JP2010209055A - Pharmaceutical composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a compound useful as an anticancer agent or anti-inflammatory agent and specifically inhibiting pro-PHBP activation. <P>SOLUTION: The compound represented by formula (24) is provided, which is obtained as a result of conducting a new screening of a Pro-PHBP activation inhibitor, with well-known compound library samples and commercialized compounds as exploring source. A pharmaceutical composition is also provided, which includes as active ingredients: a compound having a polyphenolic structure such as in tannic acids and cyanidins and the like and a compound selected from oxytetracyclines and the like. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a pharmaceutical composition and a precursor plasma hyaluronan binding protein activation inhibitor.

  As the cancer cells become malignant, they invade surrounding tissues and further metastasize to other organs to form secondary tumors. The process of invasion and metastasis is very complicated, and various factors are involved in its control. Among them, the degradation of the basement membrane and blood vessel wall is an important step in the process of invasion and metastasis. A central role in this step is a series of serine proteases and inhibitors present in plasma, and urokinase-type plasminogen activator (u-PA) plays an important role in tissue fibrinolysis. I'm in charge.

  Plasmin produced by u-PA activates matrix metalloprotease precursors (pro-MMPs) whose extracellular matrix is a direct substrate, thereby causing tissue fibrinolysis [Non-Patent Document 1]. There have been many reports on the relationship between these series of protease activities and cancer invasion / metastasis [Non-Patent Document 2], and these enzyme activities or their interactions with receptors are inhibited. Metastasis inhibitors are being studied. u-PA, like many serine proteases, exists as a precursor type that does not have enzyme activity in blood, and is activated by some factor to cause a fibrinolytic reaction. In recent years, a novel serine protease having the ability to activate prourokinase has been discovered from plasma as an enzyme that triggers the activation of u-PA.

  PHBP (plasma hyaluronan binding protein) was discovered in plasma by Miura et al. In 1994 as a protein that specifically binds to hyaluronic acid [Non-patent Document 3]. PHBP is mainly translated in the liver, and then the N-terminal signal peptide is cleaved and secreted into the blood as a precursor type pro-PHBP having no enzyme activity [Non-patent Document 4]. Pro-PHBP is a single-chain precursor of molecular weight 70 kDa consisting of 537 amino acid residues having three EGF-like domains, one kringle domain, and further a serine protease-like domain [Non-patent Document 5]. Only this precursor type is present in normal blood, and active PHBP is detected only at the time of liver injury or hepatectomy in mice [Non-Patent Document 6].

  Although the physiological role of PHBP is still unclear, it has the ability to activate fibrin and fibronectin in vitro [Non-Patent Document 6] and prourokinase activation ability to convert pro-u-PA to u-PA. It has been reported [Non-patent document 7], and it has also been clarified that fibrinolysis is promoted with the localization of PHBP on the cell surface.

  In addition, a genetic polymorphism in which the 511st amino acid named Marburg's disease is substituted from G to E has been reported for PHBP. In patients with this amino acid substitution, PHBP-dependent u-PA activity is reported. It is reduced by 50-80% compared to normal, and u-PA-dependent plasmin production is also reduced [Non-patent Document 8]. Furthermore, it has been reported that Marburg's disease is involved in the progression of arteriosclerosis and that PHBP is highly expressed in the carotid arterial wall of Marburg's disease [Non-patent Document 9]. These findings indicate that PHBP is one of the most important proteins involved in the series of tissue fibrinolysis that occurs during thrombolytic and inflammatory reactions and cancer invasion / metastasis, which is located in the most upstream of the fibrinolytic system. Show.

  The existence of an enzyme that catalyzes the conversion of Pro-PHBP to PHBP has not been reported so far, and is considered to be converted by self-activation [Non-Patent Documents 10 and 11]. A self-catalyzed cleavage between Arg290-Ile291 leads to an active double-stranded molecule (50 kDa heavy chain containing three EGF and kringle domains: Phe1-Arg290 and a 27 kDa light chain containing a serine protease domain: Ile291- Phe 537) occurs and further self-cleavage results in an inactivated four-stranded molecule. Thereafter, fragmentation proceeds by further self-cleavage [Non Patent Literature 5].

  The activation mechanism of PHBP is similar to the coagulation factor Factor XII, and its self-cleavage is promoted by charged substances such as poly-L-lysine, heparin, and dextran sulfate [Non-patent Documents 5 and 10]. It was also clarified that activation is promoted by spermidine which is one of in vivo polyamines.

