WO2006108211A1

WO2006108211A1 - Fragments of von willebrand factor a-related protein

Info

Publication number: WO2006108211A1
Application number: PCT/AU2006/000234
Authority: WO
Inventors: John Bateman; David James Fitzgerald
Original assignee: The Murdoch Childrens Research Institute
Priority date: 2005-02-25
Filing date: 2006-02-24
Publication date: 2006-10-19

Abstract

The present invention relates generally to bioactive molecules. More particularly, the present invention provides fragments of, or fractions comprising fragments of, von Willebrand Factor A-Related Protein (WARP) or a homolog, analog, derivative, mimetic or functional equivalent thereof. The fragments or fractions have a range of biochemical, physiological and/or pharmacological activities including, inter alia, activities associated with stem cell proliferation, differentiation and self-renewal, coagulation, hemeostasis, control of cancer growth and angiogenesis. The present invention further provides pharmaceutical compositions comprising the bioactive molecules or homologs, analogs, derivatives, mimetics or functional equivalents thereof. The present invention still further provides methods for preventing and/or treating a range of diseases and conditions in a subject by the administration of the bioactive molecules or homologs, analogs, derivatives, mimetics or functional equivalents thereof. In still yet further embodiments, the present invention provides diagnostic assays and coating materials for medical devices such as stents, catheters and the like.

Description

Fragments of von Willebrand Factor A-related Protein

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

DESCRIPTION OF THE PRIOR ART

Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

Bibliographic details of references provided in this document are listed at the end of the specification. The extracellular matrix (ECM) is a complex mixture of collagens, non-collagenous glycoproteins, and proteoglycans that interact to provide a structural scaffold as well as specific cues for the maintenance, growth and differentiation of cells and tissues. The protein cores of a large number of ECM molecules are composed of different combinations of a finite collection of modules (Engel et al, Development Suppl 35-42, 1994). The conservation of amino acid sequence of these modules between different ECM proteins and protein families provides us with the opportunity to identify new proteins by database homology searching to help reveal additional modular ECM proteins.

One module present in a number of proteins is the type A-domain, first described in von Willebrand factor (reviewed in Colombatti et al, Matrix 13:297-306, 1993). Members of the expanding von Willebrand factor type A-domain (VA) protein superfamily participate in a variety of functions including hemostasis, cell adhesion and protein-protein interactions between matrix molecules. ECM components that contain one or more VA domains include collagens types VI (Chu et al, EMBO J 5:1939-1946, 1989; Chu et al,

^■ EMBO J P:385-393, 1990), VII (Parente et al, Proc Natl Acad Sci USA 88:6931-6935,

1991), XII (Yamagata et al, J Cell Biol 115:209-221, 1991), XIV (Trueb et al, Eur J

Biochem 207:549-557, 1992), and XX (Koch et al, J Biol Chem 276:23120-23126, 2001), matrilins-1, -2, -3, -4 (reviewed in Deak et al, Matrix Biol 18:55-64, 1999), cochlin (Robertson et al, Genomics 4<5:345-354, 1997), polydom (Gilges et al, Biochem J 352:49- 59, 2000) and nine transmembrane α integrin chains (αl, α2, αlO, αl 1, αL, αM, αX, coD and αE) (reviewed in Lee et al, Cell 50:631-638, 1995) where they are also known as an T domain. Non-matrix proteins that contain VA domains include complement system proteins (C2, B) (Mole, J Biol Chem 259:3407-3412, 1984), inter-α-trypsin inhibitor (subunits H1-H3) (Chan et al, Biochem J 306:505-512, 1995) α2β subunit of L-type voltage-dependent Ca²⁺ channel (Ellis et al, Science 241:1661-1664, 1988) in addition to the archetypal VA domains of von Willebrand factor itself (Sadler et al, Proc Natl Acad Sci USA 52:6394-6398, 1985).

VA domains appear to play an important role in protein-protein interactions. In von Willebrand factor, they interact with subendothelial heparans, collagens I, III, (reviewed by Ruggeri, J Clin Invest 99:559-564, 1997) and collagen VI (Denis et al, Arteriosclerosis & Thrombosis 75:398-406, 1993); in integrins the I domain interacts -with several collagens (Tuckwell et al, Eur J Biochem 241:732-739, 1996); and in collagen VI VA domains interact with heparin (Specks et al, EMBO J 77:4281-4290, 1992) and collagen IV (Kuo et al, J Biol Chem 272:26522-26529, 1997). In ECM molecules; the ability of VA domains to interact with other proteins and with each other to promote higher-order structure formation may be crucial in providing a linkage between ECM structural networks. For example, in collagen VI, a specific N-terminal α3(VI) collagen VA domain (N5) is important for the assembly of collagen VI tetramers into functional microfibrils (Fitzgerald et al, J Biol Chem 275:187-193, 2001) and in matrilin-1, interchain assembly and microfilament formation is promoted by the interaction of the V A. domains in adjacent matrilin molecules (Chen et al, MoI Biol Cell 70:2149-2162, 1999).

One member of the von Willebrand factor type A-domain (VA) protein supeffamily is von Willebrand factor A-domain-related-protein (WARP; Fitzgerald et al, FEBS Letters 577:61-66, 2002). Full-length mouse WARP cDNA is 2.3 kb in size and predicts a protein of 415 amino acids which contains a signal sequence, a VA-like domain, two fibronectin type Ill-like repeats, and a short proline- and arginine-rich segment. WARP mRNA is expressed predominantly in chondrocytes and in vitro expression experiments in transfected 293 cells indicate that WARP is a secreted glycoprotein that forms disulphide- bonded oligomers (Fitzgerald et al, FEBS Letters 517:61-66, 2002).

Whilst WARP has useful properties in itself, there is a need to investigate whether WARP fragments or fractions of WARP fragments have other activities.

SUMMARY OF THE INVENTION

The present invention provides bioactive molecules in the form of fragments or fractions of fragments derived from von Willebrand Factor A-Related Protein (WARP) or a homolog, analog, derivative, mimetic or functional equivalent thereof.

One embodiment of the present invention, therefore, provides fragments or fractions of WARP or a homolog, analog, derivative, mimetic or functional equivalent thereof having at least one biochemical, physiological or pharmacological property substantially absent from the full-length WARP or homolog, analog, derivative, mimetic or functional equivalent thereof.

The fragments of WARP are peptides or small polypeptides. The peptides or polypetides of the present invention may also be referred to herein as "pepteins".

In another embodiment, the present invention provides a peptein or a multiplicity of pepteins each of from about 3 amino acid residues to about 445 amino acid residues in length following defined or random protease digestion and/or chemical or physical cleavage of WARP or a homolog, analog, derivative, mimetic or functional equivalent thereof. Examples of size ranges include from 5 and to about 445, from about 10 to about 400, from about 15 to about 390 and from about 20 to about 300 amino acid residues in length.

