Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
  • A61P13/12—Drugs for disorders of the urinary system of the kidneys
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/04—Antineoplastic agents specific for metastasis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/10—Tetrapeptides
  • C07K5/1002—Tetrapeptides with the first amino acid being neutral
  • C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

• the present invention relates to compounds which bind selectively to the adhesion molecule human P-selectin, to methods for preparing such compounds, to the use of such compounds in therapeutic or diagnostic methods and in pharmaceutical compositions, to binding molecules binding to said compounds, and to a method for determining whether a compound is capable of binding to P-selectin.
• cell surface adhesion molecules have become recognised as key mediators in numerous cellular processes including cell growth, differentiation, immune cell transmigration and response and cancer metastasis.
• CAMs immunoglobulin superfamily cell adhesion molecules
• cadherins the immunoglobulin superfamily cell adhesion molecules
• integrins the immunoglobulin superfamily cell adhesion molecules
• the selectins represent a family of presently three transmembraneous, carbohydrate- binding glycoproteins: "endothelial” E-selectin, "leukocyte” L-selectin and "platelet” P- selectin. All three selectins are divalent cation (e.g. calcium) dependent and possess an extracellular domain with a carbohydrate recognition motif, an epidermal growth factor-like motif, and some smaller domains related to complement-regulatory proteins.
• divalent cation e.g. calcium
• Human P-selectin (also referred to as GMP-140, LECAM-3, PADGEM, CD62 and CD62P) is expressed by platelets and endotheUal cells. When expressed on the surfaces of these cells, its most notable effect is the slowing of leukocytes as these leave the capillaries and enter the postcapillary venules, the latter representing the major site of leukocyte- endothelium adhesion. The slowing process is observed as leukocyte rolling, signifying an initial adhesion with relatively low affinity. The firm adhesion of rolling leukocytes is primarily mediated by integrins.
• P-selectin In endothelial cells, P-selectin is stored on Weibel-Palade bodies; in platelets, it is found in the ⁇ -granules. Following activation, P-selectin is mobilised to the cell surfaces within a few minutes in response to a variety of inflammatory or thrombogenic agents.
• the endothelial P-selectin's primarily function is to recruit leukocytes into postcapillary venules, while platelet P-selectin also results in the formation of thrombi.
• PSGL-1 P-selectin glycoprotein ligand-1
• sialoprotein expressed on the surface of leukocytes where it is concentrated at the uropod.
• selectins Conditions of the heart in which selectins are suggested to play a role include acute myocardial infarct, arterial injury, such as produced by angioplasty, and ischemia. Similarly, selectins are involved in conditions of the kidneys, such as renal injury from ischemia and reperfusion, and renal failure. Furthermore, selectins appear to play a role in organ transplant rejection, cold ischemia, hemorrhagic shock, septic shock, tumour metastasis, chrome inflammation, rheumatoid arthritis, inflammatory bowel disease, atherosclerosis, restenosis, angiogenesis, disseminated intravascular coagulation, adult respiratory stress syndrome and circulatory shock.
• P-selectin ligands or antagonists may also be used for the prevention of diseases and conditions described above. Furthermore, such ligands may also be useful in the in vivo or in vitro diagnosis of these diseases.
• rPSGL-Ig P-selectin glycoprotein ligand-1
• WO-99/41363 discloses podocalyxin-like proteins that bind to selectins.
• WO-00/41711 describes various smaller peptides or peptide sequences that bind to members of the human selectin family; most of the sequences comprise one or more units of leucine or isoleucine.
• WO-95/31210 relates to peptides and compounds that bind selectins including endothelium leukocyte adhesion molecule 1 (ELAM-1). These peptides are used for blocking adhesion of leukocytes to the selectins, i.e. especially E-selectin, but also P-selectin or L-selectin, for the purpose of inhibiting inflammation.
• selectins including endothelium leukocyte adhesion molecule 1 (ELAM-1).
• ELAM-1 endothelium leukocyte adhesion molecule 1
• a ⁇ is a D- or L-cysteine (C), D- or L-methionine (M), D- or L-valine (V) or an analogue thereof;
• a 2 is a D- or L- aspartic acid (D) or an analogue thereof;
• a 3 is a D- or L- phenylalanine (F), D- or L-tyrosine (Y) or D- or L-tryptophan (W) or an analogue thereof;
• a x is a D- or L-amino acid
• Y marks the C-terminal side of said sequence or X and Y together can form a cyclic system.
