WO2006075642A1 - Hypoxia response enhancing agent - Google Patents

Abstract

A hypoxia response enhancing agent comprising arginine as an active ingredient; and a therapeutic agent for diseases accompanied by ischemia or hypoxia condition, comprising the hypoxia response enhancing agent. This hypoxia response enhancing agent can enhance the activity of HIF-1 being a hypoxia inducing factor, thereby realizing an enhancement of hypoxia response.

Description

 Hypoxic response enhancer

 Technical field

 [0001] The present invention relates to a hypoxic response enhancer and a pharmaceutical composition containing the same, and in particular, the drug can be suitably used in the form of a pharmaceutical product and food, or in the form contained in food. I can do it.

 [0002] (Background of the Invention)

 HIF-1 (hypoxia inducible factor-1) is a protein consisting of 120 KDa subunit HIF-1 a and 91-94 KDa subunit HIF-1 β (Non-patent Document 1). It binds to the DNA element HRE (hypoxia-response element) and induces gene expression under hypoxia.

 Of these, HIF-1a responds to hypoxia and loses its activity by ubiquitination in the presence of oxygen and further degradation by the proteosome. That is, biosynthesis of proteins using HIF-1 as a transcriptional regulator is actively performed under hypoxia. In a situation where the oxygen concentration is increased, HIF-1a is degraded and HIF-1 is regarded as a transcriptional promoter. Protein biosynthesis is reduced. Proteins that use HIF-1 as a transcription promoting factor include erythrobotin, VEGF (vascular endothelial cell growth factor), tyrosine hydroxylase that controls dopamine biosynthesis, phosphofructokinase related to glycolysis, and phosphoglycease. Phosphate kinase I, lactate dehydrogenase A, aldolase, enolase I, glucose transporter (Glut-1), hemoxygenase 1, transferrin, inducible nitrogen monoxide synthase, etc. are known (Non-Patent Documents 2 and 3).

 Drugs that enhance HIF-1 activity enhance the biosynthesis of proteins that use HIF-1 as a transcriptional regulator.For example, Ivan et al. Found a compound characterized by the stability of HIF-1α (non- Patent document 4) reports that the compound enhances the biosynthesis of erythropoietin, suggesting that it is effective in the treatment of anemia (Non-patent document 5).

Arginine has been reported to be involved in the production mechanism of erythropoietin (Non-patent Documents 6 and 7), but the relationship with HIF-1, which is a hypoxia-inducing factor, has not been known so far. Diseases that occur with ischemia or hypoxia, such as kidney It is not clear whether it is effective for dysfunction, myocardial infarction, heart failure, cerebral infarction, diabetic vascular disorder, obstructive arteriosclerosis colitis, ulcer, spinal cord disorder, optic neuropathy, neuropathy, etc. When thinking about the treatment of such a disease state, the elucidation was needed.

[0003] Non-patent document 1: G. Shi Wang et al., Proc. Natl. Acad. Sci. USA, 92, 5510-5514 (1995) Non-patent document 2: Nagao et al., Protein Nucleic Acid Enzyme, Vol. 41, Νο.16, p2522-2531 (1996) Non-Patent Document 3: Sogawa et al., Protein Nucleic Acid Enzyme, Vol.44, No.15, p2472-2477 (199 9)

 Non-Patent Document 4: M. Ivan et al, Proc. Natl. Acad. Sci. USA, 99, 13459-13464 (2002) Non-Patent Document 5: Q. Wang et al., J Am Soc Nephrol. 2004; Oct 15 : 773A

 Non-Patent Document 6: S. Imagawa et.AL, blood, 96, 1716-1722 (2000)

 Non-Patent Document 7: S. Imagawa et al "Kidney Int., 61, 396-404 (2002)

 Disclosure of the invention

 [0004] An object of the present invention is to provide a hypoxic response enhancer that enhances the action of HIF-1, which is a hypoxia-inducing factor, and enhances the hypoxic response.