Hence, the present invention provides an isolated fragment or fraction of fragments derived from von Willebrand Factor A-Related Protein (WARP) said fragment or each fragment in the fraction comprises at least three amino acid residues, said fragment or fraction exhibiting at least one biochemical, physiological or pharmacological property substantially absent from full-length WARP.

The preferred pepteins or the fraction of peptides of the present invention each have an activity or property not present in the full length WARP molecule. The present invention contemplates all forms of WARP such as, but not limited to, those found in the mouse, rat and human or homologs, analogs, derivatives, mimetics or functional equivalents thereof. Preferably, the WARP or homolog, analog, derivative, mimetic or functional equivalent thereof is a human WARP or a homolog, analog, derivative, mimetic or functional equivalent thereof. The present invention also contemplates all forms of the human WARP or a homolog, analog, derivative, mimetic or functional equivalent thereof such as isoforms, splice variants and the like.

The bioactive molecules of the present invention, i.e. pepteins, also facilitate the development of pharmaceutical compositions and methods for preventing and/or treating a range of diseases and conditions in a subject and also coating materials for medical devices such as stents, catheters and the like.

TABLE 1 :SUMMARY OF SEQUENCE IDENTIFIERS

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a schematic representation showing the basic steps associated with WARP peptein production.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this specification, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Nucleotide and amino acid sequences are referred to by sequence identifier number (SEQ ID NO:). The SEQ ID NOs: correspond numerically to the sequence identifiers <400>l (SEQ ID NO:1), <400>2 (SEQ ID NO:2), etc. A summary of the sequence identifiers is provided in Table 1. A sequence listing is provided at the end of the specification.

Prior to describing the present invention in detail, it is to be understood that unless otherwise indicated, the subject invention is not limited to specific fragments or formulation or fraction components, manufacturing methods, dosage regimens, or the like, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

It must be noted that, as used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a fragment" includes a single fragment, as well as two or more fragments; reference to "an activity" includes a single activity, as well as two or more activities; and so forth.

In describing and claiming the present invention, the following terminology is used in accordance with the definitions set forth below.

The terms "fragment", "fraction", "compound", "active agent", "chemical agent", "pharmacologically active agent", "medicament", "active" and "drug" are used interchangeably herein to refer to a chemical compound that induces a desired biochemical, pharmacological and/or physiological effect. The terms also encompass pharmaceutically acceptable and pharmacologically active ingredients of those active agents specifically mentioned herein including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs and the like. When the terms "fragment", "fraction", "compound", "active agent", "chemical agent" "pharmacologically active agent", "medicament", "active" and "drug" are used, then it is to be understood that this includes the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs, etc.

Reference to a "fragment", "fraction", "compound", "active agent", "chemical agent" "pharmacologically active agent", "medicament", "active" and "drug" includes combinations of two or more actives such as two or more protein fragments. A "combination" also includes multi-part such as a two-part composition where the agents are provided separately and given or dispensed separately or admixed together prior to dispensation.

For example, a multi-part pharmaceutical pack may have two or more fragments of WARP separately maintained.

The terms "effective amount" and "therapeutically effective amount" of an agent as used herein mean a sufficient amount of the agent (e.g. a peptide derived from WARP) to provide the desired therapeutic or physiological effect or outcome. Undesirable effects, e.g. side effects, are sometimes manifested along with the desired therapeutic effect; hence, a practitioner balances the potential benefits against the potential risks in determining what is an appropriate "effective amount". The exact amount required will vary from subject to subject, depending on the species, age and general condition of the subject, mode of administration and the like. Thus, it may not be possible to specify an exact "effective amount". However, an appropriate "effective amount" in any individual case may be determined by one of ordinary skill in the art using only routine experimentation.

By "pharmaceutically acceptable" carrier, excipient or diluent is meant a pharmaceutical vehicle comprised of a material that is not biologically or otherwise undesirable, i.e. the material may be administered to a subject along with the selected active agent without causing any or a substantial adverse reaction. Carriers may include excipients and other additives such as diluents, detergents, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives, and the like.

Similarly, a "pharmacologically acceptable" salt, ester, emide, prodrug or derivative of a compound as provided herein is a salt, ester, amide, prodrug or derivative that this not biologically or otherwise undesirable.

The terms "treating" and "treatment" as used herein refer to reduction in severity and/or frequency of symptoms of the condition being treated, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms of the condition and/or their underlying cause and improvement or remediation or amelioration of damage following a condition.

"Treating" a subject may involve prevention of a condition or other adverse physiological event in a susceptible individual as well as treatment of a clinically symptomatic individual by ameliorating the symptoms of the condition.

A "subject" as used herein refers to an animal, preferably a mammal and more preferably human who can benefit from the pharmaceutical formulations and methods of the present invention. There is no limitation on the type of animal that could benefit from the presently described pharmaceutical formulations and methods. A subject regardless of whether a human or non-human animal may be referred to as an individual, patient, animal, host or recipient. The compounds and methods of the present invention have applications in human medicine, veterinary medicine as well as in general, domestic or wild animal husbandry. The fragments and compositions of fragments also have diagnostic applications.

As indicated above, the preferred animals are humans or other primates such as orangutangs, gorillas, marmosets, livestock animals, laboratory test animals, companion animals or captive wild animals, as well as avian species.

Examples of laboratory test animals include mice, rats, rabbits, guinea pigs, and hamsters. Rabbits and rodent animals, such as rats and mice, provide a convenient test system or animal model. Livestock animals include sheep, cows, pigs, goats, horses and donkeys. Non-mammalian animals such as avian species, zebrafish, and amphibians including Xenopus spp.

As used herein, the terms "WARP fragment" and "WARP-derived peptide" are used interchangeably and refer to any non-full length peptide sequence of WARP.

One embodiment of the present invention provides fragments or fractions of fragments of WARP or a homolog, analog, derivative, mimetic or functional equivalent thereof having at least one biochemical, physiological or pharmacological property substantially absent from the full-length WARP.

In particular, the present invention is directed to an isolated fragment or fraction of fragments derived from von Willebrand Factor A-Related Protein (WARP) said fragment or each fragment in the fraction comprises at least three amino acid residues, said fragment or fraction exhibiting at least one biochemical, physiological or pharmacological property substantially absent from full-length WARP.

Reference herein to "biochemical", "physiological" and "pharmacological" properties of the fragments or fractions includes, but is not limited to, properties associated with receptor binding, angiogenesis, stem cell proliferation and/or differentiation, cytokine induction, homeostatsis and gene expression modulating properties. The biochemical, physiological or pharmacological properties may also be referred to herein as "bioactivity".

The fragments or fractions of fragment are in the form of peptides or polypeptides. The peptides or polypetides of the present invention be referred to herein as "pepteins".

