WO2007053952A1 - Platelet factor-4 (pf-4) analogs and their use - Google Patents

Abstract

The present invention concerns the discovery, synthesis and use of peptidic analogs derived from platelet factor-4 chemokine (PF-4) and their pharmaceutical compositions. These compounds and their pharmaceutical compositions are useful in treating diseases such as : angiogenic related disorders ; hematopoietic disorders in which the stimulation of proliferation of hematopoietic stem cells/precursors ; the protection of hematopoietic cells from chemotherapy related drugs and/or the inhibition of differentiation of hematopoietic precursors are needed ; coagulatory related disorders requiring the inhibition of heparine or the increase in anti-coagulant activated protein C. ; and inflammation related disorders.

Description

PLATELET FACTOR-4 (PF-4) ANALOGS AND THEIR USE

FIELD OF THE INVENTION

[0001] This invention relates to the design and preparation of peptides and peptide analogs derived from chemokine platelet factor-4 (PF-4). In other aspects, the invention relates to the therapeutic use of these compounds and pharmaceutical compositions comprising the compounds.

BACKGROUND OF THE INVENTION

[0002] PF-4 is a 70-amino acid protein that belongs to the family of chemokines (Deul T. F. et al. PNAS 1977; 74: 2256-2258, and Mayo K. H. et al. Bichem. J. 1995; 312: 357-365). The complete primary structure of PF-4 is known (Poncz et al., Blood 1987; 69: 219-223). PF-4 belongs to the CXC chemokine family characterized by a Cysteine-X-Cysteine motif located in the N-terminal region. The CXC motif participates in producing the secondary and tertiary structure of native PF-4 via formation of intramolecular disulfide bonds with residues Cys-36 and Cys-51. C-terminal fragments of PF-4, including PF-4^47"70, have been reported to inhibit angiogenesis associated with cancer (Joaun et al., Blood, 94: 984-993, 1999; Hagedorn et al., FASEB J., 10: 1096, 2001 ; BeIIo et al., Clinical Cancer Research Vol. 8, 3539-3548, November 2002; Hagedorn et al., Cancer Research 62, 6884-6890, December 1 , 2002; Bikfalvi, Biochemical Pharmacology 2004; 68:1017-1021).

[0003] EP176588 (published as WO 85/04397 on 10 October 1985) discloses PF-4 derived peptides, particularly peptides that include C terminal portions or motifs of PF-4. EP281363, published 7 September 1988, describes PF- 4 related peptides, having cell growth modulating activity, including peptides having the N-terminal sequence of PF-4. EP324556, published 19 July 1989, discloses methods of expressing PF-4 from mRNAs. EP378364, published 18 July 1990, describes analogues of PF-4 and fragments thereof, for use in treating angiogenic diseases and inhibition of endothelial cell proliferation, including a 13 amino acid long C-terminal fragment of PF-4. Similarly, EP723015 and EP407122, published 9 January 1991 , describe an angiogenesis-inhibiting activity in a peptide of 13 amino acids corresponding to the C-terminal sequence of PF-4, in which two pairs of lysine residues have been replaced by two glutamic acid-glutamine pairs. WO 2002/006300, published 24 January 2002, discloses the use of PF-4, fragments and fusion peptides derived from PF-4, and their analogues, for angiogenesis- inhibiting activities, particularly peptides in which the glutamine situated at position 56 in native PF-4 is replaced by a basic amino acid, including peptide PF-4 fragments corresponding to residues 47-70 of native PF-4 (PF-4^47"70 ) or fusion peptides corresponding to fragments 17-34/47-70 of native PF-4.

SUMMARY OF THE INVENTION

[0004] The present invention is based on the discovery of platelet factor (PF-4) analogs that have improved activity. These analogs correspond to the N-terminal part (1 -16 region) linked to the C-terminal (57-70 region) by an appropriate linker. In another aspect of the invention, the C-terminal part of these peptides is cyclized between residues that correspond to position 65 and position 69, or position 66 and position 69, of native PF-4 (i.e. in Formula 1 , between residues X₂5 or X₂₆ and residue X₂g). In native PF-4, positions 65 and 66 are Lys residues, and position 69 is a GIy residue.

[0005] Accordingly, in various aspects, the invention provides PF-4 peptidic analogs selected from the group consisting of peptides or peptide analogs of PF-4 of Formula I:

R-Xθi Xθ2 Xθ3 Xθ4 Xθ5 Xθ6 Xθ7 Xθ8 Xθ9 Yθ1 Xi 1 Yθ2 Xi2 Xi3 Xi4 Xi5 Xi6 ZA Xi7 Xi8 Xi9 X20 X21 X22 X23 X24 X25 X26 X27 X28 X29 X30 (Formula I)

wherein

R is an optional N-terminal modifying group, which may be present or absent, including: PEGyI, Acetyl, or Alkyl; X01 is optional, may be present or absent, and if present may be L-or D-GIx, L- or D-Asx, L- or D-AIa, (L- or D-GIu and L- or D-GIn in selected embodiments); is any natural or non natural amino acid different from L- or D-Cys, such as: L- or D-AIa, L- or D-VaI, L- or D-IIe, L- or D-Leu, (L- or D-AIa in select embodiments); is L- or D-GIx, L- or D-Asx, L- or D-AIa, L- or D-Pro, L- or D-Phe, L- or D-Tyr, L- or D-Ser, L- or D-Thr (L-or D-GIn, L-or D-GIu or L- or D-Asp in selected embodiments); is any natural or non natural amino acid residue different from L- or D-Cys residue, such as: L- or D-GIx, L- or D-Asx, L- or D-L- or D-AIa, L- or D-VaI, L- or D-Phe (L-or D-GIn or L- or D-GIu in selected embodiments); is L- or D-Asx, L- or D-GIx, L- or D-AIa, L- or D-Phe, L- or D-Tyr (L- or D-Asn,

L- or D-Asp, or L- or D-GIu in selected embodiments); is any natural or non natural amino acid residue different from L- or D-Cys residue, such as: L- or D-AIa, L- or D-VaI, L- or D-Leu, L- or D-IIe, L- or D- Phe, L- or D-Tyr, L- or D-GIx (GIy in selected embodiments); is L- or D-Asx, L- or D-GIx, L- or D-AIa, L- or D-Phe, L- or D-Tyr (L- or D-Asn,

L- or D-Asp, L-or D-GIu in selected embodiments); is any natural or non natural amino acid residue different from L- or D-Cys residue, such as: L- or D-Leu, L- or D-AIa, L- or D-L- or D-VaI, L- or D-Leu, L- or D-IIe, L- or D-Phe, L- or D-Tyr (L- or D-Leu in selected embodiments); is L- or D-GIx, L- or D-Asx, L- or D-Arg, L- or D-Lys, L- or D-His, L- or D-Tyr, L- or D-Ser, L- or D-Thr (L- or D-GIn, L- or D-Asn in selected embodiments); is L- or D-Cys, L- or D-AIa, L- or D-Phe, L- or D-His, L- or D-Trp, L- or D-Ser, L- or D-Thr, L- or D-Lys, L- or D-His, L- or D-Tyr (L- or D-Cys, L- or D-AIa, L- or D-Phe, L- or D-Tyr, L- or D-His, or L- or D-Trp in selected embodiments); is any natural or non natural amino acid residue different from L- or D-Cys residue, such as: L- or D-GIx, L- or D-Asx, GIy, L- or D-VaI, L- or D-Leu, L- or D-IIe, L- or D-Pro, L- or D-Phe, L- or D-Tyr, L- or D-His, L- or D-Trp (L- or D- Leu in selected embodiments); is L- or D-Cys, L- or D-AIa, L- or D-Phe, L- or D-His, L- or D-Trp, L-or D-Ser, L- or D-Thr, L- or D-Lys, L- or D-His, L- or D-Tyr (L- or D-Cys, L- or D-AIa, L- or D-Phe, L- or D-Tyr, L- or D-His, or L- or D-Trp in selected embodiments); is L- or D-VaI, L- or D-Leu, L- or D-IIe, L- or D-AIa, L- or D-Phe, L- or D-Tyr (L- or D-VaI in selected embodiments); is optional, may be present or absent, and if present may be any natural or non natural amino acid residue different from L- or D-Cys residue, such as:

L- or D-Lys, L- or D-Arg, L- or D-His (L- or D-Lys or L- or D-Arg in selected embodiments); is optional, may be present or absent, and if present may be L- or D-Thr, L- or D-Ser, L- or D-Tyr, L- or D-AIa, L- or D-Phe (L- or D-Thr in selected embodiments); is optional, may be present or absent, and if present may be any natural or non natural amino acid residue different from L- or D-Cys residue, such as: L- or D-Thr, L- or D-Ser, L- or D-Tyr, L- or D-AIa, L- or D-Phe (L- or D-Thr or

L- or D-Ser in selected embodiments); is optional, may be present or absent, and if present may be any natural or non natural amino acid residue different from L- or D-Cys, such as: L- or D-

Thr, L- or D-Ser, L- or D-Tyr, L- or D-AIa, L- or D-Phe (L- or D-Thr or L- or D- Ser in selected embodiments); is a linker, such as a flexible covalent linker, for example having 1 to 20 carbon atoms, or from 2 to 200 Angstroms in length, or (CH₂)i-2o or (X_aa)i-2o; in selected embodiments, the linker may be represented by the following general formula: H₂N-X_aai-(Xaa2 Xaas X_aa4 VCO₂H, with n=0 or n=1 , where n=1 , in selected embodiments Z_A may be comprised of amino acid residues that are the same as (=) or different from (#) X_aai, represented as follows:

3" Xaa1⁼Xaa2⁼Xaa3⁼Xaa4 b- Xaa1#X_aa2=Xaa3=Xaa4

C- X_aai=X_aa2#Xaa3=Xaa4 d- X_aai=Xaa2=Xaa3#Xaa4

g- Xaa1⁼Xaa2#Xaa3#Xaa4 h- Xaa1#Xaa2#Xaa3#Xaa4 where X_aai

to 4) is any L- or D- natural or non natural amino acid residue including L- or D-Cys residue, where n=0, in selected embodiments, X_aai=11- aminoundecanoic acid, Xi₇ is any natural or non natural amino acid different from L- or D-Cys, such as: L- or D-AIa, L- or D-VaI, L- or D-IIe, L- or D-Leu (L- or D-AIa in selected embodiments); Xi₈ is L- or D-Pro, L- or D-AIa (L- or D-Pro in selected embodiments);

