JP4273293B2 - Novel anti-autoimmune disease agent by inhibiting GRF action, and screening method thereof - Google Patents

Description

【図面の簡単な説明】
【図１】図１は、実施例１における実験結果を示す図である。具体的には、ＥＡＥ発症に対するＧＲＦの関与について検討するため、ＧＲＦＲ遺伝子ホモ欠損マウス（―□―）、ＧＲＦＲヘテロ欠損マウス（―△―）、および対照として用いた野生型マウス（―●―）のそれぞれについて、人為的に誘導したＥＡＥ発症の有無を示したグラフである。
図中(Ａ)にはＥＡＥスコアの平均値の推移を示す。野生型マウスでは感作後16日目からＥＡＥの発症（スコア１）が見られ始め、感作後25日目にはスコアが１.２に達した。一方ＧＲＦＲホモ欠損マウスにおいては全観察期間（27日間）を通じて全く発症が認められなかった。またＧＲＦＲヘテロ欠損マウスではＥＡＥは発症したものの、その平均スコア値は野生型マウスの平均スコア値に比較して著しく低かった。図中（Ｂ）にはＥＡＥ発症率の推移を示す。ＧＲＦＲホモ欠損マウスは全観察期間を通じて発症率が０であった。またＧＲＦＲヘテロ欠損マウスは野生型マウスと比較してＥＡＥの発症率は著しく低かった。
【図２】 図２は、PLSJLF1マウスにMBPの感作と百日咳毒素の投与によりEAE発症を誘導し、GRFアンタゴニストであるMZ-4-71投与のEAE発症に対する影響を検討した結果を示したグラフである。
図中（Ａ）にはEAEスコアの平均の推移を示した。図中、黒い太線で示した期間はMZ-4-71または対照として生理食塩水を投与した期間を示す。生理食塩水投与群では感作後12日目から発症が見られ、感作後16日目にはスコアの平均値は4.2に達した。一方、MZ-4-71投与群では感作後11日目から発症が認められたが、感作後13日目に平均スコアが0.3に達した後に一旦寛解が導入され、感作後17日目にはすべてのマウスが寛解となり、平均スコアは0になった。感作後20日目から再発が見られたが、その後の観察期間中を通じて平均スコアの最大値は2.2（感作後23日目）にとどまった。これらの結果から、GRFアンタゴニストの投与によってEAE発症が抑制されることが示された。図中（Ｂ）には発症率の推移を示した。図中、黒い太線で示した期間はMZ-4-71または対照として生理食塩水を投与した期間を示す。生理食塩水投与群では、感作後12日目から13日目に急速な発症が見られ、感作後13日目には発症率が80%に達した。一方、MZ-4-71投与群では発症は緩慢に開始し、感作後26日目の時点でも発症率は60%にとどまった。これらの結果から、発症率の点からもGRFアンタゴニストの投与によってEAE発症が抑制されることが示された。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an anti-autoimmune disease agent, that is, an agent for preventing or treating autoimmune disease. Furthermore, the present invention relates to a method for searching for and obtaining a novel active ingredient of an anti-autoimmune disease agent.
[0002]
[Prior art]
Rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, etc. appear to be pathologically different, but all are diseases in which inflammation is observed at the lesion site. These diseases are autoimmune diseases that are caused by the pathogenesis of antibodies that should recognize foreign substances other than self, and react with antigens of their tissues and cells, that is, autoantigens. It is considered.
[0003]
It is known that antibodies against self-antigens and autoreactive T cells are usually found in healthy individuals, and their presence does not lead to autoimmune diseases. This is because an immunological tolerance (tolerance) is usually established in the immune response to the self-antigen. In contrast, in patients with autoimmune diseases, it is considered that the tolerance against self-antigen is broken for some reason. The factors involved are those involved in antigen, those involved in antigen presentation, and those involved in tolerance control. However, there are many unclear points, and the mechanism has not yet been elucidated.
[0004]
By the way, growth hormone-releasing factor (GRF) (also referred to as growth hormone-releasing hormone (GHRH)) (hereinafter also simply referred to as “GRF”) is secreted from the hypothalamus. It is a peptide hormone consisting of 44 amino acids. The GRF binds to its receptor (growth hormone-releasing factor receptor; GRFR, hereinafter also referred to as “GRF receptor”), and activates intracellular G AMP (hereinafter simply referred to as “cAMP”) through activation of the G protein. (Also called) increase the concentration and as a result promote the release of growth hormone (GH) from the pituitary gland. In addition to the above-mentioned pathway, GRF activates Protein Kinase A (PKA) by increasing intracellular cAMP concentration as an alternative pathway and increasing calcium influx into the cell to increase growth hormone. It also has the effect of promoting release.
[0005]
As described above, GRF is a hormone that is well known to be associated with the release (secretion) of growth hormone. Conventionally, GRF is tested for its ability to secrete growth hormone to produce pituitary dwarfism and primary thyroid gland. It is used as a test for diagnosing hypofunction. However, the relationship between the GRF and the autoimmune disease was not known at all.
[0006]
[Problems to be solved by the invention]
The object of the present invention is to provide an anti-autoimmune disease agent based on a novel mechanism of action. A further object of the present invention is to provide a screening method useful for searching for and obtaining candidate substances that are active ingredients of such anti-autoimmune disease agents.
[0007]
[Means for Solving the Problems]
The present inventor has developed a growth hormone releasing factor (GRF) for the onset of experimental autoimmune encephalomyelitis (hereinafter also simply referred to as “EAE”), which is an animal model of multiple sclerosis, which is one of autoimmune diseases. ), The inventors discovered a new finding that the onset of EAE can be significantly suppressed by inhibiting the action of GRF.
Specifically, homo-deficient mice (C57BL / 6J-Ghrhr) in which the GRF receptor (GRFR) gene was artificially deleted^{lit / lit}) (GRFR homo-deficient mice) and hetero-deficient mice (C57BL / 6J-Ghrhr)^{lit / +}) (GRFR hetero-deficient mice) were experimentally induced to develop EAE, GRFR homo-deficient mice did not develop EAE at all throughout the observation period, and GRFR hetero-deficient mice were also compared to wild-type mice. The result that the onset of EAE was suppressed significantly was obtained. Moreover, when a known GRF antagonist for the GRF receptor was administered to EAE mice (EAE onset induction mice), EAE onset was not induced even if experimentally induced onset of EAE, and the action of GRF via the GRF receptor was observed. It was confirmed that inhibition of EAE can be significantly suppressed by inhibition.
[0008]
Based on the above findings, that is, the fact that GRF is associated with the onset of autoimmune diseases and the inhibition of the action of GRF, the onset of autoimmune diseases can be suppressed. It was confirmed that the substance possessed can be effectively used as a preventive or therapeutic agent for autoimmune diseases. The present invention has been completed based on such findings.
[0009]
That is, the present invention is as follows:
Item 1. An anti-autoimmune disease agent comprising as an active ingredient a substance having a growth hormone releasing factor inhibitory action.
Item 2. The anti-autoimmune disease agent according to claim 1, wherein the growth hormone releasing factor inhibitory action is an action that inhibits an action of a growth hormone releasing factor (GRF) via a growth hormone releasing factor receptor (GRF receptor).
Item 3. Item 3. The anti-autoimmune disease agent according to Item 2, wherein the growth hormone releasing factor inhibitory effect is an effect of inhibiting binding between GRF and a GRF receptor.
Item 4. Item 4. The anti-autoimmune disease agent according to Item 3, wherein the substance having a growth hormone releasing factor inhibitory action is a peptide analog of GRF.
Item 5. Item 2. The anti-autoimmune disease agent according to Item 1, wherein the growth hormone releasing factor inhibitory effect is an effect of inhibiting GRF expression or secretion, or an effect of inhibiting GRF receptor expression.
Item 6. Item 6. The anti-autoimmune disease agent according to any one of Items 1 to 5, wherein the autoimmune disease is multiple sclerosis or rheumatoid arthritis.
Item 7. A screening method for an active ingredient of an anti-autoimmune disease agent, comprising a step of searching for a substance having a growth hormone releasing factor inhibitory action from among test substances.
Item 8. Item 8. The screening method according to Item 7, wherein a substance having a growth hormone releasing factor inhibitory action is searched for using inhibition of GRF action via a GRF receptor as an index.
Item 9. Item 9. The screening method according to Item 8, wherein a substance having a growth hormone releasing factor inhibitory action is searched for using binding inhibition between GRF and a GRF receptor as an index.
Item 10. Item 10. A screening method for an active ingredient of an anti-autoimmune disease agent according to Item 9, comprising the following steps (i), (ii) and (iii):
(i) contacting a GRF receptor or a homolog thereof (hereinafter referred to as “GRF receptor or the like”) with a GRF or a homolog thereof (hereinafter referred to as “GRF or the like”) in the presence of a test substance;
(ii) GRF for GRF receptor or the like obtained by measuring the binding amount of GRF or the like to GRF receptor or the like, and bringing the binding amount into contact with GRF or the like in the absence of the test substance Comparing with the amount of binding (control binding amount), etc., and
(iii) A step of selecting a test substance that decreases the binding amount compared to the control binding amount based on the result of (ii) above.
Item 11. In addition to the steps (i), (ii) and (iii) according to Item 10:
(iv) A step of selecting a test substance that does not produce the same action as GRF from the test substances selected in (iii) above.
Item 11. A screening method for an active ingredient of an anti-autoimmune disease agent according to Item 10.
Item 12. Item 8. The screening method according to Item 7, wherein a substance having a growth hormone-releasing factor inhibitory action is searched using GRF receptor expression inhibition as an index.
Item 13. Item 13. A screening method for an active ingredient of an anti-autoimmune disease agent according to Item 12, comprising the following steps (i), (ii) and (iii):
(I) contacting the test substance with a cell capable of expressing a GRF receptor;
(Ii) GRF receptor of a cell (control cell) capable of expressing a GRF receptor corresponding to the above without measuring the expression level of the cell contacted with the test substance and contacting the test substance with the expression level A step of comparing with the expression level (control expression level) of
(iii) A step of selecting a test substance that decreases the expression level of the GRF receptor as compared with the control expression level based on the comparison result of (ii) above.
Item 14. Item 8. The screening method according to Item 7, wherein a substance having a growth hormone releasing factor inhibitory action is searched for using inhibition of GRF expression or secretion as an index.
Item 15. Item 15. A screening method for an active ingredient of an anti-autoimmune disease agent according to Item 14, comprising the following steps (i), (ii) and (iii):
(I) contacting the test substance with a cell capable of expressing GRF;
(Ii) GRF expression level or secretion level of the cells contacted with the test substance is measured, and the expression level or secretion level is determined based on the GRF expression level (control expression) of the corresponding control cell not touched with the test substance. Volume) or secretion volume (control secretion volume),
(iii) A step of selecting a test substance that reduces the expression level or secretion amount of GRF as compared with the control expression level or control secretion level based on the comparison result of (ii) above.
Item 16. Item 16. The screening method according to any one of Items 7 to 15, wherein the anti-autoimmune disease agent is a prophylactic or therapeutic agent for multiple sclerosis or rheumatoid arthritis.
Item 17. Item 17. An anti-autoimmune disease agent comprising as an active ingredient a substance having a growth hormone releasing factor inhibitory action obtained by the screening method according to any one of Items 7 to 16.