The pepteins of the present invention may be produced by proteolytic digestion and chemical or physical cleavage of WARP or its homologs, analogs, derivatives, mimetics or functional equivalents. Alternatively, the pepteins may also be chemically synthesized using peptide synthetic methods well known in the art. In relation to proteolytic digestion, the digestion of WARP or its homologs, analogs, derivatives, mimetics or functional equivalents may be accomplished by a range of proteases such as but not limited to aminopeptidases (e.g. cystinyl aminopeptidase, prolyl aminopeptidase), dipeptidases (e.g. membrane dipeptidase), dipeptidyl and tipeptidyl peptidases (e.g. dipetydal peptidase I, tripeptidyl peptidase I), peptidyl dipeptidases (e.g. peptidyl dipeptidase A), serine-type carboxypeptidases (e.g. serine-type D - ala - D - ala carboxypeptidase), metallocarboxypeptidases (e.g. glutamate carboxypeptidase), omegapeptidases (e.g. acylaminoacyl peptidase, pyroglutamyl peptidase II), serine proteinases (e.g. trypsin, chymotrypsin, subtilisin), cysteine proteinases (e.g. calpain, papain, fϊcin), aspartic proteinases (e.g. pepsin) and mettallo proteinases (e.g. collagenase, gelatinase).

The steps preferably include subjecting the WARP to proteolytic digestion, screening the resulting fragments or fractions of fragments to screening for a bioactivity about in full length WARP and then selecting a derived fragment or fractions.

In another embodiment, therefore, the present invention provides a peptein or a multiplicity of pepteins each of from about 3 amino acid residues to about 445 amino acid residues in length following defined or random protease digestion and/or chemical or physical cleavage of WARP or a homolog, analog, derivative, mimetic or functional equivalent thereof.

Reference herein to "WARP" includes all forms of WARP such as, but not limited to, those found in the mouse, rat and human or homologs, analogs, derivatives, mimetics or functional equivalents thereof. Preferably, the WARP or homolog, analog, derivative, mimetic or functional equivalent thereof is a human WARP or a homolog, analog, derivative, mimetic or functional equivalent thereof. The present invention also contemplates all forms of the human WARP or a homolog, analog, derivative, mimetic or functional equivalent thereof such as isoforms, splice variants and the like. The amino acid sequence of human WARP is SEQ ID NO:2 (isoform 1) or SEQ ID NO:3 (isoform 2). The present invention also contemplates amino acid sequences having about 60% similarity to SEQ ID NO:2 or SEQ ID NO:3. Murine and other non-human WARP molecules may also be used (e.g. murine WARP-SEQ ID NO:1). The present invention contemplates, therefor, a peptein comprising at least 3 contiguous amino acids beginning from the first three amino acids of the N-terminal end to the last three amino acids at the C-terminal end. The pepteins may be from 3 amino acids in length to about 445 amino acids in length such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 3.33, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445.

Examples of ranges of sizes of fragments include from about 3 to about 445, from about 5 to about 400, from about 10 to about 400, about 15 to about 380 and from about 20 to about 300. Accordingly, in relation to pepteins of SEQ ID NO: 2 comprising at least 3 contiguous amino acids, the present invention provides a peptein or a multiplicity of pepteins each comprising an amino acid sequence (shown in single letter code format) selected from the list comprising MLP, LPW, PWT, WTA, TAL, ALG, LGL, GLA, LAL, ALS, LSL, SLR, LRL, RLA, LAL, ALA, LAR, ARS, RSG, SGA, SGA, AER, ERG, RGP, GPP, PPA, PAS, ASA, SAP, APR, PRG, RGD, GDL, DLM, LMF, MFL, FLL, LLD, LDS, DSS, SSA, SAS, ASV, SVS, VSH, SHY, HYE, YEF, EFS, FSR, SRV, RVR, VRE, REF, EFV, FVG, VGQ, GQL, QLV, LVA, VAP, APL, PLP, LPL, PLG, LGT, GTG, TGA, GAL, ALR, LRA, RAS, ASL, SLV, LVH, VHV, HVG, VGS, GSR, SRP, RPY, PYT, YTE, TEF, EFP, FPF, PFG, FGQ, GQH, QHS, HSS, SSG, SGE, GEA, EAA, AAQ, AQD, QDA, DAV, AVR, VRA, RAS, ASA, SAQ, AQR, QRM, RMG, MGD, GDT, DTH, THT, HTG, TGL, GLA, LAL, ALV, LVY, VYA, YAK, AKE, KEQ, EQL, QLF, LFA, FAE, AEA, EAS, ASG, SGA, GAR, ARP, RPG, PGV, GVP, VPK, PKV, KVL, VLV, LVW, VWV, WVT, VTD, TDG, DGG, GGS, GSS, SSD, SDP, DPV, PVG, VGP, GPP, PPM, PMQ, MQE, QEL, ELK, LKD, KDL, DLG, LGV, GVT, VTV, TVF, VFI, FIV, IVS, VST, STG, TGR, GRG, RGN, GNF, NFL, FLE, LEL, ELS, LSA, SAA, AAA, AAS, ASA, SAP, APA, PAE, AEK, EKH, KHL, HLH, LHF, HFV, FVD, VDV, DVD, VDD, DDL, DLH, LHI, HII, HV, IVQ, VQE, QEL, ELR, LRG, RGS, GSI, SIL, ILD, LDA, DAM, AMR, MRP, RPQ, PQQ, QQL, QLH, LHA, HAT, ATE, TEI, EIT, ITS, TSS, SSG, SGF, GFR, FRL, RLA, LAW, AWP, WPP, PPL, PLL, LLT, LTA, TAD, ADS, DSG, SGY, GYY, YYV, YVL, VLE, LEL, ELV, LVP, VPS, PSA, SAQ, AQP, QPG, PGA, GAA, AAR, ARR, RRQ, RQQ, QQL, QLP, LPG, PGN, GNA, NAT, ATD, TDW, DWI, WIW, IWA, WAG, AGL, GLD, LDP, DPD, PDT, DTD, TDY, DYD, YDV, DVA, VAL₅ ALV, LVP, VPE, PES, ESN, SNV, NVR, VRL, RLL, LLR,. LRP, RPQ, PQI, QIL, ILR, LRV, RVR, VRT, RTR, TRP,

RPG, PGE, GEA, EAG, AGP, GPG, PGA, GAS, ASG, SGP, GPE, PES, ESG, SGA,

GAG, AGP, GPA, PAP, APT, PTQ, TQL, QLA, LAA, AAL, ALP, LPA, PAP, APE, PEE,

EEA, EAG, AGP, GPE, PER, ERI, RIV, IVI, VIS, ISH, SHA, HAR, ARP, RPR, PRS, RSL, SLR, LRV, RVS, VSW, SWA, WAP, APA, PAL, ALG, LGS, GSA, SAA, AAA, AAL, ALG, LGY, GYH, YHV, HVQ, VQF, QFG, FGP, GPL, PLR, LRG, RGG, GGE, GEA, EAQ, AQR, QRV, RVE, VEV, EVP, VPA, PAG, AGR, GRN, RNC, NCT, CTT, TTL, TLQ, LQG, QGL, GLA, LAP, APG, PGT, GTA, TAY, AYL, YLV, LVT, VTV, TVT, VTA, TAA, AAF, AFR, FRS, RSG, SGR, GRE, RES, ESA, SAL₃ ALS, LSA₅ SAK, AKA₃ KAC, ACT, CTP, TPD, PDG, DGP₃ GPR, PRP, RPR₃ PRP, RPR₃ PRP₃ RPV, PVP₃ VPR, PRA₃ RAP₃ APT₃ PTP₃ TPG, PGT, GTA₃ TAS₃ ASR₃ SRE₃ REP, wherein the peptide or multiplicity of peptides have at least one biochemical, physiological or pharmacology property not present in full length WARP.

