその塩。
（７）テネリグリプチンまたはその塩。 Examples of DPP-IV inhibitors include nitrogen-containing heterocyclic compounds, specifically the following compounds (1) to (7).
(1) Alogliptin or a salt thereof.
(2) Trelagliptin or a salt thereof.
(3) Sitagliptin or a salt thereof.
(4) Vildagliptin or a salt thereof.
(5) Saxagliptin or a salt thereof.
(6) Linagliptin or a salt thereof.
(7) Tenerigliptin or a salt thereof.
　ＤＰＰ－ＩＶ阻害薬としては、アログリプチンまたはその塩が好ましい。
　ＤＰＰ－ＩＶ阻害薬としては、特にアログリプチン安息香酸塩が好ましい。 As the DPP-IV inhibitor, alogliptin or a salt thereof is preferable.
As the DPP-IV inhibitor, alogliptin benzoate is particularly preferable.
　上記化合物の塩としては、薬理学的に許容し得る塩、例えば、無機酸との塩、有機酸との塩、塩基性または酸性アミノ酸との塩などが挙げられる。
　無機酸との塩の好適な例としては、塩酸、臭化水素酸、硝酸、硫酸、リン酸などとの塩が挙げられる。
　有機酸との塩の好適な例としては、安息香酸、ギ酸、酢酸、トリフルオロ酢酸、フマル酸、シュウ酸、酒石酸、マレイン酸、クエン酸、コハク酸、リンゴ酸、メタンスルホン酸、ベンゼンスルホン酸、ｐ－トルエンスルホン酸などとの塩が挙げられる。
　塩基性アミノ酸との塩の好適な例としては、アルギニン、リジン、オルニチンなどとの塩が挙げられ、酸性アミノ酸との塩の好適な例としては、アスパラギン酸、グルタミン酸などとの塩が挙げられる。 Examples of the salt of the above compound include pharmacologically acceptable salts such as salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like.
Preferable examples of the salt with inorganic acid include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like.
Preferable examples of salts with organic acids include benzoic acid, formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, and benzenesulfonic acid. And salts with p-toluenesulfonic acid and the like.
Preferable examples of the salt with basic amino acid include salts with arginine, lysine, ornithine and the like, and preferable examples of the salt with acidic amino acid include salts with aspartic acid, glutamic acid and the like.
　アログリプチンの塩の好適な例としては、安息香酸、トリフルオロ酢酸、ｐ－トルエンスルホン酸、塩酸などとの塩が挙げられ、なかでも好ましくは安息香酸との塩である。
　トレラグリプチンの塩の好適な例としては、トリフルオロ酢酸、コハク酸、塩酸などとの塩が挙げられ、なかでも好ましくはコハク酸との塩である。 Preferable examples of the alogliptin salt include salts with benzoic acid, trifluoroacetic acid, p-toluenesulfonic acid, hydrochloric acid and the like, and among them, the salt with benzoic acid is preferable.
Preferable examples of the salt of trelagliptin include salts with trifluoroacetic acid, succinic acid, hydrochloric acid and the like, and among them, the salt with succinic acid is preferable.
　上記ＤＰＰ－ＩＶ阻害薬は、米国特許出願公開第2005／0261271号明細書に記載の方法、あるいはこれらに準ずる方法に従って製造することができる。 The DPP-IV inhibitor can be produced according to the method described in US Patent Application Publication No. 2005/0261271, or a method analogous thereto.
　本発明の剤は、毒性（例、急性毒性、慢性毒性、遺伝毒性、生殖毒性、心毒性、薬物相互作用、癌原性）が低く、ＤＰＰ－ＩＶ阻害薬をそのまま、または薬理学的に許容し得る担体等と混合して医薬組成物とすることにより、哺乳動物（例、ヒト、マウス、ラット、ウサギ、イヌ、ネコ、ウシ、ウマ、ブタ、サル）に対して、肺高血圧症の予防または治療剤として安全に用いることができる。 The agent of the present invention has low toxicity (eg, acute toxicity, chronic toxicity, genotoxicity, reproductive toxicity, cardiotoxicity, drug interaction, carcinogenicity), and the DPP-IV inhibitor is used as it is or pharmacologically acceptable. Prevention of pulmonary hypertension for mammals (eg, humans, mice, rats, rabbits, dogs, cats, cows, horses, pigs, monkeys) by mixing with a carrier or the like Or it can be safely used as a therapeutic agent.
　本発明の剤中のＤＰＰ－ＩＶ阻害薬の含量は、ＤＰＰ－ＩＶ阻害薬の種類、製剤の大きさなどによって異なるが、例えば１～９０重量％、好ましくは５～８０重量％である。 The content of the DPP-IV inhibitor in the agent of the present invention varies depending on the type of DPP-IV inhibitor, the size of the preparation, etc., but is, for example, 1 to 90% by weight, preferably 5 to 80% by weight.
　前記した薬理学的に許容し得る担体としては、製剤素材として慣用の各種有機あるいは無機担体物質が用いられ、固形製剤における賦形剤、滑沢剤、結合剤、崩壊剤；液状製剤における溶剤、溶解補助剤、懸濁化剤、等張化剤、緩衝剤、無痛化剤等として配合される。また必要に応じて、防腐剤、抗酸化剤、着色剤、甘味剤等の製剤添加物を用いることもできる。 As the pharmacologically acceptable carrier described above, various organic or inorganic carrier substances commonly used as pharmaceutical materials are used, and excipients, lubricants, binders, disintegrants in solid preparations; solvents in liquid preparations, It is blended as a solubilizing agent, suspending agent, isotonic agent, buffering agent, soothing agent and the like. If necessary, preparation additives such as preservatives, antioxidants, colorants, sweeteners and the like can also be used.
　賦形剤の好適な例としては、乳糖、白糖、Ｄ－マンニトール、Ｄ－ソルビトール、デンプン、α化デンプン、デキストリン、結晶セルロース、低置換度ヒドロキシプロピルセルロース、カルボキシメチルセルロースナトリウム、アラビアゴム、プルラン、軽質無水ケイ酸、合成ケイ酸アルミニウム、メタケイ酸アルミン酸マグネシウム等が挙げられる。 Preferable examples of excipients include lactose, sucrose, D-mannitol, D-sorbitol, starch, pregelatinized starch, dextrin, crystalline cellulose, low-substituted hydroxypropylcellulose, sodium carboxymethylcellulose, gum arabic, pullulan, light Anhydrous silicic acid, synthetic aluminum silicate, magnesium magnesium metasilicate, etc. are mentioned.
　滑沢剤の好適な例としては、ステアリン酸マグネシウム、ステアリン酸カルシウム、タルク、コロイドシリカ等が挙げられる。 Preferable examples of the lubricant include magnesium stearate, calcium stearate, talc, colloidal silica and the like.
　結合剤の好適な例としては、α化デンプン、ショ糖、ゼラチン、アラビアゴム、メチルセルロース、カルボキシメチルセルロース、カルボキシメチルセルロースナトリウム、結晶セルロース、白糖、Ｄ－マンニトール、トレハロース、デキストリン、プルラン、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、ポリビニルピロリドン等が挙げられる。 Preferred examples of the binder include pregelatinized starch, sucrose, gelatin, gum arabic, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, crystalline cellulose, sucrose, D-mannitol, trehalose, dextrin, pullulan, hydroxypropylcellulose, hydroxy Examples thereof include propylmethylcellulose and polyvinylpyrrolidone.
　崩壊剤の好適な例としては、乳糖、白糖、デンプン、カルボキシメチルセルロース、カルボキシメチルセルロースカルシウム、クロスカルメロースナトリウム、カルボキシメチルスターチナトリウム、軽質無水ケイ酸、低置換度ヒドロキシプロピルセルロース等が挙げられる。 Preferable examples of the disintegrant include lactose, sucrose, starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, croscarmellose sodium, carboxymethyl starch sodium, light anhydrous silicic acid, low substituted hydroxypropyl cellulose and the like.
