JP2007332120A - Novel morbidity marker and morbidity diagnosis using it - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a diagnosis marker capable of rapidly and competently judging the morbidity of live body disintegrative syndrome originated from various diseases. <P>SOLUTION: This marker peptide set of live body disintegrative syndrome comprises at least one peptide being a decomposition product of proteins existing in a live body, and detected in the biological fluid of a live body disintegrative syndrome patient and that of a healthy person with a statistically significant difference. This antibody recognizes either one of peptides of the marker peptide set. This therapeutic agent of the syndrome replenishes the parent protein of the marker peptide or contains a substance which is the marker itself or simultaneously regulates the quantity or the activity of a physiologically active peptide capable of acting in the direction of healing or worsening the disease. A judging method of the stage of live body disintegrative syndrome, by detecting at least one peptide constituting the marker peptide set in the live body sample which is derived from the patient affected a basic disease to result in live body disintegrative syndrome, an evaluation method of treatment effect, and an estimation method of the abolition of the function of part/organ of a patient and/or the danger of life continuation are also invented. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a novel biomarker for biodegradable syndrome and a method for diagnosing biodegradable syndrome by individually or comprehensively analyzing the biomarker. The present invention also replenishes the parent protein of a peptide that is a biomarker for biodegradation syndrome, or the generation of a bioactive peptide that is a biomarker and can simultaneously act on or treat the disease. Alternatively, a means for treating the disease is provided by modulating the activity. Furthermore, this invention relates to the evaluation method of the therapeutic effect of a biodegradation syndrome by analyzing this biomarker individually or exhaustively.

  Biodegradation syndrome is expected to greatly contribute to the determination of the direction of the subsequent nutritional support strategy and drug treatment strategy if the pathological condition can be accurately grasped by the biomarker group.

However, although the method of comprehensive analysis of genes and proteins has been established as a standard in biochemical research including basic medical research in recent years, there is still a considerable distance in application to the medical field. Is real. In fact, for the diagnosis of disseminated intravascular coagulation syndrome (DIC) and multiple organ failure (MOF) in which the pathological condition of biodegradation syndrome is remarkably observed, peripheral blood cell analysis, blood biochemistry, blood gas analysis, blood pressure are still present. However, unreasonable indexes such as pulse rate and body temperature are still used to accurately grasp the medical condition (see Non-Patent Document 1, for example).
Nobuo Aoki, Satoshi Hasegawa: About revision of the section “Supplementary test results for diagnosis, findings” of DIC diagnostic criteria, Ministry of Health and Welfare specific disease blood coagulation investigation research group, 1987 research report, 1988. p37- 41

  Therefore, an object of the present invention is to provide a novel biomarker that can quickly and accurately determine the pathology of biodegradation syndrome derived from various diseases, thereby enabling more accurate nutrition support strategy and drug treatment for patients. It is possible to provide a strategy.

As a result of intensive studies to achieve the above-mentioned object, the present inventors have conducted a series of processes (1 year and 6 months) from diagnosis to death outcome of a patient with crow-fukase syndrome known as an incurable disease with a poor prognosis. They succeeded in identifying various relatively low molecular weight peptides that appear in the blood as the disease progresses. Furthermore, the present inventors have found that these peptide groups are protein degradation products essential for normal maintenance activity of the living body.
As a result of further studies based on these findings, the present inventors have completed the present invention.

That is, the present invention
[1] Biodegradation syndrome marker peptide comprising one or more peptides that are degradation products of proteins present in the living body, which are detected with a statistically significant difference between the body fluid of a biodisintegration syndrome patient and the body fluid of a healthy person set;
[2] The marker peptide set according to [1] above, wherein the statistically significant difference is observed in at least one stage of stage I to stage IV of the biodegradation syndrome;
[3] The marker peptide set according to the above [1] or [2], wherein the protein is selected from the group consisting of a physiologically active carrier protein, a protease enzyme, and a nutritional assessment protein;
[4] The marker peptide set according to any one of the above [1] to [3], wherein at least one of the peptides is substantially not detected from a body fluid of a healthy person;
[5] The marker peptide set according to any one of the above [1] to [4], wherein at least one of the peptides has a molecular weight of about 50,000 or less;
[6] The marker peptide set according to any one of [1] to [5] above, wherein the protein is selected from the group consisting of α1-antitrypsin, transthyretin, apolipoprotein AI, and apolipoprotein B-100;
[7] The marker peptide set according to [6] above, which contains at least one peptide selected from the group consisting of the following (1) to (6):
(1) A peptide consisting of an amino acid sequence identical or substantially identical to the amino acid sequence represented by amino acid numbers 1 to 24 in the amino acid sequence represented by SEQ ID NO: 1.
(2) A peptide comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by amino acid numbers 81 to 103 in the amino acid sequence represented by SEQ ID NO: 2
(3) A peptide comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by amino acid numbers 62 to 83 in the amino acid sequence represented by SEQ ID NO: 3
(4) A peptide consisting of an amino acid sequence identical or substantially identical to the amino acid sequence represented by amino acid numbers 189 to 214 in the amino acid sequence represented by SEQ ID NO: 3
(5) A peptide consisting of an amino acid sequence identical or substantially identical to the amino acid sequence represented by amino acid numbers 196 to 214 in the amino acid sequence represented by SEQ ID NO: 3
(6) A peptide consisting of an amino acid sequence identical or substantially identical to the amino acid sequence represented by amino acid numbers 3218 to 3249 in the amino acid sequence represented by SEQ ID NO: 4, [8] any of the marker peptide set according to [7] above An antibody recognizing the peptide of;
[9] Any peptide constituting the marker peptide set according to any one of [1] to [7] above, wherein the peptide has a physiological activity that acts therapeutically against biodegradation syndrome;
[10] A therapeutic agent for biodegradation syndrome, comprising a substance that increases the amount or activity of the peptide according to [9] above;
[11] The substance is the peptide according to [9], an agonist of the peptide, a decomposing enzyme that releases the peptide from a protein present in a living body, and the decomposing enzyme on the N-terminal side and C-terminal side of the peptide. Producing a substrate or substrate analog molecule of the decomposing enzyme, a molecule that promotes the production of the decomposing enzyme, a molecule that promotes the activity of the decomposing enzyme, and an inhibitor of the decomposing enzyme, further comprising an amino acid sequence that is recognized and cleaved The therapeutic agent according to the above [10], which is selected from the group consisting of molecules to suppress;
[12] Any peptide constituting the marker peptide set according to any one of [1] to [7] above, which has a physiological activity that exacerbates a biodegradation syndrome;
[13] A therapeutic agent for biodegradation syndrome, comprising a substance that reduces the amount or activity of the peptide according to [12] above;
[14] The substance comprises an antagonist of the peptide according to the above [12], a molecule that suppresses the production of a degrading enzyme that releases the peptide from a protein present in a living body, an inhibitor of the decomposing enzyme, and the production of the inhibitor The therapeutic agent according to the above [113], which is selected from the group consisting of promoting molecules;
[15] A method for treating biodegradation syndrome, comprising increasing the amount or activity of the peptide according to [9] above and / or decreasing the amount or activity of the peptide according to [12] above in a patient's body;
[16] The marker peptide according to any one of [1] to [7] above, in a biological sample derived from a patient suffering from a basic disease that can result in the syndrome, in a stage determination method in a biodegradable syndrome A method comprising detecting one or more peptides constituting the set and / or the peptide according to [9] and / or [12] above;
[17] When a peptide that is a degradation product of α1-antitrypsin and / or transthyretin is detected, a peptide that is a degradation product of stage IV and apolipoprotein AI and / or apolipoprotein B-100 is detected The method according to [16] above, wherein the method comprises determining that it is in stage I;
[18] The degradation product of α1-antitrypsin is selected from the following peptide (1), the degradation product of transthyretin is the peptide of (2) below, and the degradation product of apolipoprotein AI is selected from the following (3) to (5) The method according to [17] above, wherein the one or more peptides and the degradation product of apolipoprotein B-100 are the peptides of the following (6):
(1) A peptide consisting of an amino acid sequence identical or substantially identical to the amino acid sequence represented by amino acid numbers 1 to 24 in the amino acid sequence represented by SEQ ID NO: 1.
(2) A peptide comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by amino acid numbers 81 to 103 in the amino acid sequence represented by SEQ ID NO: 2
(3) A peptide comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by amino acid numbers 62 to 83 in the amino acid sequence represented by SEQ ID NO: 3
(4) A peptide consisting of an amino acid sequence identical or substantially identical to the amino acid sequence represented by amino acid numbers 189 to 214 in the amino acid sequence represented by SEQ ID NO: 3
(5) A peptide consisting of an amino acid sequence identical or substantially identical to the amino acid sequence represented by amino acid numbers 196 to 214 in the amino acid sequence represented by SEQ ID NO: 3
(6) A peptide consisting of an amino acid sequence identical or substantially identical to the amino acid sequence represented by amino acid numbers 3218 to 3249 in the amino acid sequence represented by SEQ ID NO: 4, [19] The above [16] to [18] The method according to any one of the above;
[20] The above [19], wherein the body fluid is selected from the group consisting of blood, plasma, serum, saliva, urine, spinal fluid, bone marrow fluid, pleural effusion, ascites, joint fluid, tear fluid, aqueous humor, vitreous humor and lymph fluid ] The method according to
[21] The method according to any one of [16] to [20] above, comprising subjecting the biological sample to mass spectrometry;
[22] One or more peptides constituting the marker peptide set according to any one of [1] to [7] and / or an antibody that recognizes each of the peptides according to [9] and / or [12] are used. The method according to any one of [16] to [20] above,
[23] A biological sample is collected from a patient in time series, and one or more peptides constituting the marker peptide set according to any one of [1] to [7] in the sample and / or the above [9] and / or Or the method according to any one of [16] to [22] above, wherein the time-dependent change in the type and amount of the peptide according to [12] is examined;
[24] A method for treating biodegradation syndrome, wherein the method according to any one of [16] to [23] described above is effective for a patient in which a significant increase in a degradation product of a protein is observed. A method comprising administering an amount;
[25] A method for evaluating a therapeutic effect in a patient with biodegradation syndrome, wherein a biological sample is collected from the patient before and after treatment, and the sample according to any one of [1] to [7] above A method characterized by examining changes in the type and amount of one or more peptides constituting the marker peptide set and / or the peptides described in [9] and / or [12] above;
[26] A method for predicting the abolition of organs and organ functions and / or the risk of survival in a patient with biodegradation syndrome, and any treatment that can be taken in stage IV patients is ineffective by the method described in [25] above A method of predicting that the patient is in danger of losing organ function and / or survival, if determined to have occurred;
[27] In one or more peptides constituting the marker peptide set according to any one of [1] to [7] above, the peptides are present in vivo on the N-terminal side and / or C-terminal side of each peptide A peptide set for identifying a degrading enzyme, further comprising an amino acid sequence that is recognized and cleaved by a degrading enzyme released from the protein to be cleaved;
Etc.