Item 18. An anti-autoimmune disease agent comprising an effective amount of a substance having an inhibitory action on growth hormone releasing factor and a pharmaceutically acceptable carrier.
Item 19. Prevention or treatment of autoimmune disease, comprising administering an effective amount of an anti-autoimmune disease agent comprising a substance having a growth hormone releasing factor inhibitory action and a pharmaceutically acceptable carrier to a subject suffering from autoimmune disease Method.
Item 20. Item 20. The method for preventing or treating an autoimmune disease according to Item 19, wherein the autoimmune disease is multiple sclerosis or rheumatoid arthritis.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
In this specification, the abbreviations of amino acids, (poly) peptides, (poly) nucleotides, etc. are defined by IUPAC-IUB [IUPAC-IUB Communication on Biological Nomenclature, Eur. J. Biochem., 138: 9 (1984). ], “Guidelines for the preparation of specifications including base sequences or amino acid sequences” (edited by the Japan Patent Office), and conventional symbols in the field.
[0011]
In this specification, “gene” or “DNA” is used to include not only double-stranded DNA but also each single-stranded DNA such as a sense strand and an antisense strand constituting the DNA. The length is not particularly limited. Therefore, in this specification, unless otherwise specified, double-stranded DNA including human genomic DNA and single-stranded DNA including cDNA (positive strand) and single-stranded DNA having a sequence complementary to the positive strand. (Complementary strands) and any of these fragments are included. Further, the gene or DNA does not ask for distinction of the functional region, and can include, for example, an expression control region, a coding region, an exon, or an intron.
[0012]
In the present specification, “polynucleotide” is used to include both RNA and DNA. The DNA includes any of cDNA, genomic DNA, and synthetic DNA. The RNA includes all of total RNA, mRNA, rRNA, and synthetic RNA.
[0013]
As used herein, the term “antibody” includes a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single chain antibody, or an antibody having an antigen binding property such as a Fab fragment or a fragment generated by a Fab expression library. Some are included.
[0014]
Hereinafter, the present invention will be described in detail.
[0015]
I. Anti-autoimmune disease agent
As described above, the present inventor has found that the onset of autoimmune diseases can be suppressed or delayed by inhibiting the action of growth hormone releasing factor (GRF). From this, it is considered that a substance that inhibits the action of growth hormone releasing factor (GRF) can be effectively used for prevention or treatment of autoimmune diseases. The anti-autoimmune disease agent of the present invention has been developed based on such findings, and is characterized by containing a substance having a GRF inhibitory action as an active ingredient.
[0016]
In general, an “autoimmune disease” is a disease in which antibodies or lymphocytes that react with components that constitute the tissue of the body cause tissue damage due to continuous production in the body. Autoimmune diseases and organ non-specific autoimmune diseases can be classified into two types.
[0017]
(1) Organ-specific autoimmune diseases: Hashimoto thyroiditis, primary myxedema, thyroid poisoning, pernicious anemia, Good-pasture syndrome, acute progressive glomerulonephritis, severe myasthenia gravis, pemphigus vulgaris, bullous Pemphigoid, insulin resistant diabetes, juvenile diabetes, Addison's disease, atrophic gastritis, male infertility, premature menopause, phakogenic uveitis, exchange vasculitis, multiple sclerosis, ulcerative colitis , Primary biliary cirrhosis, chronic active hepatitis, autoimmune hemolytic anemia, paroxysmal hemoglobinuria, idiopathic thrombocytopenic purpura, Sjogren's syndrome.
[0018]
(2) Non-organ-specific autoimmune diseases: rheumatoid arthritis, systemic lupus erythematosus, discoid lupus erythematosus, polymyositis, scleroderma, mixed connective tissue disease.
[0019]
The present invention arbitrarily targets these “autoimmune diseases”. Preferably, autoimmune diseases that develop by the same or similar mechanism as “experimental autoimmune encephalomyelitis” can be targeted. Examples of such autoimmune diseases include multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus. More preferred are multiple sclerosis and rheumatoid arthritis, and particularly preferred is multiple sclerosis.
[0020]
The “anti-autoimmune disease agent” of the present invention is an action to inhibit or suppress the onset of the various autoimmune diseases, an action to delay the onset, an action to reduce or improve the symptoms after the onset. Or having at least one action of healing a disease. In that sense, the “anti-autoimmune disease agent” of the present invention can be rephrased as an autoimmune disease preventive agent, autoimmune disease symptom relieving agent, autoimmune disease ameliorating agent, or autoimmune disease therapeutic agent. Among them, the “anti-autoimmune disease agent” of the present invention is excellent in the action of inhibiting or suppressing the onset of an autoimmune disease or the action of delaying the onset, and can be used particularly effectively as an autoimmune disease preventive agent. .
[0021]
Here, the “growth hormone-releasing factor (GRF)” targeted by the present invention refers to the pituitary gland by binding to the GRF receptor as described in the section of “PRIOR ART”. It is one of the hypothalamic hormones that has the effect of promoting growth hormone (GH) release. In that sense, it is also called growth hormone-releasing hormone (GHRH).
[0022]
Its base sequence and amino acid sequence are known, for example, GenBank accession number: NM 021081 (base sequence: SEQ ID NO: 1), NP 066567 (amino acid sequence: SEQ ID NO: 2), NM 031577, NM 010285, AF 242855, U 10156, M 73486, M 31658, and M It is described in 31654 etc.
[0023]
In the present invention, the “GRF inhibitory action” broadly means an action that inhibits the functional expression of the GRF, regardless of the mechanism of action. Here, “the action of inhibiting the functional expression of GRF” includes the action of inhibiting GRF protein synthesis (including GRF gene expression) and secretion, and the action of the expressed GRF via the GRF receptor (signal The effect of inhibiting transmission) is included. In the present specification, such inhibition of GRF functional expression is sometimes simply referred to as inhibition of GRF action or inhibition of GRF.
[0024]
Specifically, these GRF inhibitory actions include 1) an action that inhibits the action of GRF (signal transduction) via the GRF receptor, 2) an action that inhibits the expression of the GRF receptor, and 3) expression of GRF or It can be roughly divided into the action of inhibiting secretion.
[0025]
In addition, the “substance having GRF inhibitory action” of the present invention may be any substance having at least one of the above-described inhibitory actions, and does not ask at all for the form or properties thereof. In addition, the “substance having the GRF inhibitory action” of the present invention may be any substance that has an inhibitory action on at least human GRF, and whether or not it has an inhibitory action on GRF derived from mammals other than humans or other biological species. There is no particular restriction.
[0026]
Hereinafter, the substance having a GRF inhibitory action will be specifically described.
[0027]
1) Substance that inhibits the action of GRF via the GRF receptor
As shown in Examples described later, the onset of autoimmune disease is suppressed by deficiency of GRF receptor, and the onset of autoimmune disease is caused by competing GRF and GRF antagonist for GRF receptor. It was found to be suppressed. This finding is that a substance that inhibits the action of GRF through the GRF receptor, in other words, a substance that inhibits signal transmission through the GRF receptor of GRF acts on the autoimmune disease in a suppressive manner. This proves that it can be an active ingredient of the agent.
[0028]
Here, the substance that inhibits the action of GRF via the GRF receptor includes the following (1) substance that inhibits the binding of GRF and GRF receptor according to the action site, and (2) downstream of GRF receptor. Can be classified into substances that act on the signal transduction system of GRF and inhibit intracellular signal transduction of GRF.
[0029]
(1) Substance that inhibits binding between GRF and GRF receptor
By inhibiting the binding between GRF and the GRF receptor, the action (signal transduction) of GRF on the GRF receptor can be inhibited. Specifically, the substance having the binding inhibitory action is (1) a substance that inhibits the binding of GRF to the GRF receptor by binding to the GRF receptor, or (2) the substance that binds to the GRF. And substances that inhibit the binding of GRF to the GRF receptor. The substance (1) has an action as an antagonist of GRF on the so-called GRF receptor, and even if it binds itself to the GRF receptor, it itself has the same qualitative and quantitative action as GRF. It is necessary not to exert (signal transmission). Here, “not exhibiting the same effect as GRF” is not only the case where it does not exhibit the same qualitative action as GRF (loss of GRF function) but also the case where it exhibits the same qualitative action as GRF. However, it also includes a case where an effect that is quantitatively reduced than that of GRF is exhibited.
[0030]
(1) Substance that binds to GRF receptor and inhibits binding of GRF to GRF receptor As such substance, specifically, a binding domain that binds to GRF receptor (Science, Vol. 218, 585 (1982 ), Biochem. Biophys. Res. Commun., Vol. 123, 854 (1984)) and GRF variants lacking GRF function, anti-GRF receptor antibodies, and the like. In addition, the substance that binds to the GRF receptor may be any substance that has at least binding ability to the human GRF receptor, and whether or not it has binding ability to the GRF receptor derived from mammals other than humans or other biological species. Is not particularly limited.
[0031]
Here, as the above-mentioned GRF variant, a GRF modified protein [or modified oligo (poly) peptide] having only a binding domain to the GRF receptor, or an amino acid sequence of a region other than the binding domain to the GRF receptor is artificially defined. A GRF modified protein [or modified oligo (poly) peptide] that loses GRF function by being deleted, substituting or inserting (adding) with other amino acids Can do. Further, as described above, the GRF variant is one that binds to at least the human GRF receptor and inhibits the binding of human GRF to the receptor, and does not exhibit the same action as GRF itself. If it exists, its origin is not particularly limited.
[0032]
For example, the amino acid sequence of the binding domain of human GRF for the GRF receptor and the base sequence encoding it are known as shown in the above-mentioned document. Therefore, a GRF variant comprising the binding domain of human GRF can be easily obtained by those skilled in the art from this information and the following modification method.
[0033]
A method for artificially modifying amino acid regions other than the binding domain of GRF to the GRF receptor is also a method known to those skilled in the art, such as Molecular Cloning 2nd Edt., Cold Spring Harbor Laboratory Press (1989). This can be done with reference to the basic book. Specifically, Cytospecific Mutagenesis [Methods in Enzymology, 154, 350, 367-382 (1987); 100, 468 (1983); Nucleic Acids Res., 12, 9441 (1984); Genetic engineering methods such as Lecture 1 “Gene Research Method II”, edited by the Chemical Society of Japan, p.105 (1986)], chemical synthesis means such as the phosphate triester method and phosphate amidite method [J. Am. Chem Soc., 89, 4801 (1967); 91, 3350 (1969); Science, 150, 178 (1968); Tetrahedron Lett., 22, 1859 (1981); 24, 245 (1983)], and these This method can be implemented by arbitrarily combining these methods.
[0034]
Whether the modified GRF protein [oligo (poly) peptide] (GRF variant) does not exhibit the same action as GRF is determined by administering the GRF variant to an animal and measuring the GH secretion ability. Can be evaluated.
[0035]
Further, as an anti-GRF receptor antibody exemplified as the substance of (1), specifically, an antibody against the extracellular domain (Nature, Vol. 360, p765-768 (1992)) of the GRF receptor should be exemplified. Can do. The anti-GRF receptor antibody only needs to recognize and bind to at least the extracellular domain of the human GRF receptor. The extracellular domain of the GRF receptor derived from mammals other than humans or other biological species. The presence or absence of the ability to recognize or bind to is not particularly limited.