As would be evident to a person skilled in the art, pepteins of greater amino acid length, such as about 4 to about 445 residues, or from other full-length WARP sequences, such as human WARP isoforms, splice variants and the like, may be similarly generated.

The pepteins of the present invention may be screened for biochemical, physiological or pharmacological activity by any method known in the art. These methods may include, but are not limited to, in vitro cell culture assays, in vivo non-human and human trials, gene expression analyses and the like.

Once pepteins have been screened, those pepteins exhibiting desirable bioactivity may be characterised by such methods including, but not limited to, mass spectrometry, electrophoresis and protein sequencing.

The present invention also contemplates mutant, derivative, homolog, analog, mimetic or functional equivalent forms of the subject pepteins.

Mutant forms may be naturally occurring or artificially generated variants of the pepteins comprising one or more amino acid substitutions, deletions or additions. Mutants may be induced by mutagenesis or other chemical methods or generated recombinantly or synthetically. Alanine scanning is a useful technique for identifying important amino acids (Wells, Methods Enzymol 202:2699-2705, 1991). In this technique, an amino acid residue is replaced by Alanine and its effect on the peptein's bioactivity is determined. Each of the amino acid residues of the peptide is analyzed in this manner to determine the important regions of the peptein.

Derivatives also include molecules having a percent amino acid sequence identity over a window of comparison after optimal alignment. Preferably, the percentage similarity between a particular sequence and a reference sequence is at least about 60% or at least about 70% or at least about 80% or at least about 90% or at least about 95% or above such as at least about 96%, 97%, 98%, 99% or greater. Preferably, the percentage similarity between species, functional or structural homologs of the instant pepteins is at least about 60% or at least about 70% or at least about 80% or at least about 90% or at least about 95% or above such as at least about 96%, 97%, 98%, 99% or greater. Percentage similarities or identities between 60% and 100% are also contemplated such as 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%.

Analogs of the pepteins contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule. This term also does not exclude modifications of the peptein, for example, glycosylations, acetylations, phosphorylations and the like. Included within the definition are, for example, peptein containing one or more analogs of an amino acid (including, for example, unnatural amino acids such as those given in Table 3) or pepteins with substituted linkages.

Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH₄; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH₄.

The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal. The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4- chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2- chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate .

Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3- hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acids, contemplated herein is shown in Table 2. TABLE 2 : CODES FOR NON-CONVENTIONAL AMINO ACIDS

^' Non-conventional Code Non-conventional Code amino acid amino acid

α-aminobutyric acid Abu L-N-methylalanine Nmala α-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine • Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-Nmethylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile

D-alanine Dal L-N-methylleucine Nmleu

D-arginine Darg L-N-methyllysine Nmlys

D-aspartic acid Dasp L-N-methylmethionine Nmmet

D-cysteine Dcys L-N-methylnorleucine Nmnle

D-glutamine DgIn L-N-methylnorvaline Nmnva

D-glutamic acid DgIu L-N-methylornithine Nmorn

D-histidine Dhis L-N-methylphenylalanine Nmphe

D-isoleucine DiIe L-N-methylproline , Nmpro

D-leucine Dleu L-N-methylserine Nmser

D-lysine Dlys L-N-methylthreonine Nnithr

D-methionine Dmet L-N-methyltryptophan Nmtrp

D-ornithine Dorn L-N-methyltyrosine Nmtyr

D-phenylalanine Dphe L-N-methylvaline Nmval

D-proline Dpro L-N-methylethylglycine Nmetg

D-serine Dser L-N-methyl-t-butylglycine Nmtbug

D-threonine Dthr L-norleucine NIe

D-tryptophan Dtrp L-norvaline Nva

D-tyrosine Dtyr α-methyl-aminoisobutyrate Maib

D-valine Dval α-methyl-γ-aminobutyrate Mgabu

D-α-methylalanine Dmala α-methylcyclohexylalanine Mchexa

D-α-methylarginine Dmarg α-methylcylcopentylalanine Mcpen

D-α-methylasparagine Dmasn α-methyl-α-napthylalanine Manap

D-α-methylaspartate Dmasp α-methylpenicillamine Mpen

D-α-methylcysteine Dmcys N-(4-aminobutyl)glycine NgIu

D-α-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg

D-α-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn

D-α-methylisoleucine Dmile N-amino-α-methylbutyrate Nmaabu

D-α-methylleucine Dmleu α-napthylalanine Anap

D-α-methyllysine Dmlys N-b,enzylglycine Nphe Non-conventional Code Non-conventional Code amino acid amino acid

D-α-methylmethionine Dmmet N-(2-carbamylethyl)glycine NgIn

D-α-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn

D-α-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine NgIu

D-α-methylproline Dmpro N-(carboxymethyl)glycine Nasp

D-α-methylserine Dmser N-cycIobutylglycine Ncbut

D-α-methylthreonine Dmthr N-cycloheptylglycine Nchep

D-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex

D-α-methyltyrosine Dmty N-cyclodecylglycine Ncdec

D-α-methylvaline Dmval N-cylcododecylglycine Ncdod

D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct

D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro

D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund

D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm

D-N-methylcysteine Dnmcys N-(3 ,3 -diphenylpropyl)glycine Nbhe

D-N-methylglutamine Dnmgln N-(3 -guanidinopropyl)glycine Narg

D-N-methylglutamate Dnmglu N-(I -hydroxyethyl)glycine Nthr

D-N-methylhistidine Dnmhis N-(hydroxyethyl)) glycine Nser

D-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis

D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp

D-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate Nmgabu

N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet

D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen

N-methylglycine NaIa D-N-methylphenylalanine Dnmphe

N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro

N-(I -methylpropyl)glycine Nile D-N-methylserine Dnmser

N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr

D-N-methyltryptophan Dnmtrp N-(I -metnylethyl)glycine Nval

D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap

D-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr

L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys

L-ethylglycine Etg penicillamine Pen

L-homophenylalanine Hphe L-α-methylalanine Mala

L-α-methylarginine Marg L-α-methylasparagine Masn

L-α-methylaspartate Masp L-α-methyl-t-butylglycine Mtbug

L-α-methylcysteine Mcys L-methylethylglycine Metg

L-α-methylglutamine MgIn L-α-methylglutamate MgIu

L-α-methylhistidine Mhis L-α-methylhomophenylalanine Mhphe

L-α-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet

L-α-methylleucine Mleu L-α-methyllysine Mlys Non-conventional Code Non-conventional Code amino acid amino acid