• the compound EWNDN described in the above-mentioned application has an affinity for P-selectin, as expressed by an ICso-value of about 2 ⁇ vl. Its tetramer on streptavidin has an IC 50 of about 2 ⁇ M.
• substances with selective affinity to P-selectin which can be used for preparing pharmaceutical compositions for the diagnosis, prevention and treatment of various diseases and conditions involving the adherence of leukocytes to vascular endothelial cells or to platelets.
• P-selectin ligands which can be used as targeting molecules or moieties in pharmaceutical compositions for the targeting of drugs or genetic material to tissues expressing P-selectin.
• a further object of the invention is the provision of methods for preparing such compounds.
• Yet another object is the presentation of uses of such compounds, and of compositions which contain the compounds.
• Figure 1 shows the chemical structures of acyl moieties 1-7 introduced to core sequence compound 8.
• Figure 2 is a schematic representation of the solid phase synthesis of compound library
• Core sequence compound 8 was synthesised using standard Fmoc chemistry on Tentagel S-NH resin with HMPA linker. The core peptide was then derivatised at the N-terminus (after removal of the Fmoc) or on the C-terminus (after selective removal of the DDE group by 2% hydrazine) with 7 different carboxylic acids (see figure 1). The peptides were liberated from the resin by TFA cleavage resulting in compound library 10.
• Fmoc N-(9- fluoroenyl)methoxy-carbonyl
• HMPA 4-hydroxymethylphenoxyacetic acid
• TFA trifluoro acetic acid
• DDE 4,4-dimethyl-2,6- dioxocyclohex-l-ylidene)ethyl.
• Figures 4A-D show the chemical structure (figure 4A) and biological activity of compound library 14 (figures B-D).
• Crude peptide library 14 (1 ⁇ M) was tested in a competition assay of TM11-PO binding to human P-selectin.
• Three different spacers between the acyl moiety and the WNDN core sequence were used (Ri : no spacer (white bars), a glycyl spacer (grey bars) and amino butyric spacer (black bars).
• the chemical structure of the introduced acyl modification (R ) is depicted below these bars.
• Figure 5 shows a graphical representation of the competition of biotin-PAA-Le a -SO 3 binding to human P-selectin ( ⁇ ), mouse P-selectin ( ⁇ ), human L-selectin (A) and human E- selectin (T) by peptide 28.
• Wells were coated with each selectin (0.3 ⁇ g/ ml) and incubated with 0.33 ⁇ g/ ml biotin-PAA-Le a -SO 3 with or without peptide 28 as described in example 3.
• Figure 6 is a graphical representation of the competition of HL-60 cell binding to CHO-P cells by peptide 28 ( ⁇ ) or EWNDN (•).
• CHO-P cells seeded in 96 wells were incubated with calcein labeled HL-60 cells in the presence of peptide 28 or EWNDN.
• Data points are means of 12 data points in quadruplicate.
• the compounds of the present invention have selective affinity to human P-selectin and are derivatives of peptides or functional equivalents thereof, such as modified peptides, peptide analogues or peptidomimetics.
• the derivatives are represented by
• - Ai is a D- or L-amino acid selected from the group consisting of cysteine (C) and valine (N), or an analogue or mimetic thereof;
• - A 2 is a D- or L- aspartic acid (D) or an analogue or mimetic thereof;
• - A 3 is a D- or L-amino acid selected from the group consisting of phenylalanine (F) and tryptophan (W), or an analogue or mimetic thereof;
• a x is any D- or L-amino acid selected from the group consisting of glutamic acid, aspartic acid, glycine, cysteine and analogues or mimetics thereof;
• - X is an ⁇ -terminal group or sequence and is hydrogen or a residue comprising 1 to 6 D- or L-amino acids or analogues thereof
• - Y is a C-terminal group or sequence and is -OH or a residue comprising 1 to 11 D- or L- amino acids or analogues thereof; and are characterised in that either X or Y, or both X and Y, are substituted with the group:
• - Z is selected from the group consisting of -CO-, -O-, - ⁇ R 2 -, and -CO- ⁇ R 2 -;
• R andR are independently selected from: - H;
• heteroaryl group which is selected from 5- or 6-membered ring systems and benzo- condensed ring systems, and has at least one heteroatom selected from the group consisting of nitrogen, oxygen and sulphur, wherein said heteroaryl group may be substituted with at least one group selected from the group consisting of a halogen, -(C ⁇ -C 6 )alkyl, -CF 3 , -OH, - O-fA-Cf alkyl, -COOH, -COO-(C 1 -C 6 )alkyl, -NO 2 , -NH 2 , -NH-(C ⁇ -C 6 )alkyl, -N-((C 1 -C 6 )alkyl) 2 and -SO 3 H; or
• an aralkyl group comprising an alkyl group as defined above and an aryl group or heteroaryl group as defined above.
