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
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/08—Peptides having 5 to 11 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates generally to peptide analogs of Ac—PHSCN—NH 2 which target tumor and endothelial cells and have anti-tumor, anti-angiogenic and anti-metasastic activity, methods of making these peptides, compositions thereof and methods of using these peptides and pharmaceutical compositions thereof to treat, prevent and detect diseases characterized by tumor growth, metastasis and angiogenesis.
• the peptide analogs may serve, inter alia, as carriers of radioactivity, PET-active compounds, toxins, fluorescent molecules and PEG molecules.
• Integrins are heterodimeric, transmembrane proteins that are involved in cell adhesion, motility and survival (Geiger et al., Nat. Rev. Mol. Cell Biol 2001, 2(11):793-805). Integrin ligands comprise the extracellular matrix (ECM) and basement membranes and include collagen, laminin, fibronectin, vitronectin, and fibrinogen (Bokel, Dev. Cell 2002, 3(3):311-21; Stupack et al., J Cell Sci 2002,115: 3729-38; Bornstein et al., Curr Opin Cell Biol 2002, 14(5): 608-16).
• ECM extracellular matrix
• basement membranes include collagen, laminin, fibronectin, vitronectin, and fibrinogen (Bokel, Dev. Cell 2002, 3(3):311-21; Stupack et al., J Cell Sci 2002,115: 3729-38; Bornstein et al., Curr Opin Cell Biol 2002, 14(5)
• the integrin ⁇ 5 ⁇ 1 is normally not expressed in quiescent endothelial cells, but is upregulated during angiogenesis (Kim et al., Am J Pathol 2000, 156(4):1345-62).
• ⁇ 5 ⁇ 1 is a receptor for fibronectin, an abundant plasma protein that may also be associated with the extracellular matrix (Labat-Robert, Semin Cancer Biol 2002, 12(3): 187-95).
• Fibronectin interacts with the ⁇ 5 ⁇ 1 integrin through several epitopes with the major adhesive interaction mediated by the RGD sequence within the 10 th type III repeat.
• the major adhesive interaction mediates cell signaling events and can be potentiated by a second epitope located in the ninth Type III repeat called the synergy region (i.e., PHSRN) (Akiyama et al., Cancer Metastasis Rev 1995, 14(3): 173-89).
• Antagonists of ⁇ 5 ⁇ 1 were able to inhibit tumor angiogenesis and cause tumor regression which demonstrates the therapeutic potential of targeting the integrin ⁇ 5 ⁇ 1 (Kim et al., supra).
• the integrin ⁇ 5 ⁇ 1 also appears to be important in the survival and metastasis of tumor cells (O'Brien et al., Exp Cell Res 1996, 224(1): 208-13; Ruoslahti, Invasion Metastasis 1994-95;14(1-6):87-9724-26); Kemperman et al., Invasion Metastasis 1994-95,14(1-6): 98-108; Tani et al., Br J Cancer 2003, 88(2): 327-33).
• the integrin ⁇ v ⁇ 3 is also a therapeutic target for the inhibition of tumor angiogenesis since inhibitors of ⁇ v ⁇ 3 (e.g., monoclonal antibodies, cyclic RGD peptides and small non-peptidic organic compounds) are efficacious in multiple pre-clinical models of cancer progression (Kumar, Curr Drug Targets 2003, 4(2): 123-31; Varner et al., Important Adv Oncol 1996, 69-87; Brooks, J Clin Invest 1995, 96(4): 1815-22).
• inhibitors of ⁇ v ⁇ 3 e.g., monoclonal antibodies, cyclic RGD peptides and small non-peptidic organic compounds
• ⁇ v ⁇ 3 is not normally expressed on epithelial cells, it is up-regulated on tumor cells, leading to tumor cell adhesion, migration and invasion (Metzner et al., J Invest Dermatol 1996, 107(4): 597-602).
• ⁇ v ⁇ 3 integrin has been implicated in melanoma progression and metastasis (Nip et al., Cancer Metastasis Rev 1995, 14(3): 241-52) and ⁇ v ⁇ 3 expression has been documented in a variety of tumor cell types including breast, prostate, pancreas, kidney, and glioma (Felding-Habermann et al., Proc Natl Acad Sci USA 2001, 98(4): 1853-8; Platten et al., Biochem Biophys Res Commun 2000, 268(2): 607-11; Lohr et al., Pancreas 1996, 12(3): 248-59; Rabb et al., Am J Nephrol 1996, 16(5): 402-8; Cooper et al., Neoplasia 2002, 4(3): 191-4). Further, the expression of ⁇ v ⁇ 3 has also been associated with metastasis to bone (Pecheur et al., FASEB J 2002,
• Ac—PHSCN—NH 2 is derived from the synergy sequence of fibronectin and has been shown to target activated ⁇ 5 ⁇ 1 and ⁇ V ⁇ 3 integrins on cell surfaces (Livant, U.S. Pat. No. 6,001,965; Livant, U.S. Pat. No. 6,472,369; Livant et al., Cancer Res 2000, 60(2): 309-20).
• Ac—PHSCN—NH 2 completely inhibits DU145 invasion and metastasis of MatLyLu cells in a rat model (Livant et al., supra) and in combination with 5-FU infusion improved survival in a CT26 colon cancer model (Stoeltzing et al., Int J Cancer 2003, 104(4): 496-503). Accordingly, Ac—PHSCN—NH 2 targets tumors and the blood vessels which nourish tumor cells as shown by visualization of a Ac—PHSCN—NH 2 derivative.
• the peptide analogs will serve, inter alia, as carriers of radioactivity for imaging and radiotherapy, PET-active compounds for PET-imaging, toxins for targeted delivery of cellular toxins, fluorescent molecules for visualization and PEG molecules for improvement of pharmacokinetic parameters.