　溶剤の好適な例としては、注射用水、生理的食塩水、リンゲル液、アルコール、プロピレングリコール、ポリエチレングリコール、ゴマ油、トウモロコシ油、オリーブ油、綿実油等が挙げられる。 Favorable examples of the solvent include water for injection, physiological saline, Ringer's solution, alcohol, propylene glycol, polyethylene glycol, sesame oil, corn oil, olive oil, cottonseed oil and the like.
　溶解補助剤の好適な例としては、ポリエチレングリコール、プロピレングリコール、Ｄ－マンニトール、トレハロース、安息香酸ベンジル、エタノール、トリスアミノメタン、コレステロール、トリエタノールアミン、炭酸ナトリウム、クエン酸ナトリウム、サリチル酸ナトリウム、酢酸ナトリウム等が挙げられる。 Preferable examples of the solubilizer include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, sodium acetate. Etc.
　懸濁化剤の好適な例としては、ステアリルトリエタノールアミン、ラウリル硫酸ナトリウム、ラウリルアミノプロピオン酸、レシチン、塩化ベンザルコニウム、塩化ベンゼトニウム、モノステアリン酸グリセリン等の界面活性剤；ポリビニルアルコール、ポリビニルピロリドン、カルボキシメチルセルロースナトリウム、メチルセルロース、ヒドロキシメチルセルロース、ヒドロキシエチルセルロース、ヒドロキシプロピルセルロース等の親水性高分子；ポリソルベート類、ポリオキシエチレン硬化ヒマシ油等が挙げられる。 Suitable examples of the suspending agent include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate; polyvinyl alcohol, polyvinylpyrrolidone And hydrophilic polymers such as sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose; polysorbates, polyoxyethylene hydrogenated castor oil, and the like.
　等張化剤の好適な例としては、塩化ナトリウム、グリセリン、Ｄ－マンニトール、Ｄ－ソルビトール、ブドウ糖等が挙げられる。 Preferable examples of the isotonic agent include sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose and the like.
　緩衝剤の好適な例としては、リン酸塩、酢酸塩、炭酸塩、クエン酸塩等の緩衝液等が挙げられる。 Suitable examples of the buffer include buffer solutions such as phosphate, acetate, carbonate and citrate.
　無痛化剤の好適な例としては、ベンジルアルコール等が挙げられる。 Benzyl alcohol etc. are mentioned as a suitable example of a soothing agent.
　防腐剤の好適な例としては、パラオキシ安息香酸エステル類、クロロブタノール、ベンジルアルコール、フェネチルアルコール、デヒドロ酢酸、ソルビン酸等が挙げられる。 Preferable examples of the preservative include p-hydroxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.
　抗酸化剤の好適な例としては、亜硫酸塩、アスコルビン酸塩等が挙げられる。 Preferable examples of the antioxidant include sulfite and ascorbate.
　着色剤の好適な例としては、水溶性食用タール色素（例、食用赤色２号および３号、食用黄色４号および５号、食用青色１号および２号等の食用色素）、水不溶性レーキ色素（例、前記水溶性食用タール色素のアルミニウム塩）、天然色素（例、β－カロチン、クロロフィル、ベンガラ）等が挙げられる。 Preferred examples of the colorant include water-soluble edible tar dyes (eg, edible dyes such as edible red Nos. 2 and 3, edible yellows Nos. 4 and 5, edible blue Nos. 1 and 2, etc.), water-insoluble lake dyes (Eg, the aluminum salt of the water-soluble edible tar dye), natural dyes (eg, β-carotene, chlorophyll, bengara) and the like.
　甘味剤の好適な例としては、サッカリンナトリウム、グリチルリチン酸二カリウム、アスパルテーム、ステビア等が挙げられる。 Suitable examples of the sweetening agent include saccharin sodium, dipotassium glycyrrhizinate, aspartame, stevia and the like.
　本発明の剤は、医薬製剤の製造法として自体公知の方法（例、日本薬局方記載の方法等）に従って、ＤＰＰ－ＩＶ阻害薬を薬理学的に許容される担体と混合することにより製造される。本発明の剤の剤形としては、例えば錠剤（糖衣錠、フィルムコーティング錠、舌下錠、口腔内崩壊錠、バッカル錠等を含む）、丸剤、散剤、顆粒剤、カプセル剤（ソフトカプセル剤、マイクロカプセル剤を含む）、トローチ剤、シロップ剤、液剤、乳剤、懸濁剤、エアゾール剤、フィルム剤（例、口腔内崩壊フィルム、口腔粘膜貼付フィルム）、注射剤（例、皮下注射剤、静脈内注射剤、筋肉内注射剤、腹腔内注射剤）、点滴剤、経皮吸収型製剤、軟膏剤、ローション剤、貼付剤、坐剤（例、肛門坐剤、膣坐剤）、ペレット、経鼻剤、経肺剤（吸入剤）、点眼剤等が挙げられ、これらは経口的または非経口的（例、静脈内、筋肉内、皮下、臓器内、鼻腔内、皮内、点眼、脳内、直腸内、膣内、腹腔内、腫瘍内部、腫瘍の近位等への投与および直接的な病巣への投与）に投与される。
　これらの製剤は、速放性製剤または徐放性製剤等の放出制御製剤（例、徐放性マイクロカプセル）であってもよい。 The agent of the present invention is produced by mixing a DPP-IV inhibitor with a pharmacologically acceptable carrier according to a method known per se as a method for producing a pharmaceutical preparation (eg, a method described in the Japanese Pharmacopoeia, etc.). The Examples of the dosage form of the agent of the present invention include tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, orally disintegrating tablets, buccal tablets, etc.), pills, powders, granules, capsules (soft capsules, microcapsules). Including capsules), lozenges, syrups, solutions, emulsions, suspensions, aerosols, films (eg, oral disintegrating films, oral mucosal film), injections (eg, subcutaneous injections, intravenous Injections, intramuscular injections, intraperitoneal injections), drops, transdermal preparations, ointments, lotions, patches, suppositories (eg, anal suppositories, vaginal suppositories), pellets, nasal Agents, pulmonary agents (inhalants), eye drops, etc., which are orally or parenterally (eg, intravenous, intramuscular, subcutaneous, intraorgan, intranasal, intradermal, ophthalmic, intracerebral, Intrarectal, intravaginal, intraperitoneal, intratumoral, proximal to the tumor, etc. It is administered to administration) to indirect lesions.
These preparations may be controlled-release preparations (eg, sustained-release microcapsules) such as immediate-release preparations or sustained-release preparations.
　以下に、製剤の具体的な製造法について詳述する。 Hereinafter, the specific production method of the preparation will be described in detail.
　例えば、経口剤は、有効成分に、例えば賦形剤（例、乳糖、白糖、デンプン、Ｄ－マンニトールなど）、崩壊剤（例、カルボキシメチルセルロースカルシウムなど）、結合剤（例、α化デンプン、アラビアゴム、カルボキシメチルセルロース、ヒドロキシプロピルセルロース、ポリビニルピロリドンなど）または滑沢剤（例、タルク、ステアリン酸マグネシウム、ポリエチレングリコール６０００など）などを添加して圧縮成形し、次いで必要により、味のマスキング、腸溶性あるいは持続性を目的として、コーティング基剤を用いて自体公知の方法でコーティングすることにより製造される。
　該コーティング基剤としては、例えば糖衣基剤、水溶性フィルムコーティング基剤、腸溶性フィルムコーティング基剤、徐放性フィルムコーティング基剤などが挙げられる。
　糖衣基剤としては、白糖が用いられ、さらに、タルク、沈降炭酸カルシウム、ゼラチン、アラビアゴム、プルラン、カルナバロウなどから選ばれる１種または２種以上を併用してもよい。
　水溶性フィルムコーティング基剤としては、例えばヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、ヒドロキシエチルセルロース、メチルヒドロキシエチルセルロースなどのセルロース系高分子；ポリビニルアセタールジエチルアミノアセテート、アミノアルキルメタアクリレートコポリマーＥ〔オイドラギットＥ（商品名）、ロームファルマ社〕、ポリビニルピロリドンなどの合成高分子；プルランなどの多糖類などが挙げられる。 For example, oral preparations contain active ingredients such as excipients (eg, lactose, sucrose, starch, D-mannitol, etc.), disintegrants (eg, carboxymethylcellulose calcium, etc.), binders (eg, pregelatinized starch, Arabic Rubber, carboxymethyl cellulose, hydroxypropyl cellulose, polyvinyl pyrrolidone, etc.) or lubricant (eg, talc, magnesium stearate, polyethylene glycol 6000, etc.), etc., and compression molded, and if necessary, taste masking, enteric Alternatively, for the purpose of durability, it is produced by coating with a coating base by a method known per se.