• indices m and n represent integers independently selected from 0 and 1. However, n is not 0 when R 1 is H. Furthermore, a compound of the invention comprises a derivative of a peptide or a molecular structure that is related to a peptide, herein referred to as functional equivalent.
• Peptides are defined as amides that are derived from two or more amino acids by combination of the amino group of one acid with the carboxyl group of another (Merriam Webster Medical Dictionary ® 2001).
• a peptide may also refer to a peptidic structure within a molecule.
• peptides are composed of naturally occurring L- ⁇ - amino acids, which are alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gin or Q), glutamic acid (Glu or
• glycine Gly or G
• histidine His or H
• isoleucine He or I
• leucine Leu or L
• lysine Lys or K
• methionine Met or M
• phenylalanine Phe or F
• proline Pro or P
• serine S
• threonine Thr or T
• tryptophan Trp or W
• tyrosine Tyr or Y
• valine Val or
• Functional equivalents of the peptides of the invention are proteinaceous molecules, comprising the same human P-selectin binding activity in kind, but not necessarily in amount, and may, for instance, be modified peptides, peptoids, peptide analogues or peptidomimetics.
• Modified peptides are molecules derived from peptides by the introduction of substituents or functional groups which are not present in naturally occurring amino acids.
• the term also includes compounds which are obtained by the reaction of peptides with molecules from other chemical categories, whether these molecules are naturally occurring or not. For instance, biotinylated peptides, glycoproteins, and lipoproteins are frequently found in nature, while peptides modified with polyethylene glycol, such as pegylated interferon ⁇ -2b (Peg-hitron ® ), are examples of chemically modified peptides that have been designed to alter some, but not all of the peptides' properties. Peptoids, like peptides, are typically amides of two or more amino acids. However, they are frequently not directly derived from naturally occurring amino acids, but rather of various types of chemically synthesised L- or D-amino acids.
• Peptidomimetics in their broadest scope, are compounds which are in their fuiictional structure more or less similar to a peptide, but which may also contain non-peptidic bonds in the backbone, or D-amino acids.
• peptidomimetics serve as substitutes for native peptides in the interaction with receptors and enzymes (Pharmaceutical Biotechnology, Ed.
• Pseudopeptides a class of peptidomimetics, are compounds containing amide bond isosteres instead of amide bonds (ibid., pp. 137-140).
• the two units of Ai witliin the sequence are selected independently; they can be identical or different from each other.
• at least one of the Ai units represents val ne (V). More preferably, both Ai units are valine (V).
• a ⁇ is an analogue or mimetic of cysteine (C), methionine (M), or valine (N).
• An approach to improve the selectivity of a peptide is to introduce con&rmational constraints to the backbone, which decreases the number of potential receptor or enzyme interactions. The constraints are most often achieved by the introduction of carbon-carbon double bonds (olefmic analogues) and ring structures (ibid.).
• a further method to identify analogues which mimic the functional structure of an amino acid such as cysteine (C), methionine (M), or valine (V) is computer-aided modeling.
• Preferred mimetic structures possess a side chain with similar charge or electronegativity, hydrophobicity and spatial orientation to said amino acids.
• one or both Ai units are D-amino acid analogues of cysteine (C), methionine (M), or valine (N).
• the present invention also encompasses derivatised functional equivalents of the said peptides. Equivalents can, among others, be found by amino acid substitution using conservative amino acid changes. Particularly, the invention also encompasses a method for determining whether a compound is capable of binding to human P-selectin, comprising substituting in a compound according to the invention, an amino acid for a conservative amino acid and determining whether the resulting compound is capable of binding to said P- selectin.
• A is a D- or L-aspartic acid with a solvent exposed side chain bearing a negative charge at physiological pH, or an analogue or mimetic thereof. For identifying appropriate analogues, the same principles apply as those that have been set forth with regard to Ai .
• a 3 is a D- or L-amino acid with an aromatic side chain selected from the group consisting of phenylalanine (F), and tryptophan (W), or an analogue or mimetic thereof. More preferably, A 3 is an L-amino acid selected from this group. Most preferred is tryptophan (W). i other embodiments, A 3 is an analogue structure related to phenylalariine (F), or tryptophan (W) as defined above.