L-α-methylmethionine Mmet L-α-methylnorleucine . MnIe

L-α-methylnorvaline Mnva L-α-methylornithine Morn

L-α-methylphenylalanine Mphe L-α-methylproline Mpro

L-α-methylserine Mser L-α-methylthreonine Mthr

L-α-methyltryptophan . Mtrp L-α-methyltyrosine Mtyr

L-α-methylvaline Mval L-N-methylhomophenylalanine Nmhphe

N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycine carbamylmethyl)glycine

1 -carboxy- 1 -(2,2-diphenyl- Nmbc ethylamino)cyclopropane

Crosslinkers. can be used, for example, to stabilize 3D conformations, using homo- bifunctional crosslinkers such as the bifunctional imido esters having (CH₂)_n spacer groups with n = 1 to n = 6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety such as maleimido or dithio moiety (SH) or carbodiimide (COOH). In addition, peptides can be conformationally constrained by, for example, incorporation of C_α and N _α-methylamino acids, introduction of double bonds between C_α and Cp atoms of amino acids and the formation of cyclic peptides or analogs by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.

The present invention also contemplates "mimetic" forms of the subject pepteins. The term "mimetic" is intended to refer to a substance which has some chemical similarity to the peptein it mimics but which antagonizes or agonizes (mimics) its interaction with a target, for example, a receptor. A peptide mimetic may be a peptide-containing molecule that mimics elements of protein secondary structure (Johnson et al, Peptide Turn Mimetics in Biotechnology and Pharmacy, Pezzuto et al, Eds., Chapman and Hall, New York, 1993). The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions such as those of antibody and antigen, enzyme and substrate or scaffolding proteins. A peptide mimetic, therefore, is designed to permit molecular interactions similar to the corresponding peptein.

The designing of mimetics to a pharmaceutically active compound, such as a peptein, is a known approach to the development of pharmaceuticals based on a "lead" compound. This might be desirable where the active compound, i.e. peptein, is difficult or expensive to synthesize or where it is unsuitable for a particular method of administration, e.g. peptides are unsuitable active agents for oral compositions, as they tend to be quickly degraded by proteases in the alimentary canal. Mimetic design, synthesis and testing is generally used to avoid randomly screening large numbers of molecules for a target bioactivity.

There are several steps commonly taken in the design of a mimetic from a peptein having a given target bioactivity. First, the particular parts of the peptein that are critical and/or important in determining the target bioactivity are determined. In the case of a peptein, this can be done by systematically varying the amino acid residues, e.g. by substituting each residue in turn. These parts or residues constituting the active region of the compound are known as its "pharmacophore".

Once the pharmacophore has been found, its structure is modelled according to its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g. spectroscopic techniques, x-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process.

In a variant of this approach, the three-dimensional structure of a receptor and ligand are modelled. This can be especially useful where the receptor and/or ligand change conformation on binding, allowing the model to take account of this in the design of the mimetic. Modelling can be used to generate agents which interact with the linear sequence or a three-dimensional configuration. • . .

A template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted onto it can conveniently be selected so that the mimetic is easy to synthesize, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound. Alternatively, where the mimetic is peptide-based, further stability can be achieved by cyclizing the peptide, increasing. its rigidity. The mimetic or mimetics found by this approach can then be screened to see whether they have the target bioactivity, or to what extent they exhibit it. Further optimization or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.

The goal of rational drug design as contemplated by the present invention is to produce structural analogs of bioactive pepteins of interest or of small molecules with which they interact (e.g. agonists, antagonists, inhibitors or enhancers) in order to fashion drugs which are, for example, more active or stable forms of the peptein, or which, for example, enhance or interfere with the function of a polypeptide in vivo (see, e.g. Hodgson,

BioTechnology 9: 19-21, 1991). An example of rational drug design is the development of HIV protease inhibitors (Erickson et al., Science 249:527-533, 1990).

The bioactive molecules, i.e. pepteins, of the present invention also facilitate the development of pharmaceutical compositions and methods for preventing and/or treating a range of diseases and conditions in a subject.

Reference herein to "treatment" may mean a reduction in the severity of an existing disease or condition. The term "treatment" is also taken to encompass "prophylactic treatment" to prevent the onset of a disease or condition. The term "treatment" does not necessarily imply that a subject is treated until total recovery. Similarly, "prophylactic treatment" does not necessarily mean that the subject will not eventually contract a disease or condition.

Subject as used herein refers to humans and non-human primates (e.g. guerilla, macaque, marmoset), livestock animals (e.g. sheep, cow, horse, donkey, pig), companion animals (e.g. dog, cat), laboratory test animals (e.g. mouse, rabbit, rat, guinea pig, hamster), captive wild animals (e.g. fox, deer), reptiles or amphibians (e.g. cane toad), fish (e.g. zebrafish) and any other organisms (e.g. C. elegans) who can benefit from the agents of the present invention. There is no limitation on the type of animal that could benefit from the presently described agents. The most preferred subject of the present invention is a human. A subject regardless of whether it is a human or non-human organism may be referred to as a patient, individual, animal, host or recipient.

The pepteins of the present invention can.be combined with one or more pharmaceutically acceptable carriers and/or diluents to form a pharmacological composition. Pharmaceutically acceptable carriers can contain a physiologically acceptable compound that acts to, e.g. stabilize, or increase or decrease the absorption or clearance rates of the pharmaceutical compositions of the invention. Physiologically acceptable compounds can include, e.g. carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of the peptides or polypeptides, or excipients or other stabilizers and/or buffers. Detergents can also used to stabilize or to increase or decrease the absorption of the pharmaceutical composition, including liposomal carriers. Pharmaceutically acceptable carriers and formulations for peptides and polypeptide are known to the skilled artisan and are described in detail in the scientific and patent literature, see e.g. Remington's Pharmaceutical Sciences, 18^th Edition, Mack Publishing Company, Easton, PA, 1990 ("Remington's").

Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives which are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, e.g. phenol and ascorbic acid. One skilled in the art would appreciate that the choice of a pharmaceutically acceptable carrier including a physiologically acceptable compound depends, for example, on the route of administration of the modulatory agent of the invention and on its particular physio-chemical characteristics.

Administration of the peptein, in the form of a pharmaceutical composition, may be performed by any convenient means known to one skilled in the art. Routes of administration include, but are not limited to, respiratorally, intratracheally, nasopharyngeal^, intravenously, intraperitoneally, subcutaneously, intracranially, intradermally, intramuscularly, intraoccularly, intrathecally, intracereberally, intranasally, orally, rectally, patch and implant.

