WO2018139477A1 - NOVEL THERAPEUTIC AGENT FOR NEPHROGENIC ANEMIA TARGETING ERYTHROPOIETIN RECEPTOR VIA NO AND NO-pathway STIMULATION - Google Patents

NOVEL THERAPEUTIC AGENT FOR NEPHROGENIC ANEMIA TARGETING ERYTHROPOIETIN RECEPTOR VIA NO AND NO-pathway STIMULATION Download PDF

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WO2018139477A1
WO2018139477A1 PCT/JP2018/002088 JP2018002088W WO2018139477A1 WO 2018139477 A1 WO2018139477 A1 WO 2018139477A1 JP 2018002088 W JP2018002088 W JP 2018002088W WO 2018139477 A1 WO2018139477 A1 WO 2018139477A1
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nitrate
isosorbide
therapeutic agent
anemia
administration
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French (fr)
Japanese (ja)
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陽介 中山
三有紀 横路
圭 深水
昌一 山岸
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学校法人 久留米大学
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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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics

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  • the present invention relates to a novel use in the clinical field of nitrate drugs, especially isosorbide nitrate. Specifically, the present invention relates to a therapeutic agent for renal anemia, which contains a nitrate drug such as isosorbide nitrate as an active ingredient.
  • Renal anemia is known to be the onset and progression factor of chronic kidney injury (CKD) and cardiovascular disease (CVD). These common risk factors for CKD and CVD interact and have attracted attention as cardiorenal linkage (CRS). Among these factors, it is speculated that vascular endothelial injury factor is an important factor in CRS. (Non-Patent Document 1). Asymmetric dimethylarginine (ADMA), which is produced by asymmetric methylation of arginine residues, is known as a vascular endothelial dysfunction factor.
  • ADMA Asymmetric dimethylarginine
  • Non-Patent Document 2 nitric oxide (vascular endothelium-derived vasorelaxant) Inhibits NO) synthase (NOS) activity, reduces NO production, causes vascular endothelial injury, and develops various diseases such as arteriosclerosis, hypertension, diabetes, and other cardiovascular and renal diseases It is known to be involved in development (Non-Patent Document 2). In recent years, the present inventors have clarified that the entire ADMA metabolic system exists in erythrocytes themselves based on previous studies that erythrocytes are rich in methylated proteins and free ADMA (Non-patent Document 3).
  • Non-Patent Document 4 Non-Patent Document 5
  • the accumulation of erythrocyte ADMA inhibits the anemia response mechanism in the body at the gene level using a renal failure model mouse, and in human chronic kidney injury (CKD) cases Reported that erythrocyte ADMA increased (Patent Document 1, FIG. 7).
  • CKD human chronic kidney injury
  • ESA erythropoietin
  • Patent Literature 2 As therapeutic agents for renal anemia other than ESA, those containing arginine as an active ingredient (Patent Literature 2, Patent Literature 3), and those using erythropoietin production promoter as a therapeutic agent for renal anemia (Patent Literature 4, Patent Literature) 5, Patent Document 6, Patent Document 7, Patent Document 8, Patent Document 9, Patent Document 10, Patent Document 11), containing a fraction extract extracted from deer rice cake as an active ingredient (Patent Document 12), bone marrow erythrocyte A progenitor cell differentiation promoter containing at least one selected from the group consisting of alanine, serine, glutamine, tyrosine and asparagine as an active ingredient (Patent Document 13), and an imidazolone derivative as an active ingredient (Patent) Reference 14) has been developed.
  • Patent Document 12 As therapeutic agents for renal anemia other than ESA, those containing arginine as an active ingredient (Patent Literature 2, Patent Literature 3), and those using erythropoiet
  • Nitrate drugs act as NO donors.
  • Nitric acid drugs such as nitroglycerin and isosorbide nitrate, can be structurally expressed as R-NO 2, and finally release NO to exert its effect.
  • the mechanism until nitric acid releases NO is as follows.
  • R-NO 2 that enters the blood is either (1) a reaction involving an enzyme (including aldehyde dehydrogenase) or (2) a reaction with a reducing substance in a living body (a thiol group). Involved in the substance possessed), it becomes R-SNO (nitrosothiol) form and moves from vascular endothelial cells to smooth muscle cells, releasing NO and exerting its action.
  • Nitrate medicine spreads the coronary arteries around the heart to increase blood flow and replenish oxygen to the heart, reducing systemic vascular resistance and reducing the burden on the heart. It is known to be effective for angina pectoris where sufficient oxygen cannot reach the muscles.
  • Nitric oxide (NO) plays an important role in maintaining blood vessel homeostasis, and it is widely known that this failure is deeply involved in the development of arteriosclerosis.
  • NO is produced from NOS using L-arginine as a substrate.
  • causes of NO production deficiency and reduced biological activity include deficiency of L-arginine, a substrate for NO, decreased expression of NOS, deficiency of coenzyme tetrahydrobioptein (BH4) essential for retention of NOS dimer (this NOS uncoupling occurs due to deficiency of coenzyme, reactive oxygen species (ROS) are produced instead of NO from NOS, NO inactivation by ROS, peroxynitrite which is a powerful radical (ONOO-) generation, competitive inhibition by methylated arginine such as ADMA, and the like.
  • NO is produced by vascular endothelial cells, not vascular smooth muscle cells.
  • the actions of NO in vascular smooth muscle are (1) activation of guanylate cyclase and (2) direct release of Ca-dependent K channels.
  • the pathway of (1) consists of (a) activation of guanylate cyclase (binding to the heme moiety) to generate cGMP from GTP, (b) cGMP activates myosin light chain dephosphorylating enzyme, and (c) myosin light Dephosphorylation from the chain, (d) weakening of myosin-actin cross-linking, (e) smooth muscle relaxation, and (f) vasodilation mechanism.
  • the pathway (2) consists of (a) activation of Ca-dependent K channel, (b) K + outflow, (c) membrane hyperpolarization, (d) potential dependence of smooth muscle cells.
  • Sexual Ca 2+ channel activity is reduced, (e) intracellular Ca 2+ concentration in smooth muscle is reduced, muscle is relaxed, and (f) is due to the mechanism of vasodilation.
  • JP2016-114606 Table 2005/089743 Chart 2006/115274 Table 2004/052859 JP2011-037841 JP2011-153105 JP2012-082181 JP2012-144571 Chart 2013/054755 JP2015-003933 JP2016-040321 JP 07-025774 JP2008-105954 JP2011-231022
  • ESA is the only effective therapeutic agent for renal anemia, and medication to ESA-resistant patients, soaring medical costs, and physical burden on patients have been problems. Under such circumstances, a novel therapeutic agent for renal anemia has been desired.
  • the present inventors have already shown that the accumulation of erythrocyte ADMA inhibits the anemia response mechanism in the body at the gene level, and the erythrocyte ADMA actually increases in human chronic kidney injury (CKD) cases using a renal failure model mouse. I found. This time, the present inventors focused on vasodilator factor NO produced by L-arginine as a substrate by endothelial NO synthase (eNOS), which acts oppositely to ADMA in vascular endothelial function, and is related to renal anemia. Verified.
  • eNOS endothelial NO synthase
  • ADMA competes with NO produced by nitric oxide synthase (NOS), and as a result of NOS inhibition by ADMA, NO concentration, which is a regulator of vascular endothelial function, decreases in CKD patients.
  • NOS nitric oxide synthase
  • isosorbide nitrate (trade name: nitrol), which is a NO-releasing agent, was administered to a renal failure model mouse that had been nephrectomized on 5/6.
  • EPO receptor expression was improved, Hb was improved without an ESA administration, and anemia was eliminated.
  • the present invention includes the following.
  • a pharmaceutical composition comprising a nitrate drug and a pharmacologically acceptable carrier.
  • a therapeutic agent for renal anemia containing a therapeutically effective amount of nitrate.
  • the therapeutic drug according to (4), wherein the nitrate drug is selected from the group consisting of nitroglycerin, isosorbide nitrate and isosorbide mononitrate.
  • the therapeutic agent for renal anemia containing the therapeutically effective amount of the nitrate drug of the present invention is a novel therapeutic agent for renal anemia in ESA-resistant patients for which there has been no therapeutic method so far. It can be offered as a treatment option for patients other than ESA resistant patients. Patients can choose treatments that take into account their side effects depending on their physical symptoms, eliminating the patient's economic and physical burden.
  • FIG. 1 is a graph showing that anemia is markedly improved by administration of a NO donor in a CKD model mouse.