Xi₉ is any natural or non natural amino acid residue different from L- or D-Cys, such as: L- or D-Leu, L- or D-AIa, L- or D-L- or D-VaI, L- or D-Leu, L- or D- Me, L- or D-Phe, L- or D-Tyr (L- or D-Leu in selected embodiments); X₂₀ is L- or D-Tyr, L- or D- Ser, L- or D-Thr, L- or D-Phe (L- or D-Tyr in selected embodiments);

X₂₁ is any natural or non natural amino acid residue different from L- or D-Cys residue, such as: L- or D-Lys, L- or D-Arg, L- or D-His (L- or D-Lys in selected embodiments);

X₂₂ is any natural or non natural amino acid residue different from L- or D-Cys residue, such as: L- or D-Lys, L- or D-Arg, L- or D-His (L- or D-Lys in selected embodiments); X₂₃ is any natural or non natural amino acid residue different from L- or D-Cys residue, such as: L- or D-GIx, L- or D-Asx, GIy, L- or D-VaI, L- or D-Leu, L- or D-IIe, L- or D-Pro, L- or D-Phe, L- or D-Tyr, L- or D-His, L- or D-Trp (L- or D- Me in selected embodiments);

X₂4 is any natural or nonnatural amino acid residue different from L- or D-Cys residue, such as: L- or D-GIx, L- or D-Asx, GIy, L- or D-VaI, L- or D-Leu, L- or D-IIe, L- or D-Pro, L- or D-Phe, L- or D-Tyr, L- or D-His, L- or D-Trp (L- or D- He in selected embodiments); X₂5 is any natural or nonnatural amino acid residue different from L- or D-Cys residue, such as: L- or D-Lys, L- or D-Arg, L- or D-His (L- or D-Lys in selected embodiments); X₂6 is any naturll or non-natural amino acid residue different from L- or D-Cys residue, such as: L- or D-Lys, L- or D-Arg, L- or D-His (L- or D-Lys in selected embodiments);

X₂7 is any natural or non natural amino acid residue different from L- or D-Cys residue, such as: L- or D-Leu, L- or D-AIa, L- or D-L- or D-VaI, L- or D-Leu, L- or D-IIe, L- or D-Phe, L- or D-Tyr (L- or D-Leu in selected embodiments); X_2S is any natural or non natural amino acid residue different from L- or D-Cys residue, such as: L- or D-Leu, L- or D-AIa, L- or D-L- or D-VaI, L- or D-Leu, L- or D-IIe, L- or D-Phe, L- or D-Tyr (L- or D-Leu in selected embodiments);

X₂₉ is L-or D-GIx, L- or D-Asx, L- or D-AIa (L- or D-GIu or L- or D-GIn in selected embodiments);

X₃₀ is any natural or non natural amino acid residue different from L- or D-Cys residue, such as: L- or D-Thr, L- or D-Ser, L- or D-Tyr, L- or D-AIa, L- or D- Phe (L- or D-Thr or L- or D-Ser in selected embodiments); and, their non-toxic acidic addition salts.

[0006] In selected embodiments, the C-termial region of the peptide, for example starting from residue 19 and finshing at residue 30, forms a stable α- helix moity.

[0007] In selected embodiments, a side chain to side chain cyclization is produced between the amino acid residue X₂₆ and the amino acide residue X₂₉, alternative types of cyclization are: lactamization, etherification, thioetherification, and cyclization generated by Mitsunubo or Ring Closing Methathesis (RCM) type of reactions. For example, in selected embodiments, the cyclization is a lactamization between a L- or D-Lys at position 26 (X₂₆) and L- or D-GIu at position 29 (X₂₉).

Alternatively, there may be a side chain to side chain cyclization between the amino acid residue X₂₅ and the amino acide residue X_2g, such as lactamization between a L- or D-Lys at position 25 and L- or D-GIu at position 29. In selected embodiments, the C-terminal residue may be amidated.

[0008] In alternative aspects, the invention relates to uses of the peptidic compounds of the invention. Specifically, compounds of the invention may be used to treat conditions or to formulate medicaments for treating conditions. For example, a peptidic compound selected from the group consisting of peptidic compounds of Formula I, and their non-toxic acidic addition salts, may be used as a drug or to prepare a drug.

[0009] Further alternative aspects of the invention relate to the use of compounds of the invention in therapeutic compositions in association with physiologically acceptable excipients, such as compounds comprising at least one peptide chosen from the group consisting of peptides of Formula I₁ and their nontoxic acidic addition salts.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 shows inhibition of human vein endothelial cell

(HUVEC) proliferation by the PF4 analog SEQ ID No.13. as determine using MTT assay. [0011] Figure 2 shows the inhibition of human endothelial cell proliferation by the PF4 analog SEQ ID No.14, as determine using MTT assay.

[0012] Figure 3 shows the inhibition of human endothelial cell proliferation by the PF4 analog SEQ ID No.15, as determine using MTT assay. [0013] Figure 4 shows the block of human erythroid leukemic cell (HEL) cell proliferation by the PF4 analog SEQ ID No.13, as determine using MTT assay.

[0014] Figure 5 shows the block of HEL cell proliferation by the PF4 analog SEQ ID No.14, as determine using MTT assay.

[0015] Figure 6 shows the block of HEL cell proliferation by the PF4 analog SEQ ID No. 15, as determine using MTT assay.

DETAILED DESCRIPTION OF THE INVENTION

[0016] In various aspects, the invention relates to the design, preparation, derivation and use of platelet factor-4 (PF-4) chemokine analogs. In one aspect, this invention is directed to the synthesis or use of PF-4 chemokine analogs. In another aspect, the invention is directed to the synthesis, design, derivation, or use of agonist or antagonist analogs of the PF-4 chemokine. The invention is not limited in its application to the details of structures and the arrangements of components set forth in the following description or illustrated in the drawings and the figures. Further, it should be understood that in any claimed list or claimed Markush group, those schooled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the list or Markush group. Additionally, any individual member of the claimed list or the claimed Markush group can be removed from the list or Markush group without affecting the patentability of the remaining members.

[0017] The sequence of platelet factor-4 (PF-4) chemokine is shown below.

Glu-Ala-Glu-Glu-Asp-Gly-Asp-Leu-Gln-Cys-Leu-Cys-Val- Lys -Thr-Thr-Ser-Gln-Val-Arg-Pro-Arg-His -Ile-Thr-Ser- Leu-Glu-Val -Ile-Lys -Ala-Gly-Pro-His-Cys -Pro-Thr-Ala- Gln-Leu- Ile-Ala-Thr-Leu-Lys-Asn-Gly-Arg-Lys-Ile-Cys - Leu-Asp-Leu-Gln-Ala-Pro-Leu-Tyr-Lys-Lys - Ile- Ile-Lys-

Lys -Leu-Leu-Glu-Ser (SEQ ID No: 1)

[0018] The N-terminal region of PF-4 chemokine is involved in the binding and activating site of its CXCR3-B receptor, as well as is the carboxy terminal region. The cluster of positive charges in the α-helix structure in the C- terminal region appears to play a roll at least in the binding to heparin sulfate proteoglycans (HSPGs) (Bikfalvi et al., Biochemical Pharmacology 2004; 68: 1017- 1021 ) by establishing an ionic interaction with the negative charges of the proteoglycans. The beta sheet structure plays a role in assuring that the distorded N- termininal region of the ligand has a proper conformation.

[0019] In selected embodiments, the PF-4 chemokine analog comprises an N-terminal region and a C-terminal region joined together by means of a linker. In someembodiments, the linker contains positively charged, natural or non natural amino acids. In further alternative embodiments, amino acid residues of the PF-4 chemokine analog are cyclized in the C-terminal region, e.g., by etherification of lysine and serine residues or by other means described infra or known in the art. In still other embodiments, the PF-4 chemokine analog comprises a sequence derived from the wild-type chemokine sequence but with one or more of the cysteines replaced with another amino acid including natural and non-natural amino acids. [0020] Selected PF-4 Chemokine analogs of the invention are useful for treating diseases, for example by means of an anti-angiogenesis effect; or by inhibiting blood clot formation (for example through the acceleration of APC formation);. or by modulating immunological responses; or by increasing the survival of hematopoietic cells (for example by protecting them from chemotherapeutic drugs); or by inhibiting the anticoagulant effect of heparin, or to treat atherosclerotic related diseases. In one aspect, the invention provides therapeutic methods that comprise administering to a patient in need of such treatment a therapeutically effective amount of a PF-4 chemokine analog of the invention.

Abreviations

[0021] Amino acids are identified in the present application by the conventional one-letter and three-letter abbreviations as indicated below, and are preceded by "L-" to indicate their L-form and by "D-" to refer to their D form. Asx represents either the amino acid Asp or the amino acid Asn. GIx represents either the amino acid GIu or the amino acid GIn. These abbreviations are generally accepted in the peptide art as recommended by the IUPAC-IUB commission in biochemical nomenclature:

Alanine A Ala Leucine L Leu

Arginine R Arg Lysine K Lys

Asparagine N Asn Methionine M Met

Aspartic acid D Asp Phenylalanine F Phe

Cysteine C Cys Proline P Pro

Glutamic acid E GIu Serine S Ser

Glutamine Q GIn Threonine T Thr

Glycine G GIy Tryptophan W Trp

Histidine H His Tyrosine Y Tyr lsoleucine I lie Valine V VaI

[0022] All of the peptide sequences set out herein are written according to the generally accepted convention whereby the N-terminal amino acid is on the left and the C-terminal amino acid is on the right. Definitions

[0023] The term "heterocyclic group" includes cyclic saturated, unsaturated and aromatic groups having from 3 to 10; from 4 to 8; or 5, 6, or 7 carbon atoms, wherein the ring structure includes about one or more heteroatoms. Heterocyclic groups include pyrrolidine, oxolane, thiolane, imidazole, oxazole, piperidine, piperazine, morpholine. The heterocyclic ring may be substituted at one or more positions with such substituents as, for example, halogens, alkyls, cycloalkyls, alkenyls, alkynyls, aryls, arylalkyls, other heterocycles, hydroxyl, amino, nitro, thiol, amines, imines, amides, phosphonates, phosphines, carbonyls, carboxyls, silyls, ethers, thioethers, sulfonyls, selenoethers, ketones, aldehydes, esters, -CF₃, -CN. Heterocycles may also be bridged or fused to other cyclic groups as described below. A linker may also link the heterocyclic group to such substituents as, for example, halogens, alkyls, cycloalkyls, alkenyls, alkynyls, aryls, arylalkyls, heterocycles, hydroxyls, aminos, nitros, thiols amines, imines, amides, phosphonates, phosphines, carbonyls, carboxyls, silyls, ethers, thioethers, sulfonyls, sulfonates, selenoethers, ketones, aldehydes, esters, -CF₃, -CN.