Examples of the coating base include sugar coating base, water-soluble film coating base, enteric film coating base, sustained-release film coating base and the like.
As the sugar coating base, sucrose is used, and one or more selected from talc, precipitated calcium carbonate, gelatin, gum arabic, pullulan, carnauba wax and the like may be used in combination.
Examples of the water-soluble film coating base include cellulose polymers such as hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, and methylhydroxyethylcellulose; polyvinyl acetal diethylaminoacetate, aminoalkyl methacrylate copolymer E [Eudragit E (trade name), Rohm Pharma Co., Ltd.], synthetic polymers such as polyvinylpyrrolidone; polysaccharides such as pullulan.
　腸溶性フィルムコーティング基剤としては、例えばヒドロキシプロピルメチルセルロース　フタレート、ヒドロキシプロピルメチルセルロース　アセテートサクシネート、カルボキシメチルエチルセルロース、酢酸フタル酸セルロースなどのセルロース系高分子；メタアクリル酸コポリマーＬ〔オイドラギットＬ（商品名）、ロームファルマ社〕、メタアクリル酸コポリマーＬＤ〔オイドラギットＬ－３０Ｄ５５（商品名）、ロームファルマ社〕、メタアクリル酸コポリマーＳ〔オイドラギットＳ（商品名）、ロームファルマ社〕などのアクリル酸系高分子；セラックなどの天然物などが挙げられる。
　徐放性フィルムコーティング基剤としては、例えばエチルセルロースなどのセルロース系高分子；アミノアルキルメタアクリレートコポリマーＲＳ〔オイドラギットＲＳ（商品名）、ロームファルマ社〕、アクリル酸エチル・メタアクリル酸メチル共重合体懸濁液〔オイドラギットＮＥ（商品名）、ロームファルマ社〕などのアクリル酸系高分子などが挙げられる。
　前記したコーティング基剤は、２種以上を適宜の割合で混合して用いてもよい。また、コーティングの際に、例えば酸化チタン、三二酸化鉄等のような遮光剤を用いてもよい。 Examples of enteric film coating bases include cellulose-based polymers such as hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, carboxymethylethylcellulose, and cellulose acetate phthalate; methacrylic acid copolymer L [Eudragit L (trade name), Acrylic polymers such as Rohm Pharma Co., Ltd., methacrylic acid copolymer LD [Eudragit L-30D55 (trade name), Rohm Pharma Co., Ltd.], and methacrylic acid copolymer S [Eudragit S (trade name), Rohm Pharma Co., Ltd.]; Examples include natural products such as shellac.
Examples of sustained-release film coating bases include cellulose polymers such as ethyl cellulose; aminoalkyl methacrylate copolymer RS [Eudragit RS (trade name), Rohm Pharma Co., Ltd.], ethyl acrylate / methyl methacrylate copolymer suspension Acrylic polymers such as suspensions (Eudragit NE (trade name), Rohm Pharma) are listed.
Two or more kinds of the above-described coating bases may be mixed and used at an appropriate ratio. Moreover, you may use light-shielding agents, such as a titanium oxide, ferric oxide, etc. in the case of coating.
　注射剤は、有効成分を分散剤（例、ポリソルベート８０、ポリオキシエチレン硬化ヒマシ油６０、ポリエチレングリコール、カルボキシメチルセルロース、アルギン酸ナトリウムなど）、保存剤（例、メチルパラベン、プロピルパラベン、ベンジルアルコール、クロロブタノール、フェノールなど）、等張化剤（例、塩化ナトリウム、グリセリン、Ｄ－マンニトール、Ｄ－ソルビトール、ブドウ糖など）などと共に水性溶剤（例、蒸留水、生理的食塩水、リンゲル液等）あるいは油性溶剤（例、オリーブ油、ゴマ油、綿実油、トウモロコシ油などの植物油、プロピレングリコール等）などに溶解、懸濁あるいは乳化することにより製造される。この際、所望により溶解補助剤（例、サリチル酸ナトリウム、酢酸ナトリウム等）、安定剤（例、ヒト血清アルブミン等）、無痛化剤（例、ベンジルアルコール等）等の添加物を用いてもよい。
　前記した各種製剤の中でも、利便性あるいはコンプライアンスに優れる経口剤が好ましい。 Injections contain active ingredients as dispersants (eg, polysorbate 80, polyoxyethylene hydrogenated castor oil 60, polyethylene glycol, carboxymethyl cellulose, sodium alginate, etc.), preservatives (eg, methyl paraben, propyl paraben, benzyl alcohol, chlorobutanol, Phenol), isotonic agents (eg, sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose, etc.), etc., and aqueous solvents (eg, distilled water, physiological saline, Ringer's solution, etc.) or oily solvents (eg, , Olive oil, sesame oil, cottonseed oil, vegetable oils such as corn oil, propylene glycol, etc.) and the like. At this time, additives such as a solubilizer (eg, sodium salicylate, sodium acetate, etc.), a stabilizer (eg, human serum albumin, etc.), a soothing agent (eg, benzyl alcohol, etc.) may be used as desired.
Among the various preparations described above, an oral preparation excellent in convenience or compliance is preferable.
　本発明の剤は、肺高血圧症の予防または治療に有用である。
　本発明の剤は、より好ましくは、肺動脈性肺高血圧症の予防または治療に有用である。 The agent of the present invention is useful for prevention or treatment of pulmonary hypertension.
The agent of the present invention is more preferably useful for the prevention or treatment of pulmonary arterial pulmonary hypertension.
　ここで、肺高血圧症の予防または治療には、肺高血圧症の進展抑制も含まれる。肺高血圧症の予防または治療効果は、例えば本発明の剤の投与対象において、生存率の改善、肺細動脈の中膜肥厚の減少、トランスフォーミング増殖因子(TGF)-βのmRNA発現の減少、TGF-βタンパク質発現の減少、塩基性線維芽細胞増殖因子（FGF2）のmRNAまたはタンパク質発現の減少、肺動脈平滑筋細胞の増殖抑制、phosphorylated-nuclear factor-kappaB p65 (p-p65) proteinレベルの抑制、p-p65の核内移行の抑制、肺病変部への免疫・炎症細胞（例、マクロファージ、T細胞等）の浸潤抑制、免疫・炎症反応に関与する細胞表面受容体またはそのリガンド（例、CTLA-4、CD86、LIGHT/TNFSF14、HVEM等）のmRNAの発現等を評価することにより確認される。 Here, prevention or treatment of pulmonary hypertension includes suppression of the progression of pulmonary hypertension. The preventive or therapeutic effect of pulmonary hypertension, for example, in the administration subject of the agent of the present invention, is improved survival rate, decreased medial thickness of pulmonary arteriole, decreased mRNA expression of transforming growth factor (TGF) -β Decreased expression of TGF-β protein, decreased expression of basic fibroblast growth factor (FGF2) mRNA or protein, suppressed proliferation of pulmonary artery smooth muscle cells, suppressed phosphorylated-nuclear factor-kappaB p65 (p-p65) protein level , Suppression of p-p65 nuclear translocation, suppression of infiltration of immune / inflammatory cells (eg, macrophages, T cells, etc.) into lung lesions, cell surface receptors involved in immune / inflammatory reactions or ligands thereof (eg, CTLA-4, CD86, LIGHT / TNFSF14, HVEM, etc.) are confirmed by evaluating the mRNA expression.