  • the numbers in FIG. 1 indicate the mean ⁇ SD. Mann-Whitney U test, * P ⁇ 0.05 vs sham, +0.05 vs 5 / 6Nx
  • FIG. 2 is a diagram showing that hemoglobin concentration was remarkably improved by administration of NO donor in CKD model mice.
  • FIG. 3 shows that gene expression of Epo receptor in hematopoietic tissue (spleen) was improved by administration of NO donor in CKD model mice.
  • FIG. 4 is a diagram showing that the expression of iron utilization-related genes in hematopoietic tissues was not changed by NO donor administration in CKD model mice.
  • FIG. 5 is a graph showing that the hepcidin expression in the liver was not significant but decreased by about 10% by administration of NO donor in CKD model mice.
  • FIG. 6 is a graph showing that anemia improvement by NO donor administration in CKD model mice is not due to Epo enhancement in the kidney.
  • FIG. 7 is a view showing erythropoietin receptor inhibition through erythropoietin receptor expression inhibition by erythropoietin signal and ADMA accumulation.
  • nitrate drugs which have been used as therapeutic drugs for heart diseases, are effective for the treatment of renal anemia.
  • NO nitrate drugs
  • Nitric acid drugs such as nitroglycerin and isosorbide nitrate, can be structurally expressed as R-NO 2, and finally release NO to exert its effect.
  • Nitric acid drugs include nitroglycerin (trade name: nitropen, nitroderm TTS, myocol spray), isosorbide nitrate (trade name: nitrol, Flandre), isosorbide mononitrate (trade name: itolol), and so on. And isosorbide mononitrate are preferred.
  • Nitroglycerin is almost lost due to the first-pass effect of the liver, and cannot be administered orally. Nitroglycerin itself has a half-life of 5 minutes, and a metabolite with a single NO 2 group has a half-life of 40 minutes. Nitroglycerin is basically used under the tongue.
  • Isosorbide nitrate is also called “isosorbide dinitrate” and is represented by the following formula. Isosorbide nitrate has a longer half-life than nitroglycerin and is more effective than nitroglycerin if the same amount. However, since it is strongly affected by the first-pass effect on the liver, it cannot be used with oral administration at present. Isosorbide nitrate has a half-life of 1 hour. When the NO 2 group at position 2 is removed, it becomes isosorbide mononitrate and the half-life is extended to 2 to 4 hours.
  • Isosorbide mononitrate (chemical name: 1,4: 3,6-Dianhydro-D-glucitol 5-nitrate) is represented by the following formula. Isosorbide mononitrate has good absorbability from the intestinal tract and is not easily affected by the first pass through the liver, so its bioavailability is almost 100%. Among the nitrate drugs, isosorbide nitrate has been marketed in a number of preparations such as tablets, sprays, and patches (tapes). Isosorbide mononitrate is a substance produced by metabolism of isosorbide nitrate, and a product obtained by formulating this metabolite as a drug is isosorbide mononitrate (trade name: Aitrol). Isosorbide mononitrate is superior in that it is less susceptible to liver metabolism than isosorbide nitrate.
  • Isosorbide mononitrate exhibits a dose-dependent vasorelaxation effect in the isolated thoracic aorta and abdominal vena cava in rabbits, increasing cGMP content in vascular tissue.
  • Such a vasorelaxant action is highly selective for venous blood vessels, and an increase in cGMP content is more pronounced in veins than in arteries.
  • Isosorbide mononitrate reduces cardiac preload by reducing venous return due to venous vasodilatation in anesthetized dogs, and afterload by reducing total peripheral vascular resistance Let Furthermore, the coronary blood flow is increased in a dose-dependent manner without directly affecting the myocardial contractile force.
  • isosorbide mononitrate When isosorbide mononitrate is orally administered to an anaesthetized dog, it exhibits a dose-dependent pulse pressure reducing action and has a high bioavailability. There is a positive correlation between plasma isosorbide mononitrate concentration and the effect of reducing pulse pressure.
  • the therapeutic agent of the present invention can be further mixed with sugars such as mannitol, glucose and lactose, salts such as sodium phosphate and sodium phosphate as additives as necessary.
  • sugars such as mannitol, glucose and lactose, salts such as sodium phosphate and sodium phosphate as additives as necessary.
  • nitrate drug that is an active ingredient
  • sublingual administration is preferred when the nitrate drug is nitroglycerin.
  • a pharmaceutical composition for parenteral administration comprises a solution of the nitrate drug of the present invention dissolved in a generally acceptable carrier, preferably an aqueous carrier.
  • a generally acceptable carrier preferably an aqueous carrier.
  • aqueous carriers can be used, all of which are known in the art, for example, water, buffered water, saline, glycine, and the like. These solutions are sterile and generally free of particulate matter.
  • These pharmaceutical compositions can be sterilized by ordinary well-known sterilization methods.
  • composition of the present invention contains generally used additives such as stabilizers (arginine, polysorbate 80, macrogol 4000, etc.), excipients (mannitol, sorbitol, sucrose), etc., and sterile filtration, dispensing.
  • stabilizers arginine, polysorbate 80, macrogol 4000, etc.
  • excipients mannitol, sorbitol, sucrose
  • sterile filtration, dispensing sterile filtration, dispensing.
  • the preparation can be prepared by treatment with freeze-drying or the like and can be administered as an injection or transmucosally (nasally, orally, sublingually).
  • the therapeutically effective dose of nitrate in the present invention varies depending on the severity of the disease state, age, weight, etc. of the subject and is ultimately determined at the discretion of the doctor, but is usually 160 ⁇ g / kg to 160,000 ⁇ / A single dose of kg / day, preferably 1,600 ⁇ g / kg to 16,000 ⁇ g / kg / day may be administered.
  • Those skilled in the art will be able to use standard pharmacological methods to determine the required treatment regimen depending on the particular disease and the severity of the condition to be treated.
  • CKD model mice C57BL6 mice were used as wild type, and CKD models were prepared by 5/6 nephrectomy. Half-nephrectomy was performed at 7 weeks of age and 2/6 nephrectomy at 8 weeks of age to create a 5/6 nephrectomy model. This model has already been established as a chronic renal failure model and exhibits erythropoietin-resistant renal anemia after 12 weeks. Control diet (CE-2) was mixed with isosorbide nitrate (ISDN), and 0.16% NO diet was orally administered.
  • CE-2 Control diet
  • ISDN isosorbide nitrate
  • Wild type + control diet, CKD mouse + control diet, CKD mouse + NO diet started at 8 weeks of age, administered for 12 weeks, and BUN, creatinine, red blood cell count, and Hb were compared at 20 weeks of age. During the study, mice consumed 3.35g on average per day.
  • FIG. 1 As is apparent from FIG. 1, anemia was markedly improved by administration of isosorbide nitrate, which is a NO donor.
  • mice Improvement of hemoglobin concentration by NO donor administration in CKD model mice
  • IDN isosorbide nitrate
  • CE-2 control diet
  • 0.16% NO diet was orally administered.
  • Wild type + control diet, CKD mouse + control diet, CKD mouse + NO diet started at 8 weeks of age and administered for 12 weeks, and hemoglobin concentrations were compared at 20 weeks of age. During the study, mice consumed 3.35g on average per day.
  • the primers and probes used for analysis of the mouse erythropoietin receptor (Epor), transferrin receptor (Tfrc-1), erythroferon (Fam132b) and hepcidin (Hamp) genes were Mm_Epor_SG, Mm_Tfrc_1_SG, Mm_Fam132b_1_SG and Mm_Hamp_1, respectively. Qiagen, Venlo, Netherlands). Hypoxanthine guanine phosphoribosyltransferase (Hprt) (Mm_Hprt_1_SG) was used as an endogenous control (Qiagen, Venlo, The Netherlands).
  • PCR cycling conditions were as follows: initial denaturation step at 95 ° C for 15 minutes followed by denaturation (94 ° C for 15 seconds), annealing (60 ° C for 30 seconds) and extension (72 ° C for 30 seconds) 45 cycles.
  • the relative amount of target gene mRNA was normalized to Hprt by the delta-delta CT method.
  • the primers and probes used for analysis of the mouse erythropoietin receptor (Epor), transferrin receptor (Tfrc-1), erythroferon (Fam132b) and hepcidin (Hamp) genes were Mm_Epor_SG, Mm_Tfrc_1_SG, Mm_Fam132b_1_SG and Mm_Hamp_1, respectively. Qiagen, Venlo, Netherlands). Hypoxanthine guanine phosphoribosyltransferase (Hprt) (Mm_Hprt_1_SG) was used as an endogenous control (Qiagen, Venlo, The Netherlands).