[0024] The term "polycyclic group" as used herein is intended to refer to two or more saturated, unsaturated or aromatic cyclic rings in which two or more carbons are common to two adjoining rings, so that the rings are "fused rings." Rings that are joined through non-adjacent atoms are termed "bridged" rings. Each of the rings of the polycyclic group may be substituted with such substituents as described above, as for example, halogens, alkyls, cycloalkyls, alkenyls, alkynyls, hydroxyl, amino, nitro, thiol, amines, imines, amides, phosphonates, phosphines, carbonyls, carboxyls, silyls, ethers, thioethers, sulfonyls, selenoethers, ketones, aldehydes, esters, -CF₃, or -CN.

[0025] The term "alkyl" refers to a saturated aliphatic groups, including straight chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In some embodiments, a straight chain or branched chain alkyl has 20 or fewer carbon atoms in its backbone (CrC≥o for straight chain, C₃-C2o for branched chain), or 10 or fewer carbon atoms. In some embodiments, cycloalkyls may have from 4- 10 carbon atoms in their ring structure, such as rings made from 5, 6 or 7. Unless the number of carbons is otherwise specified, "lower alkyl" as used herein means an alkyl group, as defined above, having from one to ten carbon atoms in its backbone structure. Likewise, "lower alkenyl" and "lower alkynyl" have chain lengths of ten or less carbons. [0026] The term "alkyl" (or "lower alkyl") as used throughout the specification and claims is intended to include both "unsubstituted alkyls" and "substituted alkyls," the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, halogen, hydroxyl, carbonyl (such as carboxyl, ketones (including alkylcarbonyl and arylcarbonyl groups)), and esters (including alkyloxycarbonyl and aryloxycarbonyl groups), thiocarbonyl, acyloxy, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, acylamino, amido, amidine, imino, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety. The moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. For instance, the substituents of a substituted alkyl may include substituted and unsubstituted forms of aminos, azidos, iminos, amidos, phosphoryls (including phosphonates and phosphinates), sulfonyls (including sulfates, sulfonamides, sulfamoyls and sulfonates), or silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), -CF₃, -CN and the like. Exemplary substituted alkyls are described below. Cycloalkyls can be further substituted with alkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl-substituted alkyls, -CF3, -CN, and the like.

[0027] The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.

[0028] The term "aralkyl," as used herein, refers to an alkyl or alkylenyl group substituted with at least one aryl group. Exemplary aralkyls include benzyl (i.e., phenylmethyl), 2-naphthylethyl, 2-(2-pyridyl)propyl, 5-dibenzosuberyl, and the like.

[0029] The term "alkylcarbonyl," as used herein, refers to -C(O)-alkyl.

Similarly, the term "arylcarbonyl" refers to -C(O)-aryl. The term "alkyloxycarbonyl," as used herein, refers to the group -C(O)-O-alkyl, and the term "aryloxycarbonyl" refers to -C(O)-O-aryl. The term "acyloxy" refers to -0-C(O)-R?, in which R₇ is alkyl, alkenyl, alkynyl, aryl, aralkyl or heterocyclyl.

[0030] The term "amino," as used herein, refers to -N(R_α)(R_β), in which R_α and R_β are each independently hydrogen, alkyl, alkyenyl, alkynyl, aralkyl, aryl, or in which R₀ and R_β together with the nitrogen atom to which they are attached form a ring having 4-8 atoms. Thus, the term "amino," as used herein, includes unsubstituted, monosubstituted (e.g., monoalkylamino or monoarylamino), and disubstituted {e.g., dialkylamino or alkylarylamino) amino groups. The term "amido" refers to -C(O)-N(R_α)(Rβ), in which R_α and Rβ are as defined above. The term "acylamino" refers to -N(R a)C(O)-R₇, in which R₇ is as defined above and R_α is alkyl.

[0031] As used herein, the term "nitro" means -NO₂; the term

"halogen" designates -F, -Cl, -Br or -I; the term "sulfhydryl" means -SH; and the term "hydroxyl" means -OH. [0032] The term "aryl" as used herein includes 5-, 6- and 7-membered aromatic groups that may include from zero to four heteroatoms in the ring, for example, phenyl, pyrrolyl, furyl, thiophenyl, imidazolyl, oxazole, thiazolyl, triazolyl, pyrazolyl, pyridyl, pyrazinyl, pyridazinyl and pyrimidinyl, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycles" or "heteroaromatics." The aromatic ring can be substituted at one or more ring positions with such substituents as described above, as for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF₃, -CN, or the like. Aryl groups can also be part of a polycyclic group. For example, aryl groups include fused aromatic moieties such as naphthyl, anthracenyl, quinolyl, indolyl, and the like.

[0033] As defined by the present invention a PF-4 peptide analog acts as an agonist or antagonist to a native chemokine. [0034] In this application, the products of the present invention are referred to by various terms, including "analogs" of the present invention, "PF-4- chemokine mimetics," "PF-4 chemokine analogs," , "PF-4 peptide analogs," "PF-4 peptidic compounds," and "PF-4 chemokine derivatives." These terms are used interchangeably and denote equivalent compounds. The term "polypeptides of the present invention," may also be used herein to refer to PF-4 chemokine analogs.

[0035] As defined by the present invention, biological activity refers to the biological activity of the native chemokine, as defined and measured by the scientific reports known to those skill in the art, and exemplified in the following review articles (Bruce, L. et al., Methods in Molecular Biology 2000; 138: 129-134, Raphaele, B. et al. Methods in Molecular Biology 2000; 138: 143-148).

[0036] PF-4 peptide analogs of the invention may include peptide analog derivatives, such as C-terminal hydroxymethyl derivatives, O-modified derivatives (e.g., C-terminal hydroxymethyl benzyl ether), N-terminally modified derivatives including substituted amides such as alkylamides and hydrazides and compounds in which a C-terminal phenylalanine residue is replaced with a phenethylamide analogue {e.g., Ser-lle-phenethylamide as an analog of the tripeptide Ser-lle-Phe), glycosylated PF-4 peptide derivatives, polyethylene glycol modified derivatives, or biotinylated derivatives.

[0037] The term "identity" as used herein refers to the measure of the identity of sequence between two proteins, two peptides or two nucleic acids molecules. Identity can be determined by comparing a position in each sequence which may be a line for the purpose of comparison. Two amino acid or nucleic acid sequences are considered substantially identical if they share at least about 75% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity and even more preferably at least 95% sequence identity and most preferably at least about 98-99% identity. [0038] As used herein, the term "homology" can be equated with

"identity". A homologous sequence is an amino acid sequence or nucleotide sequence which may be at least about 75%, or 85% or 90% or 95% identical, preferably at least about 98% or 99% identical to another amino acid sequence or nucleotide sequence. [0039] Most commercially available sequence comparison methods known to those of skill in the art produce optimal sequence alignments that take into consideration possible insertions and deletions without unduly penalizing the overall homology score. This is achieved by inserting gaps in the sequence alignment to maximize local homology. "Gap penalties" are assigned to each gap that occurs in a single alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible (which reflects a higher relatedness between the two compared sequences) will be provided with a higher score than one with many gaps. "Affine gap costs" are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

[0040] Calculation of maximum % homology or identity firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG

Wisconsin Bestfit package (University of Wisconsin, U.S.A.; Devereux). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package, FASTA and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching. [0041] There are five BLAST programs available online through the

National Center for Biotechnology Information of the National Institutes of Health (NCBI): BLASTP, BLASTN, BLASTX, TBLASTN and BLAST. The program BLASTP compares an amino acid query sequence against a protein sequence database. The program BLASTN compares a nucleotide query sequence against a nucleotide sequence database. The program BLASTX compares the six-frarnie conceptual translation products of a nucleotide query sequence (both strands) against a protein sequence database. The program TBLASTN compares a protein query sequence against a nucleotide sequence database dynamically translated in all six reading frames (both strands). The program TBLASTX compares the six- frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.

[0042] The program BLAST uses the following search parameters:

HISTOGRAM, DESCRIPTIONS, EXPECT, CUTOFF, ALIGNMENTS, MATRIX, STRAND and FILTER. HISTOGRAM: Displays a histogram of scores for each search; default is yes. (See parameter H in the BLAST Manual available online at the NCBI web-site). DESCRIPTIONS: Restricts the number of short descriptions of matching sequences reported to the number specified; default limit is 100 descriptions. (See parameter V in the manual page available online at the NCBI web-site). EXPECT: The statistical significance threshold for reporting matches against database sequences; the default value is 10, such that 10 matches are expected to be found merely by chance, according to the stochastic model of Karlin and Altschul (Altschul et al., J. MoI. Biol. 1990 Oct 5;215(3):403-10). If the statistical significance attributed to a match is greater than the EXPECT threshold, the match will not be reported. Lower EXPECT thresholds are more stringent, leading to fewer chance matches being reported. (See parameter E in the BLAST Manual available online at the NCBI web-site). CUTOFF: Cutoff score for reporting high-scoring segment pairs (HSP). The default value is calculated from the EXPECT value. HSPs are reported for a database sequence only if the statistical significance attributed to them is at least as high as would be attributed to a lone HSP having a score equal to the CUTOFF value. Higher CUTOFF values are more stringent, leading to fewer chance matches being reported. (See parameter S in the BLAST Manual available online at the NCBI web-site). ALIGNMENTS: Restricts database sequences to the number specified for which HSPs are reported; the default limit is 50. If more database sequences than this happen to satisfy the statistical significance threshold for reporting, only the matches attributed with the greatest statistical significance are reported. (See parameter B in the BLAST Manual available online at the NCBI web-site). MATRIX: Specify an alternate scoring matrix for BLASTP, BLASTX, TBLASTN and TBLASTX. The default matrix is

BLOSUM62. Alternatives include: PAM40, PAM120, PAM250 and IDENTITY. No alternate scoring matrices are available for BLASTN. STRAND: Restrict a TBLASTN search to just the top or bottom strand of the database sequences or restrict a BLASTN, BLASTX or TBLASTN search to just reading frames on the top or bottom strand of the query sequence. FILTER: Mask off segments of the query sequence that have low compositional complexity, as determined by the SEG program, or segments consisting of short-periodicity internal repeats, as determined by the XNU program or, for BLASTN, by the DUST program. Filtering can eliminate statistically significant but biologically uninteresting reports from the blast output (e.g., hits against common acidic-, basic- or proline-rich regions), leaving the more biologically interesting regions of the query sequence available for specific matching against database sequences. Default filtering is DUST for BLASTN, SEG for other programs.