• the present invention satisfies these and other needs by providing peptide analogs of Ac—PHSCN—NH 2 which target tumor and endothelial cells and have anti-tumor, anti-angiogenic and anti-metasastic activity, methods of making these peptides, compositions thereof and methods of using these peptides and pharmaceutical compositions thereof to treat, prevent and detect diseases characterized by tumor growth, metastasis and angiogenesis.
• the peptide analogs may serve, inter alia, as carriers of radioactivity, PET-active compounds, toxins, fluorescent molecules and PEG molecules.
• the present invention provides a compound of Formula (I):
• the present invention provides pharmaceutical compositions of compounds of the invention.
• the pharmaceutical compositions generally comprise one or more compounds of the invention or pharmaceutically acceptable salts, hydrates or solvates thereof and a pharmaceutically acceptable diluent, carrier, excipient and adjuvant.
• diluent, carrier, excipient and adjuvant will depend upon, among other factors, the desired mode of administration.
• the present invention provides methods for treating or preventing diseases or disorders such as cancer.
• the methods generally involve administering to a patient in need of such treatment or prevention a therapeutically effective amount of a compound of the invention and/or a pharmaceutical composition thereof.
• the present invention provides methods for detecting diseases or disorders such as cancer.
• the methods generally involve administering to a patient in need of such treatment or prevention a diagnostically effective amount of a compound of the invention and/or a pharmaceutical composition thereof.
• Compounds of the invention refers to compounds encompassed by structural formulae (I), (II), (III), (IV) and (V) disclosed herein and includes any specific compounds within that generic formula whose structure is disclosed herein.
• the compounds of the invention may be identified either by their chemical structure and/or chemical name. When the chemical structure and chemical name conflict, the chemical structure is determinative of the identity of the compound.
• the compounds of the invention may contain one or more chiral centers and/or double bonds and therefore, may exist as stereoisomers, such as double-bond isomers (i.e., geometric isomers), enantiomers or diastereomers.
• the chemical structures depicted herein encompass all possible enantiomers and stereoisomers of the illustrated compounds including the stereoisomerically pure form (e.g., geometrically pure, enantiomerically pure or diastereomerically pure) and enantiomeric and stereoisomeric mixtures.
• the compounds of the invention may also exist in several tautomeric forms. Accordingly, the chemical structures depicted herein encompass all possible tautomeric forms of the illustrated compounds.
• the compounds of the invention also include isotopically labeled compounds where one or more atoms have an atomic mass different from the atomic mass conventionally found in nature.
• isotopes examples include, but are not limited to, 2 H, 3 H, 13C, 14 C, 15 N, 17 O, 18 O, etc.
• Compounds of the invention may exist in unsolvated forms as well as solvated forms, including hydrated forms and as N-oxides. In general, the hydrated, solvated and N-oxide forms are within the scope of the present invention.
• Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention. Further, it should be understood, when partial structures of the compounds of the invention are illustrated, that brackets indicate the point of attachment of the partial structure to the rest of the molecule.
• Alkyl by itself or as part of another substituent refers to a saturated or unsaturated, branched, straight-chain or cyclic monovalent hydrocarbon radical derived by the removal of one hydrogen atom from a single carbon atom of a parent alkane, alkene or alkyne.
• Typical alkyl groups include, but are not limited to, methyl; ethyls such as ethanyl, ethenyl, ethynyl; propyls such as propan-1-yl, propan-2-yl, cyclopropan-1-yl, prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), cycloprop-1-en-1-yl; cycloprop-2-en-1-yl, prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butyls such as butan-1-yl, butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl, but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-2-yl, buta-1,
• alkyl is specifically intended to include groups having any degree or level of saturation, i.e., groups having exclusively single carbon-carbon bonds, groups having one or more double carbon-carbon bonds, groups having one or more triple carbon-carbon bonds and groups having mixtures of single, double and triple carbon-carbon bonds. Where a specific level of saturation is intended, the expressions “alkanyl,” “alkenyl,” and “alkynyl” are used.
• an alkyl group comprises from 1 to 20 carbon atoms, more preferably, from 1 to 10 carbon atoms.
• (C 1 -C 6 ) alkyl for example, refers to an alkyl group containing from 1 to 6 carbon atoms.
• Alkanyl by itself or as part of another substituent refers to a saturated branched, straight-chain or cyclic alkyl radical derived by the removal of one hydrogen atom from a single carbon atom of a parent alkane.
• Typical alkanyl groups include, but are not limited to, methanyl; ethanyl; propanyls such as propan-1-yl, propan-2-yl (isopropyl), cyclopropan-1-yl, etc.; butanyls such as butan-1-yl, butan-2-yl (sec-butyl), 2-methyl-propan-1-yl (isobutyl), 2-methyl-propan-2-yl (t-butyl), cyclobutan-1-yl, etc.; and the like.
• Alkenyl by itself or as part of another substituent refers to an unsaturated branched, straight-chain or cyclic alkyl radical having at least one carbon-carbon double bond derived by the removal of one hydrogen atom from a single carbon atom of a parent alkene.
• the group may be in either the cis or trans conformation about the double bond(s).
• Typical alkenyl groups include, but are not limited to, ethenyl; propenyls such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), prop-2-en-2-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl; butenyls such as but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, etc.; and the like.
• Alkynyl by itself or as part of another substituent refers to an unsaturated branched, straight-chain or cyclic alkyl radical having at least one carbon-carbon triple bond derived by the removal of one hydrogen atom from a single carbon atom of a parent alkyne.
• Typical alkynyl groups include, but are not limited to, ethynyl; propynyls such as prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butynyls such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like.
