WO2006106599A1

WO2006106599A1 - Pharmaceutical for preventing and/or treating disease caused by abnormal enhancement of extracellular domain shedding

Info

Publication number: WO2006106599A1
Application number: PCT/JP2006/303687
Authority: WO
Inventors: Eiichiro Nishi
Original assignee: Kyoto University
Priority date: 2005-03-01
Filing date: 2006-02-28
Publication date: 2006-10-12
Also published as: JPWO2006106599A1

Abstract

A pharmaceutical for preventing and/or treating a disease caused by the abnormal enhancement of extracellular domain shedding, containing a nardilysin inhibitor (such as a substance that inhibits the expression of nardilysin by RNAi) as an active ingredient.

Description

Drugs for prevention and z or treatment of diseases caused by abnormal enhancement of extracellular domain shedding

Technical field

[oooi] The present invention relates to a drug for the prevention and Z or treatment of diseases caused by abnormal enhancement of extracellular domain shedding, specifically, HB-EGF, TNF-a, TGF-a, or amphiregulin. The present invention relates to a medicament containing a nardilysin inhibitor that suppresses extracellular domain shedding of membrane-bound precursors such as (Amphiregulin).

Background art

Extracellular domain seeding (hereinafter sometimes referred to as “seeding” in the present specification) is an extracellular domain of a transmembrane protein by proteolysis in the vicinity of the membrane. Is a phenomenon in which is irreversibly cut. Since extracellular domain seeding is induced by various stimuli, it is considered to be under strict control in vivo and to play an important physiological role through the regulation of membrane protein activity. . In addition, it is known that some cyto force-in (TNF-α, etc.) and growth factors (TGF-a, ΗΒ-EGF, etc.) are activated and become active, causing inflammation in the body. It is considered that the abnormal increase in shedding is deeply involved.

[0003] As a cleavage enzyme responsible for shading, a group of molecules belonging to the a disintegrin and metalloprotease (ADAM) family and matrix metalloproteinase (MMP) family has been reported so far! /. Among them, TACE (TNF-a converting enzyme; ADAM 17) (Nature, 385, 729, 1997; Nature, 385, 733, 1997), identified as a cleaving enzyme for TNF-a, is derived from knockout mice. Cell studies have reported that it is involved in the shedding of a wide range of membrane proteins, including membrane-bound precursors of HB-EGF (Chem. Rev., 102, 4 627, 2002). The mechanism involved in the induction of shedding in the living body by the above cleaving enzyme such as TACE has been elucidated in detail.

[0004] Therapeutic methods that target the biological activity of growth factors or cytodynamic ins that have been activated by shading have already been applied clinically. For example, E for the treatment of colorectal cancer Antibodies that inhibit the binding of GF receptor ligands (EGF, TGF-α, HB-EGF, etc.) to the same receptor, for the treatment of lung cancer, EGF receptor phosphate inhibitors, rheumatoid arthritis, clones An antibody against TNF-a and a soluble TNF-a receptor are used for the treatment of diseases and the like. However, for example, the EGF receptor phosphate inhibitor (trade name Iressa), which was first approved in Japan as a treatment for lung cancer, has been reported to cause many deaths due to side effects (interstitial pneumonia). Leaving a problem with sex. Therefore, there is an expectation for the development of a treatment by suppressing shedding as a treatment with few side effects.

[0005] As a therapeutic method targeting seeding, a low molecular weight TACE inhibitor has been developed, but has not yet been completed. In addition, a wide variety of MMP inhibitors have been developed, and many of these MMP inhibitors suppress the activity of ADAM proteins including TACE. However, almost all clinical trials of these MMP inhibitors for malignant tumors have been discontinued due to problems such as the appearance of arthritis-like side effects, among which good results have been obtained for malignant tumors. Therefore, in order to establish a therapy that targets inflammatory site force-in or shedding of membrane-bound precursors of growth factors, TACE or There is a need to discover effective targets that can replace MMPs.