[0043] Most preferably, sequence comparisons are conducted using the simple BLAST search algorithm provided online through the National Center for Biotechnology Information of the National Institutes of Health. In some embodiments described herein, no gap penalties are used when determining sequence identity.

[0044] Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix, which is the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied. It is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.

[0045] Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result. [0046] In some embodiments, there is provided proteins, peptides and nucleic acids described herein proteins, peptides and nucleic acids having and least about 75%, more preferably at least about 85%, more preferably at least about 90% homology or identity to the sequences described herein. More preferably there is at least about 95%, more preferably at least about 98% or 99% or 100% homology or identity to a protein, peptide or nucleic acid described herein. Nucleotide homology comparisons may be conducted as described above. A preferred sequence comparison program is the GCG Wisconsin Bestfit program described above. The default scoring matrix has a match value of 10 for each identical nucleotide and -9 for each mismatch. The default gap creation penalty is - 50 and the default gap extension penalty is -3 for each nucleotide.

[0047] Proteins, peptides and nucleic acids described herein and derivatives and analogs thereof may be termed "PF-4 therapeutics" and may include proteins and peptides, nucleic acid sequences coding therefor, cells expressing such protein, peptides or nucleic acids and derivatives of such proteins and peptides. The PF-4 therapeutics may be used to ameliorate or treat diseases when administered in prophelactically or therapeutically effective dosages. PF-4 therapeutics of the invention include modifications, derivatives and analogs of PF-4 proteins, peptides and nucleic acids encoding such proteins and peptides.

[0048] Conservative substitutions of amino acids are well known. For example, Wu et al. (Proc lnt Conf lntell Syst MoI Biol. 1996;4:230-40) describe a method for identifying empirically conserved amino acid substitution groups. Conserved groups of amino acids are identified using a data structure called a conditional distribution matrix. The conditional distribution matrix considers substitutions of a group of amino acids. The matrix tabulates information from a database of protein families that contains numerous aligned positions. Each row in the matrix contains the distribution of amino acids in those aligned positions that contain a given conditioning group of amino acids. The method converts a database of protein families into a conditional distribution matrix and then examines each possible substitution group for evidence of conservation. The algorithm is applied to the BLOCKS and HSSP databases. Other known approaches include pairwise amino acid substitutions, such as substituting amino acids have liked charges or basic or acid side chains. [0049] As used herein, the term "conserved amino acid substitutions" refers to the substitution of one amino acid for another at a given location in the peptide, where the substitution can be made without loss of function. In making such changes, substitutions of like amino acid residues can be made, for example, on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the function of the peptide by routine testing.

[0050] In some embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydrophilicity value (e.g., within a value of plus or minus 2.0), where the following hydrophilicity values are assigned to amino acid residues (as detailed in United States Patent No. 4,554,101 , incorporated herein by reference): Arg (+3.0); Lys (+3.0); Asp (+3.0); GIu (+3.0); Ser (+0.3); Asn (+0.2); GIn (+0.2); GIy (0); Pro (- 0.5); Thr (-0.4); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); VaI (-1.5); Leu (-1.8); He (-1.8); Tyr (-2.3); Phe (-2.5); and Trp (-3.4).

[0051] In alternative embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydropathic index (e.g., within a value of plus or minus 2.0). In such embodiments, each amino acid residue may be assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics, as follows: He (+4.5); VaI (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); GIy (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); GIu (-3.5); GIn (-3.5); Asp (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5). [0052] In alternative embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another in the same class, where the amino acids are divided into non polar, acidic, basic and neutral classes, as follows: non-polar: Ala, VaI, Leu, He, Phe, Trp, Pro, Met; acidic: Asp, GIu; basic: Lys, Arg, His; neutral: GIy, Ser, Thr, Cys, Asn, GIn, Tyr. [0053] Retro reverso proteins or peptides of the proteins and peptides described herein are also provided. These are proteins or peptides in which D amino acid analogs are used to synthesize a protein or peptide in the reverse order. These proteins or peptides have the peptide bonds reversed as well as the N and C terminals. However, the side chains of the amino acids are in the same orientation as they would be in the L-amino acid version of the same peptide or protein. These proteins are often protected from proteolytic enzymes because the enzyme can lock onto the side chains but cannot cleave the peptide bond. These proteins or peptides will have the same activity as the L-amino acid proteins or peptides, but will also be stable in vivo, particularly with respect to degradation of the protein or peptide. Modifying Groups

[0054] In one embodiment of the invention, PF-4 chemokine analogs are designed by replacing all or part of the beta-sheet domain with a linker. In a different embodiment, all or a portion of the amino-terminal domain and all or a portion of the carboxy-terminal domain of a chemokine or chemokine analog are connected with a linker. In another embodiment, the chemokines and chemokine analogs are designed so that they are cyclized by covalent modification between residues of the peptide. In still other embodiments, the cysteines of the chemokines are replaced by other amino acids. In further embodiments, chemokines and chemokine analogs are modified by attaching modifying groups to the amino terminus.

[0055] The peptidic analogs of PF-4 in the invention may be coupled directly or indirectly to at least one modifying group. The term "modifying group" is intended to include structures that are directly attached to the peptidic structure (e.g., by covalent bonding or covalent coupling), as well as those that are indirectly attached to the peptidic structure (e.g., by a stable non-covalent bond association or by covalent coupling through a linker to additional amino acid residues). In other aspects of the invention the term "modifying group" may also refer to mimetics, analogues or derivatives thereof, which may flank the core PF-4 peptidic analog structure. For example, the modifying group can be coupled to the amino-terminus or carboxy-terminus of a PF-4 peptidic analog structure. Alternatively, the modifying group can be coupled to a side chain of at least one amino acid residue of a PF-4 peptidic structure (e.g., through the epsilon amino group of a lysyl residue(s); through the carboxyl group of an aspartic acid residue(s) or a glutamic acid residue(s); through a hydroxy group of a tyrosyl residue(s), a serine residue(s) or a threonine residue(s); or any other suitable reactive group on an amino acid side chain). In other aspects, modifying groups covalently coupled to the peptidic structure can be attached by means and using methods well known in the art for linking chemical structures, including, for example, amide, alkylamino, sulfide, carbamate or urea bonds.

[0056] In some embodiments, the modifying group may comprise a cyclic, heterocyclic or polycyclic group. The term "cyclic group," as used herein, includes cyclic saturated or unsaturated (i.e., aromatic) group having from 3 to 10; from 4 to 8; or 5, 6, or 7 carbon atoms. Exemplary non-aromatic cyclic groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. The term "heterocyclic group" includes optionally substituted, saturated or unsaturated, three- to eight-membered cyclic structures in which one or more skeletal atoms is oxygen, nitrogen, sulfur, or combinations thereof. Cyclic groups may be unsubstituted or substituted at one or more ring positions. A cyclic group may for example be substituted with halogens, alkyls, cycloalkyls, alkenyls, alkynyls, aryls, arylalkyls, heterocycles, hydroxyls, aminos, nitros, thiols amines, imines, amides, phosphonates, phosphines, carbonyls, carboxyls, silyls, ethers, thioethers, sulfonyls, sulfonates, selenoethers, ketones, aldehydes, esters, -CF₃, -CN. The cyclic group may also be linked to a substituent, such as halogens, alkyls, cycloalkyls, alkenyls, alkynyls, aryls, arylalkyls, heterocycles, hydroxyls, aminos, nitros, thiols amines, imines, amides, phosphonates, phosphines, carbonyls, carboxyls, silyls, ethers, thioethers, sulfonyls, sulfonates, selenoethers, ketones, aldehydes, esters, -CF₃, or -CN, by means of a saturated or unsaturated chain of 1 , 2, 3, 4, 5, 6, 7, 8, or more carbon atoms; additionally one or more of the carbon atoms may be replaced with an oxygen, nitrogen, or sulfur atom. Other means of linking these groups are also possible. [0057] Modifying groups may also include groups comprising biochemical labels or structures, such as biotin, fluorescent-label-containing groups, light scattering or plasmon resonant particle, a diethylene- triaminepentaacetyl group, a (O)-menthoxyacetyl group, a N-acetylneuraminyl group, a cholyl structure or an iminobiotinyl group. A chemokine analog or chemokine mimetic compound may be modified at its carboxy terminus with a cholyl group according to methods known in the art. Cholyl derivatives and analogs may also be used as modifying groups. For example, a preferred cholyl derivative is Aic (3-(O-aminoethyl-iso)-cholyl), which has a free amino group that can be used to further modify the chemokine mimetic compound. A modifying group may be a "biotinyl structure," which includes biotinyl groups and analogues and derivatives thereof (such as a 2-iminobiotinyl group). In another embodiment, the modifying group may comprise a fluorescent-label group, e.g., a fluorescein-containing group, such as a group derived from reacting a PF-4 peptidic structure with 5-(and 6-)- carboxyfluorescein, succinimidyl ester or fluorescein isothiocyanate. The chemokine analogs may also be modified by attaching other fluorescent labels including rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin and energy transfer fluorescent dyes or fluorescent ion indicators. In various other embodiments, the modifying group(s) may comprise an N- acetylneuraminyl group, a trans-4-cotininecarboxyl group, a 2-imino-1- imidazolidineacetyl group, an (S)-(-)-indoline-2-carboxyl group, a (-)- menthoxyacetyl group, a 2-norbornaneacetyl group, a γ-oxo-5- acenaphthenebutyryl, a (-)-2-oxo-4-thiazolidinecarboxyl group, a tetrahydro-3-furoyl group, a 2-iminobiotinyl group, a diethylenetriaminepentaacetyl group, a 4- morpholinecarbonyl group, a 2-thiopheneacetyl group or a 2-thiophenesulfonyl group. In other embodiments, light scattering groups, magnetic groups, nanogold, other proteins, a solid matrix, radiolabels, or carbohydrates may be attached.

[0058] In still other aspects, the modifying group may be an oligomer, for example, polyethylene glycol, an oligonucleotide, a polypeptide (which may or may not be derived from a chemokine) or one moiety of a binding pair.