  According to the present invention, since the determination of each stage in the biodegradation syndrome can be quickly and accurately determined based on the evidence, early detection and early treatment of the symptom can be performed. In particular, since there are unprecedented therapeutic drugs that are inspired by the degradation products of proteins present in the living body, development of effective early treatment methods for biodegradation syndrome, and improvement in the prognosis of organs and organs, in particular In addition to the effects, it is expected that conventional diagnostic techniques can save many patients who should die. In addition, the terminal stage of biodegradation syndrome can be determined quickly and accurately based on the evidence, so that unnecessary life-prolonging treatments can be avoided, the patient's dignity is protected, and national medical economic problems are solved. It becomes possible to do.

The present invention provides (a set of) one or more specific marker peptides for biodegradation syndrome. The peptide constituting the marker peptide set is a degradation product of a protein present in a living body, which is detected with a statistically significant difference between a body fluid of a biodegradation syndrome patient and a body fluid of a healthy person. As long as there is a statistically significant difference, it may be increased or decreased in the body fluid of the biodisintegration syndrome patient.
Here, “biological disintegration syndrome” can be defined as a pathological condition in which functional and organic disorders are manifested as a result of degradation of a protein present in the living body. The disease state is subdivided into stages I to IV according to symptoms. That is, stage I refers to a pathological condition with no subjective symptoms or mild subjective symptoms, stage II refers to a pathological condition that interferes with daily life due to decreased appetite, weight loss, general malaise, etc., stage III Refers to a marked dysfunction of one organ / organ or a pathological condition generally defined as DIC, and stage IV refers to a pathological condition approximated to the abolition of function of one organ / organ or generally defined as MOF .
Typical examples of basic diseases that lead to biodegradation syndrome include cancer, inflammatory immune diseases (such as rheumatoid arthritis), anorexia, neurodegenerative diseases (such as Alzheimer), and retinitis pigmentosa. For example, all the various diseases shown in Table 1 are included.

The marker peptide of the present invention is a degradation product of a protein present in the living body. Specific examples include carrier proteins of physiologically active substances such as hormones, vitamins and enzymes, degrading enzyme inhibitors which are regulators of proteolytic enzymes, and nutritional assessment proteins which are used as indicators of nutritional status. It is not limited to. In conventional clinical chemistry, there was no concept of acquiring pathological information by analyzing peptide fragments generated as a result of the decay of proteins constituting the living body. A unique diagnostic method for predicting “what molecules are disrupted” and “what is going to happen” by analyzing peptide fragments resulting from biological reactions to certain pathological conditions is there. A peptide serving as a marker means the destruction of the parent protein from which the peptide is derived. By accumulating the inventions of these peptides, it becomes possible to comprehensively and rapidly diagnose the pathological conditions of biodegradation syndrome. .
Further, in the process of reaching the present invention, it was found that there are innumerable disintegrating peptide fragments in a so-called “healthy” state in vivo. The living body maintains homeostasis by clever balance control of protein synthesis and disruption, and "biological disintegration syndrome" exists on the extension of the balance disruption.

  Among the marker peptides of the present invention, a further characteristic of a typical peptide group detected in the body fluid of a biodisintegration syndrome is that it is a degradation product of a protein that has not been captured in such a degraded form. is there. That is, such a peptide is not substantially detected from a biological sample of a healthy person. Here, “substantially not detected” means below the detection limit of a conventionally known detection means (for example, mass spectrometry, HPLC, gel electrophoresis, capillary electrophoresis, immunoassay, etc.).

It is estimated that there are about 10,000 marker peptides of the present invention. The ratio of marker peptides in the molecular region is about 54% when the molecular weight is 5000 or less, about 78% when the molecular weight is 10,000 or less, about 84% when the molecular weight is 20000 or less, and 100% when the molecular weight is 50000 or less. Most of these are concentrated at a molecular weight of about 5,000 or less, but are not limited thereto.
The marker peptide set of the present invention is a collection of peptides composed of one or more of these marker peptides. The marker peptide set of the present invention is comprehensively detected and quantified by, for example, mass spectrometry, preferably by mass spectrometry using a plate for mass spectrometry developed by some of the present inventors as described later. be able to.

Preferable peptides constituting the marker peptide set of the present invention include peptides that are degradation products of α1-antitrypsin, transthyretin, apolipoprotein AI, or apolipoprotein B-100. These peptides may be any degradation products as long as the conditions of the marker peptide of the present invention described above are satisfied. For example, each amino acid sequence shown in SEQ ID NOs: 1 to 4 has a chain length of about 5 to about 30 amino acids. Any fragment of a residue is mentioned. More preferably, the peptides shown in the following (1) to (6) are exemplified.
(1) A peptide consisting of an amino acid sequence identical or substantially identical to the amino acid sequence represented by amino acid numbers 1 to 24 in the amino acid sequence represented by SEQ ID NO: 1.
(2) A peptide comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by amino acid numbers 81 to 103 in the amino acid sequence represented by SEQ ID NO: 2
(3) A peptide comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by amino acid numbers 62 to 83 in the amino acid sequence represented by SEQ ID NO: 3
(4) A peptide consisting of an amino acid sequence identical or substantially identical to the amino acid sequence represented by amino acid numbers 189 to 214 in the amino acid sequence represented by SEQ ID NO: 3
(5) A peptide consisting of an amino acid sequence identical or substantially identical to the amino acid sequence represented by amino acid numbers 196 to 214 in the amino acid sequence represented by SEQ ID NO: 3
(6) A peptide comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by amino acid numbers 3218 to 3249 in the amino acid sequence represented by SEQ ID NO: 4

  The protein consisting of the amino acid sequence shown in SEQ ID NO: 1 is a precursor of α1-antitrypsin (α1-AT), which is the most major protease inhibitor in blood, which inhibits various serine proteases. The sequence consisting of 24 amino acids represented by amino acid numbers -24 to -1 is a signal peptide, and mature α1-AT is a glycoprotein consisting of 394 amino acids with a molecular weight of 51,000. α1-AT is mainly produced in hepatocytes and is one of the acute phase reactants that increase in the blood during various inflammations, and is an indicator of inflammatory diseases and malignant tumors. However, the relationship with biodegradation syndrome is not known. In addition, the existence of an α1-AT degradation product (hereinafter sometimes abbreviated as “A1AT (1-24)”) consisting of the amino acid sequence represented by amino acid numbers 1 to 24 has not been known at all. Peptide.

