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WO2003066089A1 - Ligands de la megsine - Google Patents

Ligands de la megsine Download PDF

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Publication number
WO2003066089A1
WO2003066089A1 PCT/JP2003/001316 JP0301316W WO03066089A1 WO 2003066089 A1 WO2003066089 A1 WO 2003066089A1 JP 0301316 W JP0301316 W JP 0301316W WO 03066089 A1 WO03066089 A1 WO 03066089A1
Authority
WO
WIPO (PCT)
Prior art keywords
megsin
ligand
activity
test compound
plasmin
Prior art date
Application number
PCT/JP2003/001316
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English (en)
Japanese (ja)
Inventor
Kiyoshi Kurokawa
Toshio Miyata
Original Assignee
Kiyoshi Kurokawa
Toshio Miyata
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kiyoshi Kurokawa, Toshio Miyata filed Critical Kiyoshi Kurokawa
Priority to JP2003565512A priority Critical patent/JPWO2003066089A1/ja
Priority to AU2003244385A priority patent/AU2003244385A1/en
Publication of WO2003066089A1 publication Critical patent/WO2003066089A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to ligands for proteins expressed in kidney cells.
  • the ligands of the present invention can be applied to the treatment of mesangial proliferative glomerulonephritis, and to Z or prophylaxis. Background art
  • Mesangium is located at the center of the leaflet of the capillary loop of the glomerulus and is the core tissue that connects the leaflets.
  • the mesangium is covered by the glomerular basement membrane, and the capillary lumen is connected to cells (mesangial cells) separated by endothelial cells and the inner transparent layer in the glomerular basement membrane, which consists of three layers. It is composed of intangible substances (mesangial matrix). It is known that mesangial cells play a central role in maintaining the structure and function of renal glomeruli, and in the development of glomerular diseases such as glomerulonephritis and glomerulosclerosis. It is considered to be a major factor.
  • mesangial cells also have central pathophysiological significance in various types of nephritis.
  • proliferation of mesangial cells and accumulation of extracellular glomerular matrix are important pathological findings of glomerulosclerosis in patients with various glomerular diseases such as chronic nephritis and diabetic nephropathy [D. Schlondorff, Kidney Int., 49, 1583-1585 (1996); RBSterzel et al "Glomerular mesangial cells. Immunologic Renal Diseases, pp595-626 (199
  • the present inventor isolated a gene that is particularly strongly expressed in mesangial cells by large-scale DNA sequencing and database analysis, and determined the entire nucleotide sequence thereof.
  • megsin has a biologically significant effect on mesangial function.
  • single genetic manipulation of megsin can generate experimental and early mesangial lesions present in human glomerulonephritis. Disclosure of the invention
  • the transgenic mouse of megsin which has a structural characteristic of serine protease inhibitor (SERPIN) and may be deeply involved in renal disease, progressively expands mesangial substrates and proliferates mesangial cells. , And increased immune complex deposits, indicating that nephritis may have been induced by its SERPIN activity. Therefore, finding and specifying a ligand (protease) for megsin is necessary to elucidate the biological properties of mesangial cells, to investigate the causes of diseases associated with mesangial cells, and to treat and diagnose mesangial cell-related diseases. It is effective for
  • Screening a compound that specifically inhibits the complex between megsin and a ligand can be a useful tool for the development of a therapeutic agent for nephritis.
  • the present inventors have investigated the enzymes that form a complex with megsin and the enzymes whose activities are inhibited by megsin using various serine proteases and recombinant megsin. They found that plasmin trypsin, a species, had these effects, and completed the present invention. It was known that megsin had a characteristic structure in the SERPIN superfamily, but it was not confirmed that it actually had a serine protease inhibitory action. In addition, no information has been obtained on which proteases are inhibited in many proteases.
  • the present invention relates to the following megsin ligand, and a method for treating and preventing or preventing mesangial proliferative glomerulonephritis using a megsin ligand, and a screening method.
  • a pharmaceutical composition for treating and / or preventing mesangial proliferative glomerulonephritis comprising as an active ingredient serine protease or a protein functionally equivalent to serine protease.
  • a method for evaluating the activity of a test compound that interferes with the binding between megsin and a megsin ligand comprising the following steps, wherein the megsin ligand comprises a serine protein
  • a method for inhibiting the activity of megsin comprising the step of bringing a compound obtainable by the screening method according to [5] into contact with megsin.
  • a pharmaceutical composition for treating mesangial proliferative glomerulonephritis and for preventing or preventing Z, comprising a compound obtainable by the screening method according to [5] as an active ingredient.