• a x is a D- or L-amino acid, as defined above, or an analogue or mimetic thereof.
• a x is a D- or L- glutamic acid (E), or an analogue or mimetic thereof.
• X marks the N-terminal group.
• X can simply be a hydrogen.
• X is a sequence of up to 6 amino acids, or analogues or mimetics thereof.
• the type of amino acids(s) should of course not affect e.g. by changing the configuration of the peptide or by changing the spatial orientation of the terminal carboxylic acid with respect to the core peptide the recognition by P-selectin in such a way that, the aimed effect is no longer obtained.
• Suitable analogues include amino substituted carboxylic acids, e.g. aminocyclohexanoic acid amino benzoic acid and amino caproic acid.
• Y marks the C-terminal group.
• Y may be a hydroxyl group.
• Y is a residue comprising 1 to 11 amino acids carrying a terminal hydroxyl group.
• the recognition by P-selectin is more tolerant to substituents at the C-terminus.
• at least one amino acid with a negatively charged side chain is present in Y, preferably being separated from the remaining X(A x ) rn A A 1 A ⁇ sequence by one to three amino acids, hi a particularly preferred embodiment of the invention
• Y is D- or L- lysine (K), or comprises D- or L- lysine (K).
• the compound of the invention can possess a cyclic or otherwise constrained backbone, hi that case, X+Y together may suitably be linked through an S-S-binding, although, of course, also other types of (chemical) linkages can be present, such as an amide binding, a thioether binding, a carbamate binding or an ester binding.
• the length of the group X+Y is not particularly critical as long as the core sequence is recognised by P-selectin. Generally, the minimum length of X+Y corresponds with the length of 5-6 amino acids.
• R ! -(Z) n - with which X or Y, or both X and Y independently, are substituted may be selected from R-CO groups wherein R is an alkyl, aryl or heteroaryl group.
• the alkyl group preferably has 2 to 8 carbon atoms, at least one of which maybe replaced by a nitrogen, oxygen, or sulphur moiety.
• An aryl group has preferably 6 to 14 carbon atoms, and the group may be substituted with at least one group selected from the group consisting of OH, COOH, NO 2 , NH 2 , SO 3 H and CF 3 . Also substitutions on this group selected from -NHR, -CO(NHR), -CN(NHR), -N 3 , -(CH 2 ) p -OH, and
• the heteroaryl group has the same definition as the aryl group with the exception that at least one heteroatom selected from the group consisting of nitrogen, oxygen and sulphur is incorporated instead of a carbon atom.
• the heteroaryl group is based on a 5- or 6- membered ring system or benzo-condensed ring system.
• R 1 and/or R 2 represent independently from each other the following groups: 4-hydroxyphenylcarbonyl, 3,5-dihydroxyphenylcarbonyl, 3-hydroxy-5-carbonyloxyphenylcarbonyl, 3,4,5-tri-methoxy- phenylcarbonyl, 3,5-di-nitro-phenylcarbonyl, 4-carboxyphenylcarbonyl, 3-carbonylbutyric acid, 2-hydroxy-5-sulfonylphenylcarbonyl, 3-carboxyphenylcarbonyl, 3,5-difluorophenylcarbonyl, l-sulfonyl-2-aminoethylcarbonyl,
• especially preferred groups are acyl groups and more particularly 3,5- dicarboxyphenylcarbonyl and 3,4,5-trihydroxyphenylcarbonyl groups.
• modifications based on tannic acid, caffeinic acid, oligohydroxyl aryl groups and oligocarboxy-aryl groups as well as derivatives thereof are preferred, especially on group X.
• the 3,4,5-trihydroxyphenylcarbonyl group however can be used advantageously both on group X and group Y.
• Compounds of the invention possess linear, branched, cyclic, or constrained backbones.
• peptides or functional equivalents with at least two cysteine (C) units can be cyclisised by oxidation. If a compound is cycUsised via such a disulfide bond, it is preferred that the participating cysteine units are members of X and Y, respectively.
• Cyclic structures are, in fact, an example for conformationally constrained backbones. Other types of constrained structures may also be introduced to decrease the conformational flexibility of the compound. Especially the presence of olefinic bonds or small ring structures in the backbone serves this purpose. Examples of such constraints are given in
• the compounds of the invention are provided as multimers of the derivatised peptides or functional equivalents thereof.