• “Acyl” by itself or as part of another substituent refers to a radical —C(O)R 30 , where R 30 is hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl, heteroarylalkyl as defined herein.
• Representative examples include, but are not limited to, formyl, acetyl, cyclohexylcarbonyl, cyclohexylmethylcarbonyl, benzoyl, benzylcarbonyl and the like.
• “Acyl Chelate” by itself or as part of another substituent refers to a radical —C(O)R 31 , where R 31 is alkyl, cycloalkyl, aryl as defined herein substituted with a chelating group which binds an appropriate metal.
• R 32 is a chelating group such as, for example, DOTA, TETA, a polyamino carboxylate (e.g., NODAGA, EDTA, tricine, —C(O)CH 2 -DTPA, etc.) and where the chelating group is bound to metals such as positron emitting labels (e.g., 18 F, 45 Ti, 44 Sc, 55 Co, 61 Cu, 64 Cu, 66 Ga, 68 Ga, 75 Br, 76 Br, 86 Y, 110 In, 124 I, 89 Zr, 99 Tc), radionuclides (e.g., 137 Cs, 60 Co, 131 I, 123 I, 192 Ir, 90 Y, 67 Ga, 99 Tc, 123 I, 125 I, 131 I, 111 In, 97 Ru, 67 Cu, 68 Ga, 72 As, 89 Zr, 90 Y, 201 Tl, etc.)
• positron emitting labels e.g., 18 F,
• Alkoxy by itself or as part of another substituent refers to a radical —OR 31 where R 33 represents an alkyl or cycloalkyl group as defined herein. Representative examples include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, cyclohexyloxy and the like.
• Aryl by itself or as part of another substituent refers to a monovalent aromatic hydrocarbon radical derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system.
• Typical aryl groups include, but are not limited to, groups derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picen
• Arylalkyl by itself or as part of another substituent refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp 3 carbon atom, is replaced with an aryl group.
• Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and the like.
• an arylalkyl group is (C 6 -C 30 ) arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (C 1 -C 10 ) and the aryl moiety is (C 6 -C 20 ), more preferably, an arylalkyl group is (C 6 -C 20 ) arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (C 1 -C 8 ) and the aryl moiety is (C 6 -C 12 ).
• Cycloalkyl by itself or as part of another substituent refers to a saturated or unsaturated cyclic alkyl radical. Where a specific level of saturation is intended, the nomenclature “cycloalkanyl” or “cycloalkenyl” is used.
• Typical cycloalkyl groups include, but are not limited to, groups derived from cyclopropane, cyclobutane, cyclopentane, cyclohexane and the like.
• the cycloalkyl group is (C 3 -C 10 ) cycloalkyl, more preferably (C 3 -C 7 ) cycloalkyl.
• “Cycloheteroalkyl” by itself or as part of another substituent refers to a saturated or unsaturated cyclic alkyl radical in which one or more carbon atoms (and any associated hydrogen atoms) are independently replaced with the same or different heteroatom.
• Typical heteroatoms to replace the carbon atom(s) include, but are not limited to, N, P, O, S, Si, etc. Where a specific level of saturation is intended, the nomenclature “cycloheteroalkanyl” or “cycloheteroalkenyl” is used.
• Typical cycloheteroalkyl groups include, but are not limited to, groups derived from epoxides, azirines, thiiranes, imidazolidine, morpholine, piperazine, piperidine, pyrazolidine, pyrrolidine, quinuclidine, and the like.
• Diagnostically effective amount means the amount of a compound that, when administered to a patient for detection of a disease, is sufficient to detect the disease.
• the “diagnostically effective amount” will vary depending on the compound, the disease and its severity and the age, weight, etc., of the patient to be treated.
• Heteroalkyl, Heteroalkanyl, Heteroalkeniyl and Heteroalkvnyl by themselves or as part of another substituent refer to alkyl, alkanyl, alkenyl and alkynyl groups, respectively, in which one or more of the carbon atoms (and any associated hydrogen atoms) are independently replaced with the same or different heteroatomic groups.
• Typical heteroatomic groups which can be included in these groups include, but are not limited to, —O—, —S—, —O—O—, —S—S—, —O—S—, —NR 34 R 35 , — ⁇ N—N ⁇ —, —N ⁇ N—, —N ⁇ N—, —N ⁇ N—NR 36 R 37 , —PR 38 —, —P(O) 2 —, —POR 39 —, —O—P(O) 2 —, —SO—, —SO 2 —, —SnR 40 R 41 — and the like, where R 34 , R 35 , R 36 , R 37 , R 38 , R 39 , R 40 and R 41 are independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substitute
• Heteroaryl by itself or as part of another substituent refers to a monovalent heteroaromatic radical derived by the removal of one hydrogen atom from a single atom of a parent heteroaromatic ring system.
• Typical heteroaryl groups include, but are not limited to, groups derived from acridine, arsindole, carbazole, ⁇ -carboline, chromane, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine,
• the heteroaryl group is from 5-20 membered heteroaryl, more preferably from 5-10 membered heteroaryl.
• Preferred heteroaryl groups are those derived from thiophene, pyrrole, benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole, oxazole and pyrazine
• Heteroarylalkyl by itself or as part of another substituent refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp 3 carbon atom, is replaced with a heteroaryl group. Where specific alkyl moieties are intended, the nomenclature heteroarylalkanyl, heteroarylalkenyl and/or heterorylalkynyl is used.
• the heteroarylalkyl group is a 6-30 membered heteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the heteroarylalkyl is 1-10 membered and the heteroaryl moiety is a 5-20-membered heteroaryl, more preferably, 6-20 membered heteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the heteroarylalkyl is 1-8 membered and the heteroaryl moiety is a 5-12-membered heteroaryl.