 Another object of the present invention is to provide a therapeutic agent for a disease caused by ischemia or hypoxia.

 Another object of the present invention is to provide a food containing the above enhancer and an HIF-1 action enhancer.

 [0005] As a result of intensive studies to solve the above-mentioned problems, the present inventors have caused a decrease in arginine ¾IF-1a destabilization, and the addition of arginine restored the stability of HIF-1a. As a result, the present invention has been completed.

 That is, the present invention provides a hypoxic response enhancer characterized by containing arginine as an active ingredient.

 The present invention also provides a therapeutic agent for diseases caused by ischemia or hypoxia, characterized by containing a hypoxic response enhancer.

 The present invention also provides a food characterized in that it is packaged in a package containing arginine and labeled to have a hypoxic response enhancing action.

The present invention also provides an enhanced HIF-1 action characterized by containing arginine as an active ingredient. Provide an accelerator.

 BEST MODE FOR CARRYING OUT THE INVENTION

 [0006] The drug containing arginine of the present invention has an arginine content of 100% to 0.1% by mass (hereinafter abbreviated as%) as long as the substantial effect of arginine contained in the drug is expressed. Preferably 100% to 5%. The content of algin in the drug is preferably 0.001 to 20 g, more preferably 0.1 to 10 g.

 Arginine contained as an active ingredient in the drug of the present invention may be in the form as it is, but may be various salts. Moreover, you may use the compound converted into arginine by biological reaction. Examples of the salt include organic acids such as formic acid, acetic acid, propionic acid, citrate, malonic acid and malic acid, inorganic acids such as hydrochloric acid, sulfuric acid and nitric acid, various amines and metal salts. The L-form, D-form and DL-form may be used, but the L-form and DL-form are preferred. Examples of compounds that are converted to arginine by in vivo reactions include, for example, citrulline, ornithine, arginosuccinic acid, peptides containing arginine, etc. Also included are compounds that can be converted to arginine by the above, compounds that produce arginine by proteases, esterases, transferases and the like.

 [0007] The enhancement of hypoxic response, which is a feature of the drug of the present invention, is the enhancement of the reaction carried out in vivo in an ischemia or hypoxic state (for example, oxygen concentration of 20 vol% or less, preferably 10% or less). Specifically, VEGF (vascular endothelial cell growth factor), tyrosine hydroxylase that regulates donomin biosynthesis, phosphofructokinase involved in glycolysis, phosphodarice mouth phosphate kinase I, lactate dehydrogenase A, Examples include aldolase, enolase I, or glucose transporter (Glut-1), hemoxygenase 1, transferrin, inducible nitric oxide synthase, and other proteins that are induced by hypoxia. .

The disease that occurs with ischemia or hypoxia that is a treatment target using the drug of the present invention refers to a disease in which some ischemia or hypoxia occurs at the disease site, such as renal failure, myocardial infarction, Heart failure, cerebral infarction, diabetic vascular disorder, obstructive arteriosclerosis colitis, ulcer, spinal cord disorder, optic neuropathy, neuropathy, etc. Also good.

 The drug characterized by enhancing the action of HIF-1 of the present invention means that the expression of the gene encoding the protein involved in HIF-1, more specifically, the pathological condition of HIF-1, in vivo is substantially Refers to a drug that is enhanced.

[0008] Furthermore, since arginine has been found to increase the production of erythropoietin through the enhancement of HIF-1 action, arginine has the same mechanism as a therapeutic agent for the above-mentioned diseases. Can be used in combination with drugs that do not enhance the action. Or, even if the same enhancement of HI F-1 action is possible, the action may be expressed additively, and as a therapeutic drug for the above diseases including arginine, the same mechanism of HIF-1 Can be used in combination with drugs that enhance the action.