Functional Enhancement

[0059] A PF-4 chemokine analog compound of the invention may be further modified to alter the specific properties of the compound while retaining the desired functionality of the compound. For example, in one embodiment, the compound may be modified to alter a pharmacokinetic property of the compound, such as in vivo stability, solubility, bioavailability and/or half-life. The compound may be modified to label the compound with a detectable substance. The compound may be modified to couple the compound to an additional therapeutic moiety. To further chemically modify the compound, such as to alter its pharmacokinetic properties, reactive groups can be derivatized. For example, when the modifying group is attached to the amino-terminal end of the PF-4 core domain, the carboxy-terminal end of the compound may be further modified. Potential C-terminal modifications include those that reduce the ability of the compound to act as a substrate for carboxypeptidases. Examples of C-terminal modifiers include an amide group, an ethylamide group and various non-natural amino acids, such as D-amino acids, β-alanine, C-terminal decarboxylation, and a C-terminal alcohol. Alternatively, when the modifying group is attached to the carboxy-terminal end of the aggregation core domain, the amino-terminal end of the compound may be further modified, for example, to reduce the ability of the compound to act as a substrate for aminopeptidases.

[0060] PF-4 chemokine analogs of the invention may be modified by the addition of polyethylene glycol (PEG). PEG modification may lead to improved circulation time, improved solubility, improved resistance to proteolysis, reduced antigenicity and immunogenicity, improved bioavailability, reduced toxicity, improved stability, and easier formulation (For a review see, Francis et al.,

International Journal of Hematology 68:1 -18, 1998). PEGylation may also result in a substantial reduction in bioactivity.

[0061] PF-4 chemokine analogs of the invention may also be coupled to a radioisotope such as yttrium-90 or iodine-131 for therapeutic purposes (see, e.g., DeNardo et al., "Choosing an optimal radioimmunotherapy dose for clinical response, " Cancer 94(4 Suppl): 1275-86, 2002; Kaltsas era/., "The value of radiolabeled MIBG and octreotide in the diagnosis and management of neuroendocrine tumours," Ann Oncol 12 Suppl 2.S47-50, 2001 ).

Detection Enhancement

[0062] A chemokine mimetic compound can be further modified to label the compound by reacting the compound with a detectable substance. In some aspects of the invention, suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, light scattering or plasmon resonant materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta- galactosidase, or acetylcholinesterase. Examples of suitable prosthetic groups which are members of a binding pair and are capable of forming complexes include streptavidin/biotin, avidin/biotin and an antigen/antibody complex (e.g., rabbit IgG and anti-rabbit IgG). Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin and energy transfer fluorescent dyes. An example of a luminescent material includes luminol. Examples of light scattering or plasmon resonant materials include gold or silver particles and quantum dots. Examples of suitable radioactive material include ¹⁴C, ¹²³1, ¹²⁴1, ¹²⁵1, ¹³¹I, Tc99m, ³⁵S or ³H. A chemokine mimetic compound may be radioactively labeled with ¹⁴C, either by incorporation of ¹⁴C into the modifying group or one or more amino acid structures in the PF-4 chemokine mimetic compound. Labeled PF-4 chemokine mimetic compounds may be used to assess the in vivo pharmacokinetics of the compounds, as well as to detect disease progression or propensity of a subject to develop a disease, for example for diagnostic purposes. Tissue distribution chemokine receptors can be detected using a labeled PF-4 chemokine mimetic compound either in vivo or in an in vitro sample derived from a subject. For use as an in vivo diagnostic agent, a PF-4 chemokine mimetic compound of the invention may be labeled with radioactive technetium or iodine. A modifying group can be chosen that provides a site at which a chelation group for the label can be introduced, such as the Aic derivative of cholic acid, which has a free amino group. For example, a tyrosine residue within the PF-4 chemokine analog sequence may be substituted with radioactive iodotyrosyl. Any of the various isotopes of radioactive iodine may be incorporated to create a diagnostic or therapeutic agent. ¹²³I (half-life=13.2 hours) may be used for whole body scintigraphy, ¹²⁴I (half life=4 days) may be used for positron emission tomography (PET), ¹²⁵I (half life=60 days) may be used for metabolic turnover studies and ¹³¹I (half life=8 days) may be used for whole body counting and delayed low resolution imaging studies

Prodrug

[0063] In an alternative chemical modification, a PF-4 chemokine analog compound of the invention may be prepared in a "prodrug" form, wherein the compound itself does not act as a PF-4 chemokine analog agonist or antagonist, but rather is capable of being transformed, upon metabolism in vivo, into a PF-4 chemokine analog agonist or antagonist compound as defined herein. For example, in this type of compound, the modifying group can be present in a prodrug form that is capable of being converted upon metabolism into the form of an active PF-4 chemokine analog agonist or antagonist. Such a prodrug form of a modifying group is referred to herein as a "secondary modifying group." A variety of strategies are known in the art for preparing peptide prodrugs that limit metabolism in order to optimize delivery of the active form of the peptide-based drug.

Synthesis

[0064] PF-4 chemokine analog compounds of the invention may be prepared by standard techniques known in the art. A peptide or polypeptide component of a PF-4 chemokine analog may comprise, at least in part, a peptide synthesized on resin using Standard techniques (Merrifield, R. B. et al., J. Am. Chem. Soc. 1963; 85: 2149-2154 ). Automated peptide synthesizers are commercially available (e.g., Advanced ChemTech Model 396; Milligen/Biosearch 9600, Appliedbiosystems/Pioneer). Peptides and polypeptides may be assayed for PF-4 chemokine receptor agonist or antagonist activity in accordance with standard methods. Peptides and polypeptides may be purified by HPLC and analyzed by mass spectrometry. Peptides and polypeptides may be dimerized. In one embodiment, peptides and polypeptides are dimerized via a disulfide bridge formed by gentle oxidation of the cysteines using 10% DMSO in water. Following HPLC purification, dimer formation may be verified, by mass spectrometry. One or more modifying groups may be attached to a PF-4 derived peptidic component by standard methods, for example, using methods for reaction through an amino group (e.g., the alpha-amino group at the amino-terminus of a peptide), a carboxyl group (e.g., at the carboxy terminus of a peptide), a hydroxyl group (e.g., on a tyrosine, serine or threonine residue) or other suitable reactive group on an amino acid side chain. [0065] In alternative embodiments, analogs derived from the C- terminal and N-terminal joined by a linker could be cyclized in their C-terminal moiety using side-chain to side-chain; side-chain to scaffold or, scaffold to scaffold cyclization. In some embodiments, lactamization, etherification, or RCM (Ring Closing Methatesis) are used to carry out this reaction. [0066] For instance, PF-4 chemokine analogs may be cyclized using a lactam formation procedure by joining the γ-carboxy side chain or the α-carboxy moiety of glutamate (GIu) residue to the ε-amino side chain of lysine (Lys) residue, as indicated in the following sequences by underlining of linked residues. Lactams may for example be formed between glutamic acid and lysine (Lys) in the C- terminal portion of the polypeptide (which does not correspond necessarily with the numbering of that residue in the native sequence). In further alternatives, a lysine (Lys) may be substituted by ornithine (Orn) or any other (Lor D) natural or (L or D) non-natural amino acid having an amino group on its side chain. Similarly, glutamate (GIu) may for example be substituted with aspartate (Asp), denoted by nomenclature such as (Glu-> Asp) indicating a substitution in a given position in the peptide wherein aspartate replaces glutamate.

[0067] The PF-4 chemokine analogs of the invention include chemokine polypeptide sequences wherein one or more of the amino acids have been replaced by a conservative amino acid substitution. The term "conservative amino acid substitution" refers to a polypeptide chain in which one of the amino acid residues is replaced with an amino acid residue having a side chain with similar properties. Families of amino acid residues having side chains with similar properties are well known in the art. These families include amino acids with acidic side chains {e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, an amino acid residue in a chemokine is replaced with another amino acid residue from the same side chain family.

Compositions

[0068] The invention further provides pharmaceutical compositions containing PF-4 chemokine analogs. In one embodiment, such compositions include a PF-4 chemokine analog compound in a therapeutically, diagnostically or prophylactically effective amount sufficient to be used in treating diseases or disorders selected from the group consisting of cancer, cardiovascular disease, and inflammatory disorders.

[0069] An "effective amount" of a compound of the invention includes a therapeutically effective amount or a prophylatically effective amount. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. The term "therapeutically effective amount" may also refer to that amount of active compound, prodrug or pharmaceutical agent that elicits a biological or medicinal response in a tissue, system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician in order to provide a therapeutic effect.

[0070] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as preventing or inhibiting a cytotoxic effect of a cytotoxic agent. Typically, a prophylactic dose is used in organisms prior to or at an earlier stage of disease, so that a prophylactically effective amount may be less than a therapeutically effective amount. The term "preventing" refers to decreasing the probability that an organism contracts or develops an abnormal condition.

[0071] In particular embodiments, a preferred range for therapeutically or prophylactically effective amounts of PF-4 chemokine analogs may be 0.1 nM- 0.1 M, 0.1 nM-0.05 M, 0.05 nM-15 μM or 0.01 nM-10 μM. It is to be noted that dosage values may vary with the severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgement of the person administering or supervising the administration of the compositions. Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected by medical practitioners.

[0072] The amount of active compound in the composition may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It may be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. "Dosage unit form" as used herein refers to physically discrete units suited as unitary dosages for subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

[0073] The terms "administration" or "administering" refer to a method of incorporating a compound into the cells or tissues of an animal, preferably a mammal, and still more preferably a human, in order to treat or prevent an abnormal condition. When the compound or prodrug of the invention is provided in combination with one or active agents, the terms "administration" or "administering" include sequential or concurrent introduction of the compound or prodrug with the other agent(s). For cells harbored within the organism, many techniques exist in the art to administer compounds, including (but not limited to) oral, injection, parenteral, dermal, and aerosol applications. [0074] The term "therapeutic effect" refers to the inhibition or activation of factors causing or contributing to the abnormal condition (including a disease or disorder). A therapeutic effect relieves or prevents to some extent one or more of the symptoms of the abnormal condition. In reference to the treatment of abnormal conditions, a therapeutic effect can refer to one or more of the following: (a) an increase or decrease in the number of lymphocytic cells present at a specified location, (b) an increase or decrease in the ability of lymphocytic cells to migrate, (c) an increase or decrease in the response of lymphocytic cells to a stimulus, (d) an increase or decrease in the proliferation, growth, and/or differentiation of cells; (e) inhibition (i.e., slowing or stopping) or acceleration of cell death; (f) relieving, to some extent, one or more of the symptoms associated with an abnormal condition; (g) enhancing or inhibiting the function of the affected population of cells; (h) activating an enzyme activity present in cells associated with the abnormal condition; and (i) inhibiting an enzyme activity present in cells associated with the abnormal condition.

