Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2001-06-21
2004-06-15
Russel, Jeffrey E. (Department: 1654)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C530S232000, C530S330000, C530S332000, C548S413000
Reexamination Certificate
active
06750202
ABSTRACT:
The invention relates to the use of phosphinate-peptide analogs, as inhibitors of procollagen C-proteinase (PCP), for treating fibrotic diseases.
It is known that procollagen C-proteinase (PCP) is a key enzyme in fibrogenesis. It catalyzes the hydrolytic elimination of the procollagen propeptides from procollagens I, II, III and IV and also laminin V [cf. Amano S, Takahara K; Gerecke D, Nishiyama T, Lee S, Greenspan D S, Burgeson R E: Bone morphogenetic protein-1 is the processing enzyme for laminin 5 in human keratinocytes, Mol. Biol. Cell 7 (suppl.) 58A (1996)]. Consequently, PCP is a key enzyme in collagen processing [cf. Olsen B J: Morphogenesis: collagen it takes and bone it makes, Curr. Biol. 6: 645-647 (1996)]. It was demonstrated in BMP-I knock-out mice that complete absence of PCP leads to incomplete collagen processing with the deposition of atypical, loose collagen fibrils [cf. Suzuki N, Labosky P A, Furata Y, Hargett I, Dunn R, Fogo A B, Takahara K, Peters D M, Greenspan D S, Hogan B L: Failure of ventral body wall closure in mouse embryos lacking a procollagen C-proteinase encoded by BMP-1, a mammalian gene related to Drosophila tolloid, Development 122: 3587-3595 (1996)].
PCP is probably also responsible for the hydrolytic elimination of the propeptide sequence of lysyl oxidase. The elimination of the prosequence probably leads to the activation of the catalytic lysyl oxidase activity of the mature form [cf. Pachenko M V, Stetler-Stevenson W G, Trubetskoy O V, Gacheru S N, Kagan H M: Metalloproteinase activity secreted by fibrogenic cells in the processing of prolysyl oxidase. Potential role of procollagen C-proteinase, J. Biol. Chem. 271: 7113-7119 (1996)]. Active lysyl oxidase covalently links opposing collagen fibrils to each other. In this way, PCP also indirectly increases the biological stability of the collagen towards degradation by collagenases.
PCP, or closely related proteins, appear also to play a role in the release of TGF&bgr;-type growth factors. Recent studies have shown that PCP-like proteases are able to liberate TGF&bgr;-type growth factors from an inactive complex with TGF&bgr;-binding proteins [cf. Marques G, Musacchio M, Shimell M J, Wümenberg-Stapleton K, Cho K W Y, O'Connor M B: Production of a DPP activity gradient in the early drosophila embryo through the opposing actions of the SOG and TLD proteins, Cell 91: 417-426 (1997); Blader P, Rastegar S, Fischer N, Strähle U: Cleavage of the BMP antagonist chordin by zebrafish tolloid, Science 278: 1937-1949 (1997)]. In this case, the binding partner for the TGF&bgr;-type growth factors is decomposed by means of specific proteolysis. Consequently, PCP may possibly also indirectly possess a TGF&bgr;-type agonistic activity. PCP can therefore be ascribed a crucial role in fibrogenesis.
PCP activity has its origin in splicing variants of the BMP-I gene [cf. Kessler E, Takahara K, Biniaminow L, Brusel M, Greenspan D S: Bone morphogenetic protein-1: The type I procollagen C-proteinase, Science 271: 360-362 (1996)); Reddi A H: BMP-1: Resurrection as procollagen C-proteinase, Science 217: 463 (1996); Li S W, Sieron A L, Fertala A, Hojima Y, Arnold W V, Prockop D J: The C-proteinase that processes procollagens to fibrillar collagens is identical to the protein previously identified as bone-morphogenetic protein-1. Proc. Natl. Acad. Sci. USA 93: 5127-5130 (1996)]. It is so far definitely known that splicing variants BMP-I-I and BMP-I-III (tld variant) are able to cut procollagen and pro-lysyl oxidase specifically. While relatively recent studies have identified further BMP-1 splicing variants, the biological function and substrate specificity of the latter have not yet been fully elucidated [cf. Janitz M, Heiser V, Böttcher U, Landt O, Lauster R: Three alternatively spliced variants of the gene coding for the human bone morphogenetic protein-1. J. Mol. Med. 76: 141-146 (1998)].
Although success has so far only been achieved in expression-cloning and purifying PCP in small yields, a large number of structural details of the enzyme are known. Thus, PCP belongs to the family of astacin proteases. The crystal structure of astacin is known in detail. There is a very high degree of structural homology between the catalytic domain of BMP-I and astacin such that it has been possible, on the basis of this homology, to assign to many amino acids in the PCP protease domain their probable structural and biochemical function [cf. Stöcker W, Gomis-Rüth F X, Bode W, Zwilling R: Implications of the three-dimensional structure of astacin for the structure and function of the astacin family of zinc-endopeptidases, Eur. J. Biochem. 214: 215-231 (1993)].
In the past, it has been possible, by means of computer-guided molecular modeling, to infer the binding of substate to the active center of the astacings in molecular detail [cf. Stöcker W, Grams F, Baumann U, Reinemer P, Gomis-Rüth FX, McKay DB, Bode W: The metzincin—Topological and sequential relations between the astacins, adamalysins, serralysins, and matrixins (collagenases) define a superfamily of zinc-edopeptidases, Prot Sci.: 823-840 (1995)]. These studies led to the rational design of phosphinate-peptide analogs which inhibit astacin with a high degree of potency. The complex between a phosphinate inhibitor and astacin has been strutually elucidated ([cf. Grams F, Dive V, Yiotakis A, Yiallouros I, Vassilou S, Zwilling R, Bode W, Stöcker W: Structure of astacin with transition-state analogue inhibitor, Nature Struct. Biol. 3: 671-675 (1996)].
Despite the high degree of structral homology between astacin and the catalytic domain of BMP-1, it has so far been assumed that, because of biochemical differences with regard to their reaction behavior and also in their ability to be inhibited by protease inhibitors, the two proteases are in fact markedly different. For example, there are biochemical differences between astacin and BMP-I with regard to their substrate specificity (as a crayfish digestive enzyme, astacin hydrolyzes collagen-like proteins relatively nonspecifically whereas PCP cuts highly specifically at only one site in the procollagen molecule and in pro-lysyl oxidase).
To date, only low-potency inhibitors of PCP, to which an antifibrotic effect is attributed, have been described in the literature [cf. Brenner M, Ho W B: C-proteinase inhibitors for the treatment of disorders related to the overproduction of collagen. WO 97/05865].
It has now been found, surprisingly, that phosphinate-peptide analogs of the general formula (I),
in which
R
1
represents hydrogen or methyl,
and their salts and isomers inhibit PCP with a very high degree of potency and can therefore be used for the treatment and prophylaxis of fibrotic diseases.
Within the context of the invention, preference is given to physiologically harmless salts. In general, physiologically harmless salts are salts of the compounds according to the invention with inorganic or organic acids. Preference is given to salts with inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid or sulfuric acid, or salts with organic carboxylic or sulfonic acids, such as acetic acid, maleic acid, fumaric acid, malic acid, citric acid, tartaric acid, lactic acid, benzoic acid, or methanesulfonic acid, ethanesulfonic acid, phenylsulfonic acid, toluene-sulfonic acid or naphthalenedisulfonic acid.
The compounds according to the invention can exist in stereoisomeric forms which either relate to each other as image and mirror image (enantiomers) or which do not relate to each other as image and mirror image (diastereomers). The invention also relates to the antipodes and to the racemic forms as well as to the diastereomeric mixtures.
The compounds of the general formula (I) can be present in all the enantiomeric and diastereomeric forms. Preference is given to those isomers in which the parts of the molecule formed from proline, lysine and leucine possess the L configuration, as well as to t
Burchardt Elmar-Reinhold
Lampe Thomas
Schauer Michael
Stöcker Walter
Burchardt Elmar R.
Russel Jeffrey E.
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