  The protein consisting of the amino acid sequence shown in SEQ ID NO: 2 is a precursor of transthyretin (TTR) that functions in the blood as a carrier protein for retinol-binding protein and thyroid hormone (T4). The sequence consisting of 20 amino acids represented by amino acid numbers -20 to -1 is a signal peptide, and mature TTR is a tetrameric protein having a molecular weight of 55,000. It is used for evaluation of nutritional status together with retinol binding protein. TTR has been studied in detail as a causative protein of amyloidosis, and there are reports that it prevents the accumulation of Aβ, which is the cause of Alzheimer's disease. However, the relationship with biodegradation syndrome is not known. In addition, the existence of a TTR degradation product consisting of the amino acid sequence represented by amino acid numbers 81 to 103 (hereinafter sometimes abbreviated as “TTR (81-103)”) has not been known so far, and a novel peptide It is.

  The protein consisting of the amino acid sequence shown in SEQ ID NO: 3 is a precursor of apolipoprotein A-I (ApoA-I), which is the main component (carrier) protein of HDL-cholesterol. The sequence consisting of 18 amino acids represented by amino acid numbers -24 to -7 is a signal peptide, and the pro sequence represented by amino acid numbers -6 to -1 is cleaved to become mature ApoA-I. ApoA-I is known to show high levels in arteriosclerosis and LPL deficiency. However, the relationship with biodegradation syndrome is not known. In addition, ApoA-I degradation products (hereinafter referred to as “AI (62-83)”, “AI (189-214)”, and amino acid sequences represented by amino acid numbers 62 to 83, 189 to 214, and 196-214, respectively) The existence of “AI (196-214)” (which may be abbreviated as “AI”) is a novel peptide that has not been known so far.

  The protein consisting of the amino acid sequence shown in SEQ ID NO: 4 is a precursor of apolipoprotein B-100 (ApoB-100), which is the main component (carrier) protein of LDL-cholesterol. The sequence consisting of 27 amino acids represented by amino acid numbers -27 to -1 is a signal peptide, and mature ApoB-100 is a protein consisting of 4536 amino acids with a molecular weight of about 500 kDa. ApoB-100 is known to show high values in arteriosclerosis and LPL deficiency. However, the relationship with biodegradation syndrome is not known. In addition, the existence of an ApoB-100 degradation product consisting of each amino acid sequence represented by amino acid numbers 3218 to 3249 (hereinafter sometimes abbreviated as “B100 (3218-3249)”) has not been known so far. It is a novel peptide.

The amino acid sequence represented by each amino acid number of each SEQ ID NO. And the “substantially identical amino acid sequence”
(1) A variant or polymorphism with amino acid substitution and / or deletion and / or insertion of the protein represented by each SEQ ID NO, wherein the substitution and / or deletion and / or insertion is a sequence number The amino acid sequence of the corresponding region in the variant or polymorphism present in the amino acid sequence represented by each amino acid number of the amino acid sequence represented by
(2) 1-10 amino acid residues (preferably 1-5 amino acid residues) extended from the region represented by each amino acid number to the N-terminal side and / or C-terminal side of the protein represented by each SEQ ID NO. Means a shortened amino acid sequence.
For example, a variant in which the second amino acid residue of A1AT (1-24) (ie, amino acid number 2 of SEQ ID NO: 1) is substituted from Asp to Ala; the fourth (ie, sequence of TTR (81-103) A variant in which the amino acid residue of amino acid number 84) of No. 2 is substituted from Ile to Asn or Ser, and the ninth amino acid residue (ie amino acid number 89 of SEQ ID NO. 2) is substituted from Glu to Lys or Gln A variant in which the amino acid residue at the 10th position (ie, amino acid number 90 in SEQ ID NO: 2) is substituted from His to Asn, and the amino acid residue at the 11th position (ie, amino acid number 91 in SEQ ID NO: 2) is Variant in which Ala is replaced with Ser, variant in which amino acid residue at position 17 (ie, amino acid number 97 of SEQ ID NO: 2) is substituted from Ala to Gly, position 21 (ie, amino acid number 101 in SEQ ID NO: 2) ) In which the amino acid residue is substituted from Gly to Ser, position 22 (ie, the amino acid of SEQ ID NO: 2) No. 102) Mutant in which the amino acid residue of Pro is replaced with Arg; The seventh amino acid residue of AI (62-83) (ie, amino acid number 68 of SEQ ID NO: 3) is replaced with Thy to Ile Mutants: Presence of mutants in which the amino acid residue at the 10th position of AI (189-214) (the 3rd position of AI (196-214); ie, amino acid number 198 of SEQ ID NO: 3) is substituted from Glu to Lys, etc. It has been known.
For example, AI (196-214) corresponds to a peptide in which AI (189-214) is extended by 7 amino acid residues on the N-terminal side.

  Furthermore, one or more peptides constituting the marker peptide set can also provide a drug discovery target for active biodegradation syndrome in addition to diagnosis. That is, when the marker peptide itself has a physiological function in the direction of treatment (remission) of the syndrome, the marker peptide itself can be administered by administering a substance that increases the amount or activity of the peptide to the patient. When having a physiological function in the direction of exacerbation of the syndrome, the syndrome can be treated by administering a substance that reduces the amount or activity of the peptide. Therefore, the present invention also provides any peptide constituting the marker peptide set, which itself has (1) a physiological function in the treatment (remission) direction of biodegradation syndrome (hereinafter referred to as “therapeutic peptide”). And (2) those having physiological functions in the direction of exacerbation of the syndrome (hereinafter also referred to as “exacerbation peptides”).

The present invention also provides a method of treating biodegradation syndrome by increasing the amount or activity of the therapeutic peptide and / or decreasing the amount or activity of the exacerbation peptide. Specifically, the method of treatment includes administering to a patient with biodegradation syndrome an effective amount of a substance that increases the amount or activity of the therapeutic peptide and / or a substance that decreases the amount or activity of the exacerbation peptide. Therefore, the present invention also provides a therapeutic agent for biodegradation syndrome comprising a substance that increases the amount or activity of the therapeutic peptide and / or a substance that decreases the amount or activity of the exacerbation peptide.
Specifically, the substance that increases the activity of the therapeutic peptide includes the peptide itself or a molecule having the same agonistic action. Alternatively, substances that increase the activity of a therapeutic peptide may include non-neutralizing antibodies, preferably agonist antibodies, etc. of the peptide. On the other hand, examples of the substance that reduces the activity of the exacerbation peptide include a molecule having an antagonistic action of the peptide, or a neutralizing antibody against the peptide.
In addition, substances that increase the production of therapeutic peptides include a degrading enzyme that releases the peptide from proteins present in the body, and an amino acid sequence that is recognized and cleaved by the decomposing enzyme at the N-terminal side and C-terminal side of the peptide Further comprising a substrate or a substrate analog molecule of the decomposing enzyme, a molecule (including an analogous compound) that promotes the production of the decomposing enzyme, a molecule that promotes the activity of the decomposing enzyme, or the production of an inhibitor of the decomposing enzyme. Examples include molecules. The N-terminal and C-terminal amino acid sequences of the peptide suggest the presence of a degrading enzyme that liberates the peptide, and a degrading enzyme search using the N-terminal and / or C-terminal amino acid sequences of the peptide as a probe And identification becomes possible. A substrate or substrate analog molecule of a decomposing enzyme thus identified, that is, a peptide molecule further comprising an amino acid sequence recognized and cleaved by the decomposing enzyme on the N-terminal side and C-terminal side of the peptide, In this way, the therapeutic peptide or its analog molecule is released by being cleaved by the degrading enzyme, so that the same therapeutic effect can be obtained. On the other hand, substances that promote the production and / or activity of the identified degrading enzymes can also indirectly increase the production of therapeutic peptides. If the target degrading enzyme is identified, these substances can be screened or molecularly designed by a method known per se.
On the other hand, substances that reduce the production of exacerbation peptides include molecules that suppress the production of degrading enzymes that liberate the peptides from proteins present in the body, inhibitors of the decomposing enzymes, molecules that promote the production of the inhibitors, and the like. Can be mentioned. The degrading enzyme that releases the exacerbation peptide can be searched and identified by the same method as that for the therapeutic peptide. By using a degrading enzyme thus identified, a substance that suppresses (inhibits) the production or activity of the degrading enzyme directly or indirectly can be screened or molecularly designed by a method known per se.