[0075] The term "abnormal condition" refers to a function in the cells or tissues of an organism that deviates from their normal functions in that organism and includes, but is not limited to, conditions commonly referred to as diseases or disorders. An abnormal condition can relate to cell proliferation, cell differentiation, cell survival, cell migration or movement, or the activities of enzymes within a cell. Diseases and disorders may include inflammatory disorders including rheumatoid arthritis, chronic inflammatory bowel disease, chronic inflammatory pelvic disease, multiple sclerosis, asthma, osteoarthritis, atherosclerosis, psoriasis, rhinitis, autoimmunity, organ transplant rejection, and genetic diseases.

[0076] As used herein "pharmaceutically acceptable carrier" or

"excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In one embodiment, the carrier is suitable for parenteral administration. Alternatively, the carrier can be suitable for intravenous, intraperitoneal, intramuscular, sublingual or oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions. Pharmaceutically acceptable carrier "may comprise pharmaceutically acceptable salts."

[0077] Pharmaceutical formulations for parenteral administration may include liposomes. Liposomes and emulsions are well known examples of delivery vehicles or carriers that are especially useful for hydrophobic drugs. Depending on biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed. Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with target- specific antibody. The liposomes will bind to the target protein and be taken up selectively by the cell expressing the target protein. [0078] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. 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), and suitable mixtures thereof. 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 surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin. Moreover, the chemokine analogs may be administered in a time release formulation, for example in a composition which includes a slow release polymer. The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PLG). Many methods for the preparation of such formulations are patented or generally known to those skilled in the art.

[0079] Additionally, suspensions of the compounds of the invention may be prepared as appropriate oily suspensions for injection. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil; or synthetic fatty acid esters, such as ethyl oleate or triglycerides; or liposomes. Suspensions to be used for injection may also contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[0080] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a 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 freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof. In accordance with an alternative aspect of the invention, a chemokine analog may be formulated with one or more additional compounds that enhance the solubility of the chemokine analog.

[0081] If the compounds of the invention are to be administered by inhalation, they may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, together with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of gelatin, for example, for use in an inhaler may be formulated containing a powder mix of the compound and a suitable powder base such as starch or lactose. [0082] Gene therapy is another method by which patients may be treated in accordance with various aspects of the invention. Gene constructs may be prepared using techniques know to those skilled in the art and delivered to tissues using vectors known to those skilled in the art. See, for example, Gene Therapy Protocols ed: Paul D Robbins; Humana Press (1996) and Gene Therapy in Cancer by Malcolm K Brenner; Marcel Dekker (1996).

[0083] A nucleic acid of the invention may be delivered to cells in vivo using methods such as direct injection of DNA, receptor-mediated DNA uptake or viral-mediated transfection, all of which may constitute gene therapy vectors. Direct injection has been used to introduce naked DNA into cells in vivo (see e.g., Acsadi et al. (1991) Nature 332:815-818; Wolff et al. (1990) Science 247:1465-1468). A delivery apparatus (e.g., a "gene gun") for injecting DNA into cells in vivo may be used. Such an apparatus may be commercially available (e.g., from BioRad). Naked DNA may also be introduced into cells by complexing the DNA to a cation, such as polylysine, which is coupled to a ligand for a cell-surface receptor (see for example Wu, G. and Wu, C. H. (1988) J. Biol. Chem. 263:14621 ; Wilson et al. (1992) J. Biol. Chem. 267:963-967; and U.S. Pat. No. 5,166,320). Binding of the DNA-ligand complex to the receptor may facilitate uptake of the DNA by receptor- mediated endocytosis. A DNA-ligand complex linked to adenovirus capsids which disrupt endosomes, thereby releasing material into the cytoplasm, may be used to avoid degradation of the complex by intracellular lysosomes (see for example Curiel et al. (1991) Proc. Natl. Acad. Sci. USA 88:8850; Cristiano et al. (1993) Proc. Natl. Acad. Sci. USA 90:2122-2126). [0084] Defective retroviruses are well characterized for use as gene therapy vectors (for a review see Miller, A. D. (1990) Blood 76:271). Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art. Examples of suitable packaging virus lines include .pDi.Crip, .pDi.Cre, .pDi.2 and .pDi.Am. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see for example Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014- 3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381 ; Chowdhury et al. (1991 ) Science 254:1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573).

[0085] For use as a gene therapy vector, the genome of an adenovirus may be manipulated so that it encodes and expresses a peptide compound of the invention, but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See for example Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431 -434; and Rosenfeld et al. (1992) Cell 68:143-155. Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled in the art. Recombinant adenoviruses are advantageous in that they do not require dividing cells to be effective gene delivery vehicles and can be used to infect a wide variety of cell types, including airway epithelium (Rosenfeld et al. (1992) supra), endothelial cells (Lemarchand et al. (1992) Proc. Natl. Acad. Sci. USA 89:6482-6486), hepatocytes (Herz and Gerard (1993) Proc. Natl. Acad. Sci. USA 90:2812-2816) and muscle cells (Quantin et al. (1992) Proc. Natl. Acad. Sci. USA 89:2581 -2584).

[0086] Adeno-associated virus (AAV) may be used as a gene therapy vector for delivery of DNA for gene therapy purposes. AAV is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle (Muzyczka et al. Curr. Topics in Micro, and Immunol. (1992) 158:97-129). AAV may be used to integrate DNA into non-dividing cells (see for example Flotte et al. (1992) Am. J. Respir. Cell. MoI. Biol. 7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlin et al. (1989) J. Virol. 62:1963-1973). An AAV vector such as that described in Tratschin et al. (1985) MoI. Cell. Biol. 5:3251-3260 may be used to introduce DNA into cells (see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81 :6466-6470; Tratschin et al. (1985) MoI. Cell. Biol. 4:2072-2081 ; Wondisford et al. (1988) MoI. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol. 51 :611 -619; and Flotte et al. (1993) J. Biol. Chem. 268:3781 -3790).

[0087] General methods for gene therapy are known in the art. See for example, U.S. Pat. No. 5,399,346 by Anderson et al. (incorporated herein by reference). A biocompatible capsule for delivering genetic material is described in PCT Publication WO 95/05452 by Baetge et al. Methods of gene transfer into hematopoietic cells have also previously been reported (see Clapp, D. W., et al., Blood 78: 1132-1139 (1991); Anderson, Science 288:627-9 (2000); and , Cavazzana-Calvo et al., Science 288:669-72 (2000), all of which are incorporated herein by reference). [0088] The term "modulates" refers to altering the function or activity of a chemokine receptor by contacting it with a chemokine or chemokine analog and thus increasing or decreasing the probability that a complex forms between the receptor and a natural binding partner. A chemokine or chemokine analog preferably increases the probability that such a complex forms between the chemokine receptor and the natural binding partner, more preferably increases or decreases the probability that a complex forms between the chemokine receptor and the natural binding partner depending on the concentration of the chemokine or chemokine analog exposed to the receptor, and most preferably decreases the probability that a complex forms between the chemokine receptor and the natural binding partner depending on the concentration of the chemokine or chemokine analog exposed to the polypeptide.

[0089] The term "chemokine receptor" refers to a chemokine receptor as the term is used by one skilled in the art and also refers to any other polypeptide capable of binding a chemokine or chemokine analog.

[0090] In preferred embodiments, a modulator preferably activates the catalytic activity of a chemokine receptor, more preferably activates or inhibits the catalytic activity of a chemokine receptor depending on the concentration of the chemokine or chemokine analog exposed to the chemokine receptor, or most preferably inhibits the catalytic activity of a chemokine receptor depending on the concentration of the chemokine or chemokine analog exposed to the chemokine receptor.

[0091] The term "natural binding partner" refers to G proteins, polypeptides, lipids, small molecules, or nucleic acids that bind to chemokine receptors in cells or in the extracellular environment. The term natural binding partner includes a substrate to be acted upon by the chemokine receptor. A change in the interaction between a chemokine receptor and a natural binding partner can manifest itself as an increased or decreased probability that the interaction forms, or an increased or decreased concentration of chemokine receptor/natural binding partner complex. This can result in a decreased or increased activity of the chemokine receptor.

[0092] The terms "activated," "activating," and "activation" refer to an increase in the cellular or extracellular function of a chemokine receptor. The chemokine receptor function is preferably the interaction with a natural binding partner, and most preferably catalytic activity. The term "inhibits" refers to decreasing the cellular or extracellular activity of the chemokine receptor. The cellular or extracellular activity of a chemokine receptor is preferably the interaction with a natural binding partner, and most preferably catalytic activity.

[0093] The term "complex" refers to an assembly of at least two molecules bound to one another. A signal transduction complex often contains at least two protein molecules bound to one another. For instance, a protein tyrosine receptor protein kinase, GRB2, SOS, RAF, and RAS assemble to form a signal transduction complex in response to a mitogenic ligand. Another example is a chemokine bound to a chemokine receptor. Still another example is a G protein bound to a chemokine receptor.

[0094] The term "contacting" as used herein refers to adding together a solution or a composition comprising the chemokine or chemokine analog with a liquid medium bathing the polypeptide or cells comprising a chemokine receptor. The solution comprising the chemokine or chemokine analog may also comprise another component, such as dimethyl sulfoxide (DMSO), which facilitates the uptake of the chemokine or chemokine analog into the cells of the methods. The solution comprising the chemokine or chemokine analog may be added to the medium bathing the cells by utilizing a delivery apparatus, such as a pipette-based device or syringe-based device.

[0095] As discussed supra, compounds of the present invention may be useful in threating diseases such as: angiogenic related disorders; hematopoietic disorders in which the stimulation of proliferation of hematopoietic stem cells/precursors; the protection of hematopoietic cells from chemotherapy related drugs and/or the inhibition of differentiation of hematopoietic precursors are needed; coagulatory related disorders requiring the inhibition of heparine or the increase in anti-coagulant activated protein C; and inflammation related disorders EXAMPLES

[0096] The following examples illustrate, but do not limit, the present invention.

Example 1

[0097] Peptides of the invention may be synthesized chemically using the Fmoc/tBu strategy on a continuous flow peptide synthesizer, as for example has been carried out using the following protocols:

Reagents (solvents, support, chemicals)

[0098] Main Solvent: N,N-Dimethylformamide (DMF): certified ACS spectroanalyzed from Fisher (D131-4) M.W = 73.10. The DMF is treated with activated molecular sieves, type 4A (from BDH: B54005) for at least two weeks then tested with FDNB (2,4-Dinitrofluorobenzene from Eastman).