  • PCR cycling conditions were as follows: initial denaturation step at 95 ° C for 15 minutes followed by denaturation (94 ° C for 15 seconds), annealing (60 ° C for 30 seconds) and extension (72 ° C for 30 seconds) 45 cycles.
  • the relative amount of target gene mRNA was normalized to Hprt by the delta-delta CT method.
  • FIG. 4 shows the results for transferrin receptor-1
  • FIG. 4 (b) shows the results for erythroferon.
  • hepcidin in liver in CKD model mice The same mice as in Example 1 were used, sacrificed at 20 weeks of age, and the spleen and liver were stored frozen at -80 degrees. RT-PCR was performed using the QuantiTect SYBRGreen PCR kit (Qiagen, Venlo, The Netherlands) according to the supplier's recommendations.
  • the primers and probes used for analysis of the mouse erythropoietin receptor (Epor), transferrin receptor (Tfrc-1), erythroferon (Fam132b) and hepcidin (Hamp) genes were Mm_Epor_SG, Mm_Tfrc_1_SG, Mm_Fam132b_1_SG and Mm_Hamp_1, respectively. Qiagen, Venlo, Netherlands). Hypoxanthine guanine phosphoribosyltransferase (Hprt) (Mm_Hprt_1_SG) was used as an endogenous control (Qiagen, Venlo, The Netherlands).
  • PCR cycling conditions were as follows: initial denaturation step at 95 ° C for 15 minutes followed by denaturation (94 ° C for 15 seconds), annealing (60 ° C for 30 seconds) and extension (72 ° C for 30 seconds) 45 cycles.
  • the relative amount of target gene mRNA was normalized to Hprt by the delta-delta CT method.
  • hepcidin expression in the liver was not significant but decreased by about 10% by administration of isosorbide nitrate as a NO donor. This means that NO has no effect on hepcidin expression in the liver involved in iron metabolism, or has no hepcidin inhibitory effect through anti-inflammatory action.
  • the therapeutic agent for renal anemia containing the therapeutically effective amount of the nitrate drug of the present invention is a novel therapeutic agent for renal anemia in ESA-resistant patients for which there has been no therapeutic method so far. It can be offered as a treatment option for patients other than ESA resistant patients. Patients can choose treatments that take into account their side effects depending on their physical symptoms, eliminating the patient's economic and physical burden.

Abstract

Provided is a novel therapeutic agent for nephrogenic anemia which is efficacious for ESA resistant patients too. A medicinal composition which comprises an organic nitrate and a pharmacologically acceptable carrier; and a therapeutic agent for nephrogenic anemia which comprises a therapeutically effective amount of an organic nitrate. As the organic nitrate, isosorbide nitrate or isosorbide mononitrate is preferred.

Description

ＮＯおよびＮＯ－ｐａｔｈｗａｙ刺激によるエリスロポエチン受容体を標的とした腎性貧血に対する新規治療薬Novel therapeutic agent for renal anemia targeting erythropoietin receptor by NO and NO-pathway stimulation
　本発明は、硝酸薬、とりわけ硝酸イソソルビドの臨床分野での新規な用途に関する。具体的には、本発明は、硝酸イソソルビド等の硝酸薬を有効成分として含む、腎性貧血の治療薬に関する。 The present invention relates to a novel use in the clinical field of nitrate drugs, especially isosorbide nitrate. Specifically, the present invention relates to a therapeutic agent for renal anemia, which contains a nitrate drug such as isosorbide nitrate as an active ingredient.
　腎性貧血は慢性腎障害（CKD）や心血管疾患（CVD）の発症・進展因子であることが知られている。これらCKDやCVDの共通する危険因子は相互的に作用し、心腎連関（CRS）として注目されてきており、これらの因子の中でも血管内皮障害因子はCRSにおいて重要な因子であることが推察されている（非特許文献１）。アルギニン残基の非対称的なメチル化によって生産されるADMA（asymmetric dimethylarginine）は血管内皮障害因子として知られており、生体内のADMA濃度が上昇すると、血管内皮由来血管弛緩因子である一酸化窒素（NO）の合成酵素（NOS）活性を阻害し、NO産生量を低下させ、血管内皮障害を惹起し、動脈硬化、高血圧、糖尿病を始めとする、心血管疾患や腎疾患など様々な疾患の発症や進展に関わっていることが知られている（非特許文献２)。近年、本発明者らは赤血球中にメチル化タンパク質および遊離型ADMAが豊富に存在するという先行研究をもとに赤血球自身にADMA代謝系全体が存在していることを明らかとし（非特許文献３、非特許文献４、非特許文献５)、さらに、腎不全モデルマウスを用いて赤血球ADMAの蓄積が体内の貧血対応メカニズムを遺伝子レベルで阻害すること、およびヒト慢性腎障害（CKD）症例において実際に赤血球ADMAが増加することを報告した（特許文献１、図７）。 Renal anemia is known to be the onset and progression factor of chronic kidney injury (CKD) and cardiovascular disease (CVD). These common risk factors for CKD and CVD interact and have attracted attention as cardiorenal linkage (CRS). Among these factors, it is speculated that vascular endothelial injury factor is an important factor in CRS. (Non-Patent Document 1). Asymmetric dimethylarginine (ADMA), which is produced by asymmetric methylation of arginine residues, is known as a vascular endothelial dysfunction factor. When the ADMA concentration in the body rises, nitric oxide (vascular endothelium-derived vasorelaxant) Inhibits NO) synthase (NOS) activity, reduces NO production, causes vascular endothelial injury, and develops various diseases such as arteriosclerosis, hypertension, diabetes, and other cardiovascular and renal diseases It is known to be involved in development (Non-Patent Document 2). In recent years, the present inventors have clarified that the entire ADMA metabolic system exists in erythrocytes themselves based on previous studies that erythrocytes are rich in methylated proteins and free ADMA (Non-patent Document 3). , Non-Patent Document 4, Non-Patent Document 5), and further, the accumulation of erythrocyte ADMA inhibits the anemia response mechanism in the body at the gene level using a renal failure model mouse, and in human chronic kidney injury (CKD) cases Reported that erythrocyte ADMA increased (Patent Document 1, FIG. 7).
　腎性貧血の治療薬としては、遺伝子組み換え型の人工エリスロポエチン（ESA）が使用されており、他に有効な治療薬は存在しない。しかしながら、ESA治療を行っている患者には、ESAへの抵抗性を示す患者が一定数存在する。ESA抵抗性患者は心血管合併症が有意に多いことが知られている。さらには抵抗性ではない保存期腎不全患者でもESA治療で脳卒中とがんのリスクが上昇することが報告されている（非特許文献６）。これより2010年米国FDAより腎性貧血治療をESAのみで行うことが適切ではないと報告されたが、これらの問題に対応する新規薬剤は存在しない（非特許文献７）。 Genetically engineered artificial erythropoietin (ESA) is used as a therapeutic agent for renal anemia, and there is no other effective therapeutic agent. However, there are a certain number of patients with ESA treatment who are resistant to ESA. ESA-resistant patients are known to have significantly more cardiovascular complications. Furthermore, it has been reported that ESA treatment increases the risk of stroke and cancer even in patients with conservative renal failure who are not resistant (Non-patent Document 6). From this, it was reported by the US FDA in 2010 that it is not appropriate to treat renal anemia with ESA alone, but there is no new drug that addresses these problems (Non-patent Document 7).
　ESA以外の腎性貧血の治療薬としては、アルギニンを有効成分とするもの（特許文献２、特許文献３）、エリスロポエチン産生促進剤を腎性貧血の治療薬とするもの（特許文献４、特許文献５、特許文献６、特許文献７、特許文献８、特許文献９、特許文献１０、特許文献１１）、鹿茸より抽出される分画エキスを有効成分として含有するもの（特許文献１２）、骨髄赤血球前駆細胞の分化促進剤としてアラニン、セリン、グルタミン、チロシンおよびアスパラギンからなる群から選ばれる少なくとも１種を有効成分として含有するもの（特許文献１３）、およびイミダゾロン誘導体を有効成分として含有するもの（特許文献１４）が開発されてきている。しかしながら、いずれも腎性貧血とNOとの相関については教示していない。 As therapeutic agents for renal anemia other than ESA, those containing arginine as an active ingredient (Patent Literature 2, Patent Literature 3), and those using erythropoietin production promoter as a therapeutic agent for renal anemia (Patent Literature 4, Patent Literature) 5, Patent Document 6, Patent Document 7, Patent Document 8, Patent Document 9, Patent Document 10, Patent Document 11), containing a fraction extract extracted from deer rice cake as an active ingredient (Patent Document 12), bone marrow erythrocyte A progenitor cell differentiation promoter containing at least one selected from the group consisting of alanine, serine, glutamine, tyrosine and asparagine as an active ingredient (Patent Document 13), and an imidazolone derivative as an active ingredient (Patent) Reference 14) has been developed. However, none teaches the correlation between renal anemia and NO.