Substances that increase the amount or activity of the therapeutic peptide and substances that decrease the amount or activity of the exacerbation peptide can be formulated according to conventional means.
For example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including dragees and film-coated tablets), pills, granules, powders, capsules (including soft capsules). Syrup, emulsion, suspension and the like. Such a composition is produced by a method known per se, and contains a carrier, diluent or excipient usually used in the pharmaceutical field. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.
As a composition for parenteral administration, for example, injections, suppositories and the like are used, and injections are intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, intravenous injections, intraarticular injections. Includes dosage forms such as agents. Such an injection is prepared according to a method known per se, for example, by dissolving, suspending or emulsifying the above compound or a salt thereof in a sterile aqueous or oily liquid usually used for injection. As an aqueous solution for injection, for example, isotonic solutions containing physiological saline, glucose and other adjuvants are used, and suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants [eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)] and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. The prepared injection solution is usually filled in a suitable ampoule. A suppository used for rectal administration is prepared by mixing the above-mentioned compound or a salt thereof with an ordinary suppository base.

The above oral or parenteral pharmaceutical compositions are conveniently prepared in dosage unit form to suit the dosage of the active ingredient. Examples of dosage forms of such dosage units include tablets, pills, capsules, injections (ampoules), suppositories, etc., and usually 5 to 500 mg, especially 5 to 100 mg for injections, for each dosage unit dosage form. Other dosage forms preferably contain 10 to 250 mg of the above compound.
Each of the above-described compositions contains other active ingredients unless an undesirable interaction occurs when combined with a substance that increases the amount or activity of the therapeutic peptide or a substance that decreases the amount or activity of the exacerbation peptide. May be.

Since the preparation thus obtained is safe and has low toxicity, it can be administered, for example, orally or parenterally to humans.
The dose of a substance that increases the amount or activity of a therapeutic peptide and a substance that decreases the amount or activity of an exacerbation peptide vary depending on its action, administration route, patient severity, age, weight, drug acceptability, etc. Is, for example, in the range of about 0.0008 to about 25 mg / kg, preferably about 0.008 to about 2 mg / kg, as an active ingredient amount per day for an adult, and this is administered once or in several divided doses be able to.

  The invention also relates to a stage of biodegradation syndrome in a patient (stage I˜) by detecting one or more marker peptides of the invention in a biological sample from a patient suspected of suffering from biodegradation syndrome. A method for determining (IV stage) is provided. Examples of the “patient suspected of suffering from the biodegradation syndrome” include the above-mentioned patients suffering from the underlying diseases that can result in the syndrome. In the biodegradation syndrome, characteristic changes in the appearance of protein degradation products are observed at each stage. Therefore, by measuring one or more marker peptides specific to each stage, the stage of biodegradation syndrome in the patient can be determined. For example, the degradation product of α1-antitrypsin, preferably A1AT (1-24) or the like, or the degradation product of transthyretin, preferably TTR (81-103) or the like is specifically detected in the IV phase. In contrast, for example, the degradation product of the apolipoprotein AI, preferably AI (62-83), AI (189-214), AI (196-214), or apolipoprotein B-100, preferably B100 (3218 -3249) and the like are specifically detected in stage I. In the treatment of biodegradation syndrome, since early detection and early treatment are the major principles, it is extremely significant that a specific marker peptide was found in stage I.

The patient-derived biological sample to be the test sample is not particularly limited, but is preferably one that is less invasive to the patient, for example, blood, plasma, serum, saliva, urine, tears that can be easily collected from the living body And those collected relatively easily such as cerebrospinal fluid, bone marrow fluid, pleural effusion, ascites, joint fluid, aqueous humor, and vitreous humor.
When serum or plasma is used, it can be prepared by collecting blood from a patient according to a conventional method and separating the liquid component. The marker peptide (set) of the present invention, which is a detection target, can be separated and removed in advance using a spin column or the like, if necessary, such as a high molecular weight protein fraction.

Detection of the marker peptide (set) of the present invention in a biological sample is performed by pre-treating the sample as necessary, and then individually measuring specific marker peptides or comprehensively measuring peptide groups. . Here, “individually” does not mean that each marker is separately, but if a marker to be detected is specified, a part or all of them may be measured collectively.
When a specific marker peptide is detected individually, for example, biological samples are subjected to various molecular weight measurement methods such as gel electrophoresis and various separation and purification methods (eg, ion exchange chromatography, hydrophobic chromatography, affinity chromatography). , Reverse phase chromatography, etc.), ionization methods (eg, electron impact ionization, field desorption, secondary ionization, fast atom bombardment, matrix-assisted laser desorption / ionization (MALDI), electrospray ionization, etc.) ), Mass spectrometer (eg, double-focusing mass spectrometer, quadrupole analyzer, time-of-flight mass spectrometer, Fourier transform mass spectrometer, ion cyclotron mass spectrometer, etc.) Bands or spots that match the molecular weight of the marker peptide It can be performed by detecting the click, but are not limited to. When the amino acid sequence of the marker peptide is known, a method of producing an antibody that recognizes the amino acid sequence and detecting the marker peptide by Western blotting or various immunoassays can be used more preferably.
On the other hand, when an unspecified number of marker peptides of the present invention are comprehensively detected, a biological sample is spotted on a plate for mass spectrometry, or the sample is subjected to gel electrophoresis as described in WO 2004/031759. After the electrophoresis, it is transferred onto a plate for mass spectrometry, and a peak appearing at a molecular weight of 50000 or less is identified from a mass spectrometry chart obtained by combining the above ionization method and mass spectrometry method. The marker peptide of the present invention is estimated to be present in about 10,000 species, and the abundance ratio in the molecular region is about 54% when the molecular weight is 5000 or less, about 78% when the molecular weight is 10,000 or less, and about 84% when the molecular weight is 20000 or less. It is desirable to identify a peak appearing at a molecular weight of about 5,000 or less because 100% exists at a molecular weight of 50,000 or less and most of them are concentrated at a molecular weight of about 5,000 or less.

  Particularly preferred marker peptides of the present invention are A1AT (1-24), TTR (81-103), AI (62-83), AI (189-214), AI (196-214), B100 (3218-3249) Have molecular weights of about 2690.12, about 2450.20, about 2617.27, about 2052.07, about 2864.49 and about 3688.80, respectively. Here, “molecular weight about...” Takes into consideration the range of error of each measurement method. For example, in the case of a method using a mass spectrometer, the indicated numerical value ± 0.5% (preferably ± 0.3). %, More preferably ± 0.1%) is preferably measured.

One of the particularly preferable measurement methods in the determination method of the present invention is that a test sample is brought into contact with the surface of a plate used for time-of-flight mass spectrometry, and the mass of the component captured on the plate surface is determined by a time-of-flight mass spectrometer. The method of measuring by is mentioned.
The plate compatible with the time-of-flight mass spectrometer may be any plate as long as it has a surface structure that can efficiently adsorb the marker peptide of the present invention to be detected. Such surface structures include, for example, functionalized glass, Si, Ge, GaAs, GaP, SiO ₂ , SiN ₄ , modified silicon, a wide range of gels or polymers (eg, (poly) tetrafluoroethylene, (poly And vinylidene difluoride, polystyrene, polycarbonate, or combinations thereof). Examples of surface structures having a plurality of monomer or polymer sequences include linear and cyclic polymers of nucleic acids, polysaccharides, lipids, peptides having α-, β-, or ω-amino acids, gel surfaces used in chromatography Carriers (anionic / cationic compounds, hydrophobic compounds composed of carbon chains 1-18, hydrophilic compounds (eg, carriers cross-linked with silica, nitrocellulose, cellulose acetate, agarose, etc.), artificial homopolymers) (For example, polyurethane, polyester, polycarbonate, polyurea, polyamide, polyethyleneimine, polyarylene sulfide, polysiloxane, polyimide, polyacetate, etc.) Any known drug or natural compound is bound to any of the above compounds (covalent and non-covalent bonds). ) By heteropolymer etc. Coating, and the like.

  In a preferred embodiment, the support used as the plate for mass spectrometry is a substrate coated with a thin layer of polyvinylidene difluoride (PVDF), nitrocellulose or silica gel, particularly preferably PVDF [usually a plate for mass spectrometry For example, insulators (glass, ceramics, plastics, resins, etc.), metals (aluminum, stainless steel, etc.), conductive polymers, composites thereof, etc. Although an aluminum plate is preferably used, see WO 2004/031759). The shape of the support can be appropriately devised into a shape suitable for the sample analyzer, in particular, the sample introduction port, but is not limited thereto. As such a plate for mass spectrometry coated with a thin layer with PVDF, a blot chip (registered trademark) manufactured by Protocera Inc. is preferably used.