　日本循環器学会のガイドライン（2006年改訂版）によると、安静臥位での平均肺動脈圧が25 mmHgを超える場合、または肺疾患、睡眠時無呼吸症候群、肺胞低換気症候群では平均肺動脈圧が20 mmHgを超える場合に、肺高血圧症と診断される。 According to the Japanese Circulation Society Guidelines (2006 revised edition), the average pulmonary artery pressure in resting position exceeds 25 mmHg, or in lung disease, sleep apnea syndrome, and alveolar hypoventilation syndrome Diagnosed as pulmonary hypertension if it exceeds 20 mmHg.
　本明細書中、「肺高血圧症」は、種々の形の肺高血圧症を含む。具体例としては、突発性の肺動脈性肺高血圧症；家族性の肺動脈性肺高血圧症；膠原血管病、先天性の全身ないし肺シャント、門脈圧亢進症、ＨＩＶ感染、薬物もしくは毒物に関連する肺動脈性肺高血圧症；甲状腺疾患、糖原病、ゴシェ病、遺伝性出血性毛細管拡張症、異常ヘモグロビン症、骨髄増殖性疾患もしくは脾臓除去術に関連する肺高血圧症；肺毛細血管腫症に関連する肺動脈性肺高血圧症；新生児の持続性肺高血圧症；慢性閉塞性肺疾患、間質性肺疾患、低酸素状態により引き起こされる肺胞性低換気疾患、低酸素状態により引き起こされる睡眠障害性呼吸もしくは高標高への慢性的な暴露に関連する肺高血圧症；異常発生に関連する肺高血圧症；並びに遠位の肺動脈の塞栓性の閉塞による肺高血圧症が挙げられる。 In the present specification, “pulmonary hypertension” includes various forms of pulmonary hypertension. Examples include idiopathic pulmonary arterial pulmonary hypertension; familial pulmonary arterial pulmonary hypertension; collagen vascular disease, congenital systemic or pulmonary shunt, portal hypertension, HIV infection, drugs or toxicants Pulmonary arterial hypertension; Thyroid disease, Glycogenosis, Goshe disease, Hereditary hemorrhagic telangiectasia, Abnormal hemoglobinosis, Myeloproliferative disease or Spleen removal surgery, Pulmonary hypertension; Pulmonary arterial hypertension; persistent neonatal pulmonary hypertension; chronic obstructive pulmonary disease, interstitial lung disease, alveolar hypoventilation caused by hypoxia, sleep disordered breathing caused by hypoxia or Pulmonary hypertension associated with chronic exposure to high altitude; pulmonary hypertension associated with abnormal occurrence; and pulmonary hypertension due to embolic obstruction of the distal pulmonary artery.
　本発明の剤の投与量は、投与対象、投与ルート、対象疾患などによっても異なるが、例えば成人の肺高血圧症患者に経口投与する場合、活性成分であるＤＰＰ－ＩＶ阻害薬を通常１回量として約０．０１～１００ｍｇ／ｋｇ体重、好ましくは０．０５～３０ｍｇ／ｋｇ体重、さらに好ましくは０．１～１０ｍｇ／ｋｇ体重であり、この量を１日１回～２回投与することが望ましい。ＤＰＰ－ＩＶ阻害薬がアログリプチンまたはその塩である場合、アログリプチンのフリー体換算で０．１～１ｍｇ／ｋｇ体重が好ましく、この量を１日１回投与することが望ましい。 The dose of the agent of the present invention varies depending on the administration subject, administration route, target disease, and the like. For example, when orally administered to an adult pulmonary hypertension patient, the active ingredient DPP-IV inhibitor is usually administered in a single dose. About 0.01 to 100 mg / kg body weight, preferably 0.05 to 30 mg / kg body weight, more preferably 0.1 to 10 mg / kg body weight, and this amount can be administered once or twice a day. desirable. When the DPP-IV inhibitor is alogliptin or a salt thereof, 0.1 to 1 mg / kg body weight in terms of alogliptin free body is preferable, and it is desirable to administer this amount once a day.
　本発明の剤は、他の肺高血圧症の予防または治療薬（例、プロスタグランジン製剤、ワルファリン、アスピリン等の抗凝固薬、フロセミド、スピロノラクトン等の利尿薬、ドーパミン、ジゴキシン等の強心薬、アンブリセンタン、ボセンタン、マシテンタン等のエンドセリン拮抗薬、シルデナフィル、タダラフィル等のPDE5阻害薬、エポプロステノール等のプロスタサイクリン製剤、イマチニブ等のPDGF受容体キナーゼ阻害剤等（本明細書中、併用薬剤と略記することがある））と組み合わせて用いることができる。これらの併用薬剤は、低分子化合物であってもよく、また高分子の蛋白、ポリペプチド、抗体、核酸（アンチセンス核酸、ｓｉＲＮＡ、ｓｈＲＮＡを含む）、ワクチン等であってもよい。 The agent of the present invention can be used for the prevention or treatment of other pulmonary hypertension (eg, prostaglandin preparations, anticoagulants such as warfarin and aspirin, diuretics such as furosemide and spironolactone, cardiotonic drugs such as dopamine and digoxin, and ambri. Endothelin antagonists such as sentan, bosentan, and macitentan, PDE5 inhibitors such as sildenafil and tadalafil, prostacyclin preparations such as epoprostenol, PDGF receptor kinase inhibitors such as imatinib, etc. (abbreviated as concomitant drugs in this specification) In some cases). These concomitant drugs may be low molecular weight compounds, and may be macromolecular proteins, polypeptides, antibodies, nucleic acids (including antisense nucleic acids, siRNA, shRNA), vaccines, and the like.
　ＤＰＰ－ＩＶ阻害薬および併用薬剤の投与時期は限定されず、これらを投与対象に対し、同時に投与してもよいし、時間差をおいて投与してもよい。
　投与形態としては、例えば、（１）ＤＰＰ－ＩＶ阻害薬と併用薬剤とを同時に製剤化して得られる単一の製剤の投与、（２）ＤＰＰ－ＩＶ阻害薬と併用薬剤とを別々に製剤化して得られる２種の製剤の同一投与経路での同時投与、（３）ＤＰＰ－ＩＶ阻害薬と併用薬剤とを別々に製剤化して得られる２種の製剤の同一投与経路での時間差をおいての投与、（４）ＤＰＰ－ＩＶ阻害薬と併用薬剤とを別々に製剤化して得られる２種の製剤の異なる投与経路での同時投与、（５）ＤＰＰ－ＩＶ阻害薬と併用薬剤とを別々に製剤化して得られる２種の製剤の異なる投与経路での時間差をおいての投与（例えば、ＤＰＰ－ＩＶ阻害薬および併用薬剤の順序での投与、あるいはその逆の順序での投与）等が挙げられる。 The administration timing of the DPP-IV inhibitor and the concomitant drug is not limited, and these may be administered simultaneously to the administration subject or may be administered with a time difference.
Examples of dosage forms include (1) administration of a single preparation obtained by simultaneously formulating a DPP-IV inhibitor and a concomitant drug, and (2) formulating the DPP-IV inhibitor and the concomitant drug separately. Simultaneous administration of the two preparations obtained by the same administration route, and (3) a time difference in the same administration route of the two preparations obtained by separately formulating the DPP-IV inhibitor and the concomitant drug. (4) Simultaneous administration of two preparations obtained by separately formulating a DPP-IV inhibitor and a concomitant drug through different administration routes, (5) Separately administering a DPP-IV inhibitor and a concomitant drug Administration of the two preparations obtained by formulation in different administration routes at different time intervals (for example, administration in the order of the DPP-IV inhibitor and the concomitant drug, or vice versa) Can be mentioned.