• single derivatised peptide or peptoid chains in accordance with the invention are coupled to a biocompatible protein, such as human serum albumin, humanised antibodies, liposomes, micelles, synthetic polymers, nanoparticles, and phages.
• Multimers can also represent derivatised peptides which are serially coupled to each other via spacers, i.e. concatamers, or dendrimers, or clusters.
• the compounds can generally be prepared by the methods that are known for the preparation of peptides and similar substances and derivatives thereof. Smaller compounds containing only a few amino acids or similar units, and preferably not more than 30-50 units, can be prepared by chemical and/or enzymatic ligation techniques, either using the classical approach in which the reactions take place in solution or suspension, or by employing the more modern solid phase approach, in which the peptide is assembled while being anchored to a solid surface, such as a polymeric bead. Larger compounds are typically synthesised by automatic solid phase peptide synthesisers. The compounds obtained are then modified by reaction with a suitable source, preferably a R l (Z) n - OH group. Said source reacts with a primary amine group in the peptide compound.
• a suitable source preferably a R l (Z) n - OH group. Said source reacts with a primary amine group in the peptide compound.
• the said source can be reacted with an added lysfne, which lysine can suitably be added as its DDE derivative.
• DDE is 4,4- dimethyl-2,6-dioxocyclohex-l-ylidene)-ethyl; it can selectively be removed with 2% hydrazine without affecting acid-labile side chain protecting groups.
• Salts of the derivatives of the peptides or functional equivalents thereof are prepared by known methods, which typically involve the mixing of the derivative of the peptide or peptoid with either a pharmaceutically acceptable acid to form an acid addition salt, or with a pharmaceutically acceptable base to form a base addition salt.
• acids include organic and inorganic acids, such as formic acid, acetic acid, propionic acid, lactic acid, glycolic acid, oxalic acid, pyruvic acid, succinic acid, maleic acid, malonic acid, cinnamic acid, sulfuric acid, hydrochloric acid, hydrobromic acid, nitric acid, perchloric acid, phosphoric acid and thiocyanic acid, which fonn arnmonium salts with free amino groups of the derivatised peptides and functional equivalents thereof.
• organic and inorganic acids such as formic acid, acetic acid, propionic acid, lactic acid, glycolic acid, oxalic acid, pyruvic acid, succinic acid, maleic acid, malonic acid, cinnamic acid, sulfuric acid, hydrochloric acid, hydrobromic acid, nitric acid, perchloric acid, phosphoric acid and thiocyanic acid, which fonn arnmonium salts with
• Pharmaceutically acceptable bases which form carboxylate salts with free carboxylic groups of the derivatised peptides and functional equivalents thereof, include ethylamine, methylamine, dimethylamine, triethylamine, isopropylamine, diisopropylamine, and other mono-, di- and triallcylamines, as well as arylamines.
• Multimers of the derivatised peptide or peptoid according to the invention can, for example, be prepared in a similar way as multimers of peptides, e.g. by biotinylating the N- terminus chains and subsequent complexation with streptavidin, thereby if deemed necessary adjusting the method to the specific compounds. As streptavidin is able to bind 4 biotin molecules or conjugates with high affinity, very stable tetrameric peptide complexes can be formed by this method.
• Multimers may be composed of identical or different derivitised peptides or functional equivalents. Preferably, however, the multimers of the invention are composed of two or more identical derivatised peptides or functional equivalents thereof.
• the compounds of the invention have an affinity to human P-selectin, a membrane glycoprotein expressed by vascular endothelial cell and platelets, which is involved in leukocyte adhesion to the endothelium and platelets.
• the affinity or binding characteristics of compounds to P-selectin can be quantified, for example, in terms of the IC 5 o-value.
• an ICso-value of about 50-100 ⁇ M or less would be considered as evidence for affinity and binding. More desirable for ligands are substances with IC 50 -values of about 10 ⁇ M or less. The lowest a IC 5 o-value attainable for the non-covalent type bonds playing a role in the interactions or bindings in accordance with the present invention is about 10 "15 M.
• the IC 50 -values are higher than about 10 " M and in most cases higher than about 10 "9 M.
• the compounds of the invention have low micromolar to low nanomolar IC50- values towards P-selectin, which make them about equally potent as the natural ligand P-selectin glycoprotein ligand- 1.
• N-terminally modified compounds of the invention were more effective (potent and or specific) than the corresponding C-terminally modified compounds containing the same modifications. Very good results were obtained with modification by 1,3,5-tricarboxylic acid or gallic acid.