Preferably, the coating refers to a thin layer formed by depositing on a support in a state where coating molecules are dispersed, instead of overlaying a pre-formed structure like a membrane on the support. The manner in which the coating molecules are deposited is not particularly limited, but means exemplified in a method for preparing a plate for mass spectrometry described later is preferably used.
The thickness of the thin layer can be appropriately selected within a range that does not adversely affect the transfer efficiency of the molecules contained in the tissue or cells, the measurement sensitivity of mass spectrometry, etc., for example, about 0.001 to about 100 μm , Preferably about 0.01 to about 30 μm.

Although the plate for mass spectrometry (support) can be prepared by a method known per se, for example, the above preferred mass spectrometry plate is prepared by thinly coating the surface of the support with a coating molecule such as PVDF. The Preferred examples of the coating means include application, spraying, vapor deposition, dipping, printing (printing), and sputtering.
In the case of “application”, the coating molecule is dissolved in an appropriate solvent, for example, an organic solvent such as dimethyl formamide (DMF) at an appropriate concentration (eg, about 1 to about 100 mg / mL) (coating) The molecule-containing solution) can be applied to the substrate using a suitable tool such as a brush.
In the case of “spraying”, the coating molecule-containing solution prepared in the same manner as described above may be put in a sprayer and sprayed so that PVDF is uniformly deposited on the substrate.
In the case of “evaporation”, the coating molecule (which may be solid or solution) is heated and vaporized in a vacuum tank containing the base material using a normal vacuum deposition apparatus for producing an organic thin film. A thin layer of molecules can be formed.
In the case of “immersing”, the substrate may be immersed in a coating molecule-containing solution prepared in the same manner as described above.
In the case of “printing”, various printing techniques that can be normally used depending on the material of the substrate can be appropriately selected and used. For example, screen printing or the like is preferably used.
In the case of “sputtering”, for example, a DC high voltage is applied between the substrate and the coating molecule while introducing an inert gas (eg, Ar gas) in a vacuum, and the ionized gas is caused to collide with the molecule. The repelled coating molecules can be deposited on the substrate to form a thin layer.
The coating may be applied to the entire surface of the substrate, or may be applied only to one surface subjected to mass spectrometry.

The coating molecule can be used in a suitable form depending on the coating means. For example, the coating molecule can be applied to the substrate in the form of a coating molecule-containing solution, a coating molecule-containing vapor, a solid coating molecule, etc. It is preferable to apply in the form. “Apply” refers to bringing a coating molecule into contact with the support so that the coating molecules remain and deposit on the support after contact. The amount of application is not particularly limited, and examples of the coating molecular weight include about 10 to about 100,000 μg / cm ² , preferably about 50 to about 5,000 μg / cm ² . After the application, the solvent is removed by natural drying, vacuum drying or the like.

  The substrate of the mass spectrometric plate may be modified (processed) in advance by an appropriate physical or chemical technique before coating with the coating molecule. Specifically, techniques such as polishing the plate surface, scratching, acid treatment, alkali treatment, glass treatment (tetramethoxysilane, etc.) are exemplified.

  The transfer of the test sample to the mass spectrometry plate (support) can be performed by leaving the patient-derived biological sample as the test sample untreated, or after removing and concentrating the high molecular weight protein using an antibody column or other method. -It is performed by subjecting to polyacrylamide gel electrophoresis or isoelectric focusing, and transferring (blotting) the gel after contact with the plate. A known transfer device can be used. The transfer method itself is known. Preferably electrotransfer is used. The sample developed on the gel after the electrophoresis is transferred to the plate for mass spectrometry by various methods (diffusion, electric force, etc.). As the buffer used at the time of electrotransfer, it is preferable to use a buffer having a pH of 7-9 and a low salt concentration. Specific examples include Tris buffer, phosphate buffer, borate buffer, and acetate buffer. Tris buffer as tris / glycine / methanol buffer, SDS-tris-tricine buffer, etc., phosphate buffer as ACN / NaCl / isotonic phosphate buffer, sodium phosphate / ACN, borate, etc. Examples of the buffer solution include sodium borate-hydrochloric acid buffer solution, Tris-borate / EDTA, and borate / ACN. Examples of the acetate buffer include Tris-acetate / EDTA. Preferred are tris / glycine / methanol buffer and sodium borate-hydrochloric acid buffer. Examples of the composition of the tris / glycine / methanol buffer include Tris 10-15 mM, glycine 70-120 mM, and methanol 7-13%. Examples of the composition of the sodium borate-hydrochloric acid buffer include about 5 to 20 mM sodium borate.

  Thereby, the molecules present in the test sample including the target molecules are efficiently captured on the support surface. After the plate is dried, a reagent called matrix is added to absorb the laser light and promote ionization of analyte molecules through energy transfer, which is advantageous for later mass spectrometry (when using MALDI method) You can also. As the matrix, those known in mass spectrometry can be used. For example, sinapinic acid (SPA (= 3,5-dimethoxy-4-hydoroxycinammic acid)), indoleacrylic acid (IAA), 2,5-dihydroxybenzoic acid (DHB) ), Α-cyano-4-hydroxycinammic acid (CHCA), and the like, but is not limited thereto. Preferably, it is DHB or CHCA.

The presence and amount of the marker peptide (set) of the present invention, which is the target molecule, can be identified from the information on the molecular weight by mass spectrometry of the molecules in the test sample captured on the support surface by the above method. it can.
The mass spectrometer measures the molecular weight of a substance by ionizing a gaseous sample and then putting the molecule or molecular fragment into an electromagnetic field, separating it by mass number / charge number from its movement, and obtaining the spectrum of the substance. -It is a device to detect. Matrix-assisted laser deionization (MALDI), in which a sample and a matrix that absorbs laser light are mixed, dried and crystallized, and ionized analytes are brought into vacuum by ionization by energy transfer from the matrix and instantaneous heating by laser irradiation. And the MALDI-TOFMS method using time-of-flight mass spectrometry (TOFMS), which analyzes the mass number based on the time-of-flight difference of sample molecular ions by initial acceleration. Principles of ionization, nanoelectrospray mass spectrometry (nano-ESMS), etc., in which sample solutions are electrically sprayed into the atmosphere and individual analyte multivalent ions are unfolded into the gas phase A mass spectrometer based on can be used.
A method for mass spectrometry of molecules on a plate for mass spectrometry is known per se. For example, the method described in WO 2004/031759 can be appropriately modified and used as necessary.

  From the result of mass spectrometry, the presence or absence and the amount of the target molecule in the test sample can be identified based on the molecular weight information of the target molecule. In this step, it is also possible to output information from the mass spectrometer as differential information by comparing it with mass spectrometry data in a biological sample derived from a healthy person using an arbitrary program. It will be appreciated that such programs are well known and those skilled in the art can easily construct or modify such programs using known information processing techniques.

  In a particularly preferred embodiment, each of the above steps is performed using a blot chip manufactured by Protocera as a plate for mass spectrometry, and the marker peptide of the present invention is quantitatively compared (differential analysis) using a MALDI mass spectrometer. Further, if necessary, the marker peptide remaining on the same chip can be identified. Alternatively, up to quantitative comparison (differential analysis) of test samples is performed using a blot chip system manufactured by Protocera, and the marker peptide is identified by a combination device (LC-MS) of high performance liquid chromatography and an ion spray type mass spectrometer. / MS).

  Detection of the marker peptide of the present invention in the determination method of the present invention can also be performed using an antibody against it. In such a method, if an optimized immunoassay system is constructed and a kit is made, a marker peptide can be detected with high sensitivity and high accuracy without using a special device such as the mass spectrometer. It is particularly useful in that it can.

  The antibody against the marker peptide of the present invention can be prepared, for example, by isolating and purifying the marker peptide of the present invention from a biological sample derived from a patient that expresses the marker peptide, and immunizing an animal using the peptide as an antigen. . Alternatively, when the amount of the obtained peptide is small, the peptide is partially digested with peptidase or the like, the amino acid sequence of the obtained fragment is determined by the Edman method or the like, and the nucleic acid encoding the peptide based on the sequence A hybridizable oligonucleotide is synthesized and a cDNA library derived from the patient is used as a template to obtain a cDNA encoding a protein containing the peptide, or a cDNA derived from the patient is used as a primer. By performing RT-PCR using RNA as a template, a cDNA fragment encoding the peptide is obtained, the cDNA fragment is incorporated into an appropriate expression vector, introduced into an appropriate host cell, and the resulting transformant is cultured. Thus, the peptide of the present invention can be prepared in large quantities by collecting the recombinant peptide. You can. Alternatively, the peptide of the present invention can be obtained by using a cell-free transcription / translation system using the cDNA obtained as described above as a template.