  • An inhibitor of plasmin and / or trypsin activity comprising megsin or a protein functionally equivalent to megsin as an active ingredient.
  • a method for inhibiting the activity of plasmin and Z or trypsin comprising the step of contacting Z or trypsin.
  • treatment refers to slowing or reversing the progress of any disease or condition to which the “treatment” applies, or such disease or condition. Means to relax either.
  • prevention means
  • Treatment means maintaining a situation that does not result in either the disease or condition to which it applies.
  • mesangial proliferative glomerulonephritis refers to a renal disease involving proliferation of renal mesangial cells and an increase in mesangial matrix.
  • kidney diseases include IgA nephropathy, membranous proliferative glomerulonephritis, SLE (systemic lupus erythematosus) nephropathy, diabetic nephropathy, and cryoglobulin nephropathy.
  • the present invention relates to megsin ligand.
  • a megsin ligand is a substance that binds to megsin and interferes with its function. The present inventor has found that megsin binds to a specific protease and inhibits its enzymatic activity.
  • megsin ligands discovered by the present inventors are the serine proteases plasmin and trypsin. Therefore, a megsin ligand can be defined as a substance having an activity of interfering with the inhibitory action of megsin on serine protease. Megsin ligand is useful for modulating the action of megsin. As described above, megsin has been shown to cause renal dysfunction caused by the proliferation of mesangial cells. Therefore, substances that interfere with the action of megsin are useful for treating renal dysfunction caused by overexpression of megsin.
  • the serine protease refers to a protein hydrolase having serine at the active center.
  • the amino acid sequence constituting the active center is generally Asp-Ser-Gly. Megsin force It was revealed that SEKPIN has a characteristic structure, but it is not known that it actually binds to serine protease and inhibits its enzymatic action. Plasmin and trypsin can be mentioned as desirable serine proteases in the present invention.
  • the serine protease fragment in the present invention refers to a fragment containing an amino acid sequence constituting a region necessary for binding to megsin in an amino acid sequence constituting serine protease.
  • the fragment of the serine protease in the present invention may be any This can be clarified by preparing a library of serine proteases, collecting fragments capable of binding to megsin, and analyzing the amino acid sequence.
  • a fragment of serine protease can be obtained, for example, by digestion with a protease.
  • a fragment encoding a serine protease can be obtained by isolating the cDNA encoding the serine protease and expressing the fragment.
  • the gene fragment is prepared, for example, by amplifying a target region by PCR.
  • Plasmin which is a megsin ligand of the present invention, is known as a fibrinolytic enzyme that hydrolyzes L-arginine and L-lysine peptides and esters, and particularly solubilizes fibrin.
  • plasmin was not known to have ligand activity for megsin.
  • Trypsin is a serine protease that hydrolyzes L-arginyl and L-lysyl carboxyl-linked peptides, amides and esters. Trypsin was also not known to have ligand activity for megsin.
  • the present invention relates to a pharmaceutical composition for treating and / or preventing mesangial proliferative glomerulonephritis, comprising as an active ingredient a serine protease or a protein functionally equivalent to a serine protease.
  • the present invention also relates to a method for treating mesangial proliferative glomerulonephritis, and a method for preventing or preventing Z-proliferative glomerulonephritis, comprising the step of administering serine protease or a protein functionally equivalent to serine protease.
  • the present invention relates to the use of a serine protease or a protein that is functionally equivalent to a serine protease in the manufacture of a pharmaceutical composition for the treatment and prevention of mesangial proliferative glomerulonephritis.
  • the serine protease used in the pharmaceutical composition of the present invention can be, for example, plasmin or trypsin.
  • the protein functionally equivalent to the serine protease is megsin.
  • a protein having an activity of regulating the inhibitory effect of megsin on serine protease by binding to Such a protein may be, for example, a fragment of a serine protease containing a binding domain to megsin. Fragments of serine proteases can be obtained by cleaving proteins. Methods for cleaving specific amino acid sequences by enzymatic or chemical reactions are known.
  • the stringent conditions generally indicate the following conditions. That is, hybridization is performed at 4 ⁇ SSC at 65 ° C., and the plate is washed with 0.1 ⁇ SSC at 65 ° C. for 1 hour.
  • Tm melting temperature
  • a functionally equivalent protein in the present invention preferably has an amino acid sequence constituting serine proteinase of at least 85%, more preferably at least 95%, homologous search by FASTA or BLAST homology search. Includes proteins consisting of amino acid sequences with liposomes.