　併用薬剤の投与量は、臨床上用いられている用量を基準として適宜選択することができる。また、ＤＰＰ－ＩＶ阻害薬と併用薬剤の配合比は、投与対象、投与ルート、対象疾患、症状、組み合わせ等により適宜選択することができる。例えば、投与対象がヒトである場合、ＤＰＰ－ＩＶ阻害薬１重量部に対し、併用薬剤を０．０１ないし１００重量部用いればよい。 The dose of the concomitant drug can be appropriately selected based on the clinically used dose. The mixing ratio of the DPP-IV inhibitor and the concomitant drug can be appropriately selected depending on the administration subject, administration route, target disease, symptom, combination and the like. For example, when the administration subject is a human, 0.01 to 100 parts by weight of the concomitant drug may be used per 1 part by weight of the DPP-IV inhibitor.
　上記併用薬剤は、２種以上を適宜の割合で組み合わせて用いてもよい。 The above concomitant drugs may be used in combination of two or more at an appropriate ratio.
　ＤＰＰ－ＩＶ阻害薬が併用薬剤と組み合せて使用される場合には、お互いの剤の量は、それらの剤の反対効果を考えて安全な範囲内で低減できる。従って、これらの剤により引き起こされるであろう反対効果は安全に防止できる。 When a DPP-IV inhibitor is used in combination with a concomitant drug, the amount of each agent can be reduced within a safe range considering the opposite effects of those agents. Thus, the adverse effects that would be caused by these agents can be safely prevented.
　本発明は、さらに、「哺乳動物に対して、有効量のジペプチジルペプチダーゼ－IV阻害薬を投与することを特徴とする、該哺乳動物における肺高血圧症の予防または治療方法」に関する。
　ここで、肺高血圧症およびジペプチジルペプチダーゼ－IV阻害薬としては、前記と同様のものが挙げられる。
　上記「有効量」とは、肺高血圧症の予防または治療のためのジペプチジルペプチダーゼ－IV阻害薬の治療学的に有効な量を指す。
　本発明は、さらに、「肺高血圧症の予防または治療剤として使用するための、ジペプチジルペプチダーゼ－IV阻害薬」に関する。
　ここで、肺高血圧症およびジペプチジルペプチダーゼ－IV阻害薬としては、前記と同様のものが挙げられる。 The present invention further relates to “a method for preventing or treating pulmonary hypertension in a mammal, which comprises administering an effective amount of a dipeptidyl peptidase-IV inhibitor to the mammal”.
Here, examples of the pulmonary hypertension and dipeptidyl peptidase-IV inhibitor include those described above.
The “effective amount” refers to a therapeutically effective amount of a dipeptidyl peptidase-IV inhibitor for the prevention or treatment of pulmonary hypertension.
The present invention further relates to a “dipeptidyl peptidase-IV inhibitor for use as a preventive or therapeutic agent for pulmonary hypertension”.
Here, examples of the pulmonary hypertension and dipeptidyl peptidase-IV inhibitor include those described above.
　本発明は、以下の実施例によって、さらに詳しく説明されるが、これらは本発明を限定するものではなく、また本発明の範囲を逸脱しない範囲で変化させてもよい。 The present invention will be described in more detail with reference to the following examples, but these are not intended to limit the present invention, and may be changed without departing from the scope of the present invention.
　以下の実験例に記載のアログリプチンは、アログリプチン安息香酸塩を意味する。用量について、アログリプチン(alogliptin)の投与量及び濃度はフリー体換算の値で示す。 The alogliptin described in the following experimental examples means alogliptin benzoate. Regarding the dose, the dose and concentration of alogliptin are shown in terms of free form.
実験例１
（１）動物モデル
　5週齢の雄性Sprague-Dawleyラット50匹(日本クレア；Tokyo, Japan)を用意し、1週間ストレスのない状況で飼育した。6週齢になった時点で、モノクロタリン(MCT)(和光純薬工業, Osaka, Japan) 60 mg/kgを皮下注射した。MCT投与後14日目より、アログリプチン(蒸留水に溶解) 10 mg/kg (MCT+アログリプチン群, n=25)、同量の蒸留水 (MCT群, n=25)を連日経口投与した。 Experimental example 1
(1) Animal model 50 male Sprague-Dawley rats of 5 weeks old (Claire Japan; Tokyo, Japan) were prepared and bred under no stress for 1 week. At the age of 6 weeks, monocrotaline (MCT) (Wako Pure Chemical Industries, Osaka, Japan) 60 mg / kg was subcutaneously injected. From the 14th day after MCT administration, alogliptin (dissolved in distilled water) 10 mg / kg (MCT + alogliptin group, n = 25) and the same amount of distilled water (MCT group, n = 25) were orally administered every day.
（２）生存曲線
　MCT投与後30日の時点で、それぞれの群の生存率をKaplan-Meier曲線を用いて検討した。なお、各群25匹のうち、5匹ずつサクリファイス(心臓カテーテル検査施行後、免疫組織化学染色、定量PCR用に組織を採取)に用いた。
　結果を図１に示す。MCT投与後30日での生存率は、MCT群が5%(1/20)であるのに対して、MCT+アログリプチン群は30%(6/20)であった。
　MCT投与後21日での心臓カテーテル検査において、アログリプチンは右室圧負荷を顕著に改善した（MCT群 91.4±6.9 mmHgに対して、MCT+アログリプチン群 33±4.1 mmHg）。 (2) Survival curve At 30 days after MCT administration, the survival rate of each group was examined using the Kaplan-Meier curve. Of 25 mice in each group, 5 mice were used for saccharification (tissue collected for immunohistochemical staining and quantitative PCR after cardiac catheterization).
The results are shown in FIG. The survival rate at 30 days after MCT administration was 5% (1/20) in the MCT group, compared with 30% (6/20) in the MCT + alogliptin group.
In cardiac catheterization 21 days after MCT administration, alogliptin significantly improved the right ventricular pressure load (91.4 ± 6.9 mmHg in the MCT group, 33 ± 4.1 mmHg in the MCT + alogliptin group).
（３）免疫組織化学染色
　心臓カテーテル検査を施行した日(MCT投与後21日)に、肺組織を採取し、ホルマリンにて固定し、パラフィン連続切片(1切片3 μm)を作製した。キシレンを用いて脱パラフィンを行ない、その後アルコールを用いて脱水した。次に、クエン酸バッファーを用いて55℃で一晩インキュベーションし、抗原賦活化を行なった。その翌日に、0.3%過酸化水素水を用いて内因性ペルオキシダーゼの不活性化を行なったのち、PBS(pH 7.4)にて5分×3回洗浄した。その後、一次抗体を希釈して、各切片にかけた。使用した一次抗体は平滑筋αアクチン(α-SMa)抗体(Sigma-Aldrich, St. Louis, US, 100倍希釈)および核内増殖抗原(PCNA)抗体 (Sigma-Aldrich, St. Louis, US, 50倍希釈)の2つである。抗体希釈液は1% BSA-PBSを使用した。二次抗体はシンプルステインラット MAX PO (ニチレイヒストファイン, Tokyo, Japan)を用いた。その他ヘマトキシリン・エオジン染色(HE)、エラスチカ・ワンギーソン染色(EVG)も行なった。各スライドグラスは、光学顕微鏡を用いて検鏡した。
　結果を図２に示す。免疫組織化学染色において、アログリプチンは肺細動脈の中膜肥厚を減少させることが明らかとなった。 (3) Immunohistochemical staining On the day of cardiac catheter examination (21 days after MCT administration), lung tissue was collected and fixed with formalin to prepare paraffin continuous sections (1 section 3 μm). Deparaffinization was performed using xylene, followed by dehydration using alcohol. Next, the antigen was activated by incubating overnight at 55 ° C. using a citrate buffer. The next day, after inactivation of endogenous peroxidase with 0.3% aqueous hydrogen peroxide, the plate was washed with PBS (pH 7.4) for 5 minutes × 3 times. The primary antibody was then diluted and applied to each section. The primary antibodies used were smooth muscle α-actin (α-SMa) antibody (Sigma-Aldrich, St. Louis, US, 100-fold dilution) and nuclear proliferation antigen (PCNA) antibody (Sigma-Aldrich, St. Louis, US, 50-fold dilution). The antibody diluent was 1% BSA-PBS. As the secondary antibody, simple stain rat MAX PO (Nichireihist Fine, Tokyo, Japan) was used. In addition, hematoxylin / eosin staining (HE) and elastica-Wangeson staining (EVG) were also performed. Each slide glass was examined using an optical microscope.