  Alternatively, in the mass spectrometry described above, by using the tandem mass spectrometry (MS / MS) method, the amino acid sequence of the peptide of the present invention is directly identified, and all or part of the peptide is determined based on the sequence information. It can be synthesized and used as an antigen (hapten). Peptide identification method using MS / MS method includes de novo sequencing method to determine amino acid sequence by analyzing MS / MS spectrum and partial sequence information (mass tag) contained in MS / MS spectrum A database search using the method for identifying peptides and the like can be mentioned.

As described above, a preferred peptide of the present invention is a fragment of α1-AT, TTR, ApoA-I, ApoB-100, more preferably (1) amino acid numbers 1 to 1 in the amino acid sequence represented by SEQ ID NO: 1. A peptide comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by 24 (A1AT (1-24)); (2) the amino acid sequence represented by amino acid numbers 81 to 103 in the amino acid sequence represented by SEQ ID NO: 2 (3) a peptide having the same or substantially the same amino acid sequence as (TTR (81-103)); (3) an amino acid sequence represented by amino acid numbers 62 to 83 in the amino acid sequence represented by SEQ ID NO: 3 (AI (62-83 )) A peptide comprising the same or substantially the same amino acid sequence; (4) the same or substantially the same as the amino acid sequence represented by amino acid numbers 189 to 214 (AI (189-214)) in the amino acid sequence represented by SEQ ID NO: 3 From identical amino acid sequences (5) a peptide comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by amino acid numbers 196 to 214 (AI (196-214)) in the amino acid sequence represented by SEQ ID NO: 3; and (6 ) A peptide comprising the same or substantially the same amino acid sequence as the amino acid sequence (B100 (3218-3249)) represented by amino acid numbers 3218 to 3249 in the amino acid sequence represented by SEQ ID NO: 4.
Therefore, an antibody against the marker peptide of the present invention is, for example, A1AT (1-24), TTR (81-103), AI (62-83), AI (189-214), AI (196-214) or B100 ( 3218-3249) are synthesized using a known peptide synthesis method based on the above amino acid sequence information, or isolated α1-AT, TTR, ApoA-I or ApoB-100. A1AT (1-24), TTR (81-103), AI (62-83), AI (189-214), AI (196-214) or B100 (3218-3249) cut with an appropriate peptidase or the like It is desirable to obtain a peptide fragment containing all or part of the above sequence and prepare it as an immunogen.

  The antibody against the marker peptide of the present invention (hereinafter sometimes referred to as “the antibody of the present invention”) may be either a polyclonal antibody or a monoclonal antibody, and can be prepared by a known immunological technique. The antibody includes not only a complete antibody molecule but also a fragment thereof, and examples thereof include Fab, F (ab ′) 2, ScFv, and minibody.

  For example, the polyclonal antibody may be prepared by a carrier protein such as bovine serum albumin or KLH (Keyhole Limpet Hemocyanin) prepared by any one of the above methods or other methods. (It can also be a cross-linked complex) is administered as an antigen, together with a commercially available adjuvant (for example, complete or incomplete Freund's adjuvant), subcutaneously or intraperitoneally in animals every 2 to 3 weeks, about 2 to 4 times ( The antibody titer of the partially collected serum is measured by a known antigen-antibody reaction, and the increase is confirmed.) Obtained by collecting whole blood about 3 to about 10 days after the final immunization and purifying the antiserum it can. Examples of animals to which the antigen is administered include mammals such as rats, mice, rabbits, goats, guinea pigs, and hamsters.

  Monoclonal antibodies can be obtained by cell fusion methods (for example, Takeshi Watanabe, principles of cell fusion methods and preparation of monoclonal antibodies, Akira Taniuchi, Toshitada Takahashi, “Monoclonal Antibodies and Cancer: Basic and Clinical”, 2-14). Page, Science Forum Publishing, 1985). For example, the marker peptide of the present invention or a partial peptide thereof is administered to a mouse subcutaneously or intraperitoneally 2-4 times together with a commercially available adjuvant, and the spleen or lymph node is collected about 3 days after the final administration, and white blood cells are collected. The leukocytes and myeloma cells (for example, NS-1, P3X63Ag8, etc.) are fused to obtain a hybridoma that produces a monoclonal antibody against the peptide. Cell fusion may be PEG method [J. Immunol. Methods, 81 (2): 223-228 (1985)] or voltage pulse method [Hybridoma, 7 (6): 627-633 (1988)]. A hybridoma producing a desired monoclonal antibody can be selected by detecting an antibody that specifically binds to an antigen from the culture supernatant using a known EIA or RIA method or the like. The culture of the hybridoma producing the monoclonal antibody can be performed in vitro or in vivo such as mouse or rat, or preferably mouse ascites, and the antibody can be obtained from the culture supernatant of the hybridoma and the ascites of the animal, respectively. .

  The determination method of the present invention using the antibody of the present invention is not particularly limited, and the amount of antibody, antigen or antibody-antigen complex corresponding to the amount of antigen in the test sample is determined by chemical or physical means. Any measurement method may be used as long as it is a measurement method that is detected and calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competition method, immunometric method and sandwich method are preferably used.

As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance or the like is used. As the radioisotope, for example, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C] and the like are used. The enzyme is preferably stable and has a large specific activity. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used. Furthermore, a biotin-avidin system can also be used for binding of an antibody or antigen and a labeling agent.

  For insolubilization of an antigen or antibody, physical adsorption may be used, or a method using a chemical bond usually used to insolubilize or immobilize a protein or an enzyme may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass.

  In the sandwich method, a test sample is reacted with an insolubilized antibody of the present invention (primary reaction), and another labeled antibody of the present invention is reacted (secondary reaction), followed by a labeling agent on an insolubilized carrier. By measuring the amount (activity), the amount of the peptide of the present invention in the test sample can be quantified. The primary reaction and the secondary reaction may be performed in the reverse order, or may be performed simultaneously or at different times.

The monoclonal antibody against the polypeptide of the present invention can also be used in measurement systems other than the sandwich method, such as a competitive method, an immunometric method, or nephrometry.
In the competition method, the antigen in the test sample and the labeled antigen are reacted competitively with the antibody, and then the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated ( B / F separation), measure the amount of labeled B or F, and quantify the amount of antigen in the test sample. In this reaction method, a soluble antibody is used as an antibody, a B / F separation is performed using polyethylene glycol, a liquid phase method using a second antibody against the antibody, and a solid-phased antibody is used as the first antibody, or The first antibody is soluble, and the second antibody is a solid phase method using a solid phase antibody.
In the immunometric method, the antigen of the test sample and the immobilized antigen are competitively reacted with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated, or the antigen and excess in the test sample are separated. Then, the solid phase antigen is added, and then the solid phase antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to quantify the amount of antigen in the test sample.
In nephrometry, the amount of insoluble precipitate produced as a result of the antigen-antibody reaction in a gel or solution is measured. Laser nephrometry using laser scattering is preferably used even when the amount of antigen in the test sample is small and only a small amount of precipitate is obtained.

In applying these individual immunological measurement methods to the quantification method of the present invention, special conditions, operations and the like are not required to be set. What is necessary is just to construct | assemble the measurement system of the peptide of this invention, adding the usual technical consideration of those skilled in the art to the usual conditions and operation methods in each method. For details of these general technical means, it is possible to refer to reviews, books and the like.
For example, Hiroshi Irie “Radioimmunoassay” (Kodansha, published in 1974), Hiroshi Irie “Continue Radioimmunoassay” (published in Kodansha, 1979), “Enzyme Immunoassay” edited by Eiji Ishikawa et al. 53), "Enzyme Immunoassay" edited by Eiji Ishikawa et al. (2nd edition) (Medical School, published in 1982), "Enzyme Immunoassay" edited by Eiji Ishikawa et al. (3rd edition) (Medical School, Showa) 62), “Methods in ENZYMOLOGY” Vol. 70 (Immunochemical Techniques (Part A)), ibid. Vol. 73 (Immunochemical Techniques (Part B)), ibid. Vol. 74 (Immunochemical Techniques (Part C)), ibid. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)), ibid.Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), ibid.Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies) )) (Above, published by Academic Press) It is possible to irradiation.