[0036]
The anti-GRF receptor antibody can be prepared according to a conventional method using a protein having the extracellular domain of human GRF receptor as an immunizing antigen, and may be a monoclonal antibody or a polyclonal antibody. Specifically, these antibodies are described in, for example, “Antibodies; A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)” and “Current Protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. It can be easily created with reference to basic books such as Section 11. 12-11.13 ”. Here, a protein having the extracellular domain of the human GRF receptor can be easily obtained by a person skilled in the art from the information on the amino acid sequence of the protein described in the above-mentioned document (Nature) and the base sequence encoding the protein. be able to.
[0037]
Furthermore, the substance of (1) includes other proteins and peptides, or low molecular compounds.
[0038]
Specific examples of the peptide include GRF peptide analogs that have an action of binding to a human GRF receptor and inhibiting the binding of human GRF to the receptor. The peptide analog of GRF is a so-called GRF antagonist that does not itself have the same action as GRF but binds to a human GRF receptor and has an antagonistic action on human GRF. Such a peptide analog of GRF only needs to have a binding ability to at least a human GRF receptor, and whether or not it has a binding ability to a GRF receptor derived from a mammal other than human or other biological species, and The separation of the peptide origin is not particularly limited.
[0039]
Specific examples of such GRF peptide analogs include MZ-4-71 ([Ibu⁰, D-Arg², Phe (4-Cl)⁶, Abu¹⁵, Nle²⁷] hGHRH- (1-28) Agm), MZ-4-169 ([Nac⁰, D-Arg², Phe (4-Cl)⁶, Abu¹⁵, Nle²⁷] hGHRH- (1-29) NH₂), Or MZ-4-181 ([Nac⁰-His¹, D-Arg², Phe (4-Cl)⁶, Abu¹⁵, Nle²⁷] hGHRH- (1-29) NH₂) (Above, Proc. Natl. Acad. Sci. USA, Vol.91, p.12298-12302 (1994)), MZ-5-156 ([PhAc⁰, D-Arg², Phe (4-Cl)⁶, Abu¹⁵, Nle²⁷] hGH-RH (1-28) Agm) (Proc. Natl. Acad. Sci. USA, Vol. 96, p. 226-231 (1999)), or JV-1-36 ([PhAc⁰, D-Arg², Phe (4-Cl)⁶, Arg⁹, Abu¹⁵, Nle²⁷, D-Arg²⁸, Har²⁹] hGH-RH (1-29) NH₂), JV-1-38 ([PhAc⁰, D-Arg², Phe (4-Cl)⁶, Har⁹, Tyr (Me)^Ten, Abu¹⁵, Nle²⁷, D-Arg²⁸, Har²⁹] hGH-RH (1-29) NH₂), JV-1-42 ([PhAc⁰-His¹, D-Arg², Phe (4-Cl)⁶, Arg⁹, Abu¹⁵, Nle²⁷, D-Arg²⁸, Har²⁹] hGH-RH (1-29) NH₂(Above Proc. Natl. Acad. Sci. USA, Vol. 97, p. 1218-1223 (2000)). Moreover, the peptide analogs described in International Publication No. WO95 / 16707 (particularly claims 1 to 20) or WO91 / 16923 (particularly claim 16) can also be mentioned.
[0040]
In addition, low molecular weight compounds having an action of binding to the GRF receptor and inhibiting the binding of GRF to the GRF receptor can be obtained by publicly known low molecular weight compound libraries (for example, low molecular weight compound libraries of ChemBridge Research Laboratories, etc.). On the other hand, it can be obtained by carrying out the screening method of the present invention described later.
[0041]
(2) Substance that binds to GRF and inhibits binding of GRF to GRF receptor
An anti-GRF antibody can be exemplified as the substance. The anti-GRF antibody is specifically an antibody against the GRF receptor binding domain of GRF (Science, Vol. 218, 585 (1982), Biochem. Biophys. Res. Commun., Vol. 123, 854 (1984)). Is mentioned. Such anti-GRF antibody only needs to recognize and bind to at least the receptor binding domain of human GRF, whether or not it has the ability to bind to GRF derived from mammals other than humans and other biological species, and production raw materials. The origin of the antigen (protein, polypeptide) used as is not particularly limited.
[0042]
The anti-GRF antibody can be prepared according to a conventional method using a polypeptide or protein having a receptor binding domain of human GRF as an immunizing antigen, and may be a monoclonal antibody or a polyclonal antibody. Specifically, these antibodies can be easily prepared with reference to the various basic documents mentioned above.
[0043]
Here, a protein having a receptor binding domain of human GRF can be obtained by a person skilled in the art from the information on the amino acid sequence of the receptor binding domain described in the above literature (Science, BBRC) and the base sequence encoding it. Can be easily obtained. Further, the substance (2) includes proteins, peptides, low molecular compounds, and the like.
[0044]
For example, low molecular weight compounds having an action of directly binding to GER and inhibiting the binding of GRF to the GRF receptor can be obtained by publicly known low molecular weight compound libraries (for example, low molecular weight compound libraries manufactured by ChemBridge Research Laboratories). On the other hand, it can be obtained by carrying out the screening method of the present invention described later.
[0045]
(2) Substances that inhibit intracellular signal transduction of GRF
As described in the “Prior Art” section, when GRF binds to its receptor (GRF receptor), it causes activation of G protein, which increases cAMP concentration through the activation, and as a result, Has the effect of promoting the release of growth hormone (GH) from the pituitary gland. In addition to the above-mentioned pathway, GRF also has the effect of activating protein Kinase A by increasing intracellular cAMP concentration and increasing calcium influx into the cell to promote the release of growth hormone. .
[0046]
Therefore, a substance capable of inhibiting the action of GRF by inhibiting the signal transduction (signal transduction downstream of the GRF receptor) in cells after binding of these GRF receptors is also included in the GRF of the present invention. Included in substances having an inhibitory action.
[0047]
As the substance, by inducing (enhancing) or suppressing the expression and action of various factors related to intracellular signal transmission of GRF, as a result, the action of GRF is substantially inhibited and eventually the growth hormone is released. The substance which can inhibit a promotion effect can be illustrated.
[0048]
Specific examples include proteins, peptides, and low molecular compounds that suppress the expression and action (activation) of G protein coupled to the GRF receptor.
[0049]
2) Substance that inhibits the expression of GRF receptor
As shown in Examples described later, it was found that the onset of autoimmune disease is suppressed by deleting the GRF receptor. This finding supports that a substance that inhibits the expression of the GRF receptor acts suppressively on autoimmune diseases and can be an active ingredient of anti-autoimmune disease agents.
[0050]
Any substance that inhibits the expression of the GRF receptor may be used as long as it has an action of inhibiting the production of a protein having a function as a GRF receptor regardless of the mechanism of action. For example, an antisense molecule having an action of blocking GRF receptor (protein) synthesis at the nucleic acid level by inhibiting any process such as replication, transcription or translation of the GRF receptor gene.
[0051]
In addition, the inhibitory substance made into the object here should just have the effect | action which inhibits the expression of a human GRF receptor at least, and the expression inhibitory effect with respect to the GRF receptor derived from mammals other than a human or other biological species is sufficient. The presence or absence is not particularly limited.
[0052]
Specifically, GRF receptor gene (GenBank accession number: NM 000823 [SEQ ID NO: 3], AY 008835, AY 008834, AF 184896, AB 022597, AB 022596, L 07380, L And antisense polynucleotides complementary to each other.
[0053]
The antisense molecule does not have to be a polynucleotide having a sequence that is 100% complementary to the base sequence of the GRF receptor gene, and the base sequence of the human GRF receptor gene under stringent conditions. It may be a hybridizing polynucleotide or a modified product thereof (for example, including an antisense molecule designed by a known method such as a modified poly (oligo) nucleotide or peptide nucleic acid). Specific examples thereof include those having at least 80%, preferably at least 90%, more preferably at least 95% homology with the complementary sequence of the base sequence of the GRF receptor gene.
[0054]
Substances that inhibit the expression of the GRF receptor also include proteins, peptides, low molecular compounds, and the like. For example, a low molecular compound having an action of inhibiting the expression of a human GER receptor may be obtained by screening the present invention described later against a known low molecular compound library (for example, a low molecular compound library manufactured by ChemBridge Research Laboratories). It can be obtained by carrying out the method.
[0055]
3) Substances that inhibit the expression or secretion of GRF
As described in Examples below, the onset of autoimmune disease is suppressed by deficiency of GRF receptor, and the onset of autoimmune disease by competing GRF and GRF antagonist for GRF receptor. Was found to be suppressed. This finding supports that a substance that inhibits the production of GRF, that is, a substance that inhibits the expression or secretion of GRF, can suppress an autoimmune disease and can be an active ingredient of an anti-autoimmune disease agent. is there.
[0056]
Any substance that inhibits the expression of GRF may be used as long as it has an action of inhibiting the production of a protein having a function as GRF, regardless of the mechanism of action. For example, an antisense molecule having an action of blocking GRF (protein) synthesis at the nucleic acid level by inhibiting any process such as replication, transcription or translation of the GRF gene.
[0057]
The target GRF expression inhibitor is not limited as long as it has at least an action of inhibiting the expression of human GRF. The presence or absence of an expression inhibitory action on GRF derived from mammals other than humans or other biological species. There is no particular restriction.
[0058]
Specifically, GRF gene (GenBank accession number: NM 021081 [SEQ ID NO: 1], NM 031577, NM 010285, AF 242855, U 10156, M 73486, M 31658, and M And antisense polynucleotides that are complementary to each other.
[0059]
The antisense molecule need not be a polynucleotide having a sequence that is 100% complementary to the base sequence of the GRF gene, and is a polynucleotide that hybridizes with the base sequence of the human GRF gene under stringent conditions. It may be a nucleotide or a modification thereof. Specific examples thereof include those having at least 80%, preferably at least 90%, more preferably at least 95% homology with the complementary sequence of the base sequence of the GRF gene.
[0060]
Substances that inhibit the expression or secretion of GRF include proteins, peptides, and low molecular weight compounds. For example, a low molecular weight compound having an action of inhibiting the expression or secretion of GER is performed on a low molecular weight compound library (for example, a low molecular weight compound library manufactured by ChemBridge Research Laboratories) or the like according to the present invention. Can be obtained.
[0061]
The substance having a GRF inhibitory action described above is useful as an active ingredient of an anti-autoimmune disease agent. Further, the active ingredient of the anti-autoimmune disease agent is not particularly limited to the substances specifically listed above, and can also be obtained by the screening method of the present invention described below.
[0062]
Therefore, the anti-autoimmune disease agent targeted by the present invention includes a preparation containing an effective amount of a substance having the above-mentioned GRF inhibitory action, and a substance having a GRF inhibitory action obtained by the screening method described below. Any formulation containing an effective amount is included. Here, the effective amount of a substance having a GRF inhibitory action is not particularly limited as long as it is an amount of a GRF inhibitory action substance that causes the anti-autoimmune disease agent containing the substance to exhibit the GRF inhibitory effect as a result. It is not something. The anti-autoimmune disease agent may contain a pharmaceutically acceptable carrier and various additives in addition to the GRF inhibitory agent.
[0063]
The types of carriers and additives, forms of anti-autoimmune disease agents, and usage (administration form, administration method, dose) of anti-autoimmune disease agents will be described in detail in the section (III) described later.