The results are shown in FIG. Immunohistochemical staining revealed that alogliptin reduced medial thickness of pulmonary arterioles.
（４）定量リアルタイム RT-PCR
　トランスフォーミング増殖因子(TGF)-βのmRNA発現量は、定量リアルタイム RT-PCRにより測定した。
　各群のラットから採取した肺組織のtotal RNAは、High Pure RNA Tissue Kit (Roche, Switzerland)を用いて抽出した。またcDNAは、Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Switzerland)を用いて合成した。遺伝子発現の定量には、FastStart Universal SYBR Green Master (ROX) (Roche, Switzerland)を用いた。配列特異的なPCRプライマーは、既報されているLong Lら（Long L, Crosby A, Yang X, Southwood M, Upton PD, Kim DK, et al. Altered bone morphogenetic protein and transforming growth factor-beta signaling in rat models of pulmonary hypertension: potential for activin receptor-like kinase-5 inhibition in prevention and progression of disease. Circulation 2009;119:566-576）の配列を使用した。TGF-βの配列は、Forward; TGGCGTTACCTTGGTAACC（配列番号１）, Reverse; GGTGTTGAGCCCTTTCCAG（配列番号２）である。なお、ハウスキーピング遺伝子であるβ-アクチンをコントロールとして使用した。
　結果を図３に示す。MCT+アログリプチン群では、TGF-βのmRNA発現が減少した。 (4) Quantitative real-time RT-PCR
The mRNA expression level of transforming growth factor (TGF) -β was measured by quantitative real-time RT-PCR.
Total RNA of lung tissue collected from each group of rats was extracted using High Pure RNA Tissue Kit (Roche, Switzerland). CDNA was synthesized using Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Switzerland). For quantification of gene expression, FastStart Universal SYBR Green Master (ROX) (Roche, Switzerland) was used. Sequence-specific PCR primers are described in Long L et al. (Long L, Crosby A, Yang X, Southwood M, Upton PD, Kim DK, et al. Altered bone morphogenetic protein and transforming growth factor-beta signaling in rat. The model of pulmonary hypertension: potential for activin receptor-like kinase-5 inhibition in prevention and progression of disease. Circulation 2009; 119: 566-576) was used. The sequence of TGF-β is Forward; TGGCGTTACCTTGGTAACC (SEQ ID NO: 1), Reverse; GGTGTTGAGCCCTTTCCAG (SEQ ID NO: 2). The housekeeping gene β-actin was used as a control.
The results are shown in FIG. In the MCT + alogliptin group, TGF-β mRNA expression decreased.
（５）細胞増殖アッセイ
　肺動脈平滑筋細胞 (PASMC)の細胞増殖アッセイは、Cell Counting Kit-8 (同仁化学研究所, Tokyo, Japan)を用いた。96ウエルマルチプレートにPASMCを1×103/wellとなるようにまいた。細胞を24時間血清飢餓状態とした後、各ウエルにアログリプチンを1時間投与(最終濃度 0, 0.1, 1, 5 μM)した。アログリプチンの溶媒には、100 U/mlペニシリンおよび100 mg/mlストレプトマイシンを含むDulbecco's Modified Eagle's Medium (Sigma-Aldrich)を用いた。続いて、TGF-β1タンパク (Abcam, Cambridge, UK) (最終濃度 10 ng/ml)を各ウエルに投与した (37℃, 24時間)。その後、各ウエルに、ホルマザン塩であるWST-8 (0.5 mg/ml)を加え、37℃で30分～240分インキュベーションした。Bio-Rad automated EIA analyzer (Bio-Rad Laboratories, Hercules, CA, US)を用いて、450 nmでの吸光度を測定した。
　結果を図４に示す。アログリプチンは、TGF-β刺激による肺動脈平滑筋細胞の増殖を用量依存的に阻害した。 (5) Cell proliferation assay Cell Counting Kit-8 (Dojindo Laboratories, Tokyo, Japan) was used for the cell proliferation assay of pulmonary artery smooth muscle cells (PASMC). PASMC was spread on a 96-well multiplate at 1 × 10 3 / well. Cells were serum starved for 24 hours and then alogliptin was administered to each well for 1 hour (final concentrations 0, 0.1, 1, 5 μM). As the alogliptin solvent, Dulbecco's Modified Eagle's Medium (Sigma-Aldrich) containing 100 U / ml penicillin and 100 mg / ml streptomycin was used. Subsequently, TGF-β1 protein (Abcam, Cambridge, UK) (final concentration 10 ng / ml) was administered to each well (37 ° C., 24 hours). Thereafter, WST-8 (0.5 mg / ml), which is a formazan salt, was added to each well and incubated at 37 ° C. for 30 to 240 minutes. Absorbance at 450 nm was measured using a Bio-Rad automated EIA analyzer (Bio-Rad Laboratories, Hercules, CA, US).
The results are shown in FIG. Alogliptin inhibited the proliferation of pulmonary artery smooth muscle cells induced by TGF-β in a dose-dependent manner.
（６）ウェスタンブロッティング
　6 cmディッシュに、PASMCをまいてコンフルエント (2×105/dish)にし、24時間血清飢餓状態にした。PASMCには、最終濃度1 μMのアログリプチンを1時間投与し、続いてTGF-β1タンパク(最終濃度10 ng/ml)を1時間加えた。ディッシュ各々の細胞は、Yang C-Mら（Yang C-M, Luo S-F, Hsieh H-L, Chi P-L, Lin C-C, Wu C-C, et al. Interleukin-1β induces ICAM-1 expression enhancing leukocyte adhesion in human rheumatoid arthritis synovial fibroblasts: Involvement of ERK, JNK, AP-1, and NF-κB. Journal of Cellular Physiology 2010;224:516-526）の実験方法を参考にして、細胞質タンパクと核タンパクに分けて抽出した。サンプルは、SDS-PAGEサンプルバッファー (62.5 mM Tris-HCl pH 6.8, 25% glycerol, 2% SDS, 0.5% beta-mercaptoethanol, 0.01% bromophenol blue)を用いて希釈し、95℃で10分間熱し、続いて10% SDSポリアクリルアミドゲルにて分離した。サンプルは電気泳動を行なって分離し、0.45 μmニトロセルロース膜(Bio-Rad)へ転写した。転写後、このメンブレンは、Tris-Buffered SalineとTween 20 (TTBS)を用いた5%スキムミルクで、室温で2時間ブロッキングを行なった。続いて、一次抗体phosphorylated NF-kappaB p65ポリクローナル抗体 (Cell Signaling, Danvers, US)をTTBSで1000倍に希釈し、メンブレンの上にのせ、4℃で一晩震盪させた。翌日、メンブレンをTTBSで洗浄し、二次抗体 HRP-linked anti-rabbit IgGをTTBSで5000倍に希釈し、メンブレンにのせ、室温で1時間待った。なお、positive controlとして、PCNAを用いた。タンパクの定量は、LAS-1000 (GE Healthcare, UK)を用いて行ない、得られたバンドの強度は、Image Jソフトウエアを使用して解析した。
　結果、アログリプチンは、TGF-βにより誘発されるphosphorylated-nuclear factor-kappaB p65 proteinレベルを抑制した（図５）。 (6) Western blotting PASMC was spread on a 6 cm dish to confluence (2 × 10 5 / dish), and serum-starved for 24 hours. PASMC was administered with a final concentration of 1 μM alogliptin for 1 hour, followed by addition of TGF-β1 protein (final concentration 10 ng / ml) for 1 hour. Each cell of the dish was treated with Yang CM et al. (Yang CM, Luo SF, Hsieh HL, Chi PL, Lin CC, Wu CC, et al. of ERK, JNK, AP-1, and NF-κB. Journal of Cellular Physiology 2010; 224: 516-526). Samples are diluted with SDS-PAGE sample buffer (62.5 mM Tris-HCl pH 6.8, 25% glycerol, 2% SDS, 0.5% beta-mercaptoethanol, 0.01% bromophenol blue), heated at 95 ° C for 10 minutes, and then And separated on a 10% SDS polyacrylamide gel. Samples were separated by electrophoresis and transferred to a 0.45 μm nitrocellulose membrane (Bio-Rad). After the transfer, this membrane was blocked with 5% skim milk using Tris-Buffered Saline and Tween 20 (TTBS) for 2 hours at room temperature. Subsequently, the primary antibody phosphorylated NF-kappaB p65 polyclonal antibody (Cell Signaling, Danvers, US) was diluted 1000 times with TTBS, placed on the membrane, and shaken at 4 ° C. overnight. The next day, the membrane was washed with TTBS, the secondary antibody HRP-linked anti-rabbit IgG was diluted 5000 times with TTBS, placed on the membrane, and waited at room temperature for 1 hour. PCNA was used as positive control. Protein quantification was performed using LAS-1000 (GE Healthcare, UK), and the intensity of the obtained band was analyzed using Image J software.