• “Imino” by itself or as part of another substituent refers to a radical —C ⁇ NR 42 , where R 42 is hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl, heteroarylalkyl as defined herein.
• Parent aromatic Ring System refers to an unsaturated cyclic or polycyclic ring system having a conjugated ⁇ electron system. Specifically included within the definition of “parent aromatic ring system” are fused ring systems in which one or more of the rings are aromatic and one or more of the rings are saturated or unsaturated, such as, for example, fluorene, indane, indene, phenalene, etc.
• Typical parent aromatic ring systems include, but are not limited to, aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene and the like.
• Parent Heteroaromatic Ring System refers to a parent aromatic ring system in which one or more carbon atoms (and any associated hydrogen atoms) are independently replaced with the same or different heteroatom. Typical heteroatoms to replace the carbon atoms include, but are not limited to, N, P, O, S, Si, etc. Specifically included within the definition of “parent heteroaromatic ring systems” are fused ring systems in which one or more of the rings are aromatic and one or more of the rings are saturated or unsaturated, such as, for example, arsindole, benzodioxan, benzofuran, chromane, chromene, indole, indoline, xanthene, etc.
• Typical parent heteroaromatic ring systems include, but are not limited to, arsindole, carbazole, ⁇ -carboline, chromane, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline, tetrazole, thi
• Patient includes humans.
• human and “patient” are used interchangeably herein.
• “Pharmaceutically acceptable salt” refers to a salt of a compound of the invention, which is pharmaceutically acceptable and possesses the desired pharmacological activity of the parent compound.
• Such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzen
• “Pharmaceutically acceptable vehicle” refers to a diluent, adjuvant, excipient or carrier with which a compound of the invention is administered.
• Preventing refers to a reduction in risk of acquiring a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop in a patient that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease).
• “Substituted” refers to a group in which one or more hydrogen atoms are independently replaced with the same or different substituent(s).
• Typical substituents include, but are not limited to, —M, —R 60 , —O ⁇ , ⁇ O, —OR 60 , —SR 60 , —S ⁇ , ⁇ S, —NR 60 R 61 , ⁇ NR 60 , —CF 3 , —CN, —OCN, —SCN, —NO, —NO 2 , ⁇ N 2 , —N 3 , —S(O) 2 O; —S(O) 2 OH, —S(O) 2 R 60 , —OS(O 2 )O ⁇ , —OS(O) 2 R 60 , —P(O)(O ⁇ ) 2 , —P(O)(OR 60 )(O ⁇ ), —OP(O)(OR 60 )(OR 61 ),
• substituents include —M, —R 60 , ⁇ O, —OR 60 , —SR 60 , —S ⁇ , ⁇ S, —NR 60 R 61 , ⁇ NR 60 , —CF 3 , —CN, —OCN, —SCN, —NO, —NO 2 , ⁇ N 2 , —N 3 , —S(O) 2 R 60 , —OS(O 2 )O ⁇ , —OS(O) 2 R 60 , —P(O)(O ⁇ ) 2 , —P(O)(OR 60 )(O ⁇ ), —OP(O)(OR 60 )(OR 61 ), —C(O)R 60 , —C(S)R 60 , —C(O)OR 60 , —C(O)NR 60 R 61 ,—C(O)O ⁇ , —NR 62 C(O)NR 60 R 61 ,
• Treating” or “treatment” of any disease or disorder refers, in one embodiment, to ameliorating the disease or disorder (i.e., arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment “treating” or “treatment” refers to ameliorating at least one physical parameter, which may not be discernible by the patient. In yet another embodiment, “treating” or “treatment” refers to inhibiting the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In yet another embodiment, “treating” or “treatment” refers to delaying the onset of the disease or disorder.
• “Therapeutically effective amount” means the amount of a compound that, when administered to a patient for treating a disease, is sufficient to effect such treatment for the disease.
• the “therapeutically effective amount” will vary depending on the compound, the disease and its severity and the age, weight, etc., of the patient to be treated.
• the present invention satisfies these and other needs by providing peptide analogs of Ac—PHSCN—NH 2 which target tumor and endothelial cells and have anti-tumor, anti-angiogenic and anti-metasastic activity, methods of making these peptides, compositions thereof and methods of using these peptides and pharmaceutical compositions thereof to treat, prevent and detect diseases characterized by tumor growth, metastasis and angiogenesis.
• the peptide analogs may serve, inter alia, as carriers of radioactivity, PET-active compounds, toxins, fluorescent molecules and PEG molecules.
• the present invention provides a compound of Formula (I):
• R 1 is not acetyl when R 4 and R 5 are hydrogen.
• p and q are independently an integer between and including 1 and 50. In another embodiment, p and q are independently an integer between and including 1 and 25. In still another embodiment, p and q are independently an integer between and including 1 and 10. In still another embodiment, p and q are independently an integer between and including 1 and 5. In still another embodiment, p and q are independently an integer between and including 1 and 3.
• p and q are independently an integer between and including 0 and 50. In another embodiment, p and q are independently an integer between and including 0 and 25. In still another embodiment, p and q are independently an integer between and including 0 and 10. In still another embodiment, p and q are independently an integer between and including 0 and 5. In still another embodiment, p and q are independently an integer between and including 0 and 3.
• s is 0 and r is 1. In still another embodiment, s is 1 and r is 0. In still another embodiment, at least one of s and r is not 0.
• R 1 is acyl, substituted acyl, acyl chelate, imino or substituted imino.
• R 2 is C 1 -C 6 alkyl with at least one hydrogen atom replaced by a substituent selected from the group consisting of —NR 6 R 7 , —OR 8 and —CO 2 R 9 .