  • a functionally equivalent protein comprises an amino acid sequence comprising one or more amino acids in the amino acid sequence constituting the protein, wherein the amino acid sequence has substitutions, deletions, insertions, and additions of Z or amino acids. Binds to megsin and binds megsin serine protein And proteins having an activity of regulating an inhibitory effect on the enzyme.
  • the amino acid sequence of plasminogen which is a precursor of plasmin, and the nucleotide sequence of cDNA encoding it are known (J. Biol. Chem. 1990 Apr. 15; 265 / ll: 6104-ll; GenBank Accession No.NM — 000301).
  • trypsin the amino acid sequence of trypsinogen, its precursor, and the nucleotide sequence encoding it have been clarified (Gene 1986; 41 / 2-3: 305-10; GenBank Accession No. M22612).
  • the number and location of amino acid mutations in proteins are not limited as long as their functions are maintained. The number of mutations is generally within 30%, or within 20%, for example, within 10%.
  • a more preferred number of mutations is within 5% or 3% of all amino acids.
  • a particularly preferred number of mutations can be within 2% of all amino acids or within 1% of all amino acids.
  • the functionally equivalent protein of the present invention includes a case in which a mutation of several amino acids is substituted as a plurality of amino acids. Several are, for example, 5, or even 4 or 3, or 2, even 1 amino acids.
  • the amino acid to be substituted is preferably an amino acid having properties similar to the amino acid before substitution.
  • amino acids belonging to each group as shown below are amino acids having properties similar to each other within the group. Substitution of these amino acids for other amino acids in the group often does not impair the essential function of the protein. Such amino acid substitution is called a conservative substitution and is known as a technique for converting an amino acid sequence while maintaining the function of a protein.
  • Non-polar amino acids Ala, Val, Leu, lie, Pro, Met, Phe, and Trp
  • Non-charged amino acids Gly, Ser, Thr, Cys, Tyr, Asn, and Gin Acidic amino acids: Asp, and Glu
  • Such proteins are obtained by transforming other prokaryotic or eukaryotic host cells by incorporating the gene encoding the protein into the appropriate expression vector DNA. Can be expressed.
  • expression vectors include pET-3 [Studier & Moffatt, J. Mol. Biol. 189, 113 (1986)] for Escherichia coli and pEF-BOS [Nucleic Acids Research 18, 5322 for COS cells]. (1990)], pSV2-gpt [Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78, 2072 (1981)], and in the case of CHO cells, pVYl [WO 89/03874]. Respectively.
  • proteins to be fused include a histidine tag, a c-myc tag, an MBP-tag, and a GST-tag.
  • Vectors capable of expressing inserts with these tags fused are commercially available.
  • the present invention relates to a method for evaluating the activity of a test compound in interfering with the binding between megsin and a megsin ligand, comprising the following steps.
  • the megsin ligand refers to a serine protease or a fragment thereof. Examples of serine mouth proteases include plasmin-trypsin.
  • the method of evaluation is useful for screening for compounds that interfere with the binding of megsin to the megsin ligand. That is, the present invention evaluates the effect of a test compound on the formation of a complex of megsin and a megsin ligand by the above-described evaluation method, and evaluates the compound that inhibits the formation of a complex as compared to the formation of a complex in the absence of the test compound. And a method for screening a compound having an activity of inhibiting the formation of a complex between megsin and a megsin ligand.
  • the screening method of the present invention compounds that interfere with the binding between megsin and a megsin ligand are selected.
  • Such compounds can be used to control the activity of megsin. More specifically, such a compound can be used as an activity inhibitor for megsin or a method for inhibiting megsin activity. That is, the present invention relates to a megsin activity inhibitor comprising the compound selected by the screening method.
  • the present invention includes the step of administering a compound capable you to select by the screening method, the treatment of Mesangiumu proliferative glomerulonephritis, and / 7 or a method for the prevention.
  • the present invention relates to the use of a compound obtainable by the above-mentioned screening method in the manufacture of a pharmaceutical composition for treating and preventing Z or prophylaxis of mesangial proliferative glomerulonephritis.
  • the present invention also relates to a method for inhibiting the activity of megsin, comprising the step of bringing the compound selected by the screening method into contact with megsin.
  • the present invention has revealed that megsin acts as an inhibitor on serine protease.
  • the activity of megsin refers to an activity of binding to a serine protease and inhibiting its enzymatic action.
  • megsin ligand any protein selected from serine protease or a fragment thereof can be used.