[0006] On the other hand, nardilysin, a meta-oral endopeptidase, has been purified and identified. This protein specifically binds to soluble HB-EGF, a growth factor of the EGF family, and is soluble by soluble HB-EGF. It has been reported to enhance cell migration (EMBO. J., 20, 3342, 2001). However, to date, there is no report that nardilysin is involved in extracellular domain shedding.

Disclosure of the invention

Problems to be solved by the invention

[0007] An object of the present invention is to provide a medicament for prevention and Z or treatment of a disease caused by abnormal increase in extracellular domain sieving.

Means for solving the problem

[0008] In the process of pursuing the physiological activity of nardilysin, the present inventors Newly found to bind to compound precursors, and further enhances nardilysin force HB-EGF membrane-bound precursor shedding, associates with TACE, enhances TACE activity, and HB-EGF It was found that the seeding of membrane-bound precursors of other TACE substrate proteins (such as TNF-a) was also enhanced. The present invention has been completed based on these findings.

[0009] That is, the present invention provides a medicine for prevention, Z or treatment of a disease caused by abnormal enhancement of extracellular domain seeding, comprising a nardilysin inhibitor as an active ingredient. According to a preferred embodiment of the present invention, extracellular domain seeding or extracellular domain seeding in which extracellular domain seeding involves TACE is HB-EGF, TNF-a, TGF-a, or amphiregulin. The above-mentioned medicament, which is an extracellular domain shade of a membrane-bound precursor, is provided.

According to a further preferred embodiment of the present invention, the disease caused by abnormally increased extracellular domain seeding is an inflammatory disease! /, Any of the pharmaceuticals, a nardilysin inhibitor inhibits the expression of nardi lysin. Any of the above medicaments containing the substance to be treated, and the medicament wherein the inhibition is RNAi inhibition.

[0010] According to another aspect of the present invention, there is provided a disease caused by abnormal enhancement of extracellular domain modeling, comprising a step of administering a prophylactic and Z or therapeutically effective amount of a nardilysin inhibitor to a mammal including a human. Methods of prevention and Z or treatment are provided.

According to still another aspect of the present invention, there is provided use of a nardilysin inhibitor for the manufacture of a medicament for the prevention and Z or treatment of a disease caused by abnormal enhancement of extracellular domain seeding.

Brief Description of Drawings

[0011] [Fig.l] Western blot showing seeding of membrane-bound precursor of HB-EGF by nardilysin and TACE.

FIG. 2 is a mass spectrometry spectrum of a peptide chain near the membrane of a membrane-bound precursor of HB-EGF in the presence of TACE and / or nardilysin.

FIG. 3 is a Western blot showing sieving of membrane-bound precursors of TGF-a and amphiregulin by nardilysin. FIG. 4 is an ELI SA measurement result showing seeding of a membrane-bound precursor of TNF-a by nardilysin and TACE.

[Fig. 5] Western blot showing increased nardilysin expression on the cell surface by a seeding inducer.

[Fig. 6] Western plot showing increased nardilysin-TACE complex formation by seeding inducer.

FIG. 7 is a graph showing inhibition of seeding of HB-EGF membrane-bound precursor by siRNA against nardilysin.

FIG. 8 is a graph showing suppression of HB-EGF seeding induction by anti-nardilysin polyclonal antibody.

[Fig. 9] Draft showing inhibition of HB-EGF shedding induction by nardilysin-derived peptide chain.

BEST MODE FOR CARRYING OUT THE INVENTION

[0012] Diseases resulting from abnormal enhancement of extracellular domain seeding include diseases involving an increase in site force-in and growth factors, etc., in which membrane-bound precursors undergo seeding to become active. . For example, heparin-binding EGF-like growth factor (HB-EGF), one of the members of the EGF family, is upregulated in liver cancer, spleen cancer, breast cancer, etc. It is known that cell carcinogenesis is induced (see Growth Factors, 22, 253, 2004). As used herein, the term “abnormal enhancement of extracellular domain shedding” includes a state in which extracellular domain shedding increases due to the presence of a membrane-bound precursor whose expression level is increased! It has been reported that cardiac hypertrophy was suppressed by suppressing the shedding of the membrane-bound precursor of HB-EGF (see Nat. Med., 8, 35, 2002).