  Alternatively, as another determination method of the present invention using the antibody of the present invention, the antibody is immobilized on the surface of a probe that can be adapted to a mass spectrometer as described above, and a test sample is attached to the antibody on the probe. Examples include a method of detecting a peak corresponding to the molecular weight of a marker peptide recognized by the antibody by subjecting the biological sample component captured by the antibody to mass spectrometry.

  When any of the above methods detects a specific marker peptide in any stage of biodegradation syndrome with a statistically significant difference from the level in healthy human body fluid in the subject body fluid It can be determined that the patient has a high possibility of exhibiting the pathological condition of the biodegradation syndrome.

  The determination method of the present invention is preferably carried out by collecting biological samples from patients in time series and examining changes with time in the expression of the marker peptide of the present invention in each sample. The collection interval of the biological sample is not particularly limited, but it is desirable to sample as frequently as possible within a range that does not impair the patient's QOL. For example, when plasma or serum is used as a sample, blood is collected at intervals of about 1 minute to about 12 hours. It is preferable to carry out. For each sample, the expression of the marker peptide of the present invention was examined as described above. Over time, the marker peptide specific for the more advanced stage of biodegradation syndrome was significantly different from the level in the body fluid of healthy humans. Or when the difference from the level in the body fluid of a healthy person increases, it can be determined that there is a high possibility that the pathological condition of the biodegradation syndrome has progressed in the patient. On the other hand, when the difference between the level of the marker peptide in the patient and the level in the healthy human body fluid decreases, it can be determined that there is a high possibility that the pathological condition of the biodegradation syndrome is improved in the patient.

  When an increase in a specific marker peptide is detected as a result of the above-described method for determining the stage of biodegradation syndrome, a substrate that is cleaved by a parent protein of the marker peptide (that is, a degrading enzyme that generates the marker peptide) Protein) is degraded and decreased in the patient. Therefore, an effective nutritional assessment can be achieved by supplying the parent protein into the patient. Therefore, the present invention also provides a method comprising administering an effective amount of a protein to a patient in which a significant increase in the degradation product of a protein is recognized by the method for determining the stage of biodegradation syndrome. . Such proteins include, but are not limited to albumin, retinol binding protein, transthyretin, transferrin and the like. For administration of such nutritional assessment protein, a method known per se such as intravenous infusion can be used as appropriate.

  Further, as a result of performing the above-described method for determining the stage of biodegradation syndrome, when a decrease in the level of the therapeutic peptide and / or an increase in the level of the exacerbation peptide is detected, the amount of the therapeutic peptide or An effective amount of a substance that increases activity and / or a substance that decreases the amount or activity of the exacerbation peptide can be administered. The dosage form, administration route and dosage of the substance are the same as described above.

  Furthermore, the determination method of the biodegradation syndrome by the above-mentioned time-series sampling is based on the measure when the syndrome is treated for the patient who is the subject between the previous sampling and the current sampling. It can be used to evaluate the therapeutic effect. That is, for a sample sampled before and after treatment, when it is determined that the condition after treatment is improved compared to the condition before treatment, it can be evaluated that the treatment is effective. On the other hand, when it is determined that the condition after treatment is not improved or further deteriorated as compared with the condition before treatment, it can be evaluated that the treatment has no effect.

  And even if it has the best treatment method, if it falls into a situation in which a statistically significant difference between the body fluid of the biodegradable syndrome patient and the body fluid of a healthy person cannot be resolved, the patient It can be predicted that the function of the organ is abolished or life is in danger. Therefore, the determination method of the present invention also provides that when the treatment effect is evaluated as described above and it is determined that any possible treatment is not effective in a patient with biodegradation syndrome in stage IV, It can also be used as a method for predicting that organs / organs are abandoned and / or in danger of survival. The patient who can be a subject of the prediction method is not particularly limited as long as it is a patient suffering from a basic disease that can result in biodegradation syndrome, but preferably, among the above-mentioned various basic disease patients, severe symptoms are exhibited. Patients, for example, cancer, disseminated intravascular coagulation syndrome, multiple organ failure, anorexia, and intractable diseases with uncertain prognosis. In addition, a disease that causes the abolition of function of one organ, such as a patient having retinitis pigmentosa, can also be a subject. The patient's age, sex, race, etc. are not limited at all.

  In particular, when AI (62-83), AI (189-214), AI (196-214) or B100 (3218-3249), which are preferred marker peptides of the present invention, are detected, the patient has a very prognosis. The failure can be diagnosed almost definitely. Currently, DIC and MOF are treated with anticoagulant factors (heparin, antithrombin, etc.), degradation enzyme inhibitors, blood purification therapy, etc., but there is an effective means to determine the possibility of recovery. As a result, patients who are not expected to recover are treated in the same way, which not only damages the patient's dignity but also puts pressure on the medical economy. Therefore, the novel diagnostic method for biodegradation syndrome targeting the marker peptide of the present invention provides an effective solution to such a problem.

  Hereinafter, the present invention will be described more specifically with reference to examples, but it goes without saying that the present invention is not limited thereto. Unless otherwise stated, the measurement was performed using Bruker Daltonics' Ultraflex II and Protocera's blot chip.

Example 1 Differential Profiling Analysis Using BlotChip After treating sera of Crow-Fukase syndrome patient and healthy human sera known as inferior prognosis with electrophoresis sample buffer, 4-12% gradient polyacrylamide gel (Invitorigen) was used. Applied and electrophoresed. After completion of electrophoresis, the gel was cut out and transferred to BlotChip in borate buffer at 90 mA for 120 minutes. After electrical transfer to BlotChip, rinse BlotChip with ultra-pure, apply matrix (α-Cyano-4-hydroxy cinamic acid) to the entire chip, and then perform matrix-assisted laser desorption ionization MS measurement was performed with a time-of-flight (MALDI-TOF) mass spectrometer (Ultra-FlexII manufactured by Bruker Daltnics). The measurement parameters are Detector Voltage 1685V, Supression 1000, and Laser Intensity are 28 to 35 Fuzzy modes. The chip surface where the sample is present is measured at 205 points in total, and the obtained 41 spectra are integrated. (Hereinafter referred to as integrated spectrum). Differential profiling analysis by ClinProTools (Bruker Daltonik GmbH) was performed using the obtained integrated spectrum. In addition, the integrated spectral data of human patient sera and healthy human sera were overlaid independently and the results of ClinProTools were scrutinized. A part of the results is shown in FIG. Peptide (No. 6) that is detected at a level more than twice that of a healthy person through stages I to IV, and is detected at a level of ½ times or less for a healthy person through stages I to IV Peptides (No. 2, No. 3, No. 3, No. 3, No. 3, No. 3, No. 3, No. 3, No. 3, No. 3, No. 3, No. 3, No. 3, No. 3, No. 3, No. 3, No. 3, respectively) No. 4 and No. 5) were obtained.

Example 2 Identification of peptides by de novo MS / MS analysis on BlotChip For identification, matrix-assisted laser desorption ionization Using time-of-flight (MALDI-TOF) mass spectrometer (Bruker Daltnics Ultra-FlexII), Bradykinin, AngiotensinII, AngiotensinI, SubstanceP, Bombesin, Renin Substrate, ACTH Clip {1-17}, ACTH Clip {18 -39}, mass calibration was performed using Somatostatin. After that, profiling is performed in reflectron measurement mode, and selected peptide peaks and their fragment ions are identified by MS / MS analysis through the MASCOT search engine built into Biotools (Bruker Daltonik GmbH) with NCBInr and SwissProt databases. Went.
As a result, as a peptide specific to the I phase, the amino acid sequence represented by amino acid numbers 62 to 83 (AI (62-83)) in the amino acid sequence represented by SEQ ID NO: 3, and the amino acid sequence represented by SEQ ID NO: 3 An amino acid sequence represented by amino acid numbers 189 to 214 (AI (189-214)), an amino acid sequence represented by amino acid numbers 196 to 214 in the amino acid sequence represented by SEQ ID NO: 3 (AI (196-214)), and a sequence number In the amino acid sequence shown in SEQ ID NO: 1, the peptide consisting of the amino acid sequence shown by amino acid numbers 3218 to 3249 (B100 (3218-3249)) in the amino acid sequence shown in FIG. Peptides consisting of the amino acid sequence represented by amino acid numbers 1 to 24 (A1AT (1-24)) and the amino acid sequence represented by amino acid numbers 81 to 103 (TTR (81-103)) in the amino acid sequence represented by SEQ ID NO: 2 Is identified . As a result of homology search, AI (62-83), AI (189-214) and AI (196-214) are all apolipoprotein AI degradation products, and B100 (3218-3249) is apolipoprotein B-100. It was revealed that A1AT (1-24) is a degradation product of α1-antitrypsin and TTR (81-103) is a degradation product of transthyretin.