  • serine proteases human plasmin and trypsin are known proteins. Methods for preparing these proteins are known. Purified proteins of plasmin and trypsin are commercially available.
  • proteins functionally equivalent to these serine proteases for example, fragments thereof can be used.
  • a protein fragment can be obtained by cleaving a full-length protein by enzymatic, chemical, or physical action, and purifying a fragment having a desired activity.
  • an oligopeptide having the required amino acid sequence as a fragment can be obtained by peptide synthesis.
  • a functionally equivalent protein specifically, for example, a protein containing an amino acid sequence constituting a binding domain to megsin can be shown.
  • the following peptides containing an amino acid sequence constituting the active center of plasmin and trypsin can be used as a protein functionally equivalent to serine protease.
  • the screening method of the present invention can be more easily carried out by labeling one of megsin and a megsin ligand on a solid phase and labeling the other.
  • a solid phase magnetic beads, the inner wall of a reaction vessel, or the like is used.
  • a known label component such as a fluorescent substance, a luminescent substance, or an enzyme active substance may be used.
  • the label can be directly bound to megsin (or a megsin ligand) or indirectly by utilizing an affinity reaction such as avidin-biotin binding.
  • megsin, megsin ligand, and a test compound are contacted under conditions under which megsin and the megsin ligand form a complex.
  • the conditions under which megsin and a megsin ligand can form a complex are specifically defined by, for example, physiological pH and salt concentration, and a temperature suitable for forming the complex. More specifically, if the salt concentration is about the same as that of physiological saline and the temperature is 20 to 40 ° C in a buffer giving pH 6-8, megsin and the megsin ligand can be complexed. Coalescence can be produced.
  • Megsin, a megsin ligand, and a test compound can be contacted in the order described in any of a) to c) above.
  • the test compound selected at this time has an effect of preventing the binding of megsin to the megsin ligand by modifying the megsin binding site of megsin ligand.
  • the oligopeptide P1-14 consisting of the amino acid sequence TEATAATGSNIVEK corresponding to positions 334-347 from the N-terminus in the amino acid sequence of megsin inhibits the inhibitory effect of megsin on enzyme activity by binding to plasmin. I have confirmed. The activity of such a peptide is detected by contacting the three components in the order as described in a), b) or d).
  • binding inhibitory activity is included in the activity of interfering with the binding between megsin / megsin ligand.
  • A Complex consisting of megsin and megsin ligand
  • the level of formation of these complexes can be easily measured by labeling any of the components constituting the complex.
  • megsin is immobilized on a solid phase and a megsin ligand is labeled and used, it is possible to know the level of complex formation of A and B using the activity of the solid phase and the labeling of the Z or liquid phase as an index. it can.
  • a tag can be attached to megsin or a megsin ligand, and a substance having an affinity for the tag can be bound to perform a pull-down assay.
  • a histidine tag or an HA tag is used as the tag.
  • the elements necessary for the screening of the present invention can be supplied as a pre-combined kit.
  • the screening kit according to the present invention includes megsin and a megsin ligand as essential components, and further includes a reaction vessel, a diluent, a negative control, a positive control, or a method for performing screening using each element. An operation manual describing the order can be included. Further, if necessary, a substrate compound and the like necessary for detecting the labeling component used in the screening can be combined.
  • test compound examples include natural or synthetic compounds, various organic compounds, natural or synthetic saccharides, proteins, peptides, expression products of gene libraries, cell extracts, or Cell components and the like can be mentioned.
  • a compound that can satisfy the substrate specificity of megsin by maintaining or mimicking the three-dimensional structure of the active center site of serine protease can be a candidate for a compound having a binding activity to megsin.
  • an active center of serine protease for example, oxy union hole can be shown.
  • the present invention also relates to an inhibitor of plasmin and z or trypsin activity, which comprises megsin or a protein functionally equivalent to megsin as an active ingredient.
  • the ligands of megsin are plasmin and trypsin.
  • Megsin binds to these serine proteases and inhibits their enzymatic activity. Therefore, megsin is useful as a serine protease activity inhibitor.
  • a protein functionally equivalent to megsin can also be used as a serine protease inhibitor.
  • Human 'megsin is a protein encoded by DNA having the nucleotide sequence shown in SEQ ID NO: 1.
  • the amino acid sequence of human megsin is shown in SEQ ID NO: 2.
  • the megsin of the present invention can target not only human megsin but also proteins functionally equivalent to human megsin.
  • Such a protein may be, for example, a homologue of megsin in another species.
  • the megsin homolog for example, the structure of rat megsin and mouse megsin has been elucidated by the present inventors (WO 99/15652).