[0013] Extracellular domain shedding involving TACE includes growth factor TNF-a, TGF-α, HB-EGF, amphiregulin, NRG-a2C, and membrane binding of fractal force-in (Fractalkine) Type precursors: R75 TNF-a RII, R55 TNF—a R ゝ CD30, IL—6Ra ゝ I L-1R II, GHR, ErbB4, and Notch; L-selectin as an adhesion molecule; other bAPP (b j8 -. Shiwedeingu like of amyloid precursor) are mentioned (Chem Rev., 102, 4627, 2002) 0 slave Therefore, diseases caused by abnormally increased extracellular domain seeding involving TACE include inflammatory diseases such as chronic indirect rheumatism and Crohn's disease, liver cancer, spleen cancer, Examples include tumors such as breast cancer, arteriosclerosis, hypertrophic cardiomyopathy, and Alzheimer's disease.

[0014] nardilysin (N-arginine dibasic convertase; NRDc) is a metalloproteinase belonging to the M16 family, and its protease activity is isolated and purified (J. Biol. Chem., 269, 2056, 19 94), gene sequence ( Proc. Natl. Acad. Sci. USA, 91, 6078, 1994) has already been reported. In addition, the present inventors have reported the binding activity with soluble HB-EGF as described above (EMBO. J., 20, 3342, 2001). The most homologous protein with nardilysin is an insulin-degrading enzyme that also belongs to the M16 family, but it is a major feature of nardilysin that a highly acidic domain is inserted in the enzyme domain conserved among the same family. . Nardilysin expression is extensive, but in adults it is particularly high in testis, skeletal muscle, and heart. The present inventors have shown that nardilysin enhances the shedding of membrane-bound precursors of HB-EGF in both experiments in which purified recombinant nardilysin was added to cells and in experiments where nardilysin was expressed in cells using an expression vector. heading, also make and be active to enhance Shedeingu to nardilysin variants without enzyme activity by another experiment ₀

[0015] Furthermore, the present inventors have found that nardilysin enhances the effect of TACE on seeding (Example 1), as shown in Examples described later. At that time, nardilysin, TACE, and HB-EGF formed a complex based on the present inventors' study using an immunoprecipitation method using an antibody against TACE, and a pull-down assay using purified protein was performed. Based on our study by Sei, nardilysin and TACE are bound directly.

[0016] Further, the following results in the examples described below:

• Addition of nardilysin recombinant protein increases TACE peptide cleavage activity (Example 2)

• nardilysin enhances seeding of HB-EGF as well as other TACE substrates (TGF-α, amphiregulin, TNF-a, etc.) (Examples 3 and 4).

The seeding inducer increases the expression of nardilysin on the cell surface (Example 5). 'Shieding inducer increases nardilysin-TACE complex formation (Example 6). • Suppression of nardilysin expression also inhibits HB-EGF seeding induction (Example 7). When cells are activated by seeding-inducing factors such as phorbol ester and inflammatory site force-in

(1) Cytoplasmic force Nardilysin moves to the cell surface,

(2) By combining with nardilysin force STACE on the cell surface,

(3) TACE is activated,

(4) Extracellular domain seeding of membrane proteins is thought to be enhanced.

Similarly, nardilysin may increase the effect on the shedding of enzymes other than TACE belonging to the ADAM family.

[0017] The nardilysin inhibitor used in the present invention inhibits the association of nardilysin and TACE in addition to a substance that inhibits the expression of nardilysin or a substance that acts on nardilysin to inhibit the activity and function of nardilysin. Substances, substances that inhibit the binding of nardilysin and membrane-bound protein undergoing shedding, or substances that inhibit the formation of a complex of nardilysin, TACE, and membrane-bound protein undergoing shedding. In this specification, the term “inhibition” includes the meaning of suppression or reduction.