As a result, alogliptin suppressed the phosphorylated-nuclear factor-kappaB p65 protein level induced by TGF-β (FIG. 5).
　ＤＰＰ－ＩＶは、TGF-β誘発性炎症を引き起こすことにより肺高血圧症の進行に関与していると考えられる。アログリプチンまたはその塩などのＤＰＰ－ＩＶ阻害薬は肺高血圧症の新規な予防または治療剤として有効である。 DPP-IV is thought to be involved in the progression of pulmonary hypertension by causing TGF-β-induced inflammation. DPP-IV inhibitors such as alogliptin or a salt thereof are effective as a novel preventive or therapeutic agent for pulmonary hypertension.
実験例２
　本実験例（および実験例３）では、実験例１（２）においてサクリファイスしたラットから採取した肺組織を用いて、以下の実験を行った。尚、陰性対照群(NC群、n=10)として、実験例１と同様、MCTの代わりに同量の生理食塩水を皮下注射し、2週間後より蒸留水を連日経口投与したラットを用いた。
（１）免疫組織化学染色
　各群ラットから採取した肺組織より作製したパラフィン連続切片（1切片 3 μm）を実験例１（３）と同様に前処理し、免疫組織化学染色を行った。本実験例では、一次抗体として、前述のα-SMa抗体およびPCNA抗体に加えて、phosphorylated NF-kappaB p65ポリクローナル抗体 (Cell Signaling, Danvers, US)、CD8a抗体(BD Biosciences, San Jose, US)、CD68抗体(Santa Cruz, Dallas, US)を用いた。二次抗体はシンプルステインラット MAX PO (ニチレイヒストファイン, Tokyo, Japan)を用いた。その他、α-SMa、PCNAについてはヘマトキシリン・エオジン染色(HE)およびエラスチカ・ワンギーソン染色(EVG)、CD8、CD68についてはヘマトキシリン・エオジン染色(HE)も行なった。50 μm以下の肺小動脈を各群20本ずつ任意に抽出し、強拡大(400倍; HPF)でその血管周囲のCD8、CD68の各陽性細胞数を数えた。
　結果、アログリプチンは、実験例１（３）と同様、肺細動脈の中膜肥厚を減少させた（図６）。また、アログリプチンは、phosphorylated NF-kappaB p65のレベル（従って、NFκBの核内移行）を抑制し（図７）、さらに、肺小動脈の血管周囲へのCTL（CD8陽性）およびマクロファージ（CD68陽性）の浸潤を抑制した（図８および９）。 Experimental example 2
In the present experimental example (and experimental example 3), the following experiment was performed using lung tissue collected from the rat sacrificed in experimental example 1 (2). As a negative control group (NC group, n = 10), as in Experimental Example 1, the same amount of physiological saline was subcutaneously injected instead of MCT, and distilled water was orally administered daily for 2 weeks. It was.
(1) Immunohistochemical staining Paraffin continuous sections (1 section 3 μm) prepared from lung tissues collected from each group of rats were pretreated in the same manner as in Experimental Example 1 (3), and immunohistochemical staining was performed. In this experimental example, in addition to the α-SMa antibody and PCNA antibody described above, phosphorylated NF-kappaB p65 polyclonal antibody (Cell Signaling, Danvers, US), CD8a antibody (BD Biosciences, San Jose, US), CD68 antibody (Santa Cruz, Dallas, US) was used. As the secondary antibody, simple stain rat MAX PO (Nichireihist Fine, Tokyo, Japan) was used. In addition, α-SMa and PCNA were also subjected to hematoxylin and eosin staining (HE) and elastica-Wangeson staining (EVG), and CD8 and CD68 were also subjected to hematoxylin and eosin staining (HE). 20 small pulmonary arteries of 50 μm or less were arbitrarily extracted from each group, and the number of positive cells of CD8 and CD68 around the blood vessel was counted by strong magnification (400 times; HPF).
As a result, alogliptin decreased the medial thickness of the pulmonary arteriole as in Experimental Example 1 (3) (FIG. 6). In addition, alogliptin suppresses phosphorylated NF-kappaB p65 levels (thus, NFκB translocation into the nucleus) (Fig. 7), and CTL (CD8 positive) and macrophages (CD68 positive) around the blood vessels of small pulmonary arteries Was suppressed (FIGS. 8 and 9).
（２）定量リアルタイム RT-PCR
　血管内皮における塩基性線維芽細胞増殖因子(FGF2)の過剰発現が肺高血圧症の進展に寄与することが報告されている（Izikki M, Guignabert C, Fadel E, Humbert M, Tu L, Zadigue P, et al. Endothelial-derived FGF2 contributes to the progression of pulmonary hypertension in humans and rodents. J Clin Invest 2009;119:512-523）。そこで、FGF2のmRNA発現量を、定量リアルタイム RT-PCRにより測定した。
　配列特異的なPCRプライマーとして、Izikki Mら（2009, 上述）の配列（下記）を使用した以外は、実験例１（４）と同様の方法で実施した。
FGF2増幅用プライマー
Forward; ACGGCGTCCGGGAGAA（配列番号３）
Reverse; ACACTCCCTTGATGGACACAACT（配列番号４）
　結果を図１０（右パネル）に示す。実験例１（４）のTGF-βの結果に、統計学的有意差を追記したデータを併記した（左パネル）。MCT+アログリプチン群では、TGF-βと同様、FGF2のmRNA発現も減少した。 (2) Quantitative real-time RT-PCR
Overexpression of basic fibroblast growth factor (FGF2) in vascular endothelium has been reported to contribute to the development of pulmonary hypertension (Izikki M, Guignabert C, Fadel E, Humbert M, Tu L, Zadigue P, et al. Endothelial-derived FGF2 contributes to the progression of pulmonary hypertension in humans and rodents. J Clin Invest 2009; 119: 512-523). Therefore, the mRNA expression level of FGF2 was measured by quantitative real-time RT-PCR.
The experiment was performed in the same manner as in Experimental Example 1 (4) except that the sequence (described below) of Izikki M et al. (2009, above) was used as the sequence-specific PCR primer.