[0064]
II . Screening method for active ingredient of anti-autoimmune disease agent
As shown in Examples described later, the present inventor has acquired a new finding that the onset of an autoimmune disease can be suppressed by inhibiting the functional expression of GRF. From this, it is thought that the substance which has a GRF inhibitory effect is useful as an active ingredient of an anti-autoimmune disease agent. The screening method of the present invention has been developed based on this finding, and seeks to obtain an active ingredient of an anti-autoimmune disease agent by searching for a substance having a GRF inhibitory action from test substances. It is.
[0065]
The “substance with GRF inhibitory activity” targeted by the screening method of the present invention is not limited as long as it is a substance that can inhibit GRF function expression (function exertion, action) as a result, and its action mechanism is particularly limited. is not. Specifically, the GRF inhibitory action includes 1) an action that inhibits the action (signal transduction) of GRF via the GRF receptor, 2) an action that inhibits the expression of GRF receptor, and 3) expression or secretion of GRF. The action which inhibits can be mentioned. The inhibitory action 1) includes (1) an action that inhibits the binding between GRF and a GRF receptor, and (2) an action that inhibits intracellular signal transduction of GRF. The substance having a GRF inhibitory action may be any substance having at least one of such GRF inhibitory actions.
[0066]
Candidate substances that can be active ingredients of anti-autoimmune disease agents include nucleic acids, peptides, proteins, organic compounds (including low molecular weight compounds), inorganic compounds, and the like. The screening method of the present invention can be performed on a substance that can be a candidate substance or a sample containing the substance (collectively referred to as “test substance”). The sample (test substance) containing the candidate substance includes a cell extract, a gene library expression product, a microorganism culture supernatant, and a cell component.
[0067]
The screening method of the present invention is carried out by searching for a substance having at least one of the GRF inhibitory actions from among the test substances, using the GRF inhibitory action listed above as an index. The test substance thus selected and obtained is useful as an active ingredient of an anti-autoimmune disease agent.
[0068]
Hereinafter, the screening method of the present invention will be specifically described for each GRF inhibitory action as an index.
[0069]
1) Screening method using as an index the action of inhibiting the action of GRF via the GRF receptor
In this screening method, a substance having a GRF inhibitory action is searched from test substances using the inhibition of GRF action (signal transduction) via the GRF receptor as an index, and obtained as an active ingredient of an anti-autoimmune disease agent. Is the method.
[0070]
Here, “inhibition of GRF action through GRF receptor” as an index of search includes (1) inhibition of binding between GRF and GRF receptor, and (2) inhibition of intracellular signal transmission of GRF. Can do.
[0071]
(1) Screening method using inhibition of binding between GRF and GRF receptor as an index
The screening method is a method for obtaining a substance having a GRF inhibitory action from test substances as an active ingredient of an anti-autoimmune disease agent by searching for inhibition of binding between GRF and a GRF receptor as an index.
[0072]
The screening method is not particularly limited and can be appropriately designed based on the common general technical knowledge of those skilled in the art. As an example, when the GRF is brought into contact with the GRF receptor, the GRF receptor is tested with the GRF and the test substance. And a method of screening comprising comparing and evaluating the binding of GRF to the GRF receptor. Specifically, the screening method can be carried out by performing the following steps (i), (ii) and (iii).
GRF receptor or GRF receptor homologue (collectively referred to as “GRF receptor etc.”) and GRF or GRF homologue (collectively referred to as “GRF etc.”) in the presence of the test substance Process,
The amount of binding of GRF or the like to the GRF receptor or the like is measured, and the binding amount of GRF or the like is bound to the GRF receptor or the like obtained by bringing the GRF receptor or the like into contact with GRF or the like in the absence of the test substance. Comparing the amount (control binding amount); and
A step of selecting a test substance that decreases the binding amount compared to the control binding amount based on the result of (ii) above.
[0073]
In this case, inhibition of binding between GRF receptor or GRF receptor homologue (GRF receptor, etc.) and GRF or GRF homologue (GRF, etc.) is as follows: (1) GRF receptor such as GRF is bound by binding to GRF receptor, etc. It may be of an aspect that inhibits binding to the body, etc., or (2) may be of an aspect that inhibits binding to GRF receptors, such as GRF, by binding to GRF, etc. , I don't particularly ask about that. However, in this case, particularly in the case of (1), it is necessary to select a test substance that does not exert the same action as GRF even when bound to the GRF receptor or the like. For this reason, the test substance selected by the above screening method is further:
(iv) It is preferable that the test substance selected from the above (iii) is further subjected to a step of selecting a test substance that does not exhibit the same action as GRF.
[0074]
Here, “not exhibiting the same action as GRF” is not only the case where it does not exhibit the same qualitative action as GRF (loss of GRF function) but also the case where it exhibits the same qualitative action as GRF. However, the case where an effect that is quantitatively reduced as compared with that of GRF is also included.
[0075]
The above selection method is not particularly limited, but as a specific method, for example, in vitro system or in vivo system described in Proc. Natl. Acad. Sci. USA, Vol. 91, p12298-12302 (1994) Can be mentioned.
[0076]
The GRF receptor used in the screening method of the present invention may be a natural product, a synthetic product, or a recombinant. The GRF receptor is preferably derived from humans, but GRF receptors derived from mammals other than humans such as mice and other biological species can be used as well.
[0077]
In the above screening method, a protein or poly (oligo) peptide having a binding ability to GRF (for example, a mutant or derivative of a GRF receptor having the binding ability to GRF) is used instead of the GRF receptor. You can also. In the present invention, these are referred to as “GRF receptor homologs”. For example, as a variant of such GRF receptor, the known GRF receptor (GenBank accession number: NM) 000823 [base sequence: SEQ ID NO: 3], NP 000814 [Amino acid sequence: SEQ ID NO: 4], AY 008835, AY 008834, AF 184896, AB 022597, AB 022596, L 07380, L 07379) have naturally occurring allelic variants, non-naturally occurring variants and artificially modified amino acid sequences by deletion, substitution, addition or insertion of part of the amino acid sequence of the GRF receptor Variants are included.
[0078]
In addition, the GRF receptor used in the screening method of the present invention does not necessarily need to retain the entire amino acid sequence, and at least a binding site with GRF, for example, the extracellular domain of the GRF receptor (Nature, vol. 360, p. .765-768, (1992)). That is, in the screening method of the present invention, a protein or poly (oligo) peptide having at least a binding region with GRF (for example, extracellular domain of GRF receptor) can be used instead of the GRF receptor. The “GRF receptor homolog” referred to in the present invention also includes such a protein or (poly) peptide. Furthermore, as long as the GRF receptor extracellular domain maintains its ability to bind to GRF, a part of its amino acid sequence is deleted, or is substituted, added or inserted by another amino acid residue. A protein or (poly) peptide having an extracellular domain of such a modified GRF receptor may also be included in the “GRF receptor homolog” referred to in the present invention.
[0079]
Examples of the method for obtaining the GRF receptor include the following methods.
First, the amino acid sequence information of the GRF receptor derived from humans or various biological species and the base sequence information encoding it (GenBank accession number: NM 000823 [base sequence: SEQ ID NO: 3], NP 000814 [Amino acid sequence: SEQ ID NO: 4], AY 008835, AY 008834, AF 184896, AB 022597, AB 022596, L 07380, L Based on 07379), probes or PCR primers are prepared according to conventional methods, and cDNAs encoding GRF receptors are obtained by cloning cDNA libraries derived from anterior pituitary cells of humans or various organisms. To do. These clonings can be easily carried out by those skilled in the art according to basic books such as Molecular Cloning 2nd Edt., Cold Spring Harbor Laboratory Press (1989).
[0080]
Next, the prepared GRF receptor cDNA is inserted into a known expression vector such as pCAGGS (Gene 108,193-199 (1991)) or pcDNA1.1, pcDNA3.1 derivative (both from Invitrogen). Thereafter, the obtained recombinant vector is introduced into a suitable host and cultured to produce a transformed cell in which the GRF receptor protein encoded by the introduced GRF receptor cDNA is expressed on the cell surface. it can.
[0081]
In addition, as a host here, L cell, CHO cell, C127 cell, BHK21 cell, BALB / c3T3 cell (dihydrofolate reductase, thymidine kinase, etc.) which are generally widely used as host cells. (Including deficient mutants) and COS cells are preferred, but not limited thereto, insect cells, yeast cells, bacterial cells, and the like can also be used. Moreover, as a method for introducing the GRF receptor expression vector into a host cell, any known method may be used. For example, the calcium phosphate method (J. Virol., 52, 456-467 (1973)). And a method using LT-1 (manufactured by Panvera), a method using a lipid for gene introduction (Lipofectamine, Lipofectin; manufactured by Gibco-BRL), and the like. Next, the GRF receptor is isolated from the obtained transformed cells according to a conventional method. Specifically, for example, according to the method of RG Shorr et al. (Proc. Natl. Acad. Sci. USA, 79, 2778-2782 (1982), J. Biol. Chem. 257, 12341-12350 (1982)) A crude extract containing the GRF receptor can be prepared by solubilizing the converted cells with an appropriate solubilizer (such as β-D-octylglucoside). Furthermore, in order to purify the GRF receptor from the crude extract, a conventional method in the art (for example, an anti-GRF receptor antibody such as the method of JL Benovic et al. (Biochem., 23, 4510-4518 (1984)) is used. Etc.) can be used.
[0082]
A GRF receptor variant (a variant having a modified amino acid sequence of the GRF receptor) (GRF receptor homolog) is a site based on information on the amino acid sequence of the GRF receptor and the base sequence encoding it. Specific Mutagenesis [Methods in Enzymology, 154, 350, 367-382 (1987); 100, 468 (1983); Nucleic Acids Res., 12, 9441 (1984); II ”, edited by Japan Chemical Society, p.105 (1986)), chemical synthesis means such as phosphate triester method and phosphate amidite method [J. Am. Chem. Soc., 89, 4801 (1967); 91, 3350 (1969); Science, 150, 178 (1968); Tetrahedron Lett., 22, 1859 (1981); 24, 245 (1983)], and any combination of these methods Can be prepared.
[0083]
Furthermore, the GRF receptor homolog having only the extracellular domain of the GRF receptor is obtained by using the cDNA encoding the extracellular domain of the GRF receptor in place of the cDNA of the GRF receptor in the GRF receptor preparation method described above. It can be prepared by preparing transformed cells in the same manner as described above. In this case, the GRF receptor homolog can be secreted into the culture supernatant of the transformed cells. In this case, for example, by using an affinity column with an anti-GRF receptor antibody or an antibody against the extracellular domain of the GRF receptor, the GRF receptor homolog can be isolated and purified from the culture supernatant. In addition, by constructing an expression system so that an extracellular domain is expressed and generated with a peptide tag added, a culture supernatant of transformed cells can be obtained using a column with a substance having affinity for the tag. The GRF receptor homologue can be isolated and purified from
[0084]
Here, the nucleotide sequence of the cDNA encoding the “extracellular domain” of the GRF receptor is described in Nature, Vol. 360, 765-768 (1992) as described above, and it can be easily obtained by those skilled in the art. Can be done. Furthermore, the modification of the amino acid sequence of the “extracellular domain” can be easily performed according to the above-described methods known in the art. Further, regarding the GRF receptor modified in this way (GRF receptor homolog), the presence or absence of binding ability to GRF is determined by the amount of binding between the labeled GRF and the modified GRF receptor, It can be evaluated by comparing the amount of binding with the natural GRF receptor.