For oral administration, the pepteins can be formulated into solid or liquid preparations such as capsules, pills, tablets, lozenges, powders, suspensions or emulsions. In preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, suspending agents, and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets). Due to their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar-coated or enteric-coated by standard techniques. The active agent can be encapsulated to make it stable to passage through the gastrointestinal tract while at the same time allowing for passage across the blood brain barrier, see, e.g, International Patent Publication Number WO 96/11698.

Pepteins of the present invention, when administered orally, may be protected from digestion. This can be accomplished either by complexing the agent with a composition to render it resistant to acidic and enzymatic hydrolysis or by packaging the agent in an appropriately resistant carrier such as a liposome. Means of protecting compounds from digestion are well known in the art, see, e.g. Fix, Pharm Res 73:1760-1764, 1996;

Samanen et al, J Pharm Pharmacol ¥5:119-135, 1996; U.S. Patent Number 5,391,377, describing lipid compositions for oral delivery of therapeutic agents.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion or may be in the form of a cream or other form suitable for topical application. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the pepteins in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilisation. Generally, dispersions are prepared by incorporating the various sterilised active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

For parenteral administration, the pepteins may dissolved in a pharmaceutical carrier and administered as either a solution or a suspension. Illustrative of suitable carriers are water, saline, dextrose solutions,, fructose solutions, ethanol, or oils of animal, vegetative or synthetic origin. The carrier may also contain other ingredients, for example, preservatives, suspending agents, solubilizing agents, buffers and the like. When the pepteins are being administered intrathecally, they may also be dissolved in cerebrospinal fluid.

For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated can be used for delivering the pepteins. Such penetrants are generally known in the art e.g. for transmucosal administration, bile salts and fusidic acid derivatives. In addition, detergents can be used to facilitate permeation. Transmucosal administration can be through nasal sprays or using suppositories e.g. Sayani and Chien, Crit Rev Ther Drug Carrier Syst 75:85-184, 1996. For topical, transdermal administration, the pepteins are formulated into ointments, creams, salves, powders and gels. Transdermal delivery systems can also include patches.

For inhalation, the pepteins of the invention can be delivered using any system known in the art, including dry powder aerosols, liquids delivery systems, air jet nebulizers, propellant systems, and the like, see, e.g. Patton, Nat Biotech /(5:141-143, 1998; product and inhalation delivery systems for polypeptide macromolecules by, e.g. Dura Pharmaceuticals (San Diego, CA), Aradigm (Hayward, CA), Aerogen (Santa Clara, CA), Inhale Therapeutic Systems (San Carlos, CA), and the like. For example, the pharmaceutical formulation can be administered in the form of an aerosol or mist. For aerosol administration, the formulation can be supplied in finely divided form along with a surfactant and propellant. In another aspect, the device for delivering the formulation to respiratory tissue is an inhaler in which the formulation vaporizes. Other liquid delivery systems include, for example, air jet nebulizers.

The pepteins of the invention can also be administered in sustained delivery or sustained release mechanisms, which can deliver the formulation internally. For, example, biodegradeable microspheres or capsules or other biodegradeable polymer configurations capable of sustained delivery of an agent can be included in the formulations of the invention (e.g. Putney and Burke, Nat Biotech 7(5:153-157, 1998).

In preparing pharmaceuticals of the present invention, a variety of formulation modifications can be used and manipulated to alter pharmacokinetics and biodistribution. A number of methods for altering pharmacokinetics and biodistribution are known to one of ordinary skill in the art. Examples of such methods include protection of the compositions of the invention in vesicles composed of substances such as proteins, lipids (for example, liposomes), carbohydrates, or synthetic polymers. For a general discussion of pharmacokinetics, see, e.g. Remington's. In one aspect, the pharmaceutical formulations comprising pepteins of the present invention are incorporated in lipid monolayers or bilayers such as liposomes, see, e.g. U.S. Patent Numbers 6,110,490; 6,096,716; 5,283,185 and 5,279,833. The invention also provides formulations in which water-soluble modulatory agents of the invention have been attached to the surface of the monolayer or bilayer. For example, peptides can be attached to hydrazide-PEG-(distearoylphosphatidyl) ethanolamine-containing liposomes (e.g. Zalipsky et al, Bioconjug Chern (5:705-708, 1995). Liposomes or any form of lipid membrane, such as planar lipid membranes or the cell membrane of an intact cell e.g. a red blood cell, can be used. Liposomal formulations can be by any means, including administration intravenously, transdermally (Vutla et al,, J Pharm Sci 55:5-8, 1996), transmucosally, or orally. The invention also provides pharmaceutical preparations in which the agents of the invention are incorporated within micelles and/or liposomes (Suntres and Shek, J Pharm Pharmacol 46:23-2%, 1994; Woodle et al, Pharm Res 9:260- 265, 1992). Liposomes and liposomal formulations can be prepared according to standard methods and are also well known in the art see, e.g. Remington's; Akimaru et al, Cytokines MoI Ther 7:197-210, 1995; Alving et al, Immunol Rev 145:5-31, 1995; Szoka- and Papahadjopoulos, Ann Rev Biophys Bioeng .9:467-508, 1980, U.S. Patent Numbers 4, 235,871, 4,501,728 and 4,837,028.

The pharmaceutical compositions of the invention can be administered in a variety of unit dosage forms depending upon the method of administration. Dosages for typical pharmaceutical compositions are well known to those of skill in the art. Such dosages are typically advisorial in nature and are adjusted depending on the particular therapeutic context, patient tolerance, etc. The amount of agent adequate to accomplish this is defined as the "effective amount". The dosage schedule and effective amounts for this use, i.e. the "dosing regimen" will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient's physical status, age, pharmaceutical formulation and concentration of active agent, and the like. In calculating the dosage regimen for a patient, the mode of administration also is- taken into consideration. The dosage regimen must also take into consideration the pharmacokinetics, i.e. the pharmaceutical composition's rate of absorption, bioavailability, metabolism, clearance, and the like. See, e.g. Remington's; Egleton and Davis, Peptides 75:1431-1439, 1997; Langer, Science 249:1527-1533, 1990.

In accordance with these methods, the pepteins and/or pharmaceutical compositions defined in accordance with the present invention may be co-administered with one or more other pepteins and/or pharmaceutical compositions. Reference herein to "co-administered" means simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes. Reference herein to "sequential" administration is meant a time difference of from seconds, minutes, hours or days between the administration of the two types of pepteins and/or pharmaceutical compositions. Co-administration of the pepteins and/or pharmaceutical compositions may occur in any order.

Alternatively, targeting therapies may be used to deliver the pepteins more specifically to certain types of cell, by the use of targeting systems such as, but not limited to, antibodies or cell specific ligands. Targeting may be desirable for- a variety of reasons, e.g. if the pepteins is unacceptably toxic or if it would otherwise require too high a dosage or if it would not otherwise be able to enter the target cells.