  • nucleotide sequence of rat megsin and the amino acid sequence are shown in SEQ ID NO: 3 and SEQ ID NO: 4, and the base sequence of mouse megsin and the amino acid sequence are shown in SEQ ID NO: 5 and SEQ ID NO: 6.
  • eukaryotic genes often show polymorphism, as is known for the human interferon gene. Due to this polymorphism, the protein activity is usually maintained even if one or more amino acid substitutions occur in the amino acid sequence. In general, it is known that protein activity is often maintained by modifying one or several amino acids. Therefore, SEQ ID NO: 2, SEQ ID NO: 4, and The gene encoding a protein consisting of an amino acid sequence obtained by artificially modifying the amino acid sequence shown in any of SEQ ID NO: 6 can be used as long as the protein binds to a protease and inhibits its activity. Can be used for
  • megsin derived from humans, rats, or mice and proteins having biologically equivalent functions are collectively referred to as megsins.
  • megsins megsin derived from humans, rats, or mice and proteins having biologically equivalent functions
  • megsin DNA it is advantageous to use human megsin DNA. This is because the effect on human megsin can be more faithfully reflected.
  • megsin Since plasmin is a fibrinolytic enzyme, megsin, which inhibits its activity, can be expected to promote blood coagulation. In addition, trypsin inhibitors have been used for the treatment of knee inflammation. Therefore, megsin having a trypsin inhibitory effect can be expected to be useful as a therapeutic agent for phitis.
  • a pharmaceutical composition for treating and / or preventing mesangial-proliferating glomerulonephritis comprising any one of the following active ingredients.
  • Serine protease, or a protein functionally equivalent to serine protease a compound that interferes with the formation of a complex between megsin and a megsin ligand (this compound is selected by the screening method)
  • the present invention provides a serine protease inhibitor comprising megsin or a protein functionally equivalent to megsin as an active ingredient.
  • the inhibitor of the present invention can be used for a pharmaceutical composition for controlling the activity of serine protease.
  • compositions can be used as they are or after being subjected to various treatments such as dilution with water, and can be used by being blended with pharmaceuticals, quasi-drugs and the like.
  • the compounding amount is appropriately selected according to the disease state and the product.
  • the amount is usually 0.001 to 50% by weight, particularly 0.01 to: L 0% by weight.
  • the content is less than 0.001% by weight, satisfactory preventive or therapeutic effects may not be observed, and if it exceeds 5% by weight, the stability and flavor properties of the product itself may be impaired. Is not preferred.
  • the megsin ligand of the present invention may be contained in a preparation as a pharmaceutically acceptable salt.
  • Pharmaceutically acceptable salts include, for example, salts with bases such as inorganic bases and organic bases, and acid addition salts such as inorganic acids, organic acids, basic and acidic amino acids, and the like.
  • Examples of the inorganic base include alkali metals such as sodium and potassium, alkaline earth metals such as calcium and magnesium, aluminum, and ammonium.
  • Examples of the organic base include primary amines such as ethanolamine, etc., secondary amines such as dimethylamine, diethanolamine, hexylamine, and ⁇ , ⁇ ′-dibenzylethylenediamine, and trimethylamine. And tertiary amines such as triethylamine, pyridine, picoline and triethanolamine.
  • Examples of the inorganic acid include hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like.
  • organic acids include formic acid, acetic acid, lactic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, benzoic acid, cunic acid, succinic acid, malic acid, methanesnolefonic acid, ethanesnolefonic acid, and benzenesnolefonic acid And ⁇ -tonolenesulfonic acid.
  • the basic amino acid include arginine, lysine, orditin and the like.
  • the acidic amino acid include aspartic acid and glutamic acid.
  • the administration method of the pharmaceutical composition of the present invention in addition to oral administration and intravenous administration, transmucosal administration, transdermal administration, intramuscular administration, subcutaneous administration, rectal administration, and the like can be appropriately selected, and depending on the administration method.
  • it can be used as various preparations.
  • each preparation will be described, but the dosage form used in the present invention is not limited thereto, and can be used as various preparations usually used in the field of pharmaceutical preparations.
  • the active ingredient constituting the pharmaceutical composition can be encoded by SDNA. Then, the DNA can be incorporated into a vector for gene therapy and used for gene therapy.
  • Administration can be performed by, for example, intraarterial injection, intravenous injection, intranasal administration, intrabronchial administration, intramuscular administration, subcutaneous administration, oral administration, direct administration to the affected area, and the like.
