WO2010104176A1 - Inhibiteur de clivage des méga-phosphoprotéines - Google Patents

Inhibiteur de clivage des méga-phosphoprotéines Download PDF

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Publication number
WO2010104176A1
WO2010104176A1 PCT/JP2010/054237 JP2010054237W WO2010104176A1 WO 2010104176 A1 WO2010104176 A1 WO 2010104176A1 JP 2010054237 W JP2010054237 W JP 2010054237W WO 2010104176 A1 WO2010104176 A1 WO 2010104176A1
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Prior art keywords
protein cleavage
megalin
megalin protein
inhibitor
cleavage inhibitor
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PCT/JP2010/054237
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English (en)
Japanese (ja)
Inventor
博司 岩田
英里 原田
彰 三井
亮彦 斉藤
Original Assignee
味の素株式会社
国立大学法人 新潟大学
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Application filed by 味の素株式会社, 国立大学法人 新潟大学 filed Critical 味の素株式会社
Priority to JP2011503880A priority Critical patent/JPWO2010104176A1/ja
Publication of WO2010104176A1 publication Critical patent/WO2010104176A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • 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

Definitions

  • the present invention relates to a megalin protein cleavage inhibitor useful as a reagent for conducting an experiment relating to the cleavage of megalin protein, a drug for treating a disease mediated by the cleavage of megalin protein, and the like.
  • Megalin is a member of the low-density lipoprotein (LDL) receptor family, and is a glycoprotein having a molecular weight of about 600 KDa that is localized on the plasma membrane of proximal tubular epithelial cells.
  • Megalin is a large cell membrane protein consisting of 4655 amino acids (human megalin) or 4660 amino acids (rat megalin) and located outside the N-terminal side, and is a large cell having four functional domains. It consists of an outer region followed by a short intracellular region (via a single transmembrane region).
  • Patent Literature 1 describes that megalin is excreted in the urine of a nephropathy patient and diagnoses diseases such as kidney disease by quantifying the amount.
  • Non-Patent Document 2 Non-Patent Document 2
  • Presenilin is a novel transmembrane aspa Reported to be rutile protease. (Non Patent Literature 3) Presenilin is a putative eight-transmembrane protein that regulates the production of amyloid ⁇ protein from APP.
  • Non-Patent Document 4 Non-Patent Document 4
  • ⁇ -secretase inhibitors being developed as therapeutic agents for Alzheimer's disease may have inhibitory activity not only on presenilin but also on signal peptide peptidases Reports showing sex have been made in recent years.
  • Non-patent document 2 it is not clear whether presenilin and signal peptide peptidase are involved in the cleavage of megalin protein or the relationship between megalin protein cleavage and pathological conditions.
  • An object of the present invention is to provide a therapeutic agent for renal injury based on a novel mechanism of inhibiting megalin protein cleavage.
  • Non-Patent Document 9 Based on the fact that DBZ, which has ⁇ -secretase inhibitory activity, improves urinary albumin leakage and glomerular damage in renal injury model rats, the authors of Non-Patent Document 9 show that Notch protein cleavage inhibition is a treatment for renal injury. Indicates that it is important.
  • the present inventors have found that inhibition of megalin cleavage rather than inhibition of Notch protein cleavage is important for the improvement of the associated renal damage. That is, the present inventors confirmed that the second-stage fragment of the megalin protein fragment directly induces renal tubular cell damage and inhibits ⁇ -secretase but not signal peptide peptidase.
  • Non-patent Document 8 which is also known to inhibit Notch protein cleavage
  • DAPT Non-patent Document 8
  • DBZ Non-patent Document 8
  • the present invention provides a megalin protein cleavage inhibitor comprising a signal peptide peptidase inhibitor.
  • the present invention also provides a megalin protein cleavage inhibitor comprising a ⁇ -secretase inhibitor.
  • the present invention also provides a megalin protein cleavage inhibitor comprising a megalin protein cleavage inhibitor having an amide structure in the molecule.
  • the present invention provides a therapeutic agent for renal injury characterized by containing a megalin protein cleavage inhibitor.
  • a substance that inhibits megalin protein cleavage can specifically suppress only the negative functions related to megalin protein signal transduction, so that it has few side effects on diseases associated with megalin protein cleavage, particularly kidney disease. It becomes.
  • FIG. 1 shows the daily results of Alb concentrations in urine and plasma samples by DAPT administration using a nephrotic syndrome rat model.
  • FIG. 2 shows the results of renal histology by PAS staining with DAPT administration using a rat model of nephrotic syndrome.