  However, it is unclear how these activation promoters bind to (pro-) PHBP and exert an action. As described above, the activation mechanism of pro-PHBP and its physiological role There are many unclear points. Moreover, as an inhibitor of active double-chain PHBP, C-1 inhibitor and a2-antiplasmin have been reported as endogenous inhibitors, and are inhibited by general serine protease inhibitors [Non-patent Documents] 11], none of these is PHBP-specific and has no specificity for precursor pro-PHBP.

  In the figure, uPA is a urokinase-type plasminogen activator, uPAR is a uPA receptor, PLG is plasminogen, and PM is plasmin.

Conventionally, dozens of compounds such as cyclic lipopeptides including surfactin are known as pro-PHBP activation inhibitors. [Patent Document 1] However, these compounds have problems such as low inhibitory activity and low specificity of the inhibitory action, and compounds having superior activity that specifically suppress pro-PHBP activation. Development of was desired.
Werb, Z .; (1997). ECM and cell surface protocol: regulating cellular ecology. Cell 91, 439-42 Pepper, M.M. S. (2001). Role of matrix metalloproteinase and plasminogen activator-plasmin systems in angiogenesis. Arteriosclero thrombo vas biol. 21, 1104-1117 Choi-Miura, N .; H. Tobe, T.A. Sumiya, J .; Nakano, Y. et al. Sano, Y .; Mazda, T .; and Tomita, M .; (1996). Purification and charac- terization of a novel hyaluronan-binding protein (PHBP) from human plasma, a kringle and a serine toe protease. J. et al. Biochem. 119, 1157-1165 Choi-Miura, N .; H. Yoda, M .; Saito, K .; Takahashi, K .; and Tomita, M .; (2001). Identification of the subsystems for plasma hyaluronan binding protein. Biol. Pharm. Bull. 24, 140-143 Choi-Miura, N .; H. Takahashi, K .; Yoda, M .; Saito, K .; Mazda, T .; and Tomita, M .; (2001). Proteolytic activation and inactivation of the serine protease activity of plasma hyaluronan binding protein. Biol. Pharm. Bull. 24, 448-452 Choi-Miura, N .; H. Otsuyama, K .; Sano, K .; Takahashi, K .; and Tomita, M .; (2001). Hepatic injury-specific conversion of mouse plasma hyaluronan binding protein to the active hetero-former form. Biol. Pharm. Bull. 24,892-896 Romish, J. et al. Vermohlen, S .; Feussner, A.M. Stohr, H .; A. (1999). Haemostasis. 29,292-299 Romish, J. et al. Feussner, A.M. Nerrich, C.I. Stoehr, H.C. A. and Weimer, T .; (2002). The frequent Marburg I polymorphism impulses the pro-kinase activating potency of the factor VII activating process (FSAP). Blood Coag. Fibl. 13,433-441 Ireland, H.M. Miller, G.M. J. et al. Webb, K.M. E. Cooper, J.A. A. and Humphies, S .; E. (2004). The factor VII activating process G511E (Murburg) variant and cardiovascular risk Etscheid, M.M. Hunfeld, A.M. Konig, H .; Seitz, R.A. and Dodt, J .; (2000). Activation of proPHBSP, the zymogen of a plasma hyaluronan binding serine protease, by an intermolecular molecular catalytic mechanism. Biol. Chem. 381,1223-1231 Choi-Miura, N .; H. Saito, K .; Takahashi, K .; Yoda, M .; and Tomita, M .; (2001). Regulation mechanism of the serine protease activity of plasma. Biol. Pharm. Bull. 24, 221-225 JP 2008-201699 A PHARMACEUTICAL COMPOSITION

  As a result of diligent efforts to solve the above problems, the inventor of the present invention inhibited the activation of the precursor pro-PHBP more strongly than the conventionally known compound, and the active PHBP The present invention was completed by finding out that there is no influence.

で示される化合物を有効成分として含む、医薬組成物、
抗癌剤又は抗炎症剤である、上記（１）〜（２４）に記載の医薬組成物、
前駆体型の血漿ヒアルロナン結合タンパク質の活性化に関連する疾患の治療剤である、上記（１）〜（２４）に記載の医薬組成物、及び
下記式：

で示される化合物を含む、前駆体型血漿ヒアルロナン結合タンパク質の活性化阻害剤に関する。Accordingly, the present invention provides the following:
Following formula:

A pharmaceutical composition comprising a compound represented by the formula:
The pharmaceutical composition according to the above (1) to (24), which is an anticancer agent or an anti-inflammatory agent,
The pharmaceutical composition according to (1) to (24) above, which is a therapeutic agent for a disease associated with activation of a precursor type plasma hyaluronan binding protein, and

The present invention relates to an inhibitor of precursor plasma hyaluronan binding protein activation, which comprises a compound represented by:

  The compounds of formula (1) to formula (24) are all known compounds, and can be obtained by synthesis or purification from natural products.