Instead of administering agents directly, pepteins may also be produced in a target cell, e.g. in a viral vector such as described hereinbefore or in a cell based delivery system such as described in U.S. Patent No. 5,550,050 and International Patent Publication Nos. WO 92/19195, WO 94/25503, WO 95/01203, WO 95/05452, WO 96/02286, WO 96/02646, WO 96/40871, WO 96/40959 and WO 97/12635. The vector could be targeted to the target cells. The cell based delivery system is designed to be implanted in a patient's body at the desired target site and contains a coding sequence for the target pepteins. Alternatively, the pepteins could be administered in a precursor form for conversion to the active form by an activating agent produced in, or targeted to, the cells to be treated. See, for example, European Patent Application No. 0 425 73 IA and International Patent Publication No. WO 90/07936. The pepteins of the present invention may also be used in diagnostic assays as well as in • coating materials for medical devices. Conventional medical devices that may benefit from the present invention include, for example, catheters, conventional needle syringes, hypodermic needles, biopsy needles and devices, tissue ablation devices, needle injection catheters (for endocardial, epicardial, and pericardial agent administration), filters, grafts, metallic and polymeric stents including those having a polymer coated thereon for delivery of pharmaceutically active materials, aneurysm filling coils, transmyocardial. revascularization devices, percutaneous myocardial revascularization devices, soft tissue clips, sutures, blood clot filters, implants or spikes (polymeric or metallic), microspheres or nanoparticles, and so forth.

The present invention is further described by the following non-limiting examples.

EXAMPLE 1

Basic steps associated with WARP peptein production

The basic steps associated with WARP peptein production are shown in Figure 1. Briefly, WARP pepteins are first generated by systematic fragmentation. The pepteins are then screened for biological activity using relevant genetic, biochemical, pharmacological in vitro and in vivo assays. Pepteins that exhibit desired biological activity are then characterised using standard techniques such as mass spectrometry and protein sequencing.

EXAMPLE 2

WARPpepteins

WARP pepteins are generated by defined or random protease digestion. In the case of 3 amino acid pepteins, enzymes capable of generating 3 amino acid pepteins, such as but not limited to, aminopeptidases (e.g. cystinyl aminopeptidase, prolyl aminopeptidase), dipeptidases (e.g. membrane dipeptidase), dipeptidyl and tipeptidyl peptidases {e.g. dipetydal peptidase I, tripeptidyl peptidase I), peptidyl dipeptidases {e.g. peptidyl dipeptidase A), serine-type carboxypeptidases (e.g. serine-type D - ala - D - ala carboxypeptidase), metallocarboxypeptidases (e.g. glutamate carboxypeptidase), omegapeptidases (e,g, acylaminoacyl peptidase, pyroglutamyl peptidase II), serine proteinases (e.g. trypsin, chymotrypsin, subtilisin), cysteine proteinases (e.g. calpain, papain, ficin), aspartic proteinases {e.g. pepsin) and mettallo proteinases (e.g. collagenase, gelatinase), are used to generate 3 amino acid pepteins from a WARP sequence such as SEQ ID NO:2. _.

The 3 amino acid pepteins (shown in single letter code format) include MLP, LPW, PWT, WTA, TAL, ALG, LGL, GLA, LAL, ALS, LSL, SLR, LRL, RLA, LAL₃ ALA, LAR₅

ARS, RSG, SGA, SGA, AER, ERG, RGP, GPP, PPA, PAS, ASA, SAP, APR, PRG, RGD,

GDL, DLM, LMF, MFL, FLL, LLD, LDS, DSS, SSA, SAS, ASV, SVS, VSH, SHY, HYE, YEF, EFS, FSR, SRV, RVR, VRE, REF, EFV, FVG, VGQ, GQL, QLV, LVA, VAP, APL, PLP, LPL, PLG, LGT, GTG, TGA, GAL, ALR, LRA, RAS, ASL, SLV, LVH, VHV, HVG, VGS, GSR, SRP, RPY, PYT, YTE, TEF, EFP, FPF, PFG, FGQ, GQH, QHS, HSS, SSG, SGE, GEA, EAA, AAQ, AQD, QDA, DAV, AVR, VRA, RAS, ASA, SAQ, AQR, QRM, RMG, MGD, GDT, DTH, THT, HTG, TGL, GLA, LAL, ALV, LVY, VYA, YAK, AKE, KEQ, EQL, QLF, LFA, FAE, AEA, EAS, ASG, SGA, GAR, ARP, RPG, PGV, GVP, VPK, PKV, KVL, VLV, LVW, VWV, WVT, VTD, TDG, DGG, GGS, GSS, SSD, SDP, DPV, PVG, VGP, GPP, PPM, PMQ, MQE, QEL, ELK, LKD, KDL, DLG, LGV, GVT, VTV, TVF, VFI, FIV, IVS, VST, STG, TGR, GRG, RGN, GNF, NFL, FLE, LEL, ELS, LSA, SAA, AAA, AAS, ASA, SAP, APA, PAE, AEK, EKH, KHL, HLH, LHF, HFV, FVD, VDV, DVD, VDD, DDL, DLH, LHI, HII, HV, IVQ, VQE, QEL, ELR, LRG, RGS, GSI, SIL, ILD, LDA, DAM, AMR, MRP, RPQ, PQQ, QQL, QLH, LHA, HAT, ATE, TEI, EIT, ITS, TSS, SSG, SGF, GFR, FRL, RLA, LAW, AWP, WPP, PPL, PLL, LLT, LTA, TAD, ADS, DSG, SGY, GYY, YYV, YVL, VLE, LEL, ELV, LVP, VPS, PSA, SAQ, AQP, QPG, PGA, GAA, AAR, ARR, RRQ, RQQ, QQL, QLP, LPG, PGN, GNA, NAT, ATD, TDW, DWI, WIW, IWA, WAG, AGL, GLD, LDP, DPD, PDT, DTD, TDY, DYD, YDV, DVA, VAL, ALV, LVP, VPE, PES, ESN, SNV, NVR, VRL, RLL, LLR, LRP, RPQ, PQI, QIL, ILR, LRV, RVR, VRT, RTR, TRP, RPG, PGE, GEA, EAG, AGP, GPG, PGA, GAS, ASG, SGP, GPE, PES, ESG, SGA, GAG, AGP, GPA, PAP, APT, PTQ, TQL, QLA, LAA, AAL, ALP, LPA, PAP, APE, PEE, EEA, EAG, AGP, GPE, PER, ERI, RIV, IVI, VIS, ISH, SHA, HAR, ARP, RPR, PRS, RSL, SLR, LRV, RVS, VSW, SWA, WAP, APA, PAL, ALG, LGS, GSA, SAA, AAA, AAL, ALG, LGY, GYH, YHV, HVQ, VQF, QFG, FGP, GPL, PLR, LRG, RGG, GGE, GEA, EAQ, AQR, QRV, RVE, VEV, EVP, VPA, PAG, AGR, GRN, RNC, NCT, CTT, TTL, TLQ, LQG, QGL₅ GLA, LAP, APG, PGT, GTA, TAY, AYL, YLV, LVT, VTV, TVT, VTA, TAA, AAF, AFR, FRS, RSG, SGR, GRE, RES, ESA, SAL, ALS, LSA, SAK, AKA, KAC, ACT, CTP, TPD, PDG, DGP, GPR, PRP, RPR, PRP, RPR, PRP, RPV, PVP, VPR, PRA, RAP, APT, PTP, TPG, PGT, GTA, TAS, ASR, SRE, REP.