  • the dose varies depending on conditions such as the patient's body weight, age, health degree, and administration method, but those skilled in the art can appropriately select an appropriate dose.
  • the oral dose of megsin ligand is preferably in the range of 0.03 mg / kg to 30 mg / kg, more preferably. Is between 0.1 mg / kg and 10 mg / kg.
  • the effective blood concentration is 0.2 ii g / mL to 20 / ig / mL, more preferably 0.5 / 1111 ⁇ to 10 g / mL.
  • the dosage should be adjusted so that The dose is adjusted according to age, sex, weight, and the like.
  • Dosage forms for oral administration include powders, granules, capsules, pills, tablets, elixirs, suspensions, emulsions and syrups, and can be appropriately selected.
  • these preparations can be modified such as sustained release, stabilization, easy disintegration, difficult disintegration, enteric coating, and easy absorption.
  • the dosage form for topical administration in the oral cavity includes mastication agents, sublingual agents, puccal agents, troches, ointments, patches, liquids, and the like, which can be appropriately selected.
  • these preparations can be modified such as sustained release, stabilization, easy disintegration, difficult disintegration, enteric coating, and easy absorption.
  • DDS drug delivery system
  • the DDS preparations referred to in the present specification include controlled release preparations, topically applicable preparations (troches, puccal tablets, sublingual tablets, etc.), controlled drug release preparations, enteric coating preparations, gastric coating preparations, etc. This refers to a formulation in the optimal formulation form, taking into account availability and side effects.
  • the components of a DDS basically consist of a drug, a drug release module, and an encapsulation treatment program. Each component, especially when the release is stopped, A drug with a short half-life that reduces the blood concentration is preferred, an envelope that does not react with the living tissue at the administration site is preferred, and a treatment program that maintains the best drug concentration for a set period of time is preferred. Is preferred.
  • the drug release module basically has a drug reservoir, a release controller, an energy source and a release hole or release surface. It is not necessary to have all of these basic components, and the best mode can be selected by adding or deleting as appropriate.
  • Materials that can be used for DDS include polymers, cyclodextrin derivatives, and lecithin.
  • Polymers include insoluble polymers (silicone, ethylene butyl acetate copolymer, ethylene butyl urea / reco / le copolymer, etinolace / rerose, ce / rerose acetate, etc.), water-soluble polymers Xyl gel-forming polymers (polyacrylamide, cross-linked polyhydroxyethyl methacrylate, cross-linked polyacryl, polybutyl alcohol, polyethylene oxide, water-soluble cellulose derivatives, cross-linked poloxamers, chitin, chitosan, etc.) Butyl cellulose, methyl butyl ether, partial ester of maleic anhydride copolymer, etc., gastric-soluble polymers (hydroxypropylmethylcellulose, hydroxypropylcellulose, potassium lmellose, macrogol, polybutylpyrrolidone) Dimeth
  • silicon, ethylene 'vinylinoleate copolymer, ethylene-butyl alcohol copolymer, and partial esters of methylbutyl ether / maleic anhydride copolymer can be used to control drug release, and cellulose acetate can be used for osmotic pumps.
  • material It can be used ethinoresenorelose, hydroxypropinolemethylcellulose, hydroxypropylcellulose, and methylcellulose can be used as a membrane material of a sustained-release preparation, and a crosslinked polyacryl can be used as an adhesive for oral mucosa or ocular mucosa.
  • dosage forms such as oral dosage forms, injections, suppositories, etc.
  • solvents such as disintegrants, dissolution aids, suspending agents, thickeners, emulsifiers, stabilizers, buffers, isotonic agents, soothing agents, preservatives, flavoring agents, fragrances, coloring agents, etc.
  • Additives such as disintegrants, dissolution aids, suspending agents, thickeners, emulsifiers, stabilizers, buffers, isotonic agents, soothing agents, preservatives, flavoring agents, fragrances, coloring agents, etc. Can be added.
  • additives will be exemplified with specific examples, but is not particularly limited thereto.
  • the solvent include purified water, water for injection, physiological saline, seaweed oil, ethanol, glycerin and the like.
  • excipient examples include starches, lactose, glucose, sucrose, crystalline cellulose, calcium sulfate, calcium carbonate, talc, titanium oxide, trehalose, xylitol, and the like.
  • coating agent examples include sucrose, gelatin, cellulose acetate phthalate, and the polymers described above.
  • Examples of the base include petrolatum, vegetable oil, macrogol, oil-in-water emulsion base, water-in-oil emulsion base, and the like.