Substances that inhibit the expression of nardilysin include RNAi, substances using the antisense method or ribozyme method, and the like. Although not particularly limited, siRNAs using RNAi are preferred. Examples of substances that act on nardilysin and inhibit the activity and function of nardilysin include low molecular weight compounds and antibodies. As a substance that inhibits the association of nardilysin and TACE, a substance that inhibits the binding of nardilysin and membrane-bound proteins that undergo seeding, or a substance that inhibits the formation of a complex of membrane-bound proteins that undergo nardylysin, TACE, and shedding Can be a low molecular weight compound, an antibody, or a peptide.

[0018] As the antibody, for example, an antibody prepared using a peptide having a full-length nardilysin or a peptide having a partial sequence of nardilysin described below as an immunogen can be used. As the full length nardilysin, for example, recombinant human nardilysin can be used. Antibody production should be done according to conventional methods. The antibody is preferably a monoclonal antibody.

Examples of the peptide include a partial sequence of nardilysin (for example, SEQ ID NO: 2 in the sequence listing). Peptides having the amino acid sequence from the 472th amino acid to the 492th amino acid sequence of the amino acid sequence described).

[0019] RNAi (RNA interference) means a phenomenon in which the expression of a gene having the same sequence as a double-stranded RNA introduced into a cell is suppressed. Specific examples of substances that inhibit the expression of nardilysin by RNAi include siRNA or shRNA as described below.

[0020] siRNA is an abbreviation for short interfering RNA, and refers to double-stranded RNA having a length of about 21 to 23 bases or less. The siRNA can be in any form as long as it can cause RNAi, for example, siRNA obtained by chemical or biochemical synthesis, or synthesis in an organism, or ヽ is about 40 bases or more. It may be a short double-stranded RNA of 10 base pairs or more, etc., produced by degrading the single-stranded RNA in the body. The siRNA sequence and the nardilysin mRNA partial sequence are preferably 100% -matching, but not necessarily 100% -matching.

[0021] Preferably, the region having homology between the nucleotide sequence of siRNA and the nucleotide sequence of the nardilysin gene does not include the translation initiation region of nardirilysin gene. The sequence having homology is preferably 20 bases away from the translation initiation region of the nardilysin gene, more preferably 70 bases away. The sequence having homology may be, for example, a sequence near the 3 ′ end of the nardilysin gene.

[0022] As a substance that inhibits the expression of nardilysin by RNAi, dsRNA of about 40 bases or more that produces siRNA may be used. For example, about 70% or more, preferably 75% or more, more preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, particularly preferably 95% with respect to a part of the nucleic acid sequence of the nardilysin gene. As described above, RNA containing a double-stranded portion containing a sequence having 100% homology or a variant thereof can be used most preferably. The sequence portion having homology is usually at least 15 nucleotides or more, preferably about 19 nucleotides or more, more preferably at least 20 nucleotides or more, and further preferably 21 nucleotides or more.

[0023] As a substance that inhibits the expression of nardilysin by RNAi, shRNA (short hairpin RNA) having a short hairpin structure having a protruding portion at the 3 'end can also be used. An shRNA is a single-stranded RNA that contains a partially palindromic base sequence and is double-stranded within the molecule. It is a molecule of about 20 base pairs or more that takes a structure and becomes a hairpin-like structure. The shRNA preferably has a 3 ′ protruding end. The length of the double-stranded part is not particularly limited, but is preferably 10 nucleotides or more, more preferably 20 nucleotides or more. Here, the 3 ′ protruding end is preferably DNA, more preferably DNA of at least 2 nucleotides, and further preferably DNA of 2 to 4 nucleotides.

[0024] The substance that inhibits the expression of nardilysin by RNAi may be artificially chemically synthesized, or DNA with a hairpin structure in which the DNA sequences of the sense strand and antisense strand are linked in the reverse direction is converted by T7 RNA polymerase. It can also be made by synthesizing RNA in vitro. When synthesized in vitro, antisense and sense RNAs can be synthesized from saddle-type DNA using T7 RNA polymerase and T7 promoter. When these are annealed in vitro and then introduced into cells, RNAi is induced and nardilysin expression is suppressed. Introduction into cells can be carried out, for example, by the calcium phosphate method or a method using various transfection reagents (for example, oligofectamine, Lipofectamine, lipofection, etc.).

[0025] As a substance that inhibits the expression of nardilysin by RNAi, an expression vector containing a nucleic acid sequence encoding the above-described siRNA or shRNA may be used. Further, a cell containing the expression vector may be used. The types of expression vectors and cells described above are not particularly limited, but expression vectors and cells that have already been used as pharmaceuticals are preferred.

[0026] The route of administration of the medicament of the present invention is not particularly limited, and oral administration or parenteral administration (for example, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration, rectal administration, intravaginal administration) Administration, topical administration to the affected area, skin administration, etc.). Formulation forms suitable for oral administration include solid or liquid forms, and preparation forms suitable for parenteral administration include injections, drops, suppositories, external preparations, eye drops, nasal drops, etc. Is mentioned. The medicament of the present invention may be in the form of a sustained-release preparation. The pharmaceutical agent of the present invention may be added with a pharmaceutically acceptable additive as necessary depending on the preparation form. Specific examples of pharmaceutically acceptable additives include, for example, excipients, binders, disintegrants, lubricants, antioxidants, preservatives, stabilizers, tonicity agents, coloring agents, taste masking agents. Agent, diluent, emulsifier, suspending agent, solvent, filler, extender, buffer, delivery vehicle, diluent, key Carriers, excipients and z or pharmaceutical adjuvants.

[0027] The pharmaceutical agent of the present invention in the form of an oral solid preparation includes, for example, an excipient added to the nardilysin inhibitor, which is an active ingredient, and, if necessary, a binder, a disintegrant, a lubricant, and a colorant. Or, it can be prepared as a tablet, a granule, a powder, or a capsule by a conventional method after adding additives for formulation such as a corrigent. The pharmaceutical preparation of the present invention in the form of an oral liquid preparation is prepared by adding one or more kinds of pharmaceutical additives such as a corrigent, stabilizer, or preservative to the nardilysin inhibitor that is an active ingredient, and using a conventional method. It can be prepared as an internal solution, syrup, elixir or the like.

[0028] The solvent used to formulate the drug of the present invention as a liquid preparation may be either aqueous or non-aqueous. Liquid preparations can be prepared by methods well known in the art. For example, an injection is prepared by dissolving in a saline solution, a buffer solution such as PBS, a solvent such as sterilized water, sterilizing by filtration with a filter, etc., and then filling in a sterile container (for example, an ampoule). can do. This injection may contain a conventional pharmaceutical carrier, if necessary. Alternatively, an administration method using a non-invasive catheter may be used. Examples of carriers that can be used in the present invention include neutral buffered physiological saline, physiological saline containing serum albumin, and the like.

[0029] When the active ingredient is a substance that inhibits the expression of nardilysin by RNAi, the medicament of the present invention may be in the form of a non-viral vector or a viral vector. Such administration forms are known in the art. For example, separate experimental medicine "Basic technology of gene therapy" Yodosha, 1996; separate experiment medicine "Gene transfer & expression analysis experiment method" Yodosha, References such as 1997 can be made.

Non-viral vector forms include nucleic acid molecule introduction methods using ribosomes (liposome method, HVJ-ribosome method, catonic ribosome method, lipofussion method, ribojetamine method, etc.), microinjection method, gene gun (Gene Any form that uses a method of transferring nucleic acid molecules into cells together with carriers (metal particles) in Gun) may be used. Examples of expression vectors include pCAGGS, pBJ-CMV, pcDNA3.1, pZeoSV (available from Invitrogen or Stratagene).

[0030] As a viral vector form, a virus such as a recombinant adenovirus or a retrovirus. Vectors can be used. Detoxified retroviruses, adenoviruses, adeno-associated winoles, henorepesuinores, vaccinia winoles, box winores, poliowinoles, Sindbiswinores, Sendai virus, SV40 and other DNA viruses or RNA viruses by R NAi A gene can be introduced into a cell or tissue by introducing a DNA that expresses a substance that inhibits the expression of nardilysin and infecting the cell or tissue with a recombinant virus.

A substance that inhibits the expression of nardilysin by RNAi may be directly injected into a living organ or tissue.

[0031] The dosage of the medicament of the present invention depends on the purpose of use, the severity of the disease, the patient's age, weight, sex, medical history, or the type of substance that inhibits the expression of nardilysin by RNAi as an active ingredient. In view of this, it can be determined by one skilled in the art. When the active ingredient is a substance that inhibits the expression of nardilysin by RNAi, for example, the dose of the medicament of the present invention is about 0.1 ng to about 100 mg / kg, preferably about When administered as a virus vector or non-viral vector, it is usually 0.0001 to 100 mg, preferably 0.001 to 10 mg, more preferably 0.01 to 1 mg.

[0032] The administration frequency of the medicament of the present invention may be, for example, once a day to once every several months. When a substance that inhibits the expression of nardilysin by RN Ai is used, it is generally effective for 1 to 3 days after administration, so it is preferable to administer it once every 3 days. When using an expression vector, administration once a week may be appropriate.

Example

[0033] Power to further explain the present invention by the following examples The present invention is not limited by the examples. In the examples, nardilysin prepared an expression vector so as to start at the position 283 of the cDNA of SEQ ID NO: 1 in the sequence listing (the amino acid sequence of the expressed product and the methionine at position 50 of SEQ ID NO: 2 in the sequence listing).

(Example 1) Seeding of membrane-bound precursor of HB-EGF by nardilysin and TACE

Figure 1 shows the expression vector of nardilysin, TACE with V5 tag at the C terminus and HB-EGF with H (hemadalchun) tag at the N terminus in COS7 cells. The combination was transferred. DMEM containing BSA (0.1%) after 20 hours After 4 hours, the medium and cell lysate were collected (cell lysis buffer; 10 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, protease inhibitor cocktail). The secreted HB-EGF secreted into the collected medium is partially purified with cellulose agarose beads, developed on SDS-PAGE, transferred to a nitrocellulose membrane, and anti-HA tag antibody western Blots were performed. The cell lysate was similarly subjected to Western blotting with anti-V5 tag antibody and anti-TACE antibody. The results are shown in Figure 1. Fig. 1 shows the anti-V5 antibody plot (nardilysin) of the cell lysate, the anti-TACE antibody plot (TACE) of the cell lysate, and the anti-HA antibody blot (HB-EGF) of the medium.

From the results shown in Fig. 1, the soluble HB-EGF force secreted into the medium of cells expressing nardilysin and TACE. It could be expressed in the medium of cells expressing only nardilysin or only TACE. It can be seen that there is a marked increase compared to the melted HB-EGF.

[Example 2] Cleavage of the peptide chain corresponding to the cleavage site near the membrane of the membrane-bound precursor of HB-EGF by nardilysin and TACE

A peptide chain consisting of 14 amino acids (GLSLPVENRLYTYD: SEQ ID NO: 3 in the sequence listing) (0.5 mM) corresponding to the cleavage site near the membrane of the membrane-bound precursor of HB-EGF was added to the reaction buffer only (25 mM). Tris (pH 9), 2 ^ M ZnCl) (Fig. 2, upper panel; (-)), recombinant nardilysin (100 μg / ml)

2

(Data not shown), recombinant TACE (25 μg / ml) (FIG. 2 middle; TACE), recombinant nardily sin (100 μg / ml) and recombinant TACE (25 μg / ml) (bottom of FIG. 2; TACE + nardilysin) for 8 hours at 37 ° C, and then the cleavage of the peptide chain was analyzed by mass spectrometry (MALDI-TOF). The result is shown in figure 2.

With nardilysin alone, the results were similar to those of the reaction buffer alone, and no significant peptide cleavage was observed. TACE alone has already been reported, and cleavage at the site was observed, and two types of peptide chains (indicated by arrows in Fig. 2) produced by the cleavage were detected.

In the sample to which both TACE and nardilysin were added, the cleavage of the peptide at the same site as that of TACE was significantly increased.

[0035] (Example 3) Shading of TGF-a and amphiregulin membrane-bound precursors in the presence of nardilysin

TGF-o with HA tag at the N-terminus; Amphiregulin expression vector Transfect COS7 cells, incubate with recombinant nardilysin, soluble proteins generated by extracellular domain seeding of these membrane-bound precursors, TGF-α is immunoprecipitated with anti-HA antibody, Amphiregulin was partially purified with heparin agarose beads, and then Western plotting with an anti-HA tag antibody was performed in the same manner as in Example 1. The results are shown in Figure 3. As can be seen from Fig. 3, recombinant nardilysin over InM clearly induced seeding of these membrane-bound growth factors.

[0036] (Example 4) Shedding of TNF-a membrane-bound precursor (pro TNF-a) by nardilysin and TACE

The expression vector described below was transfected into COS7 cells, and after 20 hours, the medium was replaced with BMEM (0.1%)-containing DMEM. After 4 hours, the medium was recovered and pro-TNF-α was cleaved. The soluble TNF-α concentration in the medium was measured by ELISA. The results are shown in Fig. 4. In the figure, the control indicated by diamonds is the pro TNF-α expression vector only; the square NRDc is the expression vector for pro TNF-α and nardilysin; the TACE indicated by the triangle is the expression vector for pro TNF-α and TACE; The TACE + NRDc shown is the result in COS7 cells transfected with pro TNF-a, nardilysin, and TACE expression vectors.

Similar to HB-EGF, soluble TNF-a in the medium of cells expressing both nardilysin and TACE Compared with soluble TNF-a in the medium of cells expressing nardilysin alone or TACE alone It was remarkably versatile.

[0037] (Example 5) Increased expression of nardily sin on the cell surface by phorbol ester (seeding inducer)

RAW264.7 (mouse macrophage cell line) was stimulated with phorbol ester-free (-) and with (PMA) for 5 or 30 minutes, and the cell surface was piotinized and the cell lysate was collected. The lysate was immunoprecipitated with anti-nardilysin antibody, and Western blotting was performed with streptavidin (upper part of FIG. 5: avidin) and anti-nardilysin antibody (lower part of FIG. 5: NRDc). The results are shown in Fig. 5. It can be seen that the expression of nardilysin on the cell surface (upper panel in Fig. 5) is clearly increased by phorbol ester stimulation, although the total amount of nardilysin (Fig. 5 lower panel) is not significantly changed.

[0038] (Example 6) Increased nardilysin-TACE complex formation by phorbol ester

HEK293T cells transiently expressing nardilysin and TACE (Figure 6 left panel), and MK N45 cells (gastric cancer cell line: right panel in Fig. 6) were stimulated with phorbol ester without (-) and with (PM A) for 2 hours, and then cell lysate was collected. The lysate was immunoprecipitated with anti-TACE antibody, and Western blotting was performed with anti-nardilysin antibody (upper: NRDc) and anti-TACE antibody (middle). The lower panel shows the stamp lot of the cell lysate with anti-nardilysin antibody. In both cells, the total amount of nardilysin and TACE is not changed by phorbol ester stimulation. The amount of nardilysin co-precipitated by anti-TACE antibody, that is, the amount of nardilysin- TACE complex is increased. I understand that.