　硝酸薬はNO供与体として働く。硝酸薬は、ニトログリセリン、硝酸イソソルビド等、構造的にR-NO2と表現できるものであり、最終的にNOを遊離して効果を発揮する。硝酸薬がNOを遊離するまでの機序は以下のとおりである。血液中に入ったR-NO2は、血液中または血管内皮細胞内において（１）酵素が関与した反応（アルデヒドデヒドロゲナーゼなどが関与）または（２）生体内の還元物質との反応（チオール基をもつ物質などが関与）により、R-SNO（ニトロソチオール）の形となって血管内皮細胞から平滑筋細胞に移行し、NOを放出して、作用を発揮する。 Nitrate drugs act as NO donors. Nitric acid drugs, such as nitroglycerin and isosorbide nitrate, can be structurally expressed as R-NO 2, and finally release NO to exert its effect. The mechanism until nitric acid releases NO is as follows. R-NO 2 that enters the blood is either (1) a reaction involving an enzyme (including aldehyde dehydrogenase) or (2) a reaction with a reducing substance in a living body (a thiol group). Involved in the substance possessed), it becomes R-SNO (nitrosothiol) form and moves from vascular endothelial cells to smooth muscle cells, releasing NO and exerting its action.
　硝酸薬は心臓のまわりの冠動脈を広げ血流量を増やし、心臓に酸素などを補給することで、全身の血管抵抗を減らして心臓の負担を軽くする薬であり、血管が狭くなることで心臓の筋肉に十分な酸素などが届かなくなっている狭心症に有効であることが知られている。 Nitrate medicine spreads the coronary arteries around the heart to increase blood flow and replenish oxygen to the heart, reducing systemic vascular resistance and reducing the burden on the heart. It is known to be effective for angina pectoris where sufficient oxygen cannot reach the muscles.
　一酸化窒素（NO）は血管の恒常性の維持に重要な役割を担い、この破綻が動脈硬化症の進展に深く関与していることが広く知られている。NOはL-arginineを基質としてNOSより産生される。NO産生不全や生物学的活性低下の原因として、NOの基質であるL-arginineの欠乏、NOSの発現低下、NOSの二量体の保持に必須である補酵素tetrahydrobioptein（BH4）の欠乏（この補酵素の欠乏によりNOSアンカップリングが生じ、NOSよりNOではなく活性酸素種（reactive oxygen species：ROS）が産生される）、ROSによるNOの不活性化、強力なラジカルであるパーオキシナイトライト（ONOO-）の生成、ADMAなどのメチル化アルギニンによる競合阻害などが挙げられる。NOは、血管平滑筋細胞ではなく、血管内皮細胞で産生される。 Nitric oxide (NO) plays an important role in maintaining blood vessel homeostasis, and it is widely known that this failure is deeply involved in the development of arteriosclerosis. NO is produced from NOS using L-arginine as a substrate. Causes of NO production deficiency and reduced biological activity include deficiency of L-arginine, a substrate for NO, decreased expression of NOS, deficiency of coenzyme tetrahydrobioptein (BH4) essential for retention of NOS dimer (this NOS uncoupling occurs due to deficiency of coenzyme, reactive oxygen species (ROS) are produced instead of NO from NOS, NO inactivation by ROS, peroxynitrite which is a powerful radical (ONOO-) generation, competitive inhibition by methylated arginine such as ADMA, and the like. NO is produced by vascular endothelial cells, not vascular smooth muscle cells.
　血管平滑筋におけるNOの作用は、（１）グアニル酸シクラーゼの活性化、および（２）Ca依存性Kチャネルの直接的開放である。（１）の経路は、（ａ）グアニル酸シクラーゼの活性化（ヘム部分に結合）によりGTPからcGMPが生成、（ｂ）cGMPがミオシン軽鎖脱リン酸化酵素を活性化、（ｃ）ミオシン軽鎖からの脱リン酸、（ｄ）ミオシン－アクチンの架橋形成が弱まる、（ｅ）平滑筋が弛緩、ついで（ｆ）血管拡張のメカニズムによっている。一方、（２）の経路は、（ａ）Ca依存性Kチャネルの活性化、（ｂ）K+が細胞外に流出、（ｃ）膜の過分極、（ｄ）平滑筋細胞が持つ電位依存性Ca2+チャネル活性が低下、（ｅ）平滑筋の細胞内Ca2+濃度が減少し、筋が弛緩、ついで（ｆ）血管拡張のメカニズムによっている。 The actions of NO in vascular smooth muscle are (1) activation of guanylate cyclase and (2) direct release of Ca-dependent K channels. The pathway of (1) consists of (a) activation of guanylate cyclase (binding to the heme moiety) to generate cGMP from GTP, (b) cGMP activates myosin light chain dephosphorylating enzyme, and (c) myosin light Dephosphorylation from the chain, (d) weakening of myosin-actin cross-linking, (e) smooth muscle relaxation, and (f) vasodilation mechanism. On the other hand, the pathway (2) consists of (a) activation of Ca-dependent K channel, (b) K + outflow, (c) membrane hyperpolarization, (d) potential dependence of smooth muscle cells. Sexual Ca 2+ channel activity is reduced, (e) intracellular Ca 2+ concentration in smooth muscle is reduced, muscle is relaxed, and (f) is due to the mechanism of vasodilation.
　これまで、腎性貧血とNOとの相関は確認されておらず、硝酸薬を腎性貧血の治療薬として用いることは知られていなかった。 Until now, the correlation between renal anemia and NO has not been confirmed, and it has not been known to use nitrate drugs as therapeutic agents for renal anemia.
特開2016-114606JP2016-114606 再表2005/089743Table 2005/089743 再表2006/115274Chart 2006/115274 再表2004/052859Table 2004/052859 特開2011-037841JP2011-037841 特開2011-153105JP2011-153105 特開2012-082181JP2012-082181 特開2012-144571JP2012-144571 再表2013/054755Chart 2013/054755 特開2015-003933JP2015-003933 特開2016-040321JP2016-040321 特開平07-025774JP 07-025774 特開2008-105954JP2008-105954 特開2011-231022JP2011-231022
　現時点での腎性貧血に対する有効な治療薬はESAのみであり、ESA治療抵抗性患者への投薬や医療費の高騰、患者の身体的負担が問題となっていた。こうした背景から腎性貧血の新規治療薬が望まれていた。 At this time, ESA is the only effective therapeutic agent for renal anemia, and medication to ESA-resistant patients, soaring medical costs, and physical burden on patients have been problems. Under such circumstances, a novel therapeutic agent for renal anemia has been desired.