[0085]
The GRF receptor or GRF receptor homologue (GRF receptor or the like) used in the screening method of the present invention may be an isolated or further purified GRF receptor or the like, but is not limited thereto. For example, instead of an isolated GRF receptor or the like, cells expressing the GRF receptor or the like may be used, or a cell membrane having a GRF receptor or the like may be used.
[0086]
Examples of the cells expressing GRF receptor and the like include transformed cells prepared by introducing a vector having cDNA such as the GRF receptor and the like described above into a suitable host and culturing. Since the cultured transformed cells express the GRF receptor and the like on the cell surface, they can be used as they are in the screening method of the present invention.
[0087]
A cell membrane having a GRF receptor or the like can be prepared from the above-described transformed cell. The following method can be mentioned as an example of a preparation method. First, a hypotonic homogenate buffer (10 mM Tris-HCl buffer, 1 mM EDTA, 0.5 mM PMSF (phenylmethanesulfonyl fluoride) or 1 mM AEBSF, 5 μg / ml aprotinin, 5 μg / ml leupeptin; pH 7.4) is added to the cultured transformed cells. Add and leave at 4 ° C. for about 30 minutes to disrupt the cells hypotonically, then homogenize by pipetting, and centrifuge at 50,000 × g for 30 minutes at 4 ° C. to obtain a precipitate of the cell membrane fraction . A cell membrane fraction can be obtained by suspending the precipitate in Tris-HCl buffered saline (Tris-HCl buffer, 154 mM sodium chloride; pH 7.4).
[0088]
In addition, a cell membrane fraction can be obtained from cultured transformed cells by, for example, the method of F. Pietri-Rouxel et al. (Eur. J. Biochem., 247, 1174-1179 (1997)).
[0089]
The GRF used in the screening method of the present invention may be a natural product, a synthetic product, or a recombinant. The GRF is preferably derived from humans, but GRFs derived from mammals other than humans such as mice and other biological species can be used as well. Alternatively, a protein or poly (oligo) peptide (for example, a mutant or derivative of GRF having the binding ability to the GRF receptor) having the binding ability to the GRF receptor can be used instead of GRF. In the present invention, these are referred to as “GRF homologs”. For example, such GRF variants include naturally occurring allelic variants and non-naturally occurring variants of GRF, and artificially deleting, replacing, adding or inserting part of the amino acid sequence of GRF. Variants having altered amino acid sequences are included.
[0090]
Further, the GRF used in the screening method of the present invention does not necessarily need to retain the entire amino acid sequence, and it is sufficient that it has at least a binding site for GRF to the GRF receptor. That is, in the screening method of the present invention, instead of GRF, at least the GRF receptor binding domain of GRF (Science, Vol. 218, 585 (1982); Biochem. Biophys. Res. Commun., Vol. 123, 854 (1984) )) Protein or poly (oligo) peptide can also be used. The “GRF homolog” referred to in the present invention includes such a protein or poly (oligo) peptide. In addition, as long as the GRF receptor-binding domain of the GRF is capable of binding to the GRF receptor, a part of the amino acid sequence is deleted, or is substituted, added, or inserted by another amino acid. A protein or poly (oligo) peptide having a GRF receptor binding domain of such a modified GRF may also be included in the “GRF homolog” in the present invention.
[0091]
GRF is basically a protein consisting of 44 amino acids. Therefore, amino acid sequence information of GRF derived from humans or various organisms (GenBank accession number: NP 066567 [SEQ ID NO: 2], NM 031577, NM 010285, AF 242855, U 10156, M 73486, M 31658, M 31654), and can be synthesized according to the usual peptide synthesis method. For example, peptide synthesis has been described in the literature [Peptide Synthesis, Interscience, New York, 1966; The Proteins, Vol 2, Academic Press Inc., New York, 1976; Peptide Synthesis, Maruzen ( 1975; peptide synthesis basics and experiments, Maruzen Co., Ltd., 1985; drug development continued Vol. 14, Peptide Synthesis, Hirokawa Shoten, 1991]. it can. Moreover, according to the peptide synthesis method, the aforementioned GRF homolog can be easily prepared.
[0092]
In addition, GRF or GRF homolog is GRF gene information (GenBank accession number: NM). 021081 [SEQ ID NO: 1], NM 031577, NM 010285, AF 242855, U 10156, M 73486, M 31658, M 31654), it can also be produced by genetic engineering in accordance with the method for preparing the GRF receptor or GRF receptor homolog described above. In this case, cloning of GRF cDNA can be carried out using a cDNA library derived from a human or non-human mammalian GRF-producing tumor cell.
[0093]
Further, commercially available GRF may be used. GRF can be obtained from, for example, Sumitomo Pharmaceutical Co., Ltd. (Japan).
[0094]
The GFR or GRF homologue may be used as it is, or one labeled with an arbitrary labeling substance can be used. Here, as a labeling substance, a radioisotope (for example,¹²⁵I), fluorescent substances, chemiluminescent substances, biotin, marker proteins, peptide tags, and the like. Examples of the marker protein include conventionally known marker proteins such as alkaline phosphatase (Cell 63, 185-194 (1990)), antibody Fc region (Genbank accession number M87789), or HRP (Horse radish peroxidase). Examples of peptide tags include Myc tag (Glu-Gln-Lys-Lue-Ile-Ser-Glu-Glu-Asp-Ile: SEQ ID NO: 5), His tag (His-His-His-His-His-His: Examples thereof include conventionally known peptide tags such as SEQ ID NO: 6) and FLAG tag (Asp-Tyr-Lys-Asp-Asp-Asp-Asp: SEQ ID NO: 7).
[0095]
In the step (i) of the screening method (1) of the present invention, the conditions for contacting the GRF receptor and the like with the GRF etc. in the presence of the test substance are the GRF receptor and the GRF etc. in the absence of the test substance. There is no particular limitation as long as and are combined. In addition, in the case where a cell membrane having a GRF receptor or the like or a cell membrane having a GRF receptor or the like is used in place of the isolated GRF receptor or the like, the GRF receptor or the like present on the cell or the cell membrane is GRF or the like. Any condition may be used as long as they are in contact with each other and bonded together. Specifically, in the case of cells, normal viable culture conditions, or in the case of cell membranes, physiological conditions such as Tris-HCl buffered saline can be exemplified.
[0096]
Selection of candidate substances for active ingredients of anti-autoimmune disease agents includes, for example, GRF receptor (including cells expressing GRF receptor or the like, or GRF receptor existing on the cell membrane) and GRF under the above conditions Can be carried out by searching for substances that inhibit the binding of the two. Specifically, when the GRF receptor or the like is brought into contact with GRF or the like in the presence of the test substance, the amount of binding of GRF or the like to the GRF receptor or the like is brought into contact in the same manner as described above in the absence of the test substance. In this case, the test substance can be selected as a candidate substance by using as an index a decrease in the amount of GRF binding to the GRF receptor or the like obtained in such a case (control binding amount). In addition, it does not need to be compared with the amount of control binding, and the decrease referred to here is a case where binding to GRF receptor or the like such as GRF is completely inhibited and both do not bind, that is, binding of GRF or the like to GRF receptor or the like. The case where the amount is substantially zero is also included.
[0097]
The amount of binding of GRF or the like to the GRF receptor or the like can be quantified by a known method such as Western blotting using an antibody against GRF or the like. In addition, when the aforementioned labeled GRF or the like is used as the GRF or the like, the amount of GRF or the like bound to the GRF receptor or the like is determined by a measurement method (for example, a radiation measurement method or a fluorescence detection method) according to the labeling substance used. It can also be quantified.
[0098]
(2) Screening method using inhibition of intracellular signal transduction of GRF as an index
The screening method is a method for obtaining a substance having a GRF inhibitory action from test substances as an active ingredient of an anti-autoimmune disease agent by searching for inhibition of intracellular signal transduction of GRF as an index.
[0099]
The method can be specifically carried out by performing the following steps (i), (ii) and (iii):
(i) contacting GRF or a homolog thereof (such as GRF) with a cell expressing GRF receptor or a homolog thereof (such as GRF receptor) in the presence of a test substance;
(ii) measuring the expression level or activity of an intracellular signaling substance caused by GRF in the cell, and expressing the expression level or activity between a cell expressing GRF receptor or the like in the absence of a test substance and GRF or the like Comparing the expression level or activity corresponding to the above obtained by contact (control expression level or control activity), and
(iii) A step of selecting a test substance that varies the expression level or activity relative to the control expression level or control activity based on the result of (ii) above.
[0100]
2) Screening method using as an index the action of inhibiting the expression of GRF receptor
This screening method is a method for obtaining a substance having a GRF inhibitory action from test substances as an active ingredient of an anti-autoimmune disease agent by searching for inhibition of GRF receptor expression as an index.
[0101]
The screening method is not particularly limited and can be appropriately designed based on the common general technical knowledge of those skilled in the art. As an example, a case where a test substance is brought into contact with a cell capable of expressing a GRF receptor and a test substance are used. A screening method including comparing and evaluating the expression level of the GRF receptor in the cell with and without contact can be given. Specifically, the screening method can be carried out by performing the following steps (i), (ii) and (iii).
(i) contacting the test substance with a cell capable of expressing a GRF receptor;
(ii) Measure the expression level of the GRF receptor in the cell contacted with the test substance, and compare the expression level with the expression level (control expression level) of the GRF receptor in the corresponding control cell not contacted with the test substance And the process of
(iii) A step of selecting a test substance that decreases the expression level of the GRF receptor as compared with the control expression level based on the comparison result of (ii) above.
[0102]
The cells used for such screening may be any cells that can express the GRF receptor, whether natural or recombinant. In addition, the origin of the GRF receptor is not particularly limited, and may be derived from humans, or derived from mammals such as mice other than humans or other biological species. Specific examples of such cells include anterior pituitary cells (including those derived from humans and other species) having a gene (DNA) encoding a GRF receptor, and isolated and prepared primary anterior pituitary cultures. Mention may be made of cells. In addition, transformed cells prepared by introducing an expression vector having a GRF receptor cDNA by the method described above can also be used. The category of cells used for screening includes tissues that are aggregates of cells.
[0103]
In the step (i) of the screening method 2) of the present invention, the conditions for contacting the test substance with the cell capable of expressing the GRF receptor are not particularly limited, but the cell does not die and the gene of the GRF receptor is expressed. It is preferable to select culture conditions (temperature, pH, medium composition, etc.) that can be used.
[0104]
Selection of a candidate substance for an active ingredient of an anti-autoimmune disease agent is carried out, for example, by contacting a test substance with a cell capable of expressing a GRF receptor under the above conditions and searching for a substance that suppresses the expression level of the GRF receptor. it can. Specifically, when the GRF receptor-expressing cells are cultured in the presence of the test substance, the GRF receptor-expressing cells corresponding to the above are cultured in the absence of the test substance. The test substance can be selected as a candidate substance using as an index a decrease in the expression level of the obtained GRF receptor (control expression level). In addition, it does not need to be compared with the control expression level, and the decrease referred to here is when the GRF receptor is not expressed at all (when the GRF receptor gene (mRNA) is not expressed at all, or when the GRF receptor protein is not generated at all). ), That is, the expression level of the GRF receptor is substantially zero.