• R 6 , R 7 , R 8 , R 9 , R 10 , R 11 and R 12 are independently selected from the group consisting of hydrogen, acyl, substituted acyl, acyl chelate, imino or substituted imino.
• X 1 is —NHCH 2 CO— or —NHCH(CH 3 )CO—.
• X 2 is
• l is 1. In still another embodiment, n is 1 or 2. In still another embodiment, m is 1 or 2.
• R 3 is C 1 -C 6 alkyl with at least one hydrogen atom replaced by a substituent selected from the group consisting of —NR 15 R 16 , —OR 17 and —CO 2 R 18 .
• R 15 , R 16 , R 17 , R 18 , R 19 , R 20 and R 21 are independently selected from the group consisting of hydrogen, acyl, substituted acyl, acyl chelate, imino or substituted imino.
• compounds of the invention has the structure of Formula (II):
• R 1 is
• r 0, R 1 is acetyl, R 4 is hydrogen, R 14 is hydrogen, methyl or acetyl and R 5 is
• r 0, R 4 and R 5 are hydrogen, R 14 is hydrogen, methyl or acetyl and R 1 is
• r 0, R 4 and R 5 are hydrogen, R 14 is hydrogen, methyl or acetyl and R 1 is
• r 0, R 4 and R 5 are hydrogen, R 14 is hydrogen, methyl or acetyl and R 1 is
• r 0, R 4 and R 5 are hydrogen, R 14 is hydrogen, methyl or acetyl and R 1 is and M is Cu, Ga, 111 In or 90 Y.
• r 0, R 4 and R 5 are hydrogen, R 14 is hydrogen, methyl or acetyl and R 1 is and M is Cu.
• r 0, R 4 and R 5 are hydrogen, R 14 is hydrogen, methyl or acetyl and R 1 is and M is 111 In, 90 Y or Ga.
• r 0, R 4 and R 5 are hydrogen, R 14 is hydrogen, methyl or acetyl, R 1 is and M is 111 In, 67 Ga or 68 Ga.
• r 0, R 4 and R 5 are hydrogen, R 14 is hydrogen, methyl or acetyl and R 1 is
• r 0, R 4 and R 5 are hydrogen, R 14 is hydrogen, methyl or acetyl and R 1 is
• r 0, R 4 and R 5 are hydrogen, R 14 is hydrogen, methyl or acetyl and R 1 is
• r 0, R 4 and R 5 are hydrogen, R 14 is hydrogen, methyl or acetyl and R 1 is
• r 0, R 4 and R 5 are hydrogen, R 14 is hydrogen, methyl or acetyl and R 1 is
• r 0, R 4 and R 5 are hydrogen, R 14 is hydrogen, methyl or acetyl and R 1 is
• r 0, R 4 and R 5 are hydrogen, R 14 is hydrogen, methyl or acetyl and R 1 is
• r 0, R 4 and R 5 are hydrogen, R 14 is hydrogen, methyl or acetyl and R 1 is
• r 0, R 4 and R 5 are hydrogen, R 4 is hydrogen, methyl or acetyl and R 1 is
• R 1 is acyl or substituted acyl
• R 2 is C 1 -C 4 alkyl with at least one hydrogen atom replaced by a substituent selected from the group consisting of —NR 6 R 7 , aryl and substituted aryl
• R 6 and R 7 are independently selected from the group consisting of hydrogen, acyl and substituted acyl
• X 1 is —NH(CH 2 ) h CO—
• X 2 is
• X 4 is
• X 5 is R 13 is hydrogen, acyl, substituted acyl, alkyl or substituted alkyl.
• X 6 is X 7 is —NH(CH 2 ) e CO—
• R 3 is C 1 -C 4 alkyl with at least one hydrogen atom replaced by a substituent selected from the group consisting of —NR 15 R 16 , aryl and substituted aryl
• R 15 and R 16 are independently selected from the group consisting of hydrogen, acyl and substituted acyl and R 4 and R 5 are hydrogen.
• s is 0 and r is 1
• k is 1
• R 1 is acetyl
• R 13 is hydrogen
• e is 1
• R 3 is —(CH 2 ) 4 NH 2 —.
• q is 2, 4 or 6.
• s is 0 and r is 1, k is 1, R 1 is acetyl, R 13 is hydrogen, e is 2, 4 or 6 and R 3 is —CH 2 ) 4 NHCO(CH 2 ) 2 —Ph—(4—OH).
• q is 1.
• s is 0 and r is 1, k is 1, R 1 is acetyl, R 13 is hydrogen, e is 2, 4 or 6 and R 3 is —CH 2 —Ph—(4—OH).
• q is 1.
• s is 0 and r is 1, k is 1, R 1 is acetyl, R 13 is methyl, e is 1 and R 3 is —(CH 2 ) 4 NH 2 .
• q is 2.
• s is 1 and r is 0, j is 1, R 1 is acetyl, R 13 is hydrogen, h is 1 and R 2 is —CH 2 —Ph—(4—OH).
• p is 2, 4 or 6.
• s is 1 and r is 0, j is 1, R 1 is acetyl, R 13 is hydrogen, h is 2, 4, or 6 and R 2 is —CH 2 —Ph—(4—OH).
• p is 1.
• s is 1 and r is 0, j is 0, R 1 is —CO(CH 2 ) 2 —Ph—(4—OH), R 13 is hydrogen and h is 1.
• p is 2, 4 or 6.
• s is 1 and r is 0, j is 0, R 1 is —O(CH 2 ) 2 —Ph—(4—OH), R 13 is hydrogen and h is 2, 4 or 6.
• p is 1.
• s is 0 and r is 0, R 1 is —(CH 2 ) 2 —Ph—(4—OH) and R 13 is hydrogen.