　本発明らは、既に腎不全モデルマウスを用いて、赤血球ADMAの蓄積が体内の貧血対応メカニズムを遺伝子レベルで阻害すること、およびヒト慢性腎障害（CKD）症例において実際に赤血球ADMAが増加することを見出した。今回、本発明者らは血管内皮機能においてADMAと対立的に作用する内皮型NO合成酵素（eNOS）によりL-アルギニンを基質として産生される血管拡張因子NOに着目し、腎性貧血との関連を検証した。すなわち、ADMAは前述のように一酸化窒素合成酵素（NOS）によって生産されるNOと競合することから、CKD患者において、ADMAによるNOS阻害の結果、血管内皮機能の制御因子であるNO濃度の低下により腎臓機能不全、さらには腎性貧血を引き起こすことが考えられた。これらを検証するため、5/6に腎摘出した腎不全モデルマウスにNO放出剤である硝酸イソソルビド（商品名：ニトロール）を投与した。その結果、EPO受容体発現が改善し、ESAを投与しなくてもHbが改善し、貧血が解消された。これらより、従来のESA治療によって病態の改善できなかったESA抵抗性患者において、心疾患の治療薬である硝酸薬およびその下流の可溶性グア二ル酸シクラーゼ活性やホスホジエステラーゼ5阻害薬、すなわち、NOおよびNO-pathway刺激によるエリスロポエチン受容体を標的とした新規治療薬が、ESA治療抵抗性患者を含む腎性貧血の治療薬として有用であることを見出した。 The present inventors have already shown that the accumulation of erythrocyte ADMA inhibits the anemia response mechanism in the body at the gene level, and the erythrocyte ADMA actually increases in human chronic kidney injury (CKD) cases using a renal failure model mouse. I found. This time, the present inventors focused on vasodilator factor NO produced by L-arginine as a substrate by endothelial NO synthase (eNOS), which acts oppositely to ADMA in vascular endothelial function, and is related to renal anemia. Verified. In other words, as mentioned above, ADMA competes with NO produced by nitric oxide synthase (NOS), and as a result of NOS inhibition by ADMA, NO concentration, which is a regulator of vascular endothelial function, decreases in CKD patients. Caused renal dysfunction and renal anemia. To verify these, isosorbide nitrate (trade name: nitrol), which is a NO-releasing agent, was administered to a renal failure model mouse that had been nephrectomized on 5/6. As a result, EPO receptor expression was improved, Hb was improved without an ESA administration, and anemia was eliminated. From these, in ESA resistant patients whose pathological condition could not be improved by conventional ESA treatment, nitrates and downstream guanylate cyclase activity and phosphodiesterase 5 inhibitors, i.e. NO and A novel therapeutic agent targeting erythropoietin receptor by NO-pathway stimulation was found to be useful as a therapeutic agent for renal anemia including ESA-resistant patients.
　すなわち、本発明は、以下を含む。
（１）硝酸薬および薬理学的に許容しうる担体を含む医薬組成物。
（２）硝酸薬が、ニトログリセリン、硝酸イソソルビドおよび一硝酸イソソルビドよりなる群から選ばれる、前記（１）に記載の医薬組成物。
（３）硝酸薬が、硝酸イソソルビドまたは一硝酸イソソルビドである、前記（２）に記載の医薬組成物。
（４）治療学的有効量の硝酸薬を含む、腎性貧血の治療薬。
（５）硝酸薬が、ニトログリセリン、硝酸イソソルビドおよび一硝酸イソソルビドよりなる群から選ばれる、前記（４）に記載の治療薬。
（６）硝酸薬が、硝酸イソソルビドまたは一硝酸イソソルビドである、前記（５）に記載の治療薬。 That is, the present invention includes the following.
(1) A pharmaceutical composition comprising a nitrate drug and a pharmacologically acceptable carrier.
(2) The pharmaceutical composition according to (1), wherein the nitrate drug is selected from the group consisting of nitroglycerin, isosorbide nitrate and isosorbide mononitrate.
(3) The pharmaceutical composition according to (2), wherein the nitrate drug is isosorbide nitrate or isosorbide mononitrate.
(4) A therapeutic agent for renal anemia containing a therapeutically effective amount of nitrate.
(5) The therapeutic drug according to (4), wherein the nitrate drug is selected from the group consisting of nitroglycerin, isosorbide nitrate and isosorbide mononitrate.
(6) The therapeutic agent according to (5) above, wherein the nitrate drug is isosorbide nitrate or isosorbide mononitrate.
　本発明の治療学的有効量の硝酸薬を含む、腎性貧血の治療薬は、これまで治療手段がなかったESA抵抗性患者の腎性貧血についての新規治療薬となる他、同疾患への治療についてESA抵抗性患者以外にも治療の選択肢として提供できる。患者は身体症状によってそれぞれの副作用を考慮した治療選択が可能となり、患者の経済・身体的負担が解消される。 The therapeutic agent for renal anemia containing the therapeutically effective amount of the nitrate drug of the present invention is a novel therapeutic agent for renal anemia in ESA-resistant patients for which there has been no therapeutic method so far. It can be offered as a treatment option for patients other than ESA resistant patients. Patients can choose treatments that take into account their side effects depending on their physical symptoms, eliminating the patient's economic and physical burden.
図１は、CKDモデルマウスにおけるNOドナー投与によって顕著に貧血が改善したことを示す図である。図１中の数字は平均±SDを示す。Mann-Whitney U test, ＊P<0.05 vs sham, ＋0.05 vs 5/6NxFIG. 1 is a graph showing that anemia is markedly improved by administration of a NO donor in a CKD model mouse. The numbers in FIG. 1 indicate the mean ± SD. Mann-Whitney U test, ＊ P <0.05 vs sham, ＋0.05 vs 5 / 6Nx 図２は、CKDモデルマウスにおけるNOドナー投与によってヘモグロビン濃度が顕著に改善したことを示す図である。FIG. 2 is a diagram showing that hemoglobin concentration was remarkably improved by administration of NO donor in CKD model mice. 図３は、CKDモデルマウスにおけるNOドナー投与によって造血組織（脾臓）におけるEpo受容体の遺伝子発現が改善したことを示す図である。FIG. 3 shows that gene expression of Epo receptor in hematopoietic tissue (spleen) was improved by administration of NO donor in CKD model mice. 図４は、CKDモデルマウスにおけるNOドナー投与によって造血組織における鉄利用関連遺伝子の発現は変化しなかったことを示す図である。（ａ）トランスフェリン受容体-1；（ｂ）エリスロフェロンFIG. 4 is a diagram showing that the expression of iron utilization-related genes in hematopoietic tissues was not changed by NO donor administration in CKD model mice. (A) transferrin receptor-1; (b) erythroferon 図５は、CKDモデルマウスにおけるNOドナー投与によって肝臓のヘプシジン発現が有意ではないが1割程度の低下傾向を示したことを示す図である。FIG. 5 is a graph showing that the hepcidin expression in the liver was not significant but decreased by about 10% by administration of NO donor in CKD model mice. 図６は、CKDモデルマウスにおけるNOドナー投与による貧血改善が腎臓におけるEpo亢進によるものではないことを示す図である。FIG. 6 is a graph showing that anemia improvement by NO donor administration in CKD model mice is not due to Epo enhancement in the kidney. 図７は、エリスロポエチンシグナルとＡＤＭＡ蓄積によるエリスロポエチン受容体発現阻害を介した赤血球産生障害を示す図である。FIG. 7 is a view showing erythropoietin receptor inhibition through erythropoietin receptor expression inhibition by erythropoietin signal and ADMA accumulation.
　本発明者らは、これまで心疾患の治療薬として用いられていた硝酸薬が腎性貧血の治療に有効であることを初めて見出した。腎性貧血とNOとの関連を示した報告は今までにない。 The present inventors have found for the first time that nitrate drugs, which have been used as therapeutic drugs for heart diseases, are effective for the treatment of renal anemia. To date, there has been no report showing an association between renal anemia and NO.
　本発明において使用する硝酸薬は、NO供与体として働く。硝酸薬は、ニトログリセリン、硝酸イソソルビド等、構造的にR-NO2と表現できるものであり、最終的にNOを遊離して効果を発揮する。硝酸薬としては、ニトログリセリン（商品名：ニトロペン、ニトロダームTTS、ミオコールスプレー）、硝酸イソソルビド（商品名：ニトロール、フランドル）、一硝酸イソソルビド（商品名：アイトロール）などが挙げられるが、硝酸イソソルビドおよび一硝酸イソソルビドが好ましい。 The nitrate drug used in the present invention serves as a NO donor. Nitric acid drugs, such as nitroglycerin and isosorbide nitrate, can be structurally expressed as R-NO 2, and finally release NO to exert its effect. Nitric acid drugs include nitroglycerin (trade name: nitropen, nitroderm TTS, myocol spray), isosorbide nitrate (trade name: nitrol, Flandre), isosorbide mononitrate (trade name: itolol), and so on. And isosorbide mononitrate are preferred.
　ニトログリセリンは、肝臓の初回通過効果でほぼ消失するため、経口投与はできない。ニトログリセリン自体の半減期は5分であり、ひとつNO2基がとれた代謝物の半減期は40分である。ニトログリセリンは、舌下で使用するのが基本である。 Nitroglycerin is almost lost due to the first-pass effect of the liver, and cannot be administered orally. Nitroglycerin itself has a half-life of 5 minutes, and a metabolite with a single NO 2 group has a half-life of 40 minutes. Nitroglycerin is basically used under the tongue.