  • FIG. 3 shows the expression pattern of megalin protein fragments in renal tissue by DAPT administration using a rat model of nephrotic syndrome.
  • FIG. 4 shows the daily results of Alb concentration in urine samples and plasma samples by DBZ administration using a rat model of nephrotic syndrome.
  • FIG. 5 shows the results of renal histology by PAS staining using DBZ administration using a rat model of nephrotic syndrome.
  • FIG. 6 shows the expression pattern of megalin protein fragments in renal tissue by DBZ administration using a rat model of nephrotic syndrome.
  • the megalin protein cleavage inhibitor is not particularly limited as long as it has an activity that inhibits megalin protein cleavage, and even if it has a signal peptide peptidase inhibitory activity, it has an activity to inhibit ⁇ -secretase. You may have.
  • the megalin protein cleavage inhibitor those having an amide structure in the molecule are preferred, those having a ring structure directly connected to at least one nitrogen atom of the amide structure, and those containing a nitrogen atom are more preferred.
  • a megalin protein cleavage inhibitor what has a polyamide structure is also preferable, and what further has an aromatic ring group in a molecule
  • the polyamide structure is preferably 4 or more amide structures, more preferably 4 to 25 amide structures, still more preferably 5 to 15 amide structures, and most preferably 5 to 8 amide structures. It is an amide structure.
  • megalin protein cleavage inhibitors include Naunyn Schmiedebergs Arch Pharmacol. 2008; 377 (4-6): 295-300, THE JOURNAL OF BIOLOGICAL CHEMISTRY.2007; 282 (51): 36829-36836, THE JOURNAL OF BIOLOGICAL CHEMISTRY. 2003; 278 (19): 16528-16533, and the like, but are not limited thereto.
  • DBZ Non-patent document 8
  • Compound E Non-patent document 8
  • NAP-AHW700-NX Non-patent document 11
  • LY411575 Non-patent document 11
  • L-695458 Non-patent document) Reference 11
  • TBL4K Non-Patent Document 11
  • Z-LL 2-Ketone
  • the megalin protein cleavage inhibitors are DBZ, Compound E, NAP-AHW700-NX and LY411575.
  • TBL4K and (Z-LL) 2-Ketone are also preferred as megalin protein cleavage inhibitors.
  • the megalin protein cleavage inhibitor of the present invention may be a signal peptide peptidase inhibitor and / or a ⁇ -secretase inhibitor, and is preferably a signal peptide peptidase inhibitor.
  • a commercially available product can be used as the megalin protein cleavage inhibitor of the present invention. Moreover, it can acquire by using well-known methods, such as (1) the method of synthesize
  • the chemical synthesis method include a liquid phase synthesis method and a solid phase synthesis method.
  • the synthesized signal peptide peptidase inhibitor can be purified by usual means such as ion exchange chromatography, reverse phase high performance liquid chromatography, affinity chromatography, recrystallization and the like. Such chemical synthesis methods and subsequent purification are well known in the art.
  • the megalin protein cleavage inhibitor of the present invention may contain the above-mentioned megalin protein cleavage inhibitor alone or in combination of any two or more thereof, and is further acceptable pharmaceutically, physiologically and experimentally. Any solid or liquid carrier, additive, etc. may be included.
  • the carrier examples include glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, gelatin, albumin, amino acid, water, and physiological saline. Water etc. are mentioned. Further, if necessary, conventional additives such as stabilizers, wetting agents, emulsifiers, binders, tonicity agents and the like can be appropriately added to the megalin protein cleavage inhibitor of the present invention.
  • the additive is not particularly limited as long as it is usually used for the purpose depending on the purpose. Specifically, for example, flavoring, sugar, sweetener, dietary fiber, vitamins, sodium glutamate Amino acids such as (MSG), nucleic acids such as inosine monophosphate (IMP), inorganic salts such as sodium chloride, and water.
  • the megalin protein cleavage inhibitor of the present invention can be used in any form without limitation on physical properties such as dry powder, paste, and solution.
  • the megalin protein cleavage inhibitor of the present invention can be used in medicines, quasi drugs, reagents, health foods and the like.
  • the amount of the megalin protein cleavage inhibitor of the present invention is appropriately adjusted according to each purpose.
  • the total amount of the above megalin protein cleavage inhibitors is used.
  • 0.01 g to 10 g per kg body weight is preferable, and 0.1 g to 1 g per kg body weight is more preferable.
  • the frequency of administration is not particularly limited, and can be administered once to several times per day.
  • the megalin protein cleavage inhibitor of the present invention is used as a reagent, it is preferably 0.000001 g to 10 g per formulation, more preferably 0.00001 g to 1 g per formulation.