 Examples of methods used with such other drugs include renal failure, myocardial infarction, heart failure, cerebral infarction, diabetic vasculopathy, obstructive arteriosclerosis colitis, ulcer, spinal cord injury, etc. For example, a method of using a single agent or a plurality of agents for diseases associated with ischemia such as neuropathy or neuropathy, and the agent is more preferably an agent that does not enhance the action of HIF-1. However, the drug may be a drug that enhances the action of HIF-1.

 When the drug of the present invention is used as a pharmaceutical, the form of the preparation is any form such as injection, liquid, syrup tablet, granule, fine granule, powder, capsule, cream, ointment, suppository, etc. But all possible carriers such as lactose, glucose, starch, calcium carbonate, gelatin, cellulose, hydroxymethylcellulose, etc., all possible solvents such as water, ethanol, olive oil, pharmaceuticals such as surfactants, stabilizers, etc. It may contain any possible components, seasonings such as sweeteners, fragrances, colorants and the like.

 The food of the present invention contains any carrier, solvent, surfactant, stabilizer, sweetener, seasoning, flavor, colorant, etc. that can be used as a food in any form of solution, powder, and solid. May be. Moreover, you may mix with other foodstuffs and drinks.

 Hereinafter, the present invention will be described in more detail with reference to examples. The following examples illustrate the present invention and are not intended to limit the present invention.

 Example

Example 1 Enhancement of erythropoietin (EPO) secretion by arginine using HeD3B cells Using human fetal liver cell line Hep3B, changes in erythropoietin production due to changes in arginine (Arg) concentration in the medium were measured. The medium used in the experiment was based on the D-MEM medium composition, a medium containing no amino acids (Zero medium), a medium containing all 20 amino acids in the amounts shown in Table 1 (Full medium), and Full Arginine-deficient medium (Δ Arg medium) obtained by removing only arginine from the medium was used.

 Table 1 Amino acid composition in FuU medium

[0011] Hep3B cells were seeded in a 96-well culture dish at 5 X 10 ⁴ cells / well, cultured for 24 hours, washed once with Zero medium, and arginine was added at a final concentration of 0-400 uM Δ Arg Incubate for 24 hours in normal oxygen (normoxia, 21% 0) and hypoxia (hypoxia, 3% 0).

 twenty two

 I got it. After completion of the culture, the amount of erythropoietin contained in the medium was quantified using an EPO ELISA kit (Roc, 1693417). The experiment was performed with N = 2.

 The amount of erythropoietin secreted is not secreted at all under normal oxygen (oxygen concentration 21 vol%) in the absence of arginine, but it is observed that the amount of erythropoietin secreted increases in a concentration-dependent manner with arginine supplementation. (Figure 1). In addition, the amount of erythropoietin secreted under hypoxia was higher than normal oxygen even when arginine was deficient, but increased further with 50 μM arginine supplement, and showed the maximum value with supplemented 50 μM. (Fig.

[0012] Example 2 針 ffl that targets erythropoietin promoter activity of arginine using HeD3B cells (』HTF synthesis: WT, ¾ HT HTF synthesis A HTFTF ffl) In Example 1, As shown above, arginine is a component necessary for erythropoietin (EPO) production under normal oxygen. Oxygen sensitivity involved in the regulation of EPO gene expression The involvement of HIF-1, a transcription factor, was suggested. HIF-1 is known to have a binding site in the enzyme region located downstream of the EPO gene. To investigate the involvement of HIF-1, a reporter assembly with an EPO promoter region and an enhancer region is used. We constructed a cell line and examined the effects of arginine and the involvement of HIF-1 in EPO promoter activity. The construction of the wild type (WT) EPO reporter vector was performed as follows. From the human genomic DNA (Roche, 1691112), the promoter region of the EPO gene that introduced the restriction enzyme Sail and Hindlll sites (-118 to +26 of the transcription start point), and the enhancer that introduced the restriction enzymes BamHI and Sail site. The regions (120 to 245 bp downstream of the poly A addition signal) were cloned by PCR using the primers shown in Sequence Listings 1 and 2, 3 and 4, respectively.

