Tryptase inhibitors

Details Tryptase inhibitors Tryptase inhibitors

2001-01-22

2003-09-02

Shah, Mukund J. (Department: 1624)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Heterocyclic carbon compounds containing a hetero ring...

C514S255010, C514S255050, C514S316000, C514S318000, C514S330000, C514S331000, C514S332000, C544S358000, C544S359000, C544S383000, C544S386000, C544S399000, C544S402000, C546S186000, C546S189000, C546S190000, C546S191000, C546S246000, C546S248000, C435S217000

Reexamination Certificate

active

06613769

ABSTRACT:

The invention relates to bifunctional inhibitors of human tryptase, to human tryptase in crystallized form, to a process for preparing human tryptase in crystallized form, to pharmaceutical compositions which comprise a bifunctional inhibitor of human tryptase, and to a process for developing and identifying tryptase inhibitors.
Human tryptase is a serine proteinase which is the predominant protein present in human mast cells. The term tryptase covers four closely related enzymes (&agr;, I, II/&bgr;, III; possessing 90 to 98% sequence identity) (cf. Miller et al., J. Clin. Invest. 84 (1989) 1188-1195; Miller et al., J. Clin. Invest. 86 (1990) 864-870; Vanderslice et al., Proc. Natl. Acad. Sci., USA 87 (1990) 3811-3815). With the exception of &agr;-tryptase (Schwartz et al., J. Clin. Invest. 96 (1995) 2702-2710; Sakai et al., J. Clin. Invest. 97 (1996) 988-995), the enzymes are activated intracellularly and stored in catalytically active form in secretory granules.
As compared with other known serine proteinases, such as trypsin or chymotrypsin, tryptase exhibits some exceptional properties (Schwartz et al., Methods Enzymol. 244, (1994), 88-100; G. H. Caughey, “Mast cell proteases in immunology and biology.” Marcel Dekker, Inc., New York, 1995). Tryptase obtained from human tissue has a noncovalently linked tetrameric structure which has to be stabilized by heparin or other proteoglycans in order to be proteolytically active. Furthermore, the serum is not so far known to contain any factor which inhibits tryptase. Attempts to find an endogenous inhibitor of tryptase have so far been unsuccessful. With the exception of the atypical inhibitor LDTI (leech-derived tryptase inhibitor) (Sommerhoff et al., Biol. Chem. Hoppe-Seyler 375 (1994) 685-694), tryptase is not inhibited by naturally occurring proteinase inhibitors either.
In addition, tryptase exhibits an unusual, very narrow substrate specificity, with a number of peptide substrates (Tam et al., Am. J. Respir. Cell Mol. Biol. 3 (1990) 27-32), but only a few selected proteins, being cleaved in vitro. For example, fibrinogen, fibronectin and high molecular weight kininogen are inactivated (Schwartz et al., J. Immunol., 135(4) (1985), 2762-2767; Lohi et al., J. Cell. Biochem. 50, (1992), 337-349; Little et al., Biochem. J. 307 (1995) 341-346), and the zymogens of stromelysin (proMMP-3) and the plasminogen activator of the urokinase type (pro-uPA) are activated (Gruger et al., J. Clin. Invest. 84 (1989), 1657-1662; Lees et al., Eur. J. Biochem. 223 (1994), 171-177; Stack et al., J. Biol. Chem. 269 (1994), 9416-9419). Furthermore, it has been discovered that tryptase exhibits mitogenic effects (Ruoss et al., J. Clin. Invest. 88 (1991), 493-499; Hartmann et al., Am. J. Physiol. 262 (1992), L528-L534; Brown et al., Am. J. Respir. Cell Mol. Biol. 13 (1995), 227-236).
Tryptase is released, together with other inflammation mediators, such as histamine and proteoglycans, when human mast cells are activated. It is therefore assumed that tryptase is involved in a number of diseases, in particular in allergic and inflammatory diseases, on the one hand because of the importance of mast cells in such diseases and, on the other hand, since an elevated content of tryptase has been observed in several such diseases. Thus, tryptase is thought to be linked with the following diseases, inter alia: acute and chronic (in particular inflammatory and allergenically induced) respiratory diseases of varying origin (e.g. bronchitis, allergic bronchitis, bronchial asthma and COPD); interstitial pulmonary diseases; diseases which are based on allergic reactions of the upper airways (pharynx and nose) and of the adjacent regions (e.g. paranasal sinuses and conjuctivas), such as allergic conjunctivitis and