  • the content of the megalin protein cleavage inhibitor in the megalin protein cleavage inhibitor of the present invention is not particularly limited as long as it is suitable for the amount used, and preferably 0.000001% by mass to 99.9999 per dry weight. % By mass, more preferably 0.00001% by mass to 99.999% by mass, and particularly preferably 0.0001% by mass to 99.99% by mass.
  • the megalin protein cleavage inhibitor of the present invention may contain any known substance in addition to the megalin protein cleavage inhibitor, depending on the purpose.
  • the present invention will be described with reference to examples of pharmacological tests, but these are for better understanding of the present invention and do not limit the scope of the present invention.
  • Comparative Example 1 Drug efficacy test of ⁇ -secretase inhibitor (N- [N- (3,5-difluorophenylacetyl) -L-alanyl]-(S) -phenylglycine tert-butyl ester (DAPT) using a rat model of nephrotic syndrome)
  • DAPT ⁇ -secretase inhibitor
  • the rats that were first acclimated were weighed, individuals near the median were selected, placed in metabolic cages and acclimated for 3 days, and urine collection was started for 24 hours from the 4th day after the start of metabolic cage feeding.
  • the urine weight was measured, and a part was used as a sample for measuring urinary albumin (Alb) .
  • the urine sample was mixed by inversion, centrifuged (3,000 rpm ⁇ 10 minutes, 4 ° C.), and the supernatant was collected.
  • Urinary Alb was used to measure the body weight of rats on the day before the start of puromycin aminonucleoside (PAN) administration, and heparinized blood was collected from the jugular vein under ether anesthesia.
  • PAN puromycin aminonucleoside
  • Alb second priority is weight Carried out. In the morning, the PAN administration solution was administered into the tail vein at 5 mL / kg.
  • DAPT test solution
  • 0.5% ethanol / corn oil was subcutaneously administered every day at a concentration of 50 mg / kg.
  • the dose was calculated using the body weight of the day before PAN administration.
  • One day after the start of administration of the test solution the body weight was measured, and heparinized blood was collected from the jugular vein under ether anesthesia.
  • urine collection was started and urine was collected 24 hours later.
  • body weight and urine weight were measured, and a part was used as a sample for measuring urinary Alb after administration.
  • the test solution on the second day was intraperitoneally administered.
  • urine accumulation was started, and urine was collected 24 hours later.
  • Cre creatinine
  • Alb albumin
  • Creatinine Creatinine
  • Creatinine cassette Name: CREP2, Cat No. 682965 (Roche Diagnostics Inc.) Method for Measuring Content of Plasma Alb (Albumin) Plasma Alb was measured using COBAS INTEGRA 400plus.
  • the reagent names used in the measurement are described below.
  • Albumin cassette Name: ALBIICat No. 684310 (Roche Diagnostics) Method for measuring urinary Alb content Urine Alb was measured using NEPHRATII ELISA kit.
  • Fig. 1 shows the daily results of the Alb concentration in the urine sample and plasma sample.
  • Administration of PAN caused kidney damage, resulting in increased urinary albumin and decreased blood albumin, but was further exacerbated by administration of DAPT, which is known to inhibit ⁇ -secretase but not signal peptide peptidase .
  • the kidney tissue image by PAS staining is shown in FIG. Renal tubular damage caused by PAN administration was further exacerbated by DAPT administration.
  • DAPT decreased the amount of the first-stage megalin protein and increased the amount of the second-stage megalin protein fragment (FIG. 3). .
  • Example 1 ⁇ -secretase inhibitor (S, S) -2- [2- (3,5-Difluorophenyl) acetylamino] -N- (5-methyl-6-oxo-6,7 using nephrotic syndrome model rats -Dihydro-5H-dibenzo [b, d] azepin-7-yl) propionamide (DBZ) as a test solution DBZ instead of DAPT (administration solution: 0.5% (w / v) hydroxypropyl methylcellulose / 0 1% (w / v) Tween-80, administered intraperitoneally at a dose concentration of 5 mg / kg), in the same manner as in Comparative Example 1, in the amount of megalin protein fragments in the urine In addition, the amount of albumin in blood, the amount of creatinine, and the kidney tissue image by PAS staining were examined.
  • Fig. 4 shows the daily results of the Alb concentration in the urine sample and the plasma sample.
  • Administration of PAN caused renal injury, resulting in an increase in urinary albumin and a decrease in blood albumin, but was improved by administration of DBZ, which is known to inhibit both ⁇ -secretase and signal peptide peptidase.
  • DBZ a kidney tissue image by PAS staining is shown in FIG.
  • the renal tubular damage caused by PAN administration was markedly improved by DBZ administration.
  • DBZ increased the amount of first-stage megalin protein fragments, but the second-stage fragments were not detected (FIG. 6). .