[0013] The obtained PCR fragment obtained by amplifying the two promoter regions and the enhancer region was introduced into the Bglll-Hindlll site of the luciferase reporter vector pGL3-Enhancer (Promega, E1771) by three-party ligation, and the EPO gene We constructed a wild type (WT) EPO reporter vector with a promoter region and an enhancer region upstream.

 In addition, a mutant (A HIF) EPO reporter vector to which HIF-1 cannot bind was constructed as follows. Based on the wild-type (WT) EPO reporter vector, the primers shown in Sequence Listing 5 and 6 were used to change the HIF-1 binding region (TACGTG CT) in the enhancer region to the QuikChange Site-Directed Mutagenesis kit (: STRATAGENE 200517- In 5), V and 2 mutations (TACTTGCG) were introduced to construct a mutant (ΔHIF) EPO reporter vector that could not bind HIF-1.

 Furthermore, the wild type (WT / Enh) SV40 reporter vector and the mutant type (ΔHIF / Enh) SV40 reporter vector for use as controls were constructed as follows. The wild-type EPO enzyme sequence and mutation cloned in the Sail-BamHI site of the reporter vector pGL3-Cont Promega (E1741), a luciferase gene introduced downstream of the SV40 promoter that is constitutively expressed in cells. A type (ΔHIF) enhancer region was introduced to construct a wild type (WT / Enh) SV40 reporter vector and a mutant type (ΔHIF / Enh) SV40 reporter vector.

[0014] Hep3B cells were seeded in a 96-well culture dish at 5 X 10 ⁴ cells / well, cultured for 6 hours, and then replaced with a transfection solution containing each reporter vector (plasmid solution lmg / ml) (lOOul / well) and introduce gene. Use Fugene6 (Roche) for transfection, add 1.8ul of Fugene6 to 50ul of OPTI-MEM to 1ml of medium, mix and let stand at room temperature for 5 minutes. Thereafter, 1.2 ug (lmg / ml) of the plasmid solution was added and mixed, and a solution obtained by adding 1 ml of the medium to a solution which was allowed to stand at room temperature for 20 minutes was used as a transfection solution.

 After 18 hours of gene transfer, the cells were washed once with 1 X Zero medium, and A Arg medium supplemented with arginine at a final concentration of 0 uM to 300 uM under normal oxygen (normoxia, 21% 0) and hypoxia (h

 2

The cells were cultured with ypoxia, 3% 0) for 24 hours. After culturing, Dual-GloLuciferase Assay ki

 2

Luciferase activity was measured with t (Promega, E2920). The experiment was performed at N = 3.

 As a result, EPO promoter activity increased in a concentration-dependent manner by adding arginine under normal oxygen as well as EPO protein expression level in reporter assembly, and arginine at 50uM showed the maximum value under hypoxia. (Figure 2). However, in the evaluation using V and mutant reporter vectors that cannot bind HIF-1, the increase in EPO promoter activity dependent on arginine concentration was not observed (Fig. 3). Furthermore, when the EPO enhancer region containing the HIF-1 binding region is introduced into the constitutively expressed SV40 promoter, the promoter activity increases depending on the arginine concentration, and the mutant (ΔHIF) enhancer region is introduced. It was confirmed that promoter activity did not increase even in the presence of arginine, and that arginine acts on the enhancer region including the HIF-1 binding region and increased gene expression (Fig. 4).

Example 3 Effect of arginine on HIF-1α using Hep3B cells

 HIF-1, a hypoxia-sensitive factor, consists of heterodimers of HIF-1a and HIF-1β (Arnt), and HIF-1a is degraded in the ubiquitin Z proteasome system under normal oxygen, and It is known that under low oxygen, its decomposition is suppressed and stable HIF-1a moves into the nucleus and promotes transcription. Therefore, the increase in erythropoietin mediated by HIF-1 by arginine-added potassium may be involved in the stability of HIF-1α. We examined the addition of arginine and the amount of HIF-1α protein.