• s is 0 and r is 0, R 1 is —COPh—(4—F) and R 13 is hydrogen.
• s is 0 and r is 1, k is 1, R 1 is acetyl, R 13 is methyl or hydrogen, e is 1 and R 3 is —CH 2 ) 4 NHCOPh—(4—F).
• q is 2.
• s is 0 and r is 1, k is 1, R 1 is acetyl, R 13 is hydrogen, e is 1 and R 3 is —(CH 2 ) 4 NH—8-[4 40 —fluorobenzylamino]suberoyl or —(CH 2 ) 4 NHCOCH 2 F.
• q is 2.
• s is 1 and r is 0, j is 0, R 1 is 8-[4′-fluorobenzylamino]suberoyl or —COCH 2 F, R 13 is hydrogen and h is 2.
• p is 1.
• s is 0 and r is 1, k is 1, R 1 is acetyl, R 13 is hydrogen and R 3 is —CH 2 Ph—(3-I, 4-OH) or —CH 2 Ph—(3,5-diI, 4-OH).
• q is 0.
• q is 1 and e is 2.
• q is 1 and e is 1.
• s is 1 and r is 0, j is 1, R 1 is acetyl, R 13 is hydrogen and R 2 is —CH 2 Ph—(3-I, 4-OH) or —CH 2 Ph—(3,5-diI, 4-OH).
• p is 0.
• s is 0 and r is 0, R 1 is —CO(CH 2 ) 2 Ph (4-OH, 3, 5 di-I) and R 13 is hydrogen.
• s is 1 and r is 0, j is 0, R 1 is —CO(CH 2 ) 2 Ph (4-OH, 3, 5 di-I), h is 2 and R 13 is hydrogen.
• p is 1.
• s is 1 and r is 0, j is 1, R 1 is acetyl, R 2 is —CH 2 —Ph (4-OH, 3, 5 di-I), h is 2 and R 13 is hydrogen.
• p is 1.
• s is 0 and r is 1, R 3 is —(CH 2 ) 4 NHCO(CH 2 ) 2 —Ph (4-OH, 3, 5 di-I), e is 1 and R 13 is hydrogen.
• q is 2.
• R 1 is acyl chelate
• R 2 , R 6 , R 7 , X 1 , X 2 , X 4 , X 5 , R 13 , X 6 , X 7 , R 3 , R 15 , R 16 , R 4 and R 5 are as defined in the first embodiment.
• s is 1 and r is 0, j is 0, R 1 is DOTA-In, h is 2 and R 13 is hydrogen.
• p is 1.
• s is 0 and r is 0, R 1 is DPTA or DPTA-In and R 13 is hydrogen.
• the present invention provides a compound of Formula (III):
• R 20 is acyl, substituted acyl, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, imino, substituted imino or a diagnostic agent;
• R 21 is C 1 -C 6 alkyl with at least one hydrogen atom replaced by a substituent selected from the group consisting of —NHR 22 ;
• R 22 is hydrogen, acyl, substituted acyl, alkyl, substituted alkyl or a diagnostic agent
• R 2 , X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , R 4 and R 5 are as defined in structural formula (I);
• R 20 and R 22 are diagnostic agents.
• R 2 , X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , R 4 and R 5 are as defined in the first preferred embodiment.
• R 20 is a fluorescent agent.
• R 20 is ⁇ fraction (5/6) ⁇ carboxy fluorescein, s is 1, r is 0, j is 0, e is 2 and p is 1.
• R 22 is a fluorescent agent.
• R 21 is (CH 2 ) 4 NH—
• R 22 is— ⁇ fraction (5/6) ⁇ carboxy fluorescein, s is 0, r is 1, k is 1, e is 1 and q is 2.
• R 21 is (CH 2 )4NH—
• R 22 is biotin
• s is 0, r is 1, k is 1, e is 1 and q is 2.
• the present invention provides a compound of Formula (IV):
• R 23 is acyl, substituted acyl, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, imino, substituted imino or a pegylating agent;
• R 24 is C 1 -C 6 alkyl with at least one hydrogen atom replaced by a substituent selected from the group consisting of —NHR 28 wherein R 28 is hydrogen, acyl, substituted acyl, alkyl substituted alkyl or a pegylating agent; and
• R 2 , X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , R 4 and R 5 are as defined in structural formula (I);
• R 23 or R 28 is a pegylating agent.
• R 2 , X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , R 4 and R 5 are as defined in the first preferred embodiment.
• R 23 is m-dPEG, s is 1, r is 0, j is 0, h is 2 and p is 1.
• the present invention provides a compound of Formula (V):
• R 29 is C 1 -C 6 alkyl with at least one hydrogen atom replace by —NHR 32 ;
• R 30 is acyl, substituted acyl, alkyl, substituted alkyl or a therapeutic agent.
• R 31 is hydrogen, alkyl, substituted alkyl or a therapeutic agent
• R 32 is hydrogen, acyl substituted acyl, alkyl, substituted alkyl or a therapeutic agent
• R 2 , X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 and R 4 and R 5 are as defined structural formula (I);
• R 30 , R 31 and R 32 is a therapeutic agent.
• R 2 , X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 and R 4 are as defined in the first preferred embodiment
• R 13 is methyl or acetyl
• s is 0, r is
• R 30 is acetyl
• R 31 is a therapeutic agent
• the therapeutic agent is doxorubicin.
• R 13 is methyl or hydrogen
• s is 0, r is 1
• k is 1
• e 1, q is 2
• R 30 is acetyl
• R 31 is hydrogen
• R 29 is —CH 2 ) 4 NHR 32 .
• R 32 is —CO(CH 2 ) 3 -doxorubicin or protoporphyrin.