[0105]
In step (ii) of the above screening method 2), the expression level of the GRF receptor is evaluated by measuring the expression level of the GRF receptor gene (mRNA) or by measuring the production amount of the GRF receptor protein. can do. Further, the method for measuring the expression level of the GRF receptor is not limited to the method of directly measuring the expression level of these genes (mRNA) or the amount of protein production, but may be any method that reflects these. That is, measuring the expression level of the GRF receptor in the step (ii) is a method by which the expression level of the GRF receptor gene and the generation amount of the GRF receptor protein can be evaluated regardless of direct or indirect methods. The implementation of is widely included.
[0106]
Specifically, the measurement (detection and quantification) of the expression level of the GRF receptor has a sequence complementary to the base sequence of the GRF receptor mRNA. The amount of GRF receptor protein expressed on the cell surface of a known method such as Northern blotting or RT-PCR using an oligonucleotide or a DNA array or a GRF receptor-expressing cell Can be carried out by performing a known method such as Western blotting using an anti-GRF receptor antibody. In addition, the measurement (detection and quantification) of the expression level of the GRF receptor is carried out in the GRF receptor gene control region [eg, the downstream region of the GRF receptor promoter (Genbank Accession number: AF267729, AF121969, AF127135, etc.)] A protein derived from the marker gene using a cell line into which a fusion gene connecting a reporter gene such as a luciferase gene, chloramphenicol / acetyltransferase gene, β-glucuronidase gene, β-galactosidase gene, aequorin gene, etc. It can also be carried out by measuring the activity. The screening method for an active ingredient (target substance) of an anti-autoimmune disease agent using the expression level of the GRF receptor of the present invention as an index also includes a method for searching for a target substance using the expression level of the marker gene as an index. . The preparation of the fusion gene and the measurement of the activity derived from the marker gene can be performed by known methods.
[0107]
3) Screening method using the action of inhibiting GRF expression or secretion as an index
The screening method is a method for obtaining a substance having a GRF inhibitory action from test substances as an active ingredient of an anti-autoimmune disease agent by searching for an inhibitory action on GRF expression or secretion as an index.
[0108]
The screening method is not particularly limited and can be appropriately designed based on the common general knowledge of those skilled in the art. For example, when the test substance is brought into contact with a cell capable of expressing GRF, the test substance is not brought into contact. In some cases, a screening method including comparing and evaluating the expression level or secretion level of GRF in the cell can be mentioned. Specifically, the screening method can be carried out by performing the following steps (i), (ii) and (iii).
(i) contacting a test substance with a cell capable of expressing GRF;
(ii) GRF expression level or secretion level of GRF-expressing cells contacted with a test substance is measured, and the GRF-expressing cells (control cells) corresponding to the above are not contacted with the test substance. Comparing the expression level or secretion level of GRF (control expression level or control secretion level); and
(iii) A step of selecting a test substance that reduces the expression level or secretion amount of GRF as compared with the control expression level or control secretion level based on the comparison result of (ii) above.
[0109]
The cells used for such screening may be cells that can express GRF, regardless of whether they are natural or recombinant. In addition, the origin of GRF is not particularly limited, and may be derived from a human or from a mammal other than a human such as a mouse or other biological species. Specific examples of such cells include tumor cells (including those derived from humans and other biological species) having a gene encoding GRF and primary tumor culture cells isolated and prepared. In addition, transformed cells prepared by introducing an expression vector having GRF cDNA by the above-described method can also be used. The category of cells used for screening includes tissues that are aggregates of cells.
[0110]
In the step (i) of the screening method 3) of the present invention, the conditions for contacting the test substance with the GRF-expressing cell are not particularly limited, but the cell does not die and the GRF gene (mRNA) can be expressed. It is preferable to select the culture conditions (temperature, pH, medium composition, etc.) or the culture conditions (temperature, pH, medium composition, etc.) that the produced GRF protein can secrete.
[0111]
Selection of a candidate substance for an active ingredient of an anti-autoimmune disease agent can be performed, for example, by contacting a test substance with a cell capable of expressing GRF under the above conditions and searching for a substance that suppresses the expression level or secretion level of GRF. . Specifically, the expression level or secretion amount of GRF when cells capable of expressing GRF are cultured in the presence of a test substance is the GRF obtained when cells corresponding to the above are cultured in the absence of the test substance. The test substance can be selected as a candidate substance using as an index the decrease in the expression level or secretion level (control expression level or control secretion level). In addition, it does not need to be compared with the amount of control expression or the amount of secreted secretion, and the decrease referred to here is when GRF is not expressed at all (when GRF gene (mRNA) is not expressed at all, when GRF protein is not generated at all) or The case where the GRF protein is not secreted at all, that is, the case where the expression amount or secretion amount of GRF is substantially zero is also included.
[0112]
In step (ii) of the above screening method 3), the expression level of GRF can be evaluated by measuring the expression level of the GRF gene (mRNA) or measuring the production level of the GRF protein. Moreover, the amount of GRF secretion can be evaluated by measuring the amount of GRF protein contained in a culture solution (culture supernatant) obtained by culturing cells capable of expressing GRF. The method for measuring the expression level of GRF or the secretion amount of GRF is not a method of directly measuring the expression level of these genes (mRNA) or the amount of protein production, or measuring the amount of GRF protein in the culture supernatant. However, any method that reflects these may be used. That is, in the step (ii), the expression amount or secretion amount of GRF is measured regardless of whether it is a direct or indirect method, the expression amount of GRF gene, the amount of GRF protein produced or released into the culture supernatant. Implementation of a method capable of evaluating the amount of GRF protein to be evaluated is widely included.
[0113]
The measurement (detection and quantification) of the expression level of GRF is carried out using the Northern blot method, RT-PCR, or the like using the GRF mRNA expression level of cells capable of expressing GRF using an oligonucleotide having a sequence complementary to the base sequence of the GRF mRNA. The amount of GRF produced on the cell surface of cells capable of expressing GRF is measured by a known method such as Western blotting using an anti-GRF antibody. Can be implemented. In addition, measurement (detection and quantification) of the expression level of GRF is carried out by using marker genes such as various reporter genes described above in the GRF gene control region [for example, downstream region of GRF promoter (Genbank Accession number: U10153, etc.)]. It can also be carried out by measuring the activity of the marker gene-derived protein using a cell line into which the fused gene has been introduced. The screening method for an active ingredient (target substance) of an anti-autoimmune disease agent using the expression level of GRF of the present invention as an index includes a method for searching for a target substance using the expression level of such marker gene as an index. The preparation of the fusion gene and the measurement of the activity derived from the marker gene can be performed by known methods.
[0114]
In addition, the amount of GRF secretion (detection and quantification) can be determined by culturing cells capable of expressing GRF and measuring the amount of GRF protein contained in the obtained culture supernatant, for example, Western blotting using an anti-GRF antibody. It can implement by measuring by the method of.
[0115]
The candidate substances selected from the test substances by the screening methods of 1) (1) and (2), 2) and 3) described above are, for example, pathological conditions imitating various human autoimmune diseases. It can be obtained as an active ingredient of a more practical anti-autoimmune disease agent by subjecting it to a drug efficacy test and safety test using a model animal.
[0116]
More specifically, the obtained candidate substance is subjected to a medicinal effect test using a disease model animal of multiple sclerosis (for example, an EAE-induced mouse), whereby the candidate substance has an inhibitory effect on multiple sclerosis ( Preventive effect, therapeutic effect) can be evaluated. In addition, the obtained candidate substance is used as a model animal for rheumatoid arthritis (for example, collagen-induced arthritis [Trentham DE, Townes AS, Kang AH: Autoimunity to type II collagens: An experimental model of arthritis J Exp Med 146: 857-868 , 1977], antigen-induced arthritis, or adjuvant-induced arthritis [Pearson CM: Development of arthritis, periarthritis and periotitis in rats given adjuvant. Proc Soc Expe Biolo Med 91: 95-101,1956] The inhibitory effect (preventive effect, therapeutic effect) with respect to rheumatoid arthritis can be evaluated about the said candidate substance by performing the medicinal effect test used. The substance thus selected and obtained can be industrially produced by chemical synthesis, biological synthesis (fermentation) or genetic manipulation based on the structural analysis result.
[0117]
A substance having a GRF inhibitory action selected by using the screening method of the present invention as described above is useful as an active ingredient of the anti-autoimmune disease agent as described above. Specific examples of the autoimmune disease include various diseases described in Chapter I of the present specification. In particular, the GRF inhibitory agent targeted by the present invention is a multiple sclerosis. It can be used effectively for symptom, rheumatoid arthritis, and systemic lupus erythematosus, preferably multiple sclerosis, and rheumatoid arthritis.
[0118]
The “anti-autoimmune disease agent” of the present invention is an action that inhibits or suppresses the onset of various autoimmune diseases, an action that delays the onset, an action that reduces or improves the symptoms after the onset, or a disease It has at least one action of healing. In that sense, the above-mentioned “anti-autoimmune disease agent” can also be referred to as an autoimmune disease preventive agent, autoimmune disease symptom alleviating agent, autoimmune disease ameliorating agent, or autoimmune disease therapeutic agent. The screening method can be a method for searching for these active ingredients.
[0119]
The present invention provides an anti-autoimmune disease agent comprising a substance having a GRF inhibitory action obtained by the above screening method as an active ingredient.
[0120]
III . About the usage of anti-autoimmune diseases
Hereinafter, the administration mode, administration method, dosage and the like of the anti-autoimmune disease agent containing the substance having the GRF inhibitory action of the present invention as an active ingredient will be described.
[0121]
1) When the substance having GRF inhibitory activity is in the form of a low molecular weight compound, peptide or protein
When the substance having a GRF inhibitory action is a protein such as a low molecular weight compound, a peptide or an antibody, it can be prepared in the form of a general pharmaceutical composition (pharmaceutical preparation) usually used for these substances. Depending on the dosage, it can be administered orally or parenterally. Generally, the following administration forms and administration methods are mentioned.
[0122]
Typical administration forms include solid preparations such as tablets, pills, powders, powders, granules and capsules, and liquids such as solutions, suspensions, emulsions, syrups and elixirs. Can be mentioned. These various forms can be administered orally, depending on the route of administration, as well as nasal, transdermal, rectal (suppository), sublingual, vaginal, injection (transvenous, transarterial, transdermal) It can be classified into parenteral administration agents such as muscle, subcutaneous, intradermal) and infusion. For example, as oral administration agents, for example, tablets, pills, powders, powders, granules, capsules, solutions, suspensions, emulsions, syrups, etc., or as rectal administration agents and vaginal agents, for example It can take the form of tablets, pills, capsules and the like. As a transdermal agent, for example, in addition to a liquid such as lotion, a semi-solid agent such as cream or ointment can be used.
[0123]
The injection may take the form of, for example, a solution, a suspension or an emulsion, and specifically, aseptically treated water, water-propylene glycol solution, buffered solution, 0.4% by weight physiological saline. Water etc. can be illustrated. Furthermore, the injection may be frozen or lyophilized after being prepared into a solution, and can be stored thereby. The lyophilized preparation prepared by the latter lyophilization treatment is used by re-dissolving by adding distilled water for injection at the time of use.