Hep3B cells were seeded at 9 × 10 ⁵ cells / well in a 6-well culture dish, cultured for 24 hours, and then washed once with Zero medium! In FuU medium containing 20 amino acids and Δ Arg medium The cells were cultured under normal oxygen (normoxia, 21% 02) and hypoxia (hypoxia, 3% 02) for 24 hours. After the incubation, wash once with cold PBS and let stand on ice for 5 minutes, then RIPA Buffer (150 mM NaCl, 1% NP-40, 0.5% deoxycholate ゝ 50 mM Tris.HCl pH 7.4, 0.1% SDS ゝ 50 mM β-glycerophosphate ゝ 25mM NaF ゝ 20mM EGTA, ImM DTT ゝ ImM Na VO Protease In

 twenty three,

 The crude cell extract was collected with hibitor cocktail (Nacalai Testa, 25955-11)). The collected crude cell extract was centrifuged at 15000 rpm for 10 minutes, and the supernatant protein was quantified. 20 ug of total protein in each sample was fractionated on 6-13% SDS-PAGE, transferred to a PVDF membrane, HIF-1α protein was detected by Western method using HIF-1α antibody (Becton Dickinson, 610958).

As a result, HIF-1 a protein was detected in Full medium under normal oxygen, but HIF-1 α protein disappeared in Δ Arg medium, which is an arginine-deficient medium, and HIF − 1 α protein was detected. On the other hand, under low oxygen, stable HIF-1α protein was detected equally in both Full medium and ΔArg medium. Therefore, it was confirmed that arginine is essential for the stabilization of HIF-1α protein under normal oxygen, and that HIF-1α protein becomes unstable and disappears due to arginine deficiency.

 That is, in the present invention, an effect of inducing increased erythropoietin secretion depending on the concentration of arginine alone under normal oxygen was observed. In addition, this action is shown to be via the hypoxia-sensitive factor HIF-1! /, The decrease in arginine causes destabilization of HIF-1α protein, and the addition of arginine It was shown that the stability of HIF-1 α protein was restored.

 Brief Description of Drawings

[0017] FIG. 1 shows the effect of arginine on wrinkle expression.

 FIG. 2 shows the effect of arginine on the promoter activity of sputum.

 Fig. 3 shows the effect of arginine at the HIF-1 binding site (EPO promoter activity) (3-

1: 21% 02 (normoxia); 3-2: 3% 02 (hypoxia)).

 [Fig.4] Shows the effect of arginine at the HIF-1 binding site (SV40 promoter activity) (4

-1: 21% 02 down (normoxia); 4— 2: 3% 02 down (hypoxia)).

 FIG. 5 shows the effect of arginine on HIF_1 a protein.

Claims

The scope of the claims  [1] A hypoxic response enhancer comprising arginine as an active ingredient.  [2] The hypoxic response enhancer according to claim 1, which enhances erythropoietin secretion.  [3] The hypoxic response enhancer according to claim 1 or 2, which enhances the action of HIF-1.  [4] The hypoxic response enhancer according to claim 1 or 2, which enhances the action of HIF-1a.  [5] The hypoxic response enhancer according to any one of claims 2 to 4, which is enhanced under normal oxygen.  [6] A therapeutic agent for a disease caused by ischemia or hypoxia, characterized by comprising the hypoxia response enhancer according to [1] to [4]. [7] Diseases that accompany ischemia or hypoxia include renal failure, myocardial infarction, heart failure, cerebral infarction, diabetic vasculopathy, obstructive arteriosclerosis colitis, ulcer, spinal cord injury, etc. The therapeutic agent according to claim 6, which is a neuropathy or neuropathy. [8] A food comprising an arginine and packaged in a package indicating that it has a hypoxic response enhancing action. [9] A HIF-1 action enhancer characterized by containing arginine as an active ingredient. [10] The agent for enhancing HIF-1 action according to claim 9, which enhances the action of HIF-1a.