Primer for FGF2 amplification
Forward; ACGGCGTCCGGGAGAA (SEQ ID NO: 3)
Reverse; ACACTCCCTTGATGGACACAACT (SEQ ID NO: 4)
The results are shown in FIG. 10 (right panel). Data in which statistically significant differences were added to the TGF-β results of Experimental Example 1 (4) were also written (left panel). In the MCT + alogliptin group, the mRNA expression of FGF2 decreased as well as TGF-β.
（３）細胞増殖アッセイ
　TGF-β1タンパクの代わりに、FGF2タンパク質 (Miltenyl Biotec, Auburn, US) (最終濃度 20 ng/ml) を用いて、実験例１（５）と同じ方法により、肺動脈平滑筋細胞 (PASMC)の細胞増殖アッセイを行った。
　結果を図１１（右パネル）に示す。実験例１（５）のTGF-βの結果に、統計学的有意差を追記したデータを併記した（左パネル）。アログリプチンは、FGF2刺激によるPASMCの増殖も用量依存的に阻害した。 (3) Cell Proliferation Assay Using the FGF2 protein (Miltenyl Biotec, Auburn, US) (final concentration 20 ng / ml) instead of TGF-β1 protein, the same method as in Experimental Example 1 (5) was used to obtain pulmonary artery smooth muscle. Cell proliferation assay of cells (PASMC) was performed.
The results are shown in FIG. 11 (right panel). Data in which statistically significant differences were added to the TGF-β results of Experimental Example 1 (5) were also written (left panel). Alogliptin also inhibited the growth of PASMC induced by FGF2 in a dose-dependent manner.
（４）ウェスタンブロッティング
　TGF-β1タンパクの代わりに、FGF2タンパク質 (最終濃度 20 ng/ml) でPASMCを刺激して、実験例１（６）と同じ方法により、核タンパク画分中のphosphorylated NF-kappaB p65レベルを調べた。
　結果、アログリプチンは、FGF2により誘発されるphosphorylated-nuclear factor-kappaB p65 proteinレベルを抑制した（図１２）。 (4) Western blotting Instead of TGF-β1 protein, PASMC was stimulated with FGF2 protein (final concentration 20 ng / ml), and phosphorylated NF- in the nuclear protein fraction was obtained in the same manner as in Experimental Example 1 (6). The kappaB p65 level was examined.
As a result, alogliptin suppressed the phosphorylated-nuclear factor-kappaB p65 protein level induced by FGF2 (FIG. 12).
実験例３
　実験例２（１）の結果より、アログリプチンは、肺高血圧症モデルラットの肺小動脈血管周縁へのマクロファージやT細胞の浸潤を抑制することがわかった。そこで、マクロファージなどの抗原提示細胞（APC）との相互作用やサイトカインによるT細胞の活性化に対するアログリプチンの効果を調べた。APCとT細胞との相互作用は、APCに発現するCD86と、これと相互作用するCTLA-4のmRNA発現を、また、サイトカインによるT細胞の活性化は、心不全の病変部で発現上昇し、T細胞のホーミングを誘導することが報告されているLIGHT/TNFSF14のmRNA発現を、それぞれ定量リアルタイムRT-PCRにより調べた。
　定量リアルタイムRT-PCRは、実験例１（３）と同様にして実施した。各遺伝子の配列特異的なPCRプライマーは、既報されている論文（CTLA-4: Circulation 2009;119:566-576; CD86: Am J Physiol Renal Physiol;304:F676-685; LIGHT: Eur J Heart Fail 2008;10:352-359）の配列を使用した。各mRNA増幅用プライマーの塩基配列を表１に示した。なお、ハウスキーピング遺伝子であるβアクチンをコントロールとして使用した。 Experimental example 3
From the results of Experimental Example 2 (1), it was found that alogliptin inhibits infiltration of macrophages and T cells into the pulmonary arterial vascular periphery of pulmonary hypertension model rats. Therefore, the effect of alogliptin on the interaction with antigen-presenting cells (APC) such as macrophages and the activation of T cells by cytokines was investigated. The interaction between APC and T cells is the expression of CD86 expressed in APC and the mRNA expression of CTLA-4 that interacts with it, and the activation of T cells by cytokines is increased in lesions of heart failure. LIGHT / TNFSF14 mRNA expression, which has been reported to induce T cell homing, was examined by quantitative real-time RT-PCR.
Quantitative real-time RT-PCR was performed in the same manner as in Experimental Example 1 (3). Sequence-specific PCR primers for each gene are described in published papers (CTLA-4: Circulation 2009; 119: 566-576; CD86: Am J Physiol Renal Physiol; 304: F676-685; LIGHT: Eur J Heart Fail 2008; 10: 352-359) was used. Table 1 shows the base sequences of each mRNA amplification primer. Note that β-actin, which is a housekeeping gene, was used as a control.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　結果を図１３に示す。アログリプチンの投与により、CTLA-4、CD86、LIGHTのいずれのmRNA発現も抑制された。 The results are shown in FIG. Administration of alogliptin suppressed the expression of all mRNAs of CTLA-4, CD86, and LIGHT.
実験例４
　実験例１（１）の方法に従って、新たにMCT+アログリプチン群5匹を作製して、MCT投与30日後までのラットの生存を調べ、実験例１（２）の結果と合わせて（即ち、MCT+アログリプチン群25匹、MCT群20匹）、それぞれの群の生存率をKaplan-Meier曲線で示した（図１４）。MCT投与後30日での生存率は、MCT群が5%(1/20)であるのに対して、MCT+アログリプチン群は44%(11/25)であった。 Experimental Example 4
According to the method of Experimental Example 1 (1), 5 new MCT + Alogliptin groups were prepared, and the survival of the rats up to 30 days after MCT administration was examined, and combined with the results of Experimental Example 1 (2) (that is, MCT + Alogliptin) The survival rate of each group was shown by the Kaplan-Meier curve (FIG. 14). The survival rate at 30 days after MCT administration was 5% (1/20) in the MCT group, compared with 44% (11/25) in the MCT + alogliptin group.
　本発明の剤は、ジペプチジルペプチダーゼ－IV阻害薬を含有し、肺高血圧症などの予防または治療剤として有用である。
The agent of the present invention contains a dipeptidyl peptidase-IV inhibitor and is useful as a preventive or therapeutic agent for pulmonary hypertension and the like.
　本出願は、２０１２年１０月２５日付で出願されたＰＣＴ／ＪＰ２０１２／０７７６４０を基礎としており、ここで言及することにより、その内容は全て本明細書に包含される。  This application is based on PCT / JP2012 / 077640, filed on Oct. 25, 2012, the contents of which are hereby incorporated by reference.

Claims (6)

  1. 　アログリプチンまたはその塩を含有してなる、肺高血圧症の予防または治療剤。 An agent for preventing or treating pulmonary hypertension, comprising alogliptin or a salt thereof.
  2. 　アログリプチンまたはその塩が、アログリプチン安息香酸塩である、請求項１記載の剤。 The agent according to claim 1, wherein the alogliptin or a salt thereof is alogliptin benzoate.
  3. 　肺高血圧症が、肺動脈性肺高血圧症である請求項１記載の剤。 The agent according to claim 1, wherein the pulmonary hypertension is pulmonary arterial pulmonary hypertension.
  4. 　哺乳動物に対して、有効量のアログリプチンまたはその塩を投与することを特徴とする、該哺乳動物における肺高血圧症の予防または治療方法。 A method for preventing or treating pulmonary hypertension in a mammal, comprising administering an effective amount of alogliptin or a salt thereof to the mammal.
  5. 　肺高血圧症の予防または治療剤として使用するための、アログリプチンまたはその塩。 Alogliptin or a salt thereof for use as a preventive or therapeutic agent for pulmonary hypertension.
  6. 　肺高血圧症の予防または治療剤を製造するための、アログリプチンまたはその塩の使用。
     
          Use of alogliptin or a salt thereof for producing a preventive or therapeutic agent for pulmonary hypertension.

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