allergic rhinitis; diseases which belong to the complex of arthritic diseases (e.g. rheumatoid arthritis); autoimmune diseases such as multiple sclerosis; and, in addition, periodontitis, anaphylaxis, interstitital cystitis, dermatitis, psoriasis, dermatosclerosis/systemic sclerosis, inflammatory intestinal diseases (Crohn's disease and inflammatory bowel disease) and others. Tryptase appears, in particular, to be directly associated with the pathogenesis of asthma (Caughey, Am. J. Respir. Cell Mol. Biol. 16 (1997), 621-628; R. Tanaka, “The role of tryptase in allergic inflammation” in: Protease Inhibitors, IBC Library Series, 1979, chapter 3.3.1-3.3.23).
However, in order to be able to investigate the precise function of tryptase, in particular in allergic and inflammatory diseases, it is necessary to develop selective tryptase inhibitors. To date, tryptase inhibitors have been designed and synthesized on the basis of the activity and specificity of tryptase, which are similar to those of trypsin, starting, for the most part, from a benzamidine group as substrate residue. Inhibitors of varying quality were found by the method of trial and error, with benzamidine and similar structures, in particular, being derivatized with groups which were to a greater or lesser degree rigid and hydrophobic. An example of this is 4-amidinophenylpyruvic acid (APPA; Stürzebecher et al., Biol. Chem. Hoppe-Seyler 373 (1992),
1025-1030
). However, such benzamidine-based inhibitors are not selective for tryptase, but also inhibit other physiologically important enzymes such as thrombin, factor Xa and urokinase. They cannot, therefore, be used for investigating the function of tryptase selectively.
A peptide inhibitor of tryptase, namely N-(1-hydroxy-2-naphthoyl)-L-arginyl-L-prolineamide, has also been described in the state of the art (R. Tanaka, Protease Inhibitors, IBC Series 1997, chapter 3.3; Clark et al., Drugs of the future 21(8) (1996), 811-816; WO 94/20527). However, this inhibitor is not selective for tryptase either, but also inhibits other proteinases such as trypsin and thrombin, so that it is not possible to establish unambiguously whether observed effects are being achieved due to a specific inhibition of tryptase or, rather, due to other occurrences.
Another inhibitor of tryptase which is described in the state of the art is LDTI, which is an inhibitor of the Kazal type and was isolated from leeches (LDTI, leech-derived tryptase inhibitor) (WO95/03333; Stubbs et al., J. Biol. Chem. 272 (32) (1979), 19931-19937; WO97/22626). LDTI is a proteinaceous inhibitor whose structure was determined with the aid of NMR data and using LDTI and trypsin crystals. In this connection, it was ascertained that the basic aminoterminus of LDTI probably makes an electrostatic contribution to the interaction with tryptase. While LDTI is an inhibitor which has a high affinity for tryptase (K
i
of 1.4 nM), it also inhibits trypsin and chymotrypsin in the nanomolar range.
SLPI (secretory leukocyte protease inhibitor) has been suggested as being another inhibitor of tryptase (WO96/08275 A1). This inhibitor is also proteinaceous. Finally, WO95/32945, WO96/09297 and WO98/04537 describe low molecular weight compounds which are tryptase inhibitors. At their ends, these compounds predominantly exhibit amino, guanidino or amidino groups. The activity of these compounds is likewise determined by trial and error.
One object of the present invention was therefore to provide highly specific inhibitors of human tryptase, the activity of which inhibitors can be reliably predicted using structural parameters. According to the invention, this object is achieved by means of a bifunctional inhibitor of human tryptase wherein the inhibitor comprises two head groups, H1 and H2, which are connected by a linker L, with H1 and H2 being identical or different and in each case comprising a Q group which can enter into interactions with a carboxylate group, with the linker L being able to assume such a conformation that the Q groups of the two head groups are present at a distance of from 20 to 45 Å, and with the sizes of the head groups and of the linker permitting the inhibitor to penetrate into a cavity having the dimensions 52 Å×32 Å×40 Å. In this present document,

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