  • Examples of the binder include starch and its derivatives, cellulose and its derivatives, gelatin, sodium alginate, tragacanth, natural polymer compounds such as gum arabic, synthetic high molecular compounds such as polybulpyrrolidone, dextrin, hydroxypropyl starch and the like. Can be mentioned.
  • lubricant examples include stearic acid and salts thereof, talc, waxes, wheat starch, macrogol, hydrogenated vegetable oil, sucrose fatty acid ester, polyethylene glycol and the like.
  • Disintegrators include starch and its derivatives, agar, gelatin powder, sodium hydrogencarbonate, cellulose and its derivatives, carmellose calcium, hydroxypropyl starch, carboxymethylcellulose and its salts and Bridges and low-substituted hydroxypropylcellulose can be exemplified.
  • solubilizer examples include cyclodextrin, ethanol, propylene glycol, and polyethylene glycol.
  • suspending agents include arabia gum, tragacanth, sodium alginate, aluminum monostearate, citric acid, and various surfactants.
  • Examples of the thickener include carmellose sodium, polyvinylpyrrolidone, methylcellulose, hydroxypropylmethylcellulose, polyvinylinole alcohol, tragacanth, acacia, sodium alginate and the like.
  • Examples of the emulsifier include gum arabic, cholesterol, tragacanth, methinolecellulose, various surfactants, lecithin and the like.
  • Examples of the stabilizer include sodium bisulfite, ascorbic acid, tocopherol, a chelating agent, an inert gas, a reducing substance, and the like.
  • Examples of the buffer include sodium hydrogen phosphate, sodium acetate, boric acid and the like.
  • Examples of the tonicity agent include sodium chloride, glucose and the like.
  • Examples of the soothing agent include proforce hydrochloride, lidocaine, and benzyl alcohol.
  • Examples of the preservative include benzoic acid and salts thereof, para-hydroxybenzoic acid esters, chloroptanol, inverted soap, benzyl alcohol, phenol, and tyromesal.
  • Examples of the flavoring agent include sucrose, saccharin, canzo extract, sorbitol, xylitol, glycerin and the like.
  • Examples of the fragrance, spruce tincture, rose oil and the like can be mentioned.
  • Examples of the coloring agent include a water-soluble edible dye, a lake pigment, and the like.
  • DDS preparations such as sustained-release preparations, enteric-coated preparations, or controlled-release drug preparations
  • megsin ligand may be inactivated or degraded in vivo, resulting in a diminished or diminished effect.
  • a pharmaceutical composition for treating and / or preventing mesangial proliferative glomerulonephritis
  • the effects of the ingredients can be further sustained.
  • These may be incorporated into the preparation or may be administered separately. Those skilled in the art can appropriately identify substances that inactivate or degrade megsin ligand, select substances that inhibit this, and combine or use them in combination.
  • ingredients used in the usual composition can be used as additives other than the above, and the addition amount of these ingredients should be a normal amount within a range not to impair the effects of the present invention. Can be.
  • Figure 1 is a photograph showing SDS-PAGE under non-reducing conditions followed by CBB staining (middle panel on the left) or immunoblotting with anti-t-PA antibody (right panel). Megsin was shown to have functional binding to plasmin but not to other serine proteases such as t-PA ⁇ thrombin. * Indicates the band of the complex.
  • Lane 2 o; 2-AP, 10 Lane 10: Thrombin + megsin, Lane 3: Plasmin + ⁇ 2- ⁇ , Lane 11: t-PA,
  • Lane 4 Megsin
  • Lane 1 2 PAI-1
  • Lane 5 plasmin + megsin
  • lane 13 t-PA + PAI-l
  • lane 6 thrombin
  • lane 14 megsin
  • FIG. 2 is a diagram showing the results of the MCA attestation.
  • ⁇ Substrate only
  • Mouth Plasmin
  • Jap Plasmin + antiplasmin (1: 2)
  • ⁇ Plasmin + megsin (1:10)
  • ⁇ Plasmin + inactivated megsin (1:10)
  • Ovalbumin ⁇ : anolepmin.
  • FIG. 3 is a diagram showing the results of SELDI-TOF mass spectrometry. The results of analysis of only plasmin, only megsin, and a mixture of megsin and plasmin from the top are shown. BEST MODE FOR CARRYING OUT THE INVENTION
  • megsin was obtained from the culture supernatant of Chinese hamster ovary (CHO) cells transfected with megsin transfected. C-myc tag and histidine tag were ligated to the N-terminal side of the full length coding region of megsin cDNA by mutagenesis using PCR. Tag-tagged megsin cDNA was cloned into the mammalian expression vector pREP9 (Invitrogen, Belgium, The Netherlands).