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  • Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
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Abstract

L'invention porte sur un agent d'amélioration des troubles rénaux, portant sur un nouveau mécanisme, à savoir l'inhibition du clivage d'une méga-phosphoprotéine. Une substance capable d'inhiber une peptide signale peptidase ou une gamma-sécrétase est utile en tant qu'inhibiteur de clivage des méga-phosphoprotéines, et peut supprimer uniquement les fonctions négatives liées à la signalisation d'une méga-phosphoprotéine de manière significative. Il en résulte que la substance peut être utilisée en tant qu'agent thérapeutique/prophylactique pour des maladies associées au clivage d'une méga-phosphoprotéine, en particulier des maladies rénales, tout en produisant peu d'effets secondaires indésirables.
PCT/JP2010/054237 2009-03-13 2010-03-12 Inhibiteur de clivage des méga-phosphoprotéines WO2010104176A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009035522A1 (fr) * 2007-09-14 2009-03-19 Albert Einstein College Of Medicine Of Yeshiva University Utilisation d'inhibiteurs de gamma secrétase et inhibiteurs de la voie notch pour le traitement et la prévention d'une maladie rénale

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009035522A1 (fr) * 2007-09-14 2009-03-19 Albert Einstein College Of Medicine Of Yeshiva University Utilisation d'inhibiteurs de gamma secrétase et inhibiteurs de la voie notch pour le traitement et la prévention d'une maladie rénale

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
GRIGORENKO,A.P. ET AL.: "The Caenorhabditis elegans IMPAS gene, imp-2, is essential for development and is functionally distinct from related presenilins", PROC NATL ACAD SCI U S A, vol. 101, no. 41, 2004, pages 14955 - 14960 *
LI,Y. ET AL.: "The COOH terminus of megalin regulates gene expression in opossum kidney proximal tubule cells", AM J PHYSIOL CELL PHYSIOL, vol. 295, no. 2, 2008, pages C529 - C537 *
NIRANJAN,T. ET AL.: "The Notch pathway in podocytes plays a role in the development of glomerular disease", NAT MED, vol. 14, no. 3, 2008, pages 290 - 298 *
ZOU,Z. ET AL.: "Evidence for ligand-dependent, (gamma)-secretase-mediated processing of megalin in the proximal tubule", FASEB JOURNAL, vol. 18, no. 4, 2004, pages A711 *
ZOU,Z. ET AL.: "Linking receptor-mediated endocytosis and cell signaling: evidence for regulated intramembrane proteolysis of megalin in proximal tubule", J BIOL CHEM, vol. 279, no. 33, 2004, pages 34302 - 34310 *

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