• variations of compounds invention includes, for example, the D-amino acids of the naturally occurring amino acids, ⁇ -alanine, 3-aminopropionic acid, 2,3 diaminopropionic acid, 4-aminobutyric acid, etc., sarcosine, orthinine, N-methyl glycine, citrulline, t-butyl alanine, homoarginine, etc. are within the scope of the present invention
• One or amide bonds in the compounds of the invention may be optionally replaced by isosteres such as —CH 2 —NH—, —CH 2 —S—, —CH 2 —S(O)—, CH 2 —S(O) 2 —, —COCH 2 — —CH ⁇ CH—, CH(OH)CH 2 which are well known in the art (see, e.g., Spatola, “Chemistry and Biochemistry of Amino Acids, Peptides and Proteins,” B. Weinstein, (eds.), Marcel Dekker, New York, 1983; Spatola et al., Life Sci. 1986, 38:1243-1249; Almquist et al., J. Med. Chem.
• the peptides of the invention may also contain peptide mimetics such as those described in Olson et al., J. Med. Chem. 1993, 36:3039 and Chorev et aL, Science 1979, 204:1210.
• Covalent modifications of the compounds of the invention are within the scope of the current invention and may improve the solubility, absorption, biological half life, etc. Such modifications may be effected by selective reaction of specific amino acid residues with organic reagents. For example, histidine residues may be selectively reacted with diethylpyrocarbonate at pH 5.5-7 and p-bromophenacyl bromide at pH 6.0. Residues containing free amino groups may be selectively reacted with carboxylic acid anhydrides, imidoesters, pyridoxal phosphate, trinitrobenzenesulfonic acid, O-methylisourea, 2,4 pentanedione, glyoxylate, etc.
• Arginyl residues may be selectively reacted with phenylglyoxal, and various diones. Glutaminyl and asparaginyl residues may be deaminated under mildly acidic conditions to provide the corresponding glutamyl and aspartyl residues. Proline and lysine may be selectively hydroxylated while serine and threonine residues may be selectively phosphorylated. The ⁇ -amino groups of histidine and lysine may be selectively methylated (Creighton, Proteins: Structure and Molecule Properties , W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)).
• bi-fnctional cross-linking agents e.g., 1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxy-succinimide esters, esters of 4-azidosalicylic acid, homobifunctional imidoesters (e.g., disuccinimidyl esters such as 3,3′- dithiobis(succinimidylpropionate)), bifinctional maleimides (e.g., bis-N-maleimido-1,8-octane, etc.) may be used to link compounds with water-insoluble support matrices or other macromolecular carriers.
• bi-fnctional cross-linking agents e.g., 1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxy-succinimide esters, esters of 4-azidosalicylic acid, homobifunctional imidoesters
• Photoactivatable agents such as methyl-3-[(p-azidophenyl) dithio]propioimidate may also be used to attach compounds with water-insoluble support matrices.
• compounds may be directly reacted with reactive water-insoluble matrices (e.g., cyanogen bromide-activated carbohydrates).
• the present invention also includes longer peptides comprised of repeating units of the amino acid sequences of the compounds of the invention.
• the repeating unit of such a multimer is the amino acid sequence of a compound where a, b, x, y, and z are 1.
• the repeating unit is the amino acid sequence of a compound of invention where only one of a, b, x, y, and z is 0 and the rest are 1.
• a multimer may be comprised of either the same or different combinations of repeating units comprised of amino acid sequences of compounds of structural formula (I).
• Such multimeric peptides can be made by either by chemical synthesis or by recombinant DNA techniques, followed by chemical modification of the cysteine residues.
• the synthetic multimers Preferably, have 2 to 12 repeats, more preferably, 2 to 8 repeats of the core peptide sequence. Accordingly, the total number of amino acids in the multimer should not exceed about 110 residues (or the equivalents, when including linkers or spacers).
• a preferred multimer has the formula P 1 n . where P 1 is a pentapeptide, n is 2 to 8.
• a multimer has the formula (P 1 -X m ) n -P 2 where P 1 and P 2 are pentapeptides.
• P 1 and P 2 may be the same or different and each P 1 may represent a different pentapeptide derivative of structural formula (I).
• the multimer may be optionally functionalized at both the N- and C-termini.
• Compounds of the invention may be modified by the covalent attachment of any type of molecule as long as the modification does not prevent or inhibit biological function (i.e., inhibition or prevention of angiogenesis, cell invasion, cell proliferation, etc.).
• a compound of the invention may be modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, proteolytic cleavage, linkage to cellular ligand or protein, etc.
• compounds of the invention are conjugated to a therapeutic agent or a diagnostic agent either directly or through a linking moiety.
• the linking moiety is first attached to a diagnostic or therapeutic agent to form a linking moiety intermediate which is then further attached to a compound of structural formula (I).
• the linking moiety can also be first attached to a compound of the invention to form a linking moiety intermediate which can then be attached to a diagnostic agent or therapeutic agent.
• a linking moiety will include a linker and a linking group for conjugating a therapeutic agent or diagnostic agent to a peptide.
• the nature of the linker will depend upon the particular application and the type of conjugation desired as the linker may be hydrophilic or hydrophobic, long or short, rigid or flexible.
• the linker may be optionally substituted with one ore more linking groups which may be either the same or different, accordingly providing polyvalent linking moieties which are capable of conjugating multiple therapeutic agents or diagnostic agents with a antibody.
• linkers comprised of stable bonds suitable for spacing linking groups from the amino nitro compound are known in the art, and include by way of example and not limitation, alkyl, heteroalkyl, acyclic heteroatomic bridges, aryl, arylaryl, arylalkyl, heteroaryl, heteroaryl-heteroaryl, substituted heteroaryl-heteroaryl, heteroarylalkyl, heteroaryl-heteroalkyl and the like.