　硝酸イソソルビドは、別名、二硝酸イソソルビドとも呼ばれ、下記式で表される。
Figure JPOXMLDOC01-appb-C000001
 硝酸イソソルビドはニトログリセリンよりは半減期が長く、同量であるならば、ニトログリセリンより効果が高い。ただし、肝初回通過効果の影響は強く受けるため、現状、経口投与では使えない。硝酸イソソルビドの半減期は1時間であり、2位のNO2基がとれると、一硝酸イソソルビドになり半減期は2～4時間に延長する。 Isosorbide nitrate is also called “isosorbide dinitrate” and is represented by the following formula.
Figure JPOXMLDOC01-appb-C000001
 Isosorbide nitrate has a longer half-life than nitroglycerin and is more effective than nitroglycerin if the same amount. However, since it is strongly affected by the first-pass effect on the liver, it cannot be used with oral administration at present. Isosorbide nitrate has a half-life of 1 hour. When the NO 2 group at position 2 is removed, it becomes isosorbide mononitrate and the half-life is extended to 2 to 4 hours.
　一硝酸イソソルビド（Isosorbide Mononitrate；化学名：1,4:3,6-Dianhydro-D-glucitol 5-nitrate）は、下記式で表される。
Figure JPOXMLDOC01-appb-C000002
 一硝酸イソソルビドは腸管からの吸収性もよく、肝初回通過効果も受けにくいため、バイオアベイラビリティはほぼ100％である。硝酸薬の中でも、硝酸イソソルビドは錠剤やスプレー、貼付薬（テープ剤）など、多くの製剤が発売されている。一硝酸イソソルビドは、硝酸イソソルビドが代謝されることで生成される物質であり、この代謝物を薬として製剤化したものが一硝酸イソソルビド（商品名：アイトロール）である。一硝酸イソソルビドは、硝酸イソソルビドよりも肝臓での代謝を受けにくい点で優れている。 Isosorbide mononitrate (chemical name: 1,4: 3,6-Dianhydro-D-glucitol 5-nitrate) is represented by the following formula.
Figure JPOXMLDOC01-appb-C000002
 Isosorbide mononitrate has good absorbability from the intestinal tract and is not easily affected by the first pass through the liver, so its bioavailability is almost 100%. Among the nitrate drugs, isosorbide nitrate has been marketed in a number of preparations such as tablets, sprays, and patches (tapes). Isosorbide mononitrate is a substance produced by metabolism of isosorbide nitrate, and a product obtained by formulating this metabolite as a drug is isosorbide mononitrate (trade name: Aitrol). Isosorbide mononitrate is superior in that it is less susceptible to liver metabolism than isosorbide nitrate.
　一硝酸イソソルビドの薬効薬理としては、一般に以下の作用が知られている。
１．血管拡張作用
　一硝酸イソソルビドは、ウサギの摘出胸部大動脈および腹部大静脈において用量依存的な血管弛緩作用を示し、血管組織内のcGMP含量を増加させる。このような血管弛緩作用は静脈血管に対して高い選択性を有し、cGMP含量の増加も動脈より静脈において著明である。
２．血行動態に対する作用
（１）一硝酸イソソルビドは、麻酔イヌにおいて静脈血管の拡張作用に起因する静脈還流量の減少により心臓の前負荷を減少させ、また、全末梢血管抵抗の減少により後負荷を減少させる。更に、心筋収縮力に対して直接的な影響を与えず、冠血流量を用量依存的に増加させる。
（２）無麻酔イヌに一硝酸イソソルビドを経口投与した場合、用量依存的な脈圧減少作用を示し、生物学的利用率も高い。血漿中一硝酸イソソルビド濃度と脈圧減少作用の間には正の相関がみられる。 The following actions are generally known as pharmacological effects of isosorbide mononitrate.
1. Vasodilatory action Isosorbide mononitrate exhibits a dose-dependent vasorelaxation effect in the isolated thoracic aorta and abdominal vena cava in rabbits, increasing cGMP content in vascular tissue. Such a vasorelaxant action is highly selective for venous blood vessels, and an increase in cGMP content is more pronounced in veins than in arteries.
2. Effects on hemodynamics (1) Isosorbide mononitrate reduces cardiac preload by reducing venous return due to venous vasodilatation in anesthetized dogs, and afterload by reducing total peripheral vascular resistance Let Furthermore, the coronary blood flow is increased in a dose-dependent manner without directly affecting the myocardial contractile force.
(2) When isosorbide mononitrate is orally administered to an anaesthetized dog, it exhibits a dose-dependent pulse pressure reducing action and has a high bioavailability. There is a positive correlation between plasma isosorbide mononitrate concentration and the effect of reducing pulse pressure.
　本発明の治療薬には、必要に応じさらに、マンニトール、グルコース、乳糖等の糖類、食塩、リン酸ナトリウム等の塩を添加剤として混合することができる。 The therapeutic agent of the present invention can be further mixed with sugars such as mannitol, glucose and lactose, salts such as sodium phosphate and sodium phosphate as additives as necessary.
　本発明の医薬組成物の投与方法は、有効成分である硝酸薬の種類に応じた投与方法が採用される。例えば、硝酸薬がニトログリセリンである場合は、舌下投与が好ましい。硝酸薬が硝酸イソソルビドである場合は、経口投与、腹腔内注入、気管内注入、気管支内注入および直接的な気管支内滴注、皮下注入、経皮輸送、動脈内注入、静脈内注入、経鼻投与等が例示される。硝酸薬が一硝酸イソソルビドである場合は、経口投与、腹腔内注入、気管内注入、気管支内注入および直接的な気管支内滴注、皮下注入、経皮輸送、動脈内注入、静脈内注入、経鼻投与等が例示される。非経口投与のための医薬組成物は、一般に許容しうる担体、好ましくは水性担体中に溶解した本発明の硝酸薬の溶液を含む。様々な水性担体を用いることができ、これらはすべて当該技術分野で知られたものであって、例えば、水、緩衝水、食塩水、グリシンなどを用いることができる。これら溶液は滅菌してあり、一般に粒状物質を含まない。これら医薬組成物は、通常のよく知られた滅菌法により滅菌することができる。本発明の組成物は、一般に用いられる添加剤、例えば、安定化剤（アルギニン、ポリソルベート80、マクロゴール4000など）、賦型剤（マンニトール、ソルビトール、スクロース）などを添加し、無菌濾過、分注、凍結乾燥等の処理を行い製剤化され、注射剤としてあるいは経粘膜的に投与（経鼻、経口、舌下）される製剤とすることができる。 As the method for administering the pharmaceutical composition of the present invention, a method according to the type of nitrate drug that is an active ingredient is employed. For example, sublingual administration is preferred when the nitrate drug is nitroglycerin. Oral administration, intraperitoneal injection, intratracheal injection, intrabronchial injection and direct intrabronchial instillation, subcutaneous injection, transdermal delivery, intraarterial injection, intravenous injection, nasal when the nitrate drug is isosorbide nitrate Examples are administration. When the nitrate drug is isosorbide mononitrate, oral administration, intraperitoneal injection, intratracheal injection, intrabronchial injection and direct intrabronchial instillation, subcutaneous injection, transdermal transport, intraarterial injection, intravenous injection, trans Examples include nasal administration. A pharmaceutical composition for parenteral administration comprises a solution of the nitrate drug of the present invention dissolved in a generally acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, all of which are known in the art, for example, water, buffered water, saline, glycine, and the like. These solutions are sterile and generally free of particulate matter. These pharmaceutical compositions can be sterilized by ordinary well-known sterilization methods. The composition of the present invention contains generally used additives such as stabilizers (arginine, polysorbate 80, macrogol 4000, etc.), excipients (mannitol, sorbitol, sucrose), etc., and sterile filtration, dispensing. The preparation can be prepared by treatment with freeze-drying or the like and can be administered as an injection or transmucosally (nasally, orally, sublingually).
　本発明において硝酸薬の治療上有効投与量は、被験者の病態の重篤度、年齢、体重等によって変わり、最終的には医師の裁量によって決定されるが、通常、160μg/kg～160,000μ/kg/日、好ましくは1,600μg/kg～16,000μg/kg/日の量を単回投与すればよい。当業者であれば、標準的な薬理学的方法を用い、必要な処置レジメを特定の疾患および処置すべき状態の重篤度に応じて決定することができるであろう。 The therapeutically effective dose of nitrate in the present invention varies depending on the severity of the disease state, age, weight, etc. of the subject and is ultimately determined at the discretion of the doctor, but is usually 160 μg / kg to 160,000 μ / A single dose of kg / day, preferably 1,600 μg / kg to 16,000 μg / kg / day may be administered. Those skilled in the art will be able to use standard pharmacological methods to determine the required treatment regimen depending on the particular disease and the severity of the condition to be treated.