[0124]
The above-mentioned pharmaceutical composition (pharmaceutical preparation) is prepared by blending a substance having a GRF inhibitory action and a pharmaceutically acceptable carrier by an ordinary method practiced in the art. Examples of pharmaceutically acceptable carriers include excipients, diluents, fillers, extenders, binders, disintegrants, wetting agents, lubricants, and dispersants. Further, additives usually used in the field can be blended. Such additives include, for example, stabilizers, bactericides, buffers, thickeners, pH adjusters, emulsifiers, suspending agents, preservatives, fragrances, coloring agents, depending on the form of the pharmaceutical composition to be prepared. It can be appropriately selected from a tonicity adjusting agent, a chelating agent, a surfactant and the like.
[0125]
The pharmaceutical composition having such a form can be administered by an appropriate administration route according to the target disease, target organ and the like. For example, it may be administered intravenously, artery, subcutaneously, intradermally or intramuscularly, or may be locally administered directly to the tissue where the lesion is observed, or may be administered orally or rectally.
[0126]
The dosage and frequency of administration of these pharmaceutical compositions vary depending on the dosage form, the patient's disease and symptoms, the patient's age and weight, etc., and cannot be generally specified, but are usually effective for adults per day The amount of the component can be administered in a range of about 0.0001 to about 500 mg, preferably about 0.001 to about 100 mg in one or several divided doses.
[0127]
2 ) When the substance having GRF inhibitory activity is in the form of a polynucleotide
When the substance having a GRF inhibitory action has a form of a polynucleotide such as an antisense polynucleotide having a base sequence complementary to the base sequence of the GRF gene or GRF receptor gene, It can take the form of a prophylactic agent. In recent years, reports of gene therapy using various genes have been made, and gene therapy has become an established technique.
[0128]
A gene therapy agent can be prepared by introducing a target polynucleotide into a vector, or by transforming appropriate cells with the vector. Here, the administration form to a patient is roughly classified into two cases: (1) when a non-viral vector is used as a vector for introducing the target polynucleotide, and (2) when a viral vector is used. The preparation method and administration method of gene therapy agents are explained in detail in the experiment manual for cases where a viral vector is used as a vector and a non-viral vector is used (for example, “Experimental Medicine for Separate Volume, Basic Technology of Gene Therapy”, Yodosha, 1996, separate volume experimental medicine: “Gene transfer and expression analysis experiment method”, Yodosha, 1997, “Gene Therapy Development Handbook edited by the Japanese Society for Gene Therapy”, NTS, 1999, etc.). Hereinafter, the preparation method and administration method of the gene therapy agent will be specifically described for each vector to be used.
[0129]
(1) When using non-viral vectors as vectors
As the non-viral vector, any expression vector capable of expressing a gene (polynucleotide) encoding a target polypeptide or protein in vivo, preferably in a human organism, can be used. Although it does not restrict | limit in particular, For example, pCAGGS (Gene 108,193-200 (1991)), pBK-CMV, pcDNA3, pZeoSV (above, Invitrogen, Stratagene) etc. are mentioned.
[0130]
Introduction of a polynucleotide into a patient can be performed by introducing the desired polynucleotide into these non-viral vectors (expression vectors) according to a conventional method and administering the resulting recombinant expression vector. Thereby, the objective polynucleotide can be introduce | transduced into a test subject's cell and tissue.
[0131]
More specifically, examples of the method for introducing a polynucleotide into a cell include a phosphate-calcium coprecipitation method and a direct injection method of DNA (polynucleotide) using a micro glass tube.
[0132]
In addition, as a method for introducing a polynucleotide into a tissue, a polynucleotide introduction method using an internal type liposome or an electrostatic type liposome, an HVJ-liposome method, an improved HVJ-liposome method (HVJ-AVE Examples include liposome method), receptor-mediated polynucleotide introduction method, method of transferring polynucleotide together with carrier (metal particles) with a particle gun, direct introduction of naked-DNA, introduction method using positively charged polymer, etc. be able to.
[0133]
(2) When using viral vectors as vectors
Examples of viral vectors include vectors derived from recombinant adenoviruses and retroviruses. More specifically, for example, detoxified retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus, shinbis virus, Sendai virus, SV40, immunodeficiency virus (HIV), etc. And DNA virus or RNA virus vectors. Among these, adenoviral vectors are known to have much higher infection efficiency than other viral vectors, and from this viewpoint, it is preferable to use adenoviral vectors.
[0134]
Introduction of a polynucleotide into a patient can be performed by introducing the desired polynucleotide into these viral vectors according to a conventional method, and infecting the resulting recombinant virus with desired cells. Thereby, it is possible to introduce the target polynucleotide into the cell.
[0135]
The gene therapy agent thus prepared can be administered to a patient by an in vivo method in which the gene therapy agent is directly introduced into the body, or by removing certain cells from a human and supplying the gene therapy agent outside the body. There is an ex vivo method that introduces cells into cells and returns them to the human body ("Nikkei Science", April 1994, pages 20-45; "Monthly Pharmaceutical Affairs", 36 (1), 23-48, 1994; “Experimental Medicine Extra Number”, 12 (15), 1994; “Gene Therapy Development Research Handbook”, NTS, 1999). Among the autoimmune diseases targeted by the present invention, when used for the prevention or treatment of systemic autoimmune diseases, it is preferably introduced into the body by an in vivo method.
[0136]
Administration by the in vivo method can be performed by an appropriate administration route according to the target disease, target organ and the like. For example, it can be administered intravenously, arterially, subcutaneously, intradermally, intramuscularly, or directly locally to the tissue in which the lesion is observed.
[0137]
As the form of the gene therapy agent, various preparation forms can be adopted depending on the above administration forms. For example, when it has the form of an injection, the injection can be prepared by a conventional method. For example, after dissolving a polynucleotide as an active ingredient in a solvent such as a buffer solution such as PBS, physiological saline, or sterilized water, If necessary, it can be prepared by sterilizing by filtration with a filter or the like and then filling in an aseptic container. A conventional carrier or the like may be added to the injection as necessary. Moreover, in liposomes, such as HVJ-liposome, it can be set as the form of various liposome preparations, such as a suspension agent, a freezing agent, and a centrifugation concentration freezing agent.
[0138]
In addition, in order to facilitate the presence of genes around the diseased site, it is possible to prepare sustained-release preparations (mini-pellet preparations, etc.) and implant them near the affected area, or use an osmotic pump etc. It is also possible to administer gradually and continuously.
[0139]
The content of the polynucleotide in the gene therapy agent can be appropriately adjusted depending on the disease to be treated, the age, weight, etc. of the patient, but is usually about 0.0001 to about 100 mg, preferably about 0 as each polynucleotide. 0.001 to about 10 mg, which is preferably administered once every few days to several months.
[0140]
Further, the present invention described above is a method for preventing or treating autoimmune diseases using an anti-autoimmune disease agent. This method can be carried out by administering an effective amount of the anti-autoimmune disease agent of the present invention to a subject suffering from an autoimmune disease (autoimmune disease patient). Examples of the autoimmune disease include various autoimmune diseases described in Chapter I. Preferred are multiple sclerosis, rheumatoid arthritis and systemic lupus erythematosus, and more preferred are multiple sclerosis and rheumatoid arthritis.
[0141]
In addition, not only subjects who have developed a specific pathology of autoimmune disease but also the possibility of developing autoimmune disease, even if it has not, Also included are subjects who have Therefore, prevention of autoimmune disease as referred to in the present invention includes prevention of the onset of autoimmune disease and suppression of the onset of a subject suffering from autoimmune disease. The treatment of an autoimmune disease in the present invention includes alleviation or improvement of the pathological condition (symptoms) of the autoimmune disease that has occurred in a subject suffering from the autoimmune disease.
[0142]
The administration form, administration method, and dosage of the anti-autoimmune disease agent are as described above.
[0143]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited at all by these Examples.
[0144]
Example 1    Examination of the effect of growth hormone releasing factor receptor (GRFR) gene deficiency on the development of experimental autoimmune encephalomyelitis (EAE)
Experimental autoimmune encephalomyelitis (EAE), a model of human multiple sclerosis, in mice lacking the GRF receptor gene to investigate the involvement of growth hormone releasing factor (GRF) in the development of autoimmune diseases The presence or absence of onset was examined. Experimental methods and results are shown below.
[0145]
<Experiment method>
In the experiment, GRFR gene homo-deficient mice (C57BL / 6J-Ghrhr^{lit / lit}), GRFR heterozygous mice (C57BL / 6J-Ghrhr)^{lit / +}) And wild type mice (C57BL / 6J) (all female, both purchased from The Jackson Laboratory) as controls. All the mice were purchased at 5 weeks of age, and were bred so that the lights were turned on every day from 8 am to 8 pm, and water and solid feed (CE-2; Nippon Claire) were freely available.
[0146]
These mice (five mice in each group, 15 mice in total) were artificially induced to develop experimental autoimmune encephalomyelitis (EAE). Specifically, in each of these mice, 2 mg / ml myelin oligodendrocyte glycoprotein (MOG) 35-55 synthetic peptide (amino acid sequence: Met-Glu) dissolved in physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.). -Val-Gly-Trp-Tyr-Arg-Ser-Pro-Phe-Ser-Arg-Val-Val-His-Leu-Tyr-Arg-Asn-Gly-Lys: SEQ ID NO: 8) (Accord) solution 100 μl of emulsion mixed with Freund's complete Freund's adjuvant (CFA) (manufactured by Difco) in 100 μl subcutaneously in the thigh and immediately after sensitization and 2 days later, physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.) 200 ng pertussis toxin (manufactured by List Biological Laboratories) was intraperitoneally administered to induce experimental autoimmune encephalomyelitis (EAE). All mice were allowed to freely consume feed and water throughout the entire breeding period, including during the experiment.
[0147]
Body weight was measured daily for 27 days, EAE symptoms were observed, and EAE symptom scores were recorded based on the following criteria.
[0148]
[EAE Symptom Score]
0: asymptomatic, 1: tail paralysis, 2: weak hind leg paralysis, 3: moderate to strong hind leg paralysis or weak front leg paralysis or both, 4: complete hind leg paralysis or moderate to strong Paralysis of the forelimbs of the child, or both, 5: Paralysis of the extremities or in the death war, 6: Death.
[0149]
<Result>
The transition of the average score of the obtained EAE symptom scores is shown in FIG. As a result, in the GRFR homo-deficient mice, experimental autoimmune encephalomyelitis development (score 1) was not observed throughout the entire observation period (27 days). In addition, in the GRFR hetero-deficient mouse, a significant decrease in the EAE score was recognized as compared with the wild-type mouse.
[0150]
The transition of the incidence of EAE is shown in FIG. As a result, the incidence of GRFR homo-deficient mice was 0 throughout the entire observation period (27 days). In addition, the incidence rate of GRFR hetero-deficient mice was also lower than that of wild-type mice. From these results, it became clear that the onset of EAE is strongly suppressed by the deletion of the GRFR gene.
[0151]
Example 2    Study of the effects of growth hormone releasing factor (GRF) antagonists on the development of experimental autoimmune encephalomyelitis (EAE)
In order to investigate the effect of GRF antagonist administration on the development of autoimmune diseases, the study was conducted using experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis. Experimental methods and results are shown below.