Pepteins of greater amino acid length, such as about 4 to about 445 residues, or from other full-length WARP sequences, such as human WARP isoforms, splice variants and the like, are generated using similar methodology. Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

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Claims

CLAIMS:

1. An isolated fragment or fraction of fragments derived from von Willebrand Factor A-Related Protein (WARP) said fragment or each fragment in the fraction comprises at least three amino acid residues, said fragment or fraction exhibiting at least one biochemical, physiological or pharmacological property substantially absent from full- length WARP. .

2. The isolated fragment or fraction of Claim 1 wherein each fragment is from about 5 to about 445 amino acid residues in length.

3. The isolated fragment or fraction of Claim 2 wherein each fragment is from about 5 to about 400 amino acid residues in length.

4.. The isolated fragment or fraction of Claim 1 derived from human WARP isoform 1 (SEQ ID NO:2).

5. The isolated fragment or fraction of Claim 1 derived from human WARP isoform 2 (SEQ ID NO:3).

6. The isolated fragment or fraction of Claim 1 derived from murine WARP (SEQ ID NO:1).

7. The isolated fragment or fraction of Claim 4 wherein the amino acid sequence of the fragment is selected from the list comprising MLP, LPW, PWT, WTA, TAL, ALG, LGL, GLA, LAL, ALS, LSL, SLR, LRL₅ RLA, LAL, ALA, LAR, ARS, RSG, SGA, SGA, AER, ERG, RGP, GPP, PPA, PAS₃ ASA, SAP, APR, PRG, RGD, GDL, DLM, LMF, MFL, FLL, LLD, LDS, DSS, SSA, SAS, ASV, SVS, VSH, SHY, HYE, YEF, EFS, FSR, SRV, RVR, VRE, REF, EFV, FVG, VGQ, GQL, QLV, LVA, VAP, APL, PLP, LPL, PLG, LGT, GTG, TGA, GAL, ALR, LRA, RAS, ASL, SLV, LVH, VHV, HVG, VGS, GSR, SRP, RPY, PYT, YTE, TEF, EFP, FPF, PFG, FGQ, GQH, QHS, HSS, SSG, SGE, GEA, EAA, AAQ₅ AQD, QDA, DAV, AVR, VRA, RAS, ASA, SAQ, AQR, QRM, RMG, MGD, GDT, DTH, THT, HTG, TGL, GLA, LAL, ALV, LVY, VYA, YAK, AKE, KEQ, EQL, QLF, LFA, FAE, AEA, EAS, ASG, SGA, GAR, ARP, RPG, PGV, GVP, VPK, PKV, KVL, VLV, LVW, VWV, WVT, VTD, TDG, DGG, GGS, GSS, SSD, SDP, DPV, PVG, VGP, GPP, PPM, PMQ, MQE, QEL, ELK, LKD, KDL, DLG, LGV, GVT, VTV, TVF, VFI, FIV, IVS, VST, STG, TGR, GRG, RGN, GNF, NFL, FLE, LEL, ELS, LSA, SAA, AAA, AAS, ASA, SAP, APA, PAE, AEK, EKH, KHL, HLH, LHF, HFV, FVD, VDV, DVD, VDD, DDL, DLH, LHI, HII, HV, IVQ, VQE, QEL, ELR, LRG, RGS, GSI, SIL, ILD, LDA, DAM, AMR, MRP, RPQ, PQQ, QQL, QLH, LHA, HAT, ATE, TEI, EIT, ITS, TSS, SSG, SGF, GFR, FRL, RLA, LAW, AWP, WPP, PPL, PLL, LLT, LTA, TAD, ADS, DSG, SGY, GYY, YYV, YVL, VLE, LEL, ELV, LVP, VPS, PSA, SAQ, AQP, QPG, PGA, GAA, AAR, ARR, RRQ, RQQ, QQL, QLP, LPG, PGN, GNA, NAT, ATD, TDW, DWI, WIW, IWA, WAG, AGL, GLD, LDP, DPD, PDT, DTD, TDY, DYD, YDV, DVA, VAL, ALV, LVP, VPE, PES, ESN, SNV, NVR, VRL, RLL, LLR, LRP, RPQ, PQI, QIL, ILR, LRV, RVR, VRT, RTR, TRP, RPG, PGE, GEA, EAG, AGP, GPG, PGA, GAS, ASG, SGP, GPE, PES, ESG, SGA, GAG, AGP, GPA, PAP, APT, PTQ, TQL, QLA, LAA, AAL, ALP, LPA, PAP, APE, PEE, EEA, EAG, AGP, GPE, PER, ERI, RIV, IVI, VIS, ISH, SHA, HAR, ARP, RPR, PRS, RSL, SLR, LRV, RVS, VSW, SWA, WAP, APA, PAL, ALG, LGS, GSA, SAA, AAA, AAL, ALG, LGY, GYH, YHV, HVQ, VQF, QFG, FGP, GPL, PLR, LRG, RGG, GGE, GEA, EAQ, AQR, QRV, RVE, VEV, EVP, VPA, PAG, AGR, GRN, RNC, NCT, CTT, TTL, TLQ, LQG, QGL, GLA, LAP, APG, PGT, GTA, TAY, AYL, YLV, LVT, VTV, TVT, VTA, TAA, AAF, AFR, FRS, RSG, SGR, GRE, RES, ESA, SAL, ALS, LSA, SAK, AKA, KAC, ACT, CTP, TPD, PDG, DGP, GPR, PRP, RPR, PRP, RPR, PRP, RPV, PVP, VPR, PRA, RAP, APT, PTP, TPG, PGT, GTA, TAS, ASR, SRE, REP.

8. The isolated fragment or fraction of Claim 1 wherein the biochemical, , physiological or- pharmacological property is selected from receptor binding, angiogenesis stem cell proliferation and/or differentiation, cytokine induction, ^'homeostasis and gene expression.

9. A pharmaceutical composition comprising an isolated fragment or fraction of fragments as defined in any one of Claims 1 to 8.

10. A method for generating a fragment or fraction of fragment of any one of Claims 1 to 8 by the method comprising subjecting a WARP to proteolytic digestion, screening isolated fragments or fractions of fragments for bioactivity absent in full length WARP and selecting said bioactive fragment or fractions of fragments.

11. Use of WARP in the manufacture of a medicament comprising a fragment or fraction of fragments of WARP for generating a fragment or fraction within a bioactivity absent from full-length WARP.