• the compounds according to the invention may render the peptides more stable against proteases, particularly against N-exopeptidases, due to the derivatisation.
• the compounds further may have an enhanced specificity for P-selectin and therefore modulate a pharmacological action such as interaction between monocyte and platelet, that between monocyte and endothelial cells, that between platelet cells and platelet cells. Potential side effects may thus also be diminished.
• the compounds would be cross- reactive in many species.
• a further aspect of the invention refers to the uses of the disclosed compounds. Since the compounds bind selectively to P-selectin, they can, depending on their type of interaction with P-selectin after binding, function as antagonists, partial antagonists, or as mere targeting means to target conjugated substances to cells and tissues expressing P-selectin. Thus, the compounds can be advantageously used in pharmaceutical compositions.
• compositions containing the compounds of the invention may be adapted for various routes of administration, such as parenteral, oral, transmucosal, nasal, or pulmonary. They may further contain drug targeting agents, bioavailability enhancement agents, or active ingredients other than compounds of the invention, and provide for immediate or modified release.
• the term "pharmaceutical composition” refers to therapeutic and diagnostic compositions, as well as to medicaments and diagnostics containing such compositions.
• Therapeutic compositions and medicaments are used for the prevention or treatment of diseases and other conditions of mammals of which conditions improvement is desirable. Diagnostics and diagnostic compositions are used for the diagnosis of such diseases in vivo and in vitro.
• a preferred use of the compounds is for preparing therapeutic compositions or medicaments to prevent or improve diseases and conditions involving the adhesion of leukocytes, such as monocytes and neutrophils, to the vascular endo heUum and to platelets.
• compositions containing one or more compounds of the invention can contribute to controlling leukocyte-mediated mflammatory processes. It is known that activated leukocytes release toxic molecules which can damage normal tissue.
• inflammatory responses are part of several pathological conditions, such as transplant rejection, cold ischemia, hemorrhagic shock, septic shock, tumour metastasis, thrombosis, chronic inflammation, rheumatoid arthritis, inflammatory bowel disease, atherosclerosis, restenosis, angiogenesis, disseminated intravascular coagulation, adult respiratory stress syndrome, circulatory shock, severe traumatic brain injury, relapsing-remitting multiple sclerosis, cerebral artery occlusion, ischemia, stroke, acute myocardial infarct, deep vein thrombosis, arterial injury, such as produced by angioplasty, myocardial ischemia, renal injury from ischemia and reperfusion, and renal failure.
• pathological conditions such as transplant rejection, cold ischemia, hemorrhagic shock, septic shock, tumour metastasis, thrombosis, chronic inflammation, rheumatoid arthritis, inflammatory bowel disease, atherosclerosis, restenosis, an
• the compounds are used in the preparation of diagnostic compositions or products.
• Such compositions can be used for in vitro tests to quantify P- selectin concentrations in body fluids as markers for the diseases and conditions described above. They may be also used for in vivo diagnostic imaging procedures to monitor P- selectin mediated atherosclerosis, aneurisms, restenosis following percutaneous fransluminal coronary angioplasty (post-PTCA restenosis) and other conditions selected from those in which P-selectin is mobilised.
• a compound according to the invention may be conjugated with a chelator, which is subsequently complexed with an isotropic label that is detectable by the chosen monitoring system.
• Another use of the compounds is as that of a tool in research. For instance, they can be used to test the binding affinity of molecules to P-selectin or functional equivalents of P- selectin.
• P-selectin or a functional equivalent of P-selectin would be contacted and incubated with a molecule to be tested for bmding affinity and with a compound of the invention.
• a reduced binding of the compound of the invention would indicate an affinity of the molecule to P-selectin.
• the compounds can also be used as targeting molecules or conjugates in pharmaceutical compositions for the targeting of drugs or genetic material to tissues that express P-selectin.
• conjugates the compounds can be directly coupled with active molecules or nucleic acids that are to be delivered to such tissues. Alternatively, they can be incorporated into or anchored onto the surface of liposomes or other lipid vesicles, emulsion droplets, polymers, nano- or microparticles to obtain targeted vehicles for drugs or genetic material which is delivered to P-selectin expressing tissues.
• compositions preferably contain one or more compounds with P- selectin affinity as disclosed herein and at least one carrier or excipient.