  • CHO cells cultured in the beta one 3 7 ° C plated on 6 ⁇ El plates were transformed with the Lipofecta mine reagent (GIBCO) and pREP9 containing Hitomegushin cDNA. Stable transformants were selected using 0.5 mg / mL geneticin disulfate (Wako Pure Chemical Industries). Then, a C-myc-histidine tag-conjugated megsin recombinant was purified from the culture supernatant using a (His) 6 abundity column.
  • the P17-P8 sequence of megsin (EGTEATAAT) is conserved as a consensus sequence for inhibitory SERPIN [T. Miyata, J. CUn. Invest., 102, 828-836 (1998)]. Therefore, the activity of megsin on serine proteases by in vitro complex formation was evaluated. Plasmin, kallikrein, elastase, trypsin (Sigma, St Louis, MO), tissue plasminogen activator (t-PA: Biopool, Ventu) are used as serine proteases to test complex formation with megsin. ra, CA) and thrombin (Calbiochem, La Jolla, CA) were used.
  • the purified megsin obtained above was combined with a 2: 1 molar ratio of serine protease in phosphate buffered saline (pH 7.4), respectively. Incubate with C for 30 minutes, perform SDS-PAGE analysis under non-reducing conditions, stain with Coomassie brilliant blue (CBB), or Imno-plot analysis using anti-human t-PA antibody (Cedarlane, Ontario, Canada) (For t-PA).
  • CBB Coomassie brilliant blue
  • Imno-plot analysis using anti-human t-PA antibody (Cedarlane, Ontario, Canada) (For t-PA).
  • reaction mixture was also applied to a reverse phase chip (H4 Protein Chip TM: Ciphergen Bio systems, Fremont, CA) and air dried. After the chip was reacted with sinapinic acid, mass spectrometry was performed using a surface-modified / time-of-flight mass spectrometer (SELDI-TOF-MASS) (Ciphergen SELDI Protein Biology System II).
  • H4 Protein Chip TM Ciphergen Bio systems, Fremont, CA
  • mass spectrometry was performed using a surface-modified / time-of-flight mass spectrometer (SELDI-TOF-MASS) (Ciphergen SELDI Protein Biology System II).
  • ⁇ ⁇ X indicates the depth of the band in the order of X, and X indicates that no band was observed.
  • FIG. 2 and Table 2 show the inhibitory effect of megsin on the enzyme activity of plasmin using MCA Atsee.
  • ⁇ indicates the strength of the inhibition in order
  • X indicates that no inhibition was observed.
  • Ovalbumin especially as evident from Figure 2.
  • the present invention provides a pharmaceutical composition for treating and / or preventing mesangial proliferative glomerulonephritis.
  • the present invention also provides a method for screening the ability of a compound to inhibit the binding of megsin to megsin ligand, wherein the test compound and the megsin ligand form a complex with the test compound and the megsin ligand.
  • the above method is provided by the present invention, comprising the step of contacting under conditions.

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Abstract

L'invention concerne l'identification de protéases à sérine en tant que ligands de la megsine. La megsine se lie avec la plasmine et la trypsine, qui sont des protéases à sérine, et inhibe ainsi les activités de ces enzymes. L'hyperexpression de megsine provoque l'insuffisance rénale. L'insuffisance rénale peut par conséquent être traitée par la régulation de la fonction de la megsine à l'aide des ligands décrits.
PCT/JP2003/001316 2002-02-08 2003-02-07 Ligands de la megsine WO2003066089A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
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WO1999015652A1 (fr) * 1997-09-22 1999-04-01 Kurokawa, Kiyoshi Proteine megsine
WO2000057189A1 (fr) * 1999-03-19 2000-09-28 Kurokawa, Kiyoshi Procede de detection de proteine megsine et son utilisation
WO2001024628A1 (fr) * 1999-10-06 2001-04-12 Kurokawa, Kiyoshi Modele animal pour nephrite proliferative a cellules mesangiales

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999015652A1 (fr) * 1997-09-22 1999-04-01 Kurokawa, Kiyoshi Proteine megsine
WO2000057189A1 (fr) * 1999-03-19 2000-09-28 Kurokawa, Kiyoshi Procede de detection de proteine megsine et son utilisation
WO2001024628A1 (fr) * 1999-10-06 2001-04-12 Kurokawa, Kiyoshi Modele animal pour nephrite proliferative a cellules mesangiales

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