　以下、実施例により本発明をより具体的に説明するが、本発明はこれら実施例により何ら限定されるものではない。 Hereinafter, the present invention will be described more specifically by way of examples. However, the present invention is not limited to these examples.
CKDモデルマウスにおけるNOドナー投与による貧血の改善
　ワイルドタイプとしてC57BL6マウスを用い、CKDモデルは5/6腎摘を行い作成した。7週令で半腎摘、8週令でさらに2/6腎摘を行い、5/6腎摘モデルを作成した。このモデルは慢性腎不全モデルとして既に確立しており、12週後にエリスロポエチン抵抗性の腎性貧血を呈する。コントロール食(CE-2)に硝酸イソソルビド(ISDN)を配合し、0.16%NO食を経口投与した。配合剤を摂取ワイルドタイプ＋コントロール食、CKDマウス＋コントロール食、CKDマウス＋NO食を8週令より開始し12週間投与、20週令でBUN、クレアチニン、赤血球数、Hbを比較した。なお当研究中マウスは1日平均3.35g摂取した。 Improvement of anemia by NO donor administration in CKD model mice C57BL6 mice were used as wild type, and CKD models were prepared by 5/6 nephrectomy. Half-nephrectomy was performed at 7 weeks of age and 2/6 nephrectomy at 8 weeks of age to create a 5/6 nephrectomy model. This model has already been established as a chronic renal failure model and exhibits erythropoietin-resistant renal anemia after 12 weeks. Control diet (CE-2) was mixed with isosorbide nitrate (ISDN), and 0.16% NO diet was orally administered. Wild type + control diet, CKD mouse + control diet, CKD mouse + NO diet started at 8 weeks of age, administered for 12 weeks, and BUN, creatinine, red blood cell count, and Hb were compared at 20 weeks of age. During the study, mice consumed 3.35g on average per day.
　その結果を図１に示す。図１から明らかなように、NOドナーである硝酸イソソルビドの投与により貧血が顕著に改善した。 The result is shown in FIG. As is apparent from FIG. 1, anemia was markedly improved by administration of isosorbide nitrate, which is a NO donor.
CKDモデルマウスにおけるNOドナー投与によるヘモグロビン濃度の改善
　実施例１と同様のマウスを用い、コントロール食(CE-2)に硝酸イソソルビド(ISDN)を配合し、0.16%NO食を経口投与した。配合剤を摂取ワイルドタイプ＋コントロール食、CKDマウス＋コントロール食、CKDマウス＋NO食を8週令より開始し12週間投与、20週令でヘモグロビン濃度を比較した。なお当研究中マウスは1日平均3.35g摂取した。 Improvement of hemoglobin concentration by NO donor administration in CKD model mice The same mice as in Example 1 were used, and isosorbide nitrate (ISDN) was mixed with the control diet (CE-2), and 0.16% NO diet was orally administered. Wild type + control diet, CKD mouse + control diet, CKD mouse + NO diet started at 8 weeks of age and administered for 12 weeks, and hemoglobin concentrations were compared at 20 weeks of age. During the study, mice consumed 3.35g on average per day.
　その結果を図２に示す。図２から明らかなように、NOドナーである硝酸イソソルビドの投与によりヘモグロビン濃度が顕著に改善した。 The result is shown in FIG. As is clear from FIG. 2, the hemoglobin concentration was significantly improved by administration of isosorbide nitrate, which is a NO donor.
CKDモデルマウスにおけるNOドナー投与による造血組織（脾臓）におけるEpo受容体の遺伝子発現の改善
　実施例１と同様のマウスを用い、20週令で屠殺し、脾臓、肝臓を－80度で冷凍保存した。RT-PCRは、QuantiTect SYBRGreen PCRキット（Qiagen、Venlo、オランダ）を使用して、供給者の推奨に従って実施した。マウスエリスロポエチン受容体（Epor）、トランスフェリン受容体（Tfrc-1）、エリスロフェロン（Fam132b）およびヘプシジン（Hamp）遺伝子の分析に用いたプライマーおよびプローブは、それぞれMm_Epor_SG、Mm_Tfrc_1_SG、Mm_Fam132b_1_SGおよびMm_Hamp_1であった（Qiagen、Venlo、オランダ）。ヒポキサンチングアニンホスホリボシルトランスフェラーゼ（Hprt）（Mm_Hprt_1_SG）を内因性対照として用いた（Qiagen、Venlo、オランダ）。PCRサイクリング条件は以下の通りであった：95℃で15分間の初期変性ステップ、続いて変性（94℃で15秒間）、アニーリング（60℃で30秒間）および伸長（72℃で30秒間）の45サイクル。標的遺伝子mRNAの相対量は、デルタ－デルタCT法によってHprtに正規化し検討した。 Improvement of gene expression of Epo receptor in hematopoietic tissue (spleen) by NO donor administration in CKD model mice Using the same mice as in Example 1, they were sacrificed at 20 weeks of age and the spleen and liver were stored frozen at -80 degrees . RT-PCR was performed using the QuantiTect SYBRGreen PCR kit (Qiagen, Venlo, The Netherlands) according to the supplier's recommendations. The primers and probes used for analysis of the mouse erythropoietin receptor (Epor), transferrin receptor (Tfrc-1), erythroferon (Fam132b) and hepcidin (Hamp) genes were Mm_Epor_SG, Mm_Tfrc_1_SG, Mm_Fam132b_1_SG and Mm_Hamp_1, respectively. Qiagen, Venlo, Netherlands). Hypoxanthine guanine phosphoribosyltransferase (Hprt) (Mm_Hprt_1_SG) was used as an endogenous control (Qiagen, Venlo, The Netherlands). PCR cycling conditions were as follows: initial denaturation step at 95 ° C for 15 minutes followed by denaturation (94 ° C for 15 seconds), annealing (60 ° C for 30 seconds) and extension (72 ° C for 30 seconds) 45 cycles. The relative amount of target gene mRNA was normalized to Hprt by the delta-delta CT method.
　その結果を図３に示す。図３から明らかなように、NOドナーである硝酸イソソルビドの投与により造血組織（脾臓）におけるEpo受容体の遺伝子発現が改善した。 The result is shown in FIG. As is clear from FIG. 3, the gene expression of the Epo receptor in the hematopoietic tissue (spleen) was improved by administration of isosorbide nitrate, which is a NO donor.
CKDモデルマウスにおける造血組織における鉄利用関連遺伝子の発現
　実施例１と同様のマウスを用い、20週令で屠殺し、脾臓、肝臓を－80度で冷凍保存した。RT-PCRは、QuantiTect SYBRGreen PCRキット（Qiagen、Venlo、オランダ）を使用して、供給者の推奨に従って実施した。マウスエリスロポエチン受容体（Epor）、トランスフェリン受容体（Tfrc-1）、エリスロフェロン（Fam132b）およびヘプシジン（Hamp）遺伝子の分析に用いたプライマーおよびプローブは、それぞれMm_Epor_SG、Mm_Tfrc_1_SG、Mm_Fam132b_1_SGおよびMm_Hamp_1であった（Qiagen、Venlo、オランダ）。ヒポキサンチングアニンホスホリボシルトランスフェラーゼ（Hprt）（Mm_Hprt_1_SG）を内因性対照として用いた（Qiagen、Venlo、オランダ）。PCRサイクリング条件は以下の通りであった：95℃で15分間の初期変性ステップ、続いて変性（94℃で15秒間）、アニーリング（60℃で30秒間）および伸長（72℃で30秒間）の45サイクル。標的遺伝子mRNAの相対量は、デルタ－デルタCT法によってHprtに正規化し検討した。 Expression of iron utilization-related genes in hematopoietic tissues in CKD model mice The same mice as in Example 1 were used, sacrificed at 20 weeks of age, and the spleen and liver were stored frozen at -80 degrees. RT-PCR was performed using the QuantiTect SYBRGreen PCR kit (Qiagen, Venlo, The Netherlands) according to the supplier's recommendations. The primers and probes used for analysis of the mouse erythropoietin receptor (Epor), transferrin receptor (Tfrc-1), erythroferon (Fam132b) and hepcidin (Hamp) genes were Mm_Epor_SG, Mm_Tfrc_1_SG, Mm_Fam132b_1_SG and Mm_Hamp_1, respectively. Qiagen, Venlo, Netherlands). Hypoxanthine guanine phosphoribosyltransferase (Hprt) (Mm_Hprt_1_SG) was used as an endogenous control (Qiagen, Venlo, The Netherlands). PCR cycling conditions were as follows: initial denaturation step at 95 ° C for 15 minutes followed by denaturation (94 ° C for 15 seconds), annealing (60 ° C for 30 seconds) and extension (72 ° C for 30 seconds) 45 cycles. The relative amount of target gene mRNA was normalized to Hprt by the delta-delta CT method.