[0099] Procedure: Mix equal volumes of FDNB solution (1 mg/ml in 95% EtOH) and DMF; Let stand 30 minutes; read the absorbance at 381 nm over a FDNB blank (0.5ml FDNB + 0.5ml 95% EtOH). If the absorbance ~ 0.2, the DMF is suitable to be used for the synthesis.

[00100] Deblocking Agent: 20% Piperidine (from Aldrich Chemical company, catalog No: 10,409-4) in DMF containing 0.5 % Triton X100 v/v ( from Sigma , catalg No: T-9284).

[00101 ] Activating Agents: 2-(H-benzotriazol-lyl) 1 , 1 ,3,3- tetramethyluronium tetrafluoroborate (TBTU: M.W.=321.09. from Quantum Richilieu, catalog No: R0139)/ Hydroxybenzotriazole (HOBt M.W.=135.1 from Quantum Richilieu, catalog No.: R0166-100) respectively, 0.52 M in DMF and 4- Methylmorpholine (NMM ; M.W.=101.15, d=0.926 from Aldrich, catalog No.:

M5, 655-7): 0.9 M in DMF or in the case of sensitive amino acids to racemization like Cys, we use 2,4,6-Collidine, 99% ( M.W.=121.18,d=0.917, from Aldrich, catalog No: 14,238-7): 0.78M in DMF/DCM, 1/1 v/v. [00102] Support: TentaGel R RAM (90 μm), RinK-type Fmoc (from Peptides International, catalog No.: RTS -9995-PI): 0.21 mmol/g, 0.5g for 0.1 mmol of peptide.

[00103] Fmoc-L-amino derivative, side-chains protected with: Boc; tBu; Trt groups: with 4 fold excess (from Peptides International, Bachem, Novabiochem, Chem-lmpex Inc). Glu24 and Lys24 are Allyl-protected (from Millipore/Perseptive Biosystems).

Initial Amino Loading and Peptide Synthesis Procedure

[00104] The C-terminal amino acid Ser and the remaining residues are double coupled at room temperature or at 45⁹C automatically with 4-fold excess in each coupling. The synthesis is interrupted after residue Leu19. The peptide- bound support is removed from the synthesizer column and placed in a react-vial containing a small magnetic bar for gentle stirring.

Removal of The AIIyI Groups [00105] A solution of tetrakis(triphenylphosphine)Palladium(0)

Pd(PPh3)4 (from Sigma-Aldrich, catalog No: 21 ,666-6); M.W.=1155.58 x 0.1 mmol peptide x 3 fold = 347mg dissolved in 5% Acetic Acid; 2.5% NMM in CHCI3 to 0.14 M, under argon. The solution is added to the support-bound peptide previously removed from the coulmn in a reactvial containing a small mangnetic bar for gentle stirring. The mixture is flushed with argon, sealed and stirred at room temperature for 6 hours. The support-bound peptide is transferred to a filter funnel, washed with 30 ml of a solution made of 0.5% Sodium Diethyldithiocarbonate/ in DMF, then DCM; DCM/DMF (1 : 1 ) and DMF. A positive Kaiser test indicate the deprotection of the amino side chaine of the Lys20. Lactam Formation:

[00106] Activating agent: 7-Azabenztriazol-1 -yloxytris (pyrrolindino) phosphonium-hexafluorophosphate (PyAOP: M.W.=521.7 from PerSeptive Biosystems GmbH, catalog No: GEN076531) , 1.4-fold: 0.105mmol x 1.4 x 521.7 = 76.6mg and NMM 1.5-fold: 0.105 x 1.4 x 1.5 = 0.23 mmol v = 0.23/0.9 M NMM solution = 263 μl). [00107] The cyclisation may be carried out in an amino acid vial at room temperature overnight (-16 hours) with gentle agitation. The completion of cyclization may be indicated by a negative kaiser test. The support-bound peptide may be poured into the column, washed with DMF and the synthesis continues to completion, with a cyclic amide bridge thereby introduced into the peptide.

Final Product Removal From The Support:

[00108] The support-bound peptide is removed from the synthesizer in to a medium filter funnel, washed with DCM to replace the non-volatile DMF and thoroughly dried under high vacuum for at least two hours, or preferably, overnight. Cleavage Mixture (reagent K):

TFA/Phenol/Water/Thio-Anisol/EDT (82/5/5/5/2.5) ; 7.5ml

Support: 0.5g resin-peptide.

TFA 6.15ml ( Biograde from Halocarbon)

Phenol 0.375ml ( Aldrich) Water 0.375ml ( MiIIQ)

Thio-Anisol 0.375ml (Aldrich)

EDT 0.187ml ( Aldrich)

Total 7.5ml [00109] The cleavage may be performed at room temperature for 4 hours with gentle agitation on a rocker.

Precipitation of the Peptide

[00110] The cleaved peptide solution is filtered through a filter funnel in a 50 ml round bottom flask. The support is rinsed twice with 4 ml TFA. The TFA solution is concentrated on a rotavap and added drop wise into a cold diethyl ether previously treated with activated neutral aluminum oxide to make it free of peroxide. Approximately 10-fold excess of ether are used. The beads are stored until the yield is determined and peptide characterized. The precipitate is collected at room temperature in screw capped 50 ml polypropylene vial by centrifugation at 2K rpm using a top bench centrifuge (4 minutes run time). The pellet is washed 3x with cold ether, centrifuged and dried with a flow of argon. The precipitate is dissolved in 20 % acetonitrile, 0.1% TFA and lyophilized.

Crude Product Characterization:

[00111] The product is characterized by analytical HPLC. Experimental conditions: Column: Vydac 218TP54: C18 reversed-phase 5μm, 4.6 mm ID x 150 mm L Eluants: 0.1% TFA/H₂O (solvant A); 0.1% TFA/acetonitrile (solvent B). Elution Conditions: 20-50% B (40 min); 60-90% B (5 min); 90-20% B (5 min); 20% B (10 min). At 1.0 ml/min and A214 nm = 0.5 absorbance unit full scale.

Sample Preparation:

[00112] An aliquot of the product is weighed and dissolved in 20% acetonitrile 0.1% TFA at a concentration of 2 mg/ml. The solution is microfuged and 20μl is applied onto the column. The main peak or the major peaks are collected, SpeedVac dried and molecular weight determined by mass spectrometry.

[00113] Some examples of peptides of the invention that have been prepared by solid phase peptide synthesis are shown in Table 1 (with their SEQ ID Nos).

Table 1

cyclized sequences are shown in bold and underlined, with cyclization between positions at either end of the underlined sequence, positions that correspond in native PF-4 to residues 65 or 66 (corresponding to positions X₂₅ and X₂β in Formula 1 ) and residue 69 (corresponding to position X₂g in Formula 1).

Example 2

[00114] As illustrated in Figures 1 through 3, the efficacy of PF4 analogs of the invention as PF4 agonists is demonstrated through their inhibition of human endothelial cell growth. The inhibition of endothelial cell growth is an important function of angiostatic compounds. The growth and survival of endothelial cells is tightly regulated by growth factors. The present examples illustrate the ability of PF4 analogs to inhibit the growth stimulating effects of basic Fibroblast Growth Factor (bFGF) on HUVEC cells. The three analogs tested inhibited the growth of HUVEC cells at a concentration of 0.1 ug/ml as determined using the MTT assay. Briefly, the colorimetric MTT assay measures cellular proliferation by determining the amount of yellow MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) reduced to purple formazan spectrophotometrically. This reduction is indicative of mitochondrial reductase enzyme activity, and is therefore related to the number of viable cells (Mosmann, T., Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays. J. Immunol. Meth. 1983, 65, 55-63). In the present examples and the Figures, the measured reduction in maximal absorbance attributable to purple formazan is indicative of the degree of inhibition of cellular proliferation.

Example 3

[00115] The efficacy of PF4 analogs of the invention as PF4 agonists is also demonstrated by their ability to block the proliferation of a human erythroleukemia cell line (HEL) with megakaryocyte phenotype. The three analogs tested inhibited the growth of HEL cells at a concentration of 0.1 ug/ml, as determine using the MTT assay, as illustrated in Figures 4 to 6.

ALTERNATIVE EMBODIMENTS

[00116] As evidenced in the foregoing Examples, in some embodiments, the compounds of the invention have an activity that is greater than that of a native PF-4 peptide. Accordingly the I₅₀ (concentration of compound that makes it possible to obtain 50% inhibition) may be less than the I₅₀ value for native PF-4. In some embodiments, for example, the I₅₀ of the compound of the invention may be 2 to 20 fold less, or 4 to 15-fold less, or 5 to 10-fold less than the I₅₀ value for native PF-4. In this context, the I₅₀ may for example be measured by conventional techniques (Jouan V et al., 1999, Blood, 94: 984-93). The activity to be measured may for example be any physiological or therapeutic activity of interest, such as an anti-prliferative effect or an anti-angiogenic effect.

[00117] The invention illustratively described herein can suitably be practiced in the absence of any element or elements, limitation or limitations that is not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," etc., shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalent of the invention shown or portion thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modifications and variations of the inventions embodied herein disclosed can be readily made by those skilled in the art, and that such modifications and variations are considered to be within the scope of the inventions disclosed herein. The inventions have been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form the part of these inventions. This includes within the generic description of each of the inventions a proviso or negative limitation that will allow removing any subject matter from the genus, regardless or whether or not the material to be removed was specifically recited. In addition, where features or aspects of an invention are described in terms of the Markush group, those schooled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. Further, when a reference to an aspect of the invention lists a range of individual members, as for example, 'SEQ ID NO:2 to SEQ ID NO:9' it is intended to be equivalent to listing every member of the list individually, and additionally it should be understood that every individual member may be excluded or included in the claim individually.

[00118] The steps depicted and/or used in methods herein may be performed in a different order than as depicted and/or stated. The steps are merely exemplary of the order these steps may occur. The steps may occur in any order that is desired such that it still performs the goals of the claimed invention. [00119] The invention contemplates various alternatives and equivalents can be used to implement the concepts of the present invention without departing from its scope. While the invention has been described with specific reference to certain embodiments, changes can be made in form and detail without departing from the spirit and the scope of the invention. The described embodiments are considered in all respects as illustrative and not restrictive. It should also be understood that the invention is not limited to the particular embodiments described herein, but is capable of many equivalents, rearrangements, modifications, and substitutions without departing from the scope of the invention. Thus, additional embodiments are within the scope of the invention and within the following claims. [00120] Further, all patents and publications described herein are hereby incorporated by reference to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.