  The novel biodegradation syndrome diagnostic system of the present invention is useful in that the pathological condition of the syndrome can be determined quickly and accurately, so that early detection and early treatment of the symptoms are possible. In addition, it is possible to develop therapeutic drugs inspired by the degradation products of proteins present in the living body. With the development of effective treatment methods for the biodegradation syndrome, as well as the effect of improving the prognosis of organs and organs, conventional diagnostic techniques It is expected that many patients who will die can be saved. Furthermore, the terminal stage in biodegradable syndrome can be determined quickly and accurately based on the evidence, so it is possible to discontinue useless treatment for patients who are unlikely to recover, and there is a problem among developed countries. It is expected to contribute to the improvement of the medical economy not only in Japan but also around the world.

It is a figure which shows the detection of a marker specific to each clinical stage of biodegradation syndrome. FIG. 1A shows a part of a mass spectrometry chart of serum samples derived from patients with various clinical stages (stage I to IV) of biodegradation syndrome and healthy individuals, and FIG. 1B shows a quantitative comparison of the obtained biomarkers.

Claims (27)

  A marker peptide set for biodegradation syndrome, comprising one or more peptides that are degradation products of proteins present in the living body, which are detected with a statistically significant difference between the biodisintegration syndrome patient body fluid and the healthy human body fluid.   The marker peptide set according to claim 1, wherein the statistically significant difference is observed in at least one stage of stage I to stage IV of the biodegradation syndrome.   The marker peptide set according to claim 1 or 2, wherein the protein is selected from the group consisting of a physiologically active substance carrier protein, a protease inhibitor, and a nutritional assessment protein.   The marker peptide set according to any one of claims 1 to 3, wherein at least one of the peptides is substantially undetectable from body fluid of a healthy person.   The marker peptide set according to any one of claims 1 to 4, wherein at least one of the peptides has a molecular weight of about 50,000 or less.   The marker peptide set according to any one of claims 1 to 5, wherein the protein is selected from the group consisting of α1-antitrypsin, transthyretin, apolipoprotein AI, and apolipoprotein B-100. The marker peptide set according to claim 6, comprising at least one peptide selected from the group consisting of the following (1) to (6).
(1) A peptide consisting of an amino acid sequence identical or substantially identical to the amino acid sequence represented by amino acid numbers 1 to 24 in the amino acid sequence represented by SEQ ID NO: 1.
(2) A peptide comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by amino acid numbers 81 to 103 in the amino acid sequence represented by SEQ ID NO: 2
(3) A peptide comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by amino acid numbers 62 to 83 in the amino acid sequence represented by SEQ ID NO: 3
(4) A peptide consisting of an amino acid sequence identical or substantially identical to the amino acid sequence represented by amino acid numbers 189 to 214 in the amino acid sequence represented by SEQ ID NO: 3
(5) A peptide consisting of an amino acid sequence identical or substantially identical to the amino acid sequence represented by amino acid numbers 196 to 214 in the amino acid sequence represented by SEQ ID NO: 3
(6) A peptide comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by amino acid numbers 3218 to 3249 in the amino acid sequence represented by SEQ ID NO: 4   An antibody that recognizes any peptide of the marker peptide set according to claim 7.   A peptide having the physiological activity that therapeutically acts on the biodegradation syndrome, which is any peptide constituting the marker peptide set according to any one of claims 1 to 7.   A therapeutic agent for biodegradation syndrome, comprising a substance that increases the amount or activity of the peptide according to claim 9.   The substance is recognized and cleaved by the peptide according to claim 9, an agonist of the peptide, a decomposing enzyme that releases the peptide from a protein present in a living body, and the N-terminal side and C-terminal side of the peptide by the decomposing enzyme. A substrate or substrate analog molecule of the decomposing enzyme, a molecule that promotes the production of the decomposing enzyme, a molecule that promotes the activity of the decomposing enzyme, and a molecule that suppresses the production of an inhibitor of the decomposing enzyme The therapeutic agent of Claim 10 selected from the group which consists of.   The peptide which comprises the marker peptide set in any one of Claims 1-7, Comprising: The peptide which has the physiological activity which exacerbates a biodegradation syndrome.   A therapeutic agent for biodegradation syndrome, comprising a substance that reduces the amount or activity of the peptide according to claim 12.   The substance is an antagonist of the peptide according to claim 12, a molecule that suppresses the production of a degrading enzyme that liberates the peptide from a protein present in a living body, an inhibitor of the decomposing enzyme, and a molecule that promotes the production of the inhibitor. The therapeutic agent of Claim 13 selected from the group which consists of.   A method of treating biodegradation syndrome, comprising increasing the amount or activity of the peptide of claim 9 and / or decreasing the amount or activity of the peptide of claim 12 in a patient.   A method for determining a stage in a biodegradable syndrome, comprising one or more marker peptide sets according to any one of claims 1 to 7 in a biological sample derived from a patient suffering from a basic disease that can result in the syndrome And / or detecting the peptide of claim 9 and / or 12.   Phase IV when a peptide that is a degradation product of α1-antitrypsin and / or transthyretin is detected, and I when a peptide that is a degradation product of apolipoprotein AI and / or apolipoprotein B-100 is detected The method of claim 16, comprising determining that it is a period. The degradation product of α1-antitrypsin is the peptide of (1) below, the degradation product of transthyretin is the peptide of (2) below, and the degradation product of apolipoprotein AI is one or more selected from (3) to (5) below The method according to claim 17, wherein the peptide and the degradation product of apolipoprotein B-100 are peptides of the following (6).
(1) A peptide consisting of an amino acid sequence identical or substantially identical to the amino acid sequence represented by amino acid numbers 1 to 24 in the amino acid sequence represented by SEQ ID NO: 1.
(2) A peptide comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by amino acid numbers 81 to 103 in the amino acid sequence represented by SEQ ID NO: 2
(3) A peptide comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by amino acid numbers 62 to 83 in the amino acid sequence represented by SEQ ID NO: 3
(4) A peptide consisting of an amino acid sequence identical or substantially identical to the amino acid sequence represented by amino acid numbers 189 to 214 in the amino acid sequence represented by SEQ ID NO: 3
(5) A peptide consisting of an amino acid sequence identical or substantially identical to the amino acid sequence represented by amino acid numbers 196 to 214 in the amino acid sequence represented by SEQ ID NO: 3
(6) A peptide comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by amino acid numbers 3218 to 3249 in the amino acid sequence represented by SEQ ID NO: 4   The method according to any one of claims 16 to 18, wherein the biological sample is a body fluid.   20. The method of claim 19, wherein the body fluid is selected from the group consisting of blood, plasma, serum, saliva, urine, spinal fluid, bone marrow fluid, pleural effusion, ascites, joint fluid, tear fluid, aqueous humor, vitreous humor and lymph. .   21. A method according to any of claims 16 to 20, comprising subjecting the biological sample to mass spectrometry.   An antibody that recognizes one or more peptides constituting the marker peptide set according to any one of claims 1 to 7 and / or the peptide according to claims 9 and / or 12, respectively, is used. The method in any one of -20.   A biological sample is collected from a patient in time series, and one or more peptides constituting the marker peptide set according to any one of claims 1 to 7 and / or types of peptides according to claims 9 and / or 12 in the sample. 23. The method according to any one of claims 16 to 22, wherein the change over time and the amount is examined.   24. A method for treating biodegradation syndrome, comprising administering an effective amount of a protein to a patient in which a significant increase in a degradation product of a protein is recognized by the method according to any one of claims 16 to 23. Including methods.   A method for evaluating a therapeutic effect in a patient with biodegradation syndrome, wherein a biological sample is collected from the patient before and after treatment, and the marker peptide set according to any one of claims 1 to 7 is configured in the sample 13. A method comprising examining changes in the type and amount of one or more peptides and / or peptides according to claim 9 and / or 12.   A method for predicting the abolition of organs and organs and / or the risk of survival in a patient with biodegradation syndrome, and according to the method according to claim 25, it has been determined that any treatment that can be taken in patients with stage IV was ineffective In some cases, the method predicts that the patient is at risk of organ abolition and / or survival.   8. One or more peptides constituting the marker peptide set according to any one of claims 1 to 7, wherein the peptide is decomposed at the N-terminal side and / or C-terminal side of each peptide from a protein present in the living body. The peptide set for identifying a degrading enzyme, further comprising an amino acid sequence that is recognized and cleaved by an enzyme.