• a carrier or excipient is any pharmaceutically acceptable substance or mixture of substances having no substantial pharmacological activity, which can be used as a vehicle or as an auxiliary substance to formulate a compound into dosage fo ⁇ n which is stable and easy to administer. Examples of pharmaceutically acceptable excipients are found in the monographs of all major pharmacopoeias.
• the composition is formulated and processed for parenteral injection, preferably for intravascular injection, such as intravenous or intra-arterial, but also for intramuscular, subcutaneous, intralesional, intraperitoneal or other routes of parenteral administration.
• parenteral injection preferably for intravascular injection, such as intravenous or intra-arterial, but also for intramuscular, subcutaneous, intralesional, intraperitoneal or other routes of parenteral administration.
• intravascular injection such as intravenous or intra-arterial
• intramuscular, subcutaneous, intralesional, intraperitoneal or other routes of parenteral administration will also teach those skilled in the arts on how to prepare such compositions.
• parenteral dosage forms is their sterility.
• Other requirements are described in all major pharmacopoeias, such as in USP 24, in the monograph "General Requirements for Tests and Assays. 1. Injections'', p. 1775-
• a parenteral formulation it maybe necessary to provide a dried dosage form which must be reconstituted before it can be administered.
• An example of such a dosage form is a freeze-dried or lyophilised formulation.
• Prolonged release may be provided by solid implants, nanoparticles, nanocapsules, microparticles, microcapsules, emulsions, suspensions, oily solutions, liposomes, or similar structures.
• Excipients that are particularly useful for the preparation of parenteral formulations are solvents, cosolvents and liquid or semisolid carriers, such as sterile water, ethanol, glycerol, propylene glycol, polyethylene glycol, butanediol, fatty oils, short- and medium chain triglycerides, lecithin, polyoxyethylene castor oil derivatives; substances to adjust the osmolality and pH, such as sugars, especially glucose, sugar alcohols, especially manriitol, sodium chloride, sodium carbonate, citric acid, acetate, phosphate, phosphoric acid, hydrochloric acid, sodium hydroxide etc.; stabilizers, antioxidants, and preservatives, such as ascorbic acid, sodium sulfite or -hydrogen sulfite, EDTA, benzyl alcohol etc.; other excipients and lyophilization aids, such as albumin, dextran etc.
• solvents, cosolvents and liquid or semisolid carriers such as
• the pharmaceutical compositions may be designed for oral administration and processed accordingly.
• Appropriate oral dosage forms include tablets, hard capsules, soft capsules, powders, granules, orally disintegrating dosage forms, syrups, drops, suspensions, effervescent tablets, chewable tablets, oral films, lyophilised dosage fonns, sustained release dosage forms and controlled release dosage forms.
• the oral dosage form is an enterically coated solid dosage fo ⁇ n to provide protection of the compound from the acidic and proteolytic environment of the stomach. It may also be advantageous to administer a compound of the invention in a transmucosal dosage form.
• Transmucosal administration is possible via, for instance, nasal, buccal, sublingual, gingival or vaginal dosage forms.
• These dosage forms can be prepared by known techniques; they can be formulated to represent nasal drops or sprays, inserts, films, patches, gels, ointments or tablets.
• the excipients used for a transmucosal dosage form include one or more substances providing for mucoadhesion, thus prolonging the contact time of the dosage form with the site of absorption and thereby potentially increasing the extent of absorption.
• the compounds are administered via the pulmonary route, using a metered dose inhaler, a nebuliser, an aerosol spray, or a dry powder inhaler.
• a metered dose inhaler a nebuliser, an aerosol spray, or a dry powder inhaler.
• Appropriate formulations can be prepared by known methods and techniques. Transdermal, rectal, or ocular administration may also be feasible in some cases.
• an appropriate dosage form may contain a bioavailability enhancing agent, which may be any substance or mixture of substances which increases the availability of the compound. This maybe achieved, for instance, by the protection of the compound from degradation, such as by an enzyme inhibitor or an antioxidant. More preferably, the enhancing agent increases the bioavailability of the compound by increasing the permeability of the absorption barrier, which is typically a mucosa. Permeation enhancers can act via various mechanisms; some increase the fluidity of mucosal membranes, while others open or widen the gap junctions between mucosal cells.
• bioavailability enhancers are amphiphilic substances such as cholic acid derivatives, phosphohpids, ethanol, fatty acids, oleic acid, fatty acid derivatives, EDTA, carbomers, polycarbophil, and chitosan.
• amphiphilic substances such as cholic acid derivatives, phosphohpids, ethanol, fatty acids, oleic acid, fatty acid derivatives, EDTA, carbomers, polycarbophil, and chitosan.