　その結果を図４に示す。図４から明らかなように、NOドナーである硝酸イソソルビドの投与により造血組織における鉄利用関連遺伝子の発現は変化しなかった。図４（ａ）はトランスフェリン受容体-1、図（ｂ）はエリスロフェロンの結果を示す。 The result is shown in FIG. As is apparent from FIG. 4, the expression of iron utilization-related genes in the hematopoietic tissue was not changed by administration of isosorbide nitrate, which is a NO donor. FIG. 4 (a) shows the results for transferrin receptor-1, and FIG. 4 (b) shows the results for erythroferon.
　以上より、NOはCKDにおけるEpo受容体低下を有意に改善し、血球新生に繋がるエリスロフェロンの発現を改善した。鉄関連因子はCKDによる炎症の影響を受ける。NOが貧血を改善する機序はEpo受容体発現低下を抑制することが主な作用であることが証明された。もしくは赤血球数、Hbが十分に改善し、生理的なフィードバックにより亢進する必要がないことを意味している。 From the above, NO significantly improved Epo receptor decline in CKD, and improved the expression of erythroferon that leads to hematopoiesis. Iron-related factors are affected by inflammation caused by CKD. It was proved that the mechanism by which NO improves anemia is mainly to suppress the decrease in Epo receptor expression. Or it means that the red blood cell count and Hb are sufficiently improved, and there is no need to increase by physiological feedback.
CKDモデルマウスにおける肝臓のヘプシジン発現
　実施例１と同様のマウスを用い、20週令で屠殺し、脾臓、肝臓を－80度で冷凍保存した。RT-PCRは、QuantiTect SYBRGreen PCRキット（Qiagen、Venlo、オランダ）を使用して、供給者の推奨に従って実施した。マウスエリスロポエチン受容体（Epor）、トランスフェリン受容体（Tfrc-1）、エリスロフェロン（Fam132b）およびヘプシジン（Hamp）遺伝子の分析に用いたプライマーおよびプローブは、それぞれMm_Epor_SG、Mm_Tfrc_1_SG、Mm_Fam132b_1_SGおよびMm_Hamp_1であった（Qiagen、Venlo、オランダ）。ヒポキサンチングアニンホスホリボシルトランスフェラーゼ（Hprt）（Mm_Hprt_1_SG）を内因性対照として用いた（Qiagen、Venlo、オランダ）。PCRサイクリング条件は以下の通りであった：95℃で15分間の初期変性ステップ、続いて変性（94℃で15秒間）、アニーリング（60℃で30秒間）および伸長（72℃で30秒間）の45サイクル。標的遺伝子mRNAの相対量は、デルタ－デルタCT法によってHprtに正規化し検討した。 Expression of hepcidin in liver in CKD model mice The same mice as in Example 1 were used, sacrificed at 20 weeks of age, and the spleen and liver were stored frozen at -80 degrees. RT-PCR was performed using the QuantiTect SYBRGreen PCR kit (Qiagen, Venlo, The Netherlands) according to the supplier's recommendations. The primers and probes used for analysis of the mouse erythropoietin receptor (Epor), transferrin receptor (Tfrc-1), erythroferon (Fam132b) and hepcidin (Hamp) genes were Mm_Epor_SG, Mm_Tfrc_1_SG, Mm_Fam132b_1_SG and Mm_Hamp_1, respectively. Qiagen, Venlo, Netherlands). Hypoxanthine guanine phosphoribosyltransferase (Hprt) (Mm_Hprt_1_SG) was used as an endogenous control (Qiagen, Venlo, The Netherlands). PCR cycling conditions were as follows: initial denaturation step at 95 ° C for 15 minutes followed by denaturation (94 ° C for 15 seconds), annealing (60 ° C for 30 seconds) and extension (72 ° C for 30 seconds) 45 cycles. The relative amount of target gene mRNA was normalized to Hprt by the delta-delta CT method.
　その結果を図５に示す。図５から明らかなように、NOドナーである硝酸イソソルビドの投与により肝臓のヘプシジン発現が有意ではないが1割程度の低下傾向を示した。このことは、NOは鉄代謝に関与する肝臓でのヘプシジン発現に対しては影響を及ぼさない、もしくは抗炎症作用を介したヘプシジン抑制効果はないことを意味する。 The result is shown in FIG. As is clear from FIG. 5, hepcidin expression in the liver was not significant but decreased by about 10% by administration of isosorbide nitrate as a NO donor. This means that NO has no effect on hepcidin expression in the liver involved in iron metabolism, or has no hepcidin inhibitory effect through anti-inflammatory action.
CKDモデルマウスにおけるNOドナー投与による貧血改善
　実施例１と同様のマウスを用い、20週令時にトサツし採血を行った。血漿エリスロポエチン濃度を、市販のEPO ELISA（R&D systems, Minneapolis, MN）を用いて評価した。 Improvement of anemia by administration of NO donor in CKD model mice Using the same mice as in Example 1, blood was sampled at 20 weeks of age. Plasma erythropoietin concentrations were evaluated using a commercially available EPO ELISA (R & D systems, Minneapolis, Minn.).
　その結果を図６に示す。図６から明らかなように、NOドナーである硝酸イソソルビドの投与による貧血改善は腎臓におけるEpo亢進によるものではないことが示された。 The result is shown in FIG. As is clear from FIG. 6, it was shown that the improvement of anemia by administration of isosorbide nitrate, which is a NO donor, was not due to Epo enhancement in the kidney.
　本発明の治療学的有効量の硝酸薬を含む、腎性貧血の治療薬は、これまで治療手段がなかったESA抵抗性患者の腎性貧血についての新規治療薬となる他、同疾患への治療についてESA抵抗性患者以外にも治療の選択肢として提供できる。患者は身体症状によってそれぞれの副作用を考慮した治療選択が可能となり、患者の経済・身体的負担が解消される。 The therapeutic agent for renal anemia containing the therapeutically effective amount of the nitrate drug of the present invention is a novel therapeutic agent for renal anemia in ESA-resistant patients for which there has been no therapeutic method so far. It can be offered as a treatment option for patients other than ESA resistant patients. Patients can choose treatments that take into account their side effects depending on their physical symptoms, eliminating the patient's economic and physical burden.

Claims (6)

  1. 　硝酸薬および薬理学的に許容しうる担体を含む医薬組成物。 A pharmaceutical composition comprising a nitric acid drug and a pharmacologically acceptable carrier.
  2. 　硝酸薬が、ニトログリセリン、硝酸イソソルビドおよび一硝酸イソソルビドよりなる群から選ばれる、請求項１に記載の医薬組成物。 The pharmaceutical composition according to claim 1, wherein the nitric acid drug is selected from the group consisting of nitroglycerin, isosorbide nitrate and isosorbide mononitrate.
  3. 　硝酸薬が、硝酸イソソルビドまたは一硝酸イソソルビドである、請求項２に記載の医薬組成物。 The pharmaceutical composition according to claim 2, wherein the nitric acid drug is isosorbide nitrate or isosorbide mononitrate.
  4. 　治療学的有効量の硝酸薬を含む、腎性貧血の治療薬。 A therapeutic agent for renal anemia, including a therapeutically effective amount of nitrate.
  5. 　硝酸薬が、ニトログリセリン、硝酸イソソルビドおよび一硝酸イソソルビドよりなる群から選ばれる、請求項４に記載の治療薬。 The therapeutic drug according to claim 4, wherein the nitric acid drug is selected from the group consisting of nitroglycerin, isosorbide nitrate and isosorbide mononitrate.
  6. 　硝酸薬が、硝酸イソソルビドまたは一硝酸イソソルビドである、請求項５に記載の治療薬。 The therapeutic agent according to claim 5, wherein the nitric acid drug is isosorbide nitrate or isosorbide mononitrate.
PCT/JP2018/002088 2017-01-30 2018-01-24 NOVEL THERAPEUTIC AGENT FOR NEPHROGENIC ANEMIA TARGETING ERYTHROPOIETIN RECEPTOR VIA NO AND NO-pathway STIMULATION WO2018139477A1 (en)

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