[0152]
<Experiment method>
Wild type mice (PLSJLF1 / J) (female, purchased from The Jackson Laboratory) were used for the experiment. Mice were purchased at the age of 5 weeks and were bred daily from 8:00 am to 8:00 pm, with free access to water and solid feed (CE-2; Japan Claire). First, to these mice (10 mice in total), 2 mg / ml rabbit myelin basic protein (MBP) (manufactured by Sigma) dissolved in physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.) and complete Freund's adjuvant (CFA) ( Difco's 1: 1 emulsion was sensitized to the thigh subcutaneously in 100 μl aliquots, and 200 ng pertussis toxin (List Biological Laboratories, Inc.) dissolved in physiological saline immediately after and 2 days after sensitization. Produced intraperitoneally to induce experimental autoimmune encephalomyelitis (EAE). Then, administration of the GRF antagonist was performed according to the following procedure. Mice treated with MBP sensitization and pertussis toxin (10 mice in total) were divided into 2 groups, and 1 group (5 mice) had 30 μg of GRF antagonist (MZ-4-71: US Calbioquet per mouse). Mnova Biochem) was administered once daily from the 8th day to the 15th day after MBP sensitization (GRF antagonist (MZ-4-71) administration group). In another group (5 animals), the same amount of physiological saline was similarly administered as a control once daily from the 8th to 15th day after MBP sensitization to the cervical dorsal subcutaneous region (saline administration group). ). All mice were allowed free access to feed and water throughout the entire breeding period including the experimental time. Body weight was measured daily, EAE symptoms were observed, and EAE symptom scores were recorded based on the following criteria.
[0153]
<EAE Symptom Score>
0: asymptomatic, 1: tail paralysis, 2: weak hind leg paralysis, 3: moderate to strong hind leg paralysis or weak front leg paralysis or both, 4: complete hind leg paralysis or moderate to strong Paralysis of the forelimbs of the child, or both, 5: Paralysis of the extremities or in the death war, 6: Death.
[0154]
<Result>
The results are shown in FIG. FIG. 2A shows the transition of the average value of the EAE score. In the GRF antagonist (MZ-4-71) administration group, a decrease in EAE score was observed as compared to the physiological saline administration group. FIG. 2B shows the transition of the incidence of EAE. In the GRF antagonist (MZ-4-71) administration group, the onset of EAE onset was delayed compared to the physiological saline administration group. From these results, it became clear that the onset of EAE is suppressed by administration of the GRF antagonist.
[0155]
Example 3    Screening of active ingredients of anti-autoimmune diseases using GRF receptor (GRFR) -expressing cells
Using an expression vector containing the GRF receptor cDNA, CHO cells and COS cells are transformed by a conventional method to obtain transformed cells that stably express the GRF receptor on the cell surface. Alternatively, rat anterior pituitary cells are isolated and prepared by a conventional method (see Proc. Natl. Acad. Sci. USA, Vol. 91, p12298-12302 (1994)). On the other hand, GRF (manufactured by Sumitomo Pharmaceutical Co., Ltd.) is biotin or radioisotope (manufactured by conventional methods).¹²⁵Label with I).
[0156]
Next, (i) labeled GRF or (ii) labeled GRF and a test substance are added to the GRF receptor-expressing cells (transformed cells or rat anterior pituitary cells), and after a predetermined time, The binding of labeled GRF to the GRF receptor-expressing cells in the reaction system (i) and the reaction system (ii) is measured and compared. In the reaction system of (ii), a test substance that is not binding or is less binding than that in the reaction system of (i) is a candidate substance for an active ingredient of an anti-autoimmune disease agent. Choose as.
[0157]
Further, the obtained candidate substance is further subjected to another screening system for detecting a GRF inhibitory action and administration experiment for an EAE model animal. Thereby, it can confirm that it can become an active ingredient of an anti-autoimmune disease agent about the said candidate substance.
[0158]
Example 4    Screening of active ingredients of anti-autoimmune diseases using cells capable of expressing GRF
A group in which (i) a test substance is not added and (ii) a group in which a test substance is added are prepared for GRF-producing tumor cells. Each group is cultured for a certain period, and then the cells are collected and lysed by a conventional method, and the expression level of GRF mRNA is compared by Northern blot analysis. Alternatively, the cell culture supernatant is collected, and the amount of GRF protein secreted into the culture solution is compared by Western blot analysis. By these screenings, when GRF expression or GRF secretion is not observed in the group (ii), or the GRF expression level or GRF secretion level in the group (ii) is less than the result obtained in the group (i) In this case, the test substance used in (ii) is selected as a candidate substance for the active ingredient of the anti-autoimmune disease agent.
[0159]
Further, the obtained candidate substance is further subjected to another screening system for detecting a GRF inhibitory action and administration experiment for an EAE model animal. Thereby, it can confirm that it can become an active ingredient of an anti-autoimmune disease agent about the said candidate substance.
[0160]
【The invention's effect】
According to the present invention, a novel anti-autoimmune disease agent based on a new mechanism of action called GRF inhibitory action can be provided. Furthermore, the present invention provides a screening method for obtaining an active ingredient of an anti-autoimmune disease agent that exerts an effect based on a new action mechanism called GRF inhibitory action. According to the screening method using the GRF inhibitory action of the present invention as an index, it is possible to obtain an active ingredient capable of preventing or treating the development of an autoimmune disease based on a new action mechanism. Therefore, the screening method of the present invention can be suitably used for the development of new drugs effective for the prevention or treatment of autoimmune diseases.
[0161]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a diagram showing experimental results in Example 1. FIG. Specifically, to investigate the involvement of GRF in the development of EAE, GRFR gene homo-deficient mice (-□-), GRFR hetero-deficient mice (-△-), and wild-type mice used as controls (-●-) It is the graph which showed the presence or absence of EAE onset induced artificially about each of these.
(A) in the figure shows the transition of the average value of the EAE score. In wild-type mice, the onset of EAE (score 1) began to appear on the 16th day after sensitization, and the score reached 1.2 on the 25th day after sensitization. On the other hand, no onset was observed in the GRFR homo-deficient mice throughout the entire observation period (27 days). Although GRAE hetero-deficient mice developed EAE, the average score value was significantly lower than that of wild-type mice. (B) in the figure shows the transition of the incidence of EAE. The GRFR homo-deficient mice had an onset rate of 0 throughout the observation period. In addition, the incidence rate of EAE was significantly lower in GRFR hetero-deficient mice than in wild-type mice.
FIG. 2 is a graph showing the results of inducing EAE onset in PLSJLF1 mice by MBP sensitization and pertussis toxin administration, and examining the effects of GRF antagonist MZ-4-71 administration on EAE onset. It is.
(A) in the figure shows the average transition of the EAE score. In the figure, the period indicated by the black thick line indicates the period when MZ-4-71 or physiological saline was administered as a control. In the saline administration group, onset was observed from day 12 after sensitization, and the average score reached 4.2 on day 16 after sensitization. On the other hand, in the MZ-4-71 administration group, onset was observed from the 11th day after the sensitization, but after 13 days after the sensitization, the average score reached 0.3, and once the remission was introduced, 17 days after the sensitization All mice were in remission with an average score of 0. Recurrence was observed on day 20 after sensitization, but the maximum average score remained at 2.2 (23 days after sensitization) throughout the subsequent observation period. From these results, it was shown that the onset of EAE is suppressed by administration of the GRF antagonist. (B) in the figure shows the transition of the incidence. In the figure, the period indicated by the black thick line indicates the period when MZ-4-71 or physiological saline was administered as a control. In the physiological saline administration group, rapid onset was observed from the 12th day to the 13th day after the sensitization, and the onset rate reached 80% on the 13th day after the sensitization. On the other hand, in the MZ-4-71 administration group, the onset started slowly, and the onset rate remained at 60% even on the 26th day after sensitization. From these results, it was shown that the administration of GRF antagonists also suppressed the onset of EAE from the point of incidence.

Claims (9)

 An anti-autoimmune disease agent characterized by searching for a substance having a growth hormone releasing factor inhibitory action among test substances using as an index the inhibition of the action of the growth hormone releasing factor via a growth hormone releasing factor receptor A method for screening candidate substances for active ingredients.  2. The screening method according to claim 1, wherein a substance having a growth hormone releasing factor inhibitory action is searched using as an index the binding inhibition between the growth hormone releasing factor and the growth hormone releasing factor receptor. A method for screening a candidate substance for an active ingredient of an anti-autoimmune disease agent according to claim 2, comprising the following steps (i), (ii) and (iii):
(i) in the presence of a test substance, contacting the growth hormone-releasing factor receptor or a homologue grayed and growth hormone releasing factor or a homologue grayed,
(ii) The amount of growth hormone releasing factor or its homologue bound to the growth hormone releasing factor receptor or its homolog is measured, and the amount of binding is grown with the growth hormone releasing factor receptor or its homolog in the absence of the test substance. and comparing the amount of binding of growth hormone releasing factor or homologue thereof to growth hormone releasing factor receptor or homolog thereof obtained (control binding amount) by contacting the hormone releasing factor or its homologs and,
(iii) A step of selecting a test substance that decreases the binding amount compared to the control binding amount based on the result of (ii) above.  In addition to steps (i), (ii) and (iii) according to claim 3, (iv) a test which does not exhibit the same action as a growth hormone releasing factor among the test substances selected in (iii) above The method of screening the candidate substance of the active ingredient of the anti-autoimmune disease agent of Claim 3 including the process of selecting a substance. A screening method for an active ingredient of an anti-autoimmune disease agent, which comprises searching for a substance having a growth hormone releasing factor inhibitory action from among test substances, using expression inhibition of growth hormone releasing factor receptor as an index. A method for screening a candidate substance for an active ingredient of an anti-autoimmune disease agent according to claim 5, comprising the following steps (i), (ii) and (iii):
(I) contacting the test substance with a cell capable of expressing a growth hormone releasing factor receptor;
(Ii) The expression level of the growth hormone releasing factor receptor in the cell contacted with the test substance is measured, and the expression level is not contacted with the test substance and the cell capable of expressing the corresponding growth hormone releasing factor receptor (control) Comparing the expression level of the growth hormone releasing factor receptor (control expression level) of the cell),
(iii) A step of selecting a test substance that reduces the expression level of the growth hormone releasing factor receptor compared to the control expression level based on the comparison result of (ii) above. A candidate substance for an active ingredient of an anti-autoimmune disease agent, characterized in that a substance having a growth hormone releasing factor inhibitory action is searched from test substances using as an index the inhibition of expression or secretion of growth hormone releasing factor. How to screen. A method for screening a candidate substance for an active ingredient of an anti-autoimmune disease agent according to claim 7, comprising the following steps (i), (ii) and (iii):
(I) contacting the test substance with a cell capable of expressing a growth hormone releasing factor;
(Ii) measuring the expression level or secretion level of the growth hormone releasing factor of the cells contacted with the test substance, and measuring the expression level or secretion level of the growth hormone releasing factor of the corresponding control cell not contacting the test substance Comparing the expression level (control expression level) or secretion level (control secretion level) of
(iii) A step of selecting a test substance that reduces the expression level or secretion amount of the growth hormone releasing factor compared to the control expression level or control secretion level based on the comparison result of (ii).  The screening method according to any one of claims 1 to 8, wherein the anti-autoimmune disease agent is a prophylactic or therapeutic agent for multiple sclerosis, rheumatoid arthritis or systemic lupus erythematosus.

Images