  In the present invention, the disease associated with the activation of the precursor plasma hyaluronan binding protein refers to a disease state caused by the presence of the active plasma hyaluronan binding protein, and examples thereof include cancer and inflammation. The medicament of the present invention can be used as a preventive or therapeutic agent for cancer and inflammation.

  The administration form of the crystals of the present invention can be, for example, oral administration by tablets, capsules, granules, powders, syrups, etc., or parenteral administration by injections or suppositories. Furthermore, the crystals of the invention can be administered pulmonary as a powder, solution or suspension. Formulations for these are produced by well-known methods using additives such as excipients, lubricants, binders, disintegrants, stabilizers, flavoring agents, and diluents.

  The amount of the pharmaceutical composition of the present invention varies depending on symptoms, age, administration method, etc. For example, in the case of oral administration, the lower limit is 0.1 mg (preferably 1 mg, more preferably 1 day) per day for adults. 5 mg), and as an upper limit, 1000 mg (preferably 100 mg, more preferably 50 mg) is desirably divided into 1 or several times and administered according to the symptoms. In the case of intravenous administration, the lower limit is 0.01 mg (preferably 0.1 mg) and the upper limit is 100 mg (preferably 10 mg) per day for adults. Is desirable.

  The pharmaceutical composition of the present invention has the effect of suppressing the activation of Pro-PHBP, thereby reducing side effects and suppressing the tissue fibrosis during inflammatory reaction and cancer invasion / metastasis.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

Purification of Pro-PHBP and preparation of PHBP Pro-PHBP was prepared by the method of Etscheid et al. [Biol. Chem. 381, 1223-1231 (2000)] and subjected to two-step anion column chromatography in the presence of 6M urea. After protein quantification by the biciconic acid (BCA) method, it was stored at −80 ° C. until use, and after freezing and thawing, it was stored at 4 ° C. and used within one week. The activated double-stranded PHBP is obtained by adding 0.2 mM pro-PHBP in a processing buffer (50 mM Tris-HCl, pH 6.0 (25 ° C.), 0.15 M NaCl, 10 mM CaCl ₂ , 0.1% Tween-20) at 37 ° C., 20 Prepared by incubating for minutes.

Pro-PHBP activation Pro-PHBP activation was measured by one of the following methods. All were measured at 37 ° C. in a 50 ml Substrate buffer (50 mM Tris-HCl, pH 7.4 (25 ° C.), 75 mM NaCl, 5 mM CaCl ₂ , 0.05% Tween-20) using a round bottom 96-well plate. The absorption wavelength at 405 nm was measured continuously for 60 minutes or 20 minutes, and the liberated p-nitroaniline was quantified. The concentrations of the enzyme (precursor), the synthetic chromogenic substrate and the pro-PHBP activation promoter added to the evaluation system are as shown below.

(1) pro-PHBP activation (one-step assay):
Pro-PHBP 5 nM, Spectrozyme TH 0.1 mM, spermidine trihydrochloride 5 mM was added as an activation promoter.
(2) pro-PHBP activation (two step assay):
Pro-PHBP 5 nM and spermidine trihydrochloride 5 mM as an activation promoter were added and incubated at 37 ° C. for 20 minutes. After preincubation, Spectrozyme TH was added to a final concentration of 0.1 mM.
(3) Amidolytic activity of PHBP:
PHBP 2 nM, Spectrozyme TH 0.1 mM;
Each measurement was performed in triplicate.

Screening method A known compound library sample owned by the inventor and a commercially available compound were used as search sources. The pro-PHBP activation inhibitory activity was measured using the method described above.

Results As a result of conducting a new screening of a Pro-PHBP activation inhibitor, the following compounds showed pro-PHBP activation inhibitory activity.

Claims (4)

Following formula:

A pharmaceutical composition comprising a compound represented by the formula:   The pharmaceutical composition according to claim 1, which is an anticancer agent or an anti-inflammatory agent.   The pharmaceutical composition according to claim 2, which is a therapeutic agent for a disease associated with activation of precursor plasma hyaluronan binding protein. Following formula:

An activation inhibitor of precursor plasma hyaluronan-binding protein comprising the compound represented by: