Family of protease inhibitors, and other biologic active...

Details Family of protease inhibitors, and other biologic active... Family of protease inhibitors, and other biologic active...

1998-03-27

2001-05-29

Saidha, Tekchand (Department: 1652)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C435S212000, C435S213000, C435S214000, C435S215000, C435S216000, C435S217000, C435S218000, C435S219000, C435S069100, C435S252300, C435S320100, C536S023200, C530S324000, C530S350000

Reexamination Certificate

active

06239106

ABSTRACT:

The present invention relates to certain novel compounds which have protease inhibitory activity, as well as compounds derived therefrom and compositions comprising such compounds, and other biologically active substances. More specifically the invention relates to such compounds which are polypeptide-like or of a proteinaceous nature and of polysaccharide derivates and/or glyco-poly saccharide. The invention especially relates to such compounds and compositions and uses thereof obtainable from leeches.
The invention further relates to therapeutic uses of the novel protease inhibitors. An illustrative example of such a use is given below.
Several diseases, like emphysema, arthritis, gingivitis, periodontitis and other inflammatory conditions are associated with tissue destruction caused by the enzyme human neutrophil elastase (HNE). HNE is a serine protease which is capable of solubilising fibrous proteins like elastin and collagen. HNE is mainly present in the azurophilic granules of neutrophil leucocytes. Under normal physiological conditions, the proteolytic activity of the enzyme is kept under control by an excess of inhibitors present in plasma and other secretions. However, some disorders result in a local deficiency or inactivation of inhibitor which leads to an imbalance in the ratio of inhibitor to elastase, resulting in tissue destruction.
The balance may be restored employing protease inhibitors, for instance those provided by the invention.
Furthermore, in the replication cycle of HIV, the proteolytic cleavage of the gag and env precursors is an important step. Development of inhibitors of these proteases is a rationale in anti-viral drug development. Study-objectives evaluating anti-viral capacity of potential substances is therefore valuable.
Various substances extracted from leeches are known to have useful biological activity. These were reviewed by Sawyer, (Sawyer, 1990). Essentially two groups of activity can be recognised. The first group comprises antithrombotic and fibrinolytic activities, the second group comprises enzymes and inhibitors. Well known representatives of the first group include for instance Hirudin, a thrombin inhibitor, (Markwardt, 1956; 1988; Petersen, et al, 1984); Hementin, a fibrinolytic agent (Budzynski, et al, 1981; Kirschbaum & Budzynski, 1990); Antistasin, an inhibitor of coagulation factor Xa (Gasic, et al, 1983), which was reported to have antimetastatic properties as well; Gilanten, another factor X inhibitor (Condra, et al, 1989; Blankenship, et al, 1990). Representatives of the second group are: Bdellin, an inhibitor of trypsin and plasmin (Fritz & Krejci, 1976); Eglin, an inhibitor of chymotrypsin, elastase and Cathepsin G (Seemuller, 1979); Orgelase, an hyaluronidase (Sawyer, 1986).
More recently several additions in this field have been published in patent literature: A fibrinolytic enzyme isolated from Hirudo medicinalis, which splits Glutamyl-Lysin sequences (EP 0502876); a platelet adhesion inhibitor, isolated from Hirudo medicinalis, which inhibits collagen-induced platelet aggregation (EP 0552269); a thrombin inhibitor from the leech Hirudinaria manillensis (PCT/GB89/01345); an inhibitor of platelet aggregation from leeches from the Hirudinidae family (EP 0348208); an anticoagulant/modulator factor isolated from Hirudo medicinalis (EP 0352903); A chymotrypsin- and elastase inhibitor from Hirudinaria manillensis (PCT/NL90/00046).
This invention provides novel protease-inhibitors and other biologically active substances, as well as pharmaceutical and cosmetic preparations containing one or more of these compounds. In one aspect the invention provides substances having protease inhibiting activity obtainable from Limnatis Nilotica or fragments or derivatives of such substance having similar activity.
Such substances can be derived from all body parts and secretions of the leech, inclusive saliva and gut-, intestinal-and skin secretions and mucus. These novel Elastase/chymotrypsin- and trypsin-inhibitors according to part of this invention can be typically isolated from leech tissue by solvent extraction-techniques; alternatively they may be isolated from leech secretions (such as leech saliva), although the invention is not limited to specific ways of obtaining the novel protease-inhibitors. In a further embodiment the invention provides such a novel protease inhibitor which is of a proteinaceous or polypeptide-like nature. Preferably said proteinaceous substance includes at least a part of the following amino acid sequence: (1; SEQ ID NO:2)
                              5                   10
(N-terminal) Asp-Asp-Asn-Cys-Gly-Gly-Lys-Val-Cys-Ser-Lys-Gly-Gln-
(1; SEQ ID NO:2)

     15                  20                  25
Leu-Cys-His-Asp-Gly-His-Cys-Glu-Cys-Thr-Pro-Ile-Arg-Cys-Leu-

     45                  50
Leu-Pro-Cys-Ser-Cys-Lys-His-Gln: (Carboxy-terminal) or
(2; SEQ ID NO:)

  1               5                   10                  15
Asp-
Asp
-
Asp
-Cys-Gly-Gly-Gln-Val-Cys-Ser-Lys-Gly-Gln-Leu-Cys-
    Gly Asn

 16              20                  25                  30
Val-Asp-Gly-Gln-Cys-Lys-Cys-Thr-Pro-Ile-Arg-Cys-Arg-Ile-Tyr-

 31              35                   40                  45
Cys-Pro-Lys-Gly-Phe-Glu-Val-Asp?-Glu-Asn-Gly-Cys-Glu-Leu-Pro-

 46               50
Cys-Thr-Cys-Lys?-Gln?
although it will be clear that the activities are the really important features of this invention, so that mutations, isoforms, derivatives, such as salts, fragments or even peptidomimetics and anti-idiotypic or catalytic antibodies are also a part of this invention.
For the definition of isoforms or mutants, one has to understand that by biologic evolution enzymatic and other systems active molecules are subject to continuous phylogenetic development. It is in this understanding, that we define isoforms or mutant forms of these molecules. The primary structure (1) as reported here is one of three or four isoforms, which have large conformity, and are being defined by molecule weight and amino acid sequence. The same may be true for (2).
A number of isoforms of (1) are identified below. The novel protease-inhibitors can be applied in the known applications for such substances. They can be suitably formulated into pharmaceutical compositions, which may comprise suitable excipients for administration. Administration may be accomplished in any suitable manner, although for proteinaceous substances in systemic applications parenteral routes are preferred. Dosages for these substances can be taken from the literature and designed on the basis of specific activities of the substan

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