• molecules capable of binding to the compounds disclosed above are within the scope of the invention. For instance, standard hybridysation technology can be applied to prepare specific monoclonal antibodies to a compound. Other techniques are available to design and prepare smaller molecules capable of binding to a compound of the invention.
• Lys(DDE)-GABA-HMPA-resin see fig. 1
• Lys(DDE)-GABA-HMPA-resin see fig. 1
• HMPA 4-hydroxymethylphenoxyacetic acid
• Fmoc-GABA-OH (10 eq.) was attached to the HMPA resin 8 under the agency of N,N'-dicyclohexylcarbodiimide (DCC, 5 eq.) and 4- dimethylaminopyridine (DMAP, 0.5 eq.) All other amino acids, with acid labile side chain protection if necessary, were attached by coupling in the presence of HOBt/TBTU Dipea (4/4/8 eq.). After coupling, the resin was washed with DMF, isopropanol and diethylether and subsequently dried.
• DCC N,N'-dicyclohexylcarbodiimide
• DMAP 4- dimethylaminopyridine
• the solid phase synthesis of the compound library 10 was performed using a Flexchem ® system (Robbins Scientific, Sunnyvale U.S.A.). After removal of the N- terminal Fmoc group of compound 9 by 20% piperidine in DMF, the resin was washed
• TM11-PO a tetrameric TMll/strepPO complex, which has previously been shown to bind with high affinity and specificity to human P-selectin (Molenaar et al., Blood 2002, 100, 3570-3577), was freshly prepared by incubating streptavidin-peroxidase (strep-PO (Amersham Life Science, Little Chalfont, United Kingdom), 8.4 ⁇ l, 2.0 ⁇ M) and TMll-biotin (biotin-CDVEWNDVSSLEWDLPC (synthesised by Dr.
• wells were washed with assay buffer and incubated for 1 hour at 37°C with blocking buffer (3% BSA in assay buffer). After washing with assay buffer, the wells were incubated for 2 hours at 37°C with the human P-selectin IgG-Fc chimera (R&D Systems Europe Ltd., Abingdon, United Kingdom) (0.3 ⁇ g/ml). Subsequently, wells were washed with assay buffer and incubated for 1 hour at 4°C with the TM11-PO complex. The wells were washed six times with washing buffer (0.1% Tween 20 in assay buffer).
• the derivatised peptides are coded HPzj ' , wherein i and / refer to the acyl moieties R 1 and R 2 attached to the N- and C-terminus, respectively, of formula 10 (Fig.l). Unmodified amino group is indicated by 0. The acyl moieties 1 to 7 are shown in Fig. 2.
• the affinity of the compounds was confirmed by a cell adhesion assay in which Chinese hamster ovary cells expressing human P-selectin were incubated with HL60 cells expressing PSGL-1 in the presence of titered amounts of the compounds.
• Example 6 Inhibition of dynamic interactions between HL60 cells and human P-selectin expressing Chinese hamster ovary cells
• CHO cells stably transfected with human P-selectin were kindly donated by Dr. Modderman (University of Amsterdam, Amsterdam, the Netherlands). Cells were grown in DMEM (BioWhittaker Europe, Venders, Belgium) containing 10% foetal calf serum (FCS) (BioWitthaker), 5 mM L-glutamine, 20,000 units penicillin/streptomycin (BioWhittaker) and 5 mM non-essential amino acids (Gibco, Paisley, United Kingdom). Flasks with cells were incubated at 37°C in 5% CO for 3 or 4 days until cells had grown nearly confluent.
• FCS foetal calf serum
• HL60 cells were from ATCC and grown in RPMI 1640 medium (BioWhittaker) with 10%) FCS, 5mM L-glutamine and 20,000 units penicillin/streptomycin. HL-60 cells were fluorescently labeled by incubation for 30 min at 37°C with 5 ⁇ M calcein-AM (Molecular Probes, Leiden, The Netherlands) in RPMI.
• CHO-P cells cultured in DMEM with 10% FCS, 5 mM non-essential amino acids, 5 mM L-glutamine and 20.000 U penicillin/streptomycin
• P-selectin CHO-P cells, cultured in DMEM with 10% FCS, 5 mM non-essential amino acids, 5 mM L-glutamine and 20.000 U penicillin/streptomycin
• CHO-P associated fluorescence was measured ( ⁇ eXC 485/ ⁇ em 530 nm).

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