Claims

CLAIMSWe claim:

1. A peptidic compound of Formula I:

R-Xθ1 Xθ2 Xθ3 Xθ4 Xθ5 Xθ6 Xθ7 Xθ8 Xθ9 Yθ1 Xi1 Yθ2 Xi2 Xi3 Xi4 Xi5 Xi6 ZA Xi7 X18 Xi9 X20 X21 X22 X23 X24 X25 X26 X27 X28 X29 X30

(Formula I)

wherein

R is an optional N-terminal modifying group, which may be present or absent; X₀₁ is optional, may be present or absent, and if present is L-or D-GIx, L- or D-

Asx, or L- or D-AIa;

X₀₂ is L- or D-AIa, L- or D-VaI, L- or D-IIe, L- or D-Leu; X₀₃ is L- or D-GIx, L- or D-Asx, L- or D-AIa, L- or D-Pro, L- or D-Phe, L- or D-Tyr,

L- or D-Ser, L- or D-Thr;

Xo4 is L- or D-GIx, L- or D-Asx, L- or D-L- or D-AIa, L- or D-VaI, or L- or D-Phe; X₀₅ is L- or D-Asx, L- or D-GIx, L- or D-AIa, L- or D-Phe, L- or D-Tyr; X06 is L- or D-AIa, L- or D-VaI, L- or D-Leu, L- or D-IIe, L- or D-Phe, L- or D-Tyr, or L- or D-GIx;

Xo7 is L- or D-Asx, L- or D-GIx, L- or D-AIa, L- or D-Phe, L- or D-Tyr;

X08 is L- or D-Leu, L- or D-AIa, L- or D-L- or D-VaI, L- or D-Leu, L- or D-IIe, L- or

D-Phe, L- or D-Tyr;

X₀₉ is L- or D-GIx, L- or D-Asx, L- or D-Arg, L- or D-Lys, L- or D-His, L- or D-Tyr, L- or D-Ser, L- or D-Thr;

Y01 is L- or D-Cys, L- or D-AIa, L- or D-Phe, L- or D-His, L- or D-Trp, L- or D-Ser,

L- or D-Thr, L- or D-Lys, L- or D-His, L- or D-Tyr; Xn is L- or D-GIx, L- or D-Asx, GIy, L- or D-VaI, L- or D-Leu, L- or D-IIe, L- or D-

Pro, L- or D-Phe, L- or D-Tyr, L- or D-His, L- or D-Trp; Y02 is L- or D-Cys, L- or D-AIa, L- or D-Phe, L- or D-His, L- or D-Trp, L-or D-Ser, L- or D-Thr, L- or D-Lys, L- or D-His, L- or D-Tyr; X₁₂ is L- or D-VaI, L- or D-Leu, L- or D-IIe, L- or D-AIa, L- or D-Phe, or L- or D-

Tyr; Xi₃ is optional, may be present or absent, and if present is L- or D-Lys, L- or D-

Arg, L- or D-His; X₁₄ is optional, may be present or absent, and if present is L- or D-Thr, L- or D-

Ser, L- or D-Tyr, L- or D-AIa, L- or D-Phe; Xi₅ is optional, may be present or absent, and if present is L- or D-Thr, L- or D-

Ser, L- or D-Tyr, L- or D-AIa, or L- or D-Phe;

Xi₆ is optional, may be present or absent, and if present is L- or D-Thr, L- or D- Ser, L- or D-Tyr, L- or D-AIa, or L- or D-Phe;

Z_A is a flexible covalent linker; Xi7 is L- or D-AIa, L- or D-VaI, L- or D-IIe, L- or D-Leu; X₁₈ is L- or D-Pro, L- or D-AIa;

X₁₉ is L- or D-Leu, L- or D-AIa, L- or D-L- or D-VaI, L- or D-Leu, L- or D-IIe, L- or D-Phe, or L- or D-Tyr;

X20 is L- or D-Tyr, L- or D- Ser, L- or D-Thr, or L- or D-Phe; X₂1 is L- or D-Lys, L- or D-Arg, L- or D-His; X22 is L- or D-Lys, L- or D-Arg, or L- or D-His;

X₂₃ is L- or D-GIx, L- or D-Asx, GIy, L- or D-VaI, L- or D-Leu, L- or D-IIe, L- or D- Pro, L- or D-Phe, L- or D-Tyr, L- or D-His, L- or D-Trp;

X24 is L- or D-GIx, L- or D-Asx, GIy, L- or D-VaI, L- or D-Leu, L- or D-IIe, L- or D-

Pro, L- or D-Phe, L- or D-Tyr, L- or D-His, or L- or D-Trp; X₂₅ is L- or D-Lys, L- or D-Arg, L- or D-His; X₂₆ is L- or D-Lys, L- or D-Arg, L- or D-His; X27 is L- or D-Leu, L- or D-AIa, L- or D-L- or D-VaI, L- or D-Leu, L- or D-IIe, L- or

D-Phe, or L- or D-Tyr; X_2S is L- or D-Leu, L- or D-AIa, L- or D-L- or D-VaI, L- or D-Leu, L- or D-IIe, L- or

D-Phe, or L- or D-Tyr;

X₂₉ is L-or D-GIx, L- or D-Asx, or L- or D-AIa; X30 is L- or D-Thr, L- or D-Ser, L- or D-Tyr, L- or D-AIa, L- or D-Phe; and, non-toxic acidic addition salts thereof.

2. The compound of claim 1 , wherein R is present, and R is selected from the group consisting of PEGyI, Acetyl, and Alkyl.

3. The compound of claim 1 or 2, wherein X_Oi is present, and is L- or D-GIu or L- or D-GIn.

4. The compound of any one of claims 1 to 3, wherein X₀2 is L- or D-AIa.

5. The compound of any one of claims 1 to 4, wherein X₀₃ is L-or D-GIn, L-or D- GIu or L- or D-Asp.

6. The compound of any one of claims 1 to 5, wherein X₀₄ is L-or D-GIn or L- or D-GIu.

7. The compound of any one of claims 1 to 6, wherein X₀₅ is L- or D-Asn, L- or D-Asp, or L- or D-GIu.

8. The compound of any one of claims 1 to 7, wherein X₀₆ is GIy.

9. The compound of any one of claims 1 to 8, wherein X₀₇ is D-GIu.

10. The compound of any one of claims 1 to 9, wherein Xoβ is L- or D-Leu.

11. The compound of any one of claims 1 to 10, wherein X₀₉ is L- or D-GIn or L- or D-Asn.

12. The compound of any one of claims 1 to 11 , wherein Y_Oi is L- or D-Cys, L- or D-AIa, L- or D-Phe, L- or D-Tyr, L- or D-His, or L- or D-Trp.

13. The compound of any one of claims 1 to 12, wherein Xn is L- or D-Leu.

14. The compound of any one of claims 1 to 13, wherein Y₀₂ is L- or D-Cys, L- or D-AIa, L- or D-Phe, L- or D-Tyr, L- or D-His, or L- or D-Trp.

15. The compound of any one of claims 1 to 14, wherein X1₂ is L- or D-VaI.

16. The compound of any one of claims 1 to 15, wherein X₁₃ is L- or D-Lys or L- or D-Arg.

17. The compound of any one of claims 1 to 16, wherein X₁₄ is L- or D-Thr.

18. The compound of any one of claims 1 to 17, wherein X₁₅ is L- or D-Thr or L- or D-Ser.

19. The compound of any one of claims 1 to 18, wherein X₁₆ is L- or D-Thr or L- or D-Ser in selected embodiments.

20. The compound of any one of claims 1 to 19, wherein Z_A is (CH₂)i-2o or (X_aa)i-

20-

21. The compound of any one of claims 1 to 20, wherein ZA is represented by the formula: H2N-X_aai-(Xaa2 Xaa3 Xaa4 )n"CO2H, with n=0 or n=1 , where X_aai 0=1 to 4) is any L- or D- natural or non natural amino acid.

22. The compound of any one of claims 1 to 21 , wherein Z_A is 11 - aminoundecanoic acid.

23. The compound of any one of claims 1 to 22, wherein X₁₇ is L- or D-AIa.

24. The compound of any one of claims 1 to 23, wherein X₁₈ is L- or D-Pro.

25. The compound of any one of claims 1 to 24, wherein X₁₉ is L- or D-Leu.

26. The compound of any one of claims 1 to 25, wherein X₂o is L- or D-Tyr.

27. The compound of any one of claims 1 to 26, wherein X₂₁ is L- or D-Lys.

28. The compound of any one of claims 1 to 27, wherein X₂₂ is L- or D-Lys.

29. The compound of any one of claims 1 to 28, wherein X₂₃ is L- or D-IIe.

30. The compound of any one of claims 1 to 29, wherein X₂₄ is L- or D-IIe.

31. The compound of any one of claims 1 to 30, wherein X₂₅ is L- or D-Lys.

32. The compound of any one of claims 1 to 31 , wherein X₂₆ is L- or D-Lys.

33. The compound of any one of claims 1 to 32, wherein X₂₇ is L- or D-Leu.

34. The compound of any one of claims 1 to 33, wherein X₂₈ is L- or D-Leu.

35. The compound of any one of claims 1 to 34, wherein X₂₉ is L- or D-GIu or L- or D-GIn.

36. The compound of any one of claims 1 to 35, wherein X₃₀ is L- or D-Thr or L- or D-Ser.

37. The compound of any one of claims 1 to 36, wherein the region from X_igto X₃O forms a stable α-helix.

38. The compound of any one of claims 1 to 37, wherein there is a a side chain to side chain cyclization between X₂₆ and X₂₉, or between X₂₅ and X₂₉.

39. The compound of claim 38, wherein the cyclization is a lactamization between a L- or D-Lys and L- or D-GIu.

40. The compound of claim 1 selected from the group consisting of peptides or peptide analogs of SEQ ID No:2, 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 and SEQ ID No:29.

41. An antibody directed toward the compound of any one of claims 1 to 40.

42. The use of the compound of any one of claims 1 to 40 to prepare a medicament.

43. The use of the compound of any one of claims 1 to 40 to prepare a medicament to treat a condition selected from the group consisting of: angiogenic disorders, hematopoietic disorders, coagulatory disorders, inflammation, and cardiovascular disease.

44. A therapeutic composition comprising the compound of any one of claims 1 to 40, and a physiologically acceptable excipient.