[Example 7] Suppression of HB-EGF membrane-bound precursor seeding inhibition by nardilysin expression Stable expression of HB-EGF precursor with alkaline phosphatase (AP) tag attached to the N-terminus The 293T cells were transfected with a siRNA cocktail against nardilysin (Dharmacon, siGEN OME SMARTpool). As a control, 293T cells transfected with siRNA against cyclophilin were similarly prepared. After 72 hours, the medium was changed to BMEM (0.1%)-containing DMEM, stimulated with phorbol ester (PMA: 100 nM) for 1 hour, and AP activity in the medium (proportional to the level of HB-EGF shedding) It was measured. Figure 7 shows the results. As can be seen from FIG. 7, the AP activity of PMA is clearly increased compared to the unstimulated control. Cells transfected with siRNA against nardilysin are cells transfected with siRNA against cyclophilin. Compared with, the degree of increase decreased. When Western blotting was performed on cells treated in the same manner, it was confirmed that the expression of nardilysin protein was reduced by 30-40% by siRNA. From these results, it was shown that the shedding of the membrane-bound precursor of HB-EGF can be suppressed by suppressing the expression of nardilysin.

[0040] (Example 8) Inhibition of HB-EGF seeding induction by anti-nardilysin polyclonal antibody

HEK293T cells stably expressing an HB-EGF membrane-bound precursor with an alkaline phosphatase (AP) tag added to the N-terminus were treated with control mouse IgG (control IgG; 10 μg / ml) or mouse anti-nardilysin polyclonal antibody ( In the presence of anti-NRDc (poly); 10 g / ml), no phorbol ester (black), with (white) was stimulated for 2 hours, and the medium was collected and AP activity was measured. AP activity corresponds to the amount of HB-EGF released into the medium, ie, the amount of shedding of membrane bound precursors. The results are shown in FIG. The data should be displayed as the average of 7 independent experiments plus the standard error! From FIG. 8, it is clear that the anti-nardilysin antibody significantly suppresses the shelving induction of HB-EGF by phorbol ester.

[0041] (Example 9) Suppression of HB-EGF shedding induced by nardilysin-derived peptide chain

Streptavidin agarose beads alone (-), or part of human nardilysin in the medium of HEK293T cells stably expressing HB-EGF membrane-bound precursor with alkaline phosphatase (AP) tag added to the N-terminus The peptide chain (peptide 41; SGSG SILSFLRKKCWALALFGGNGE: SEQ ID NO: 4 in the sequence listing) having a sequence (the amino acid sequence from the 472th position to the 492th position of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing) was subjected to a thiotin modification. After binding the sample to the above beads, the sample was further stimulated for 2 hours without phorbol ester (black) or with (white), and the medium was collected and AP activity was measured. The results are shown in FIG. Data are expressed as the mean plus standard deviation (n = 4) of 4 independent experiments with similar results. Peptide chain (41) significantly inhibited HB-EGF shedding induction by phorbol ester. Peptide chain (41) is one of the strongest anti-shedding effects of HB-EGF, which was screened for 92 types of peptide chains covering the entire length of human nardilysin.

Industrial applicability

[0042] According to the present invention, there is provided a medicament for preventing and / or treating a disease caused by abnormal increase in extracellular domain shedding. This drug is useful as a drug for the prevention and Z or treatment of inflammatory diseases.

Claims

The scope of the claims

[1] A drug for the prevention and Z or treatment of a disease caused by abnormal increase in extracellular domain shedding, comprising a nardilysin inhibitor as an active ingredient.

[2] Extracellular domain seeding force The medicament according to claim 1, which is extracellular domain seeding involving TACE.

[3] The pharmaceutical according to claim 1 or 2, wherein the extracellular domain shedding is extracellular domain shedding of a membrane-bound precursor of HB-EGF, TNF-a, TGF-a, or amphiregulin.

[4] The disease caused by abnormally increased extracellular domain seeding is an inflammatory disease.

The medicine according to any one of 1 to 3.

[5] The medicine according to any one of [1] to [4], wherein the nardilysin inhibitor comprises a substance that inhibits the expression of nardilysin.

6. The medicine according to claim 5, wherein the inhibition is inhibition by RNAi.

[7] The medicament according to any one of [1] to [4], wherein the nardilysin inhibitor comprises an antibody produced by using a full-length nardilysin or a peptide having a partial sequence of nardilysin as an immunogen.

[8] The medicament according to any one of [1] to [4], wherein the nardilysin inhibitor comprises a peptide having a partial sequence of nardilysin.