• the linker may include single, double, triple or aromatic carbon-carbon bonds, nitrogen-nitrogen bonds, carbon-nitrogen, carbon-oxygen bonds and/or carbon-sulfur bonds.
• finctionalities such as carbonyls, ethers, thioethers, carboxarnides, sulfonamides, ureas, urethanes, hydrazines, etc. may be included in a linker.
• linker may be rigid polyunsaturated alkyl or an aryl, biaryl, heteroaryl, etc.
• the linker may be a flexible peptide such as Gly-Gly-Gly or a flexible saturated alkanyl or heteroalkanyl.
• Hydrophilic linkers may be, for example, polyalcohols or polyethers such as polyalkyleneglycols.
• Hydrophobic linkers may be, for example, alkyls or aryls.
• a linking group is capable of mediating formation of a covalent bond with complementary reactive functionality of, for example, peptide to provide the therapeutic agent or diagnostic agent conjugated to the peptide.
• the linking group may be any reactive functional group known to those of skill in the art that will react with common chemical groups found in peptides (e.g., amino, sulfhydryl, hydroxyl, carboxylate, imidizaloyl, guandinium, amide, etc.).
• the linking group may be, for example, a photochemically activated group, an electrochemically activated group, a free radical donor, a free radical acceptor, a nucleophilic group or an electrophilic group.
• the linking group may be —NHR 1 , —NH 2 , —OH, —SH, halogen, —CHO, —R 1 CO, —SO 2 H, —PO 2 H, —N 3 , —CN, —CO 2 H, —SO 3 H, —PO 3 H, —PO 2 (O R 1 )H, —CO 2 R 1 , —SO 3 R 1 or —PO(OR 1 ) 2 where R 1 is alkyl.
• the linking group is —NHR 1 , —NH 2 , —OH, —SH, —CHO, —CO 2 H, R 1 CO—, halogen and —CO 2 R 1 .
• linker and the linking group include, for example, compounds where the linker is —(CH 2 ) n —, n is an integer between 1 and 8, the linking group is —NH 2 , —OH, —CO 2 H, and —CO 2 R 1 and the corresponding analogues where any suitable hydrogen is substituted.
• Other embodiments of the linking moiety include any amino acid, which may be, for example, a D or L amino acid.
• the linking moiety may be a dipeptide, a tripeptide or a tetrapeptide comprised of any combination of amino acids.
• the polarity of the peptide bond in these peptides may be either C—N or N—C.
• Therapeutic agents and diagnostic agents may be linked to peptides directly using a variety of conventional reactions known to the skilled artisan.
• condensation reagents e.g., carbodiimides, carbonyldiimidazoles, etc.
• residues such as glutamic acid, aspartic acid and the C-terminal carboxyl group of a compound of structural formula (I).
• diagnostic agents and therapeutic agents containing a linker and linking group may be attached to the amino group of lysine, the carboxylic acid groups of glutamic acid and aspartic acid, the sulfhydryl group of cysteine, the hydroxyl groups of threonine and serine and the various moieties of aromatic amino acids of peptides using conventional approaches known to the skilled artisan.
• selection of an appropriate strategy for conjugating diagnostic agents or therapeutic agents to a peptide either directly or through a linker and linking group is well within the ambit of the skilled artisan.
• Therapeutic agents which can be conjugated to peptides include, but are not limited to, radionuclides, porphyrins and porphyrin derivatives for photodynamic therapy (e.g., protoporphyrin, benzoporphyrin derivative monoacid A, tin-etio purpurin, meta-tetrahydroxyphenylchlorin, HPD, photofrin, protoporhyrin IX, Pc4, mono aspartyl chlorin e 6 , for others see T. Hassan et al, “PhotoDynamic Therapy of Cancer” in Cancer medicine, fiflh edition, R. C. Blast et al., Ed., B. C. Decker Inc, Canada, 2000, p.
• photodynamic therapy e.g., protoporphyrin, benzoporphyrin derivative monoacid A, tin-etio purpurin, meta-tetrahydroxyphenylchlorin, HPD, photofrin, pro
• protein toxins e.g., ricin, Pseudomonas exotoxin, diptheria toxin, saporin, pokeweed antiviral protein, bouganin, etc.
• cytotoxic cancer agents e.g., camptothecins (e.g., 9-nitrocamptothecin (9NC), 9-aminocamptothecin (9AC), 10-aminocamptothecin, 9-chlorocamptothecin, 10,11-methylendioxycamptothecin, irinothecin, aromatic camptothecin esters, alkyl camptothecin esters, topotecan, (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy4-methyl-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1 ,2-b]quinoline-10,13(9H,15H)-d
• the therapeutic agent is a cytotoxic cancer agent, such as, for example, a taxane, a camptothecin, an epithilone or a anthracycline.
• the therapeutic agent is doxorubicin.
• the therapeutic agent is a radionuclide.
• peglyating agents include, but are not limited to, a-methoxy-w-carboxy-PEG 2K & 5K1, a-methoxy-w-N-succinimidylglutarate-PEG 2K & 5K1, a-methoxy-w-glutarate-PEG 2K, 5K, 20K, 30K2, a-methoxy-w-GGGglutarate-PEG 2K & 5K1, mPEG-Succinimidyl propionate 2K, 5K, 20K, 30K2 and m-PEG-ButyrALD 2K, 5K, 20K, 30K2 (for other peglyating agents see Li et al., Biomacromolecules, 2003, 4, 1055.1067). Common pegylating agents are also available from commercial supplies such as Nektar Therapeutics, San Carlos, Calif. Methods for attachment of various PEG groups
• diagnostically labeled means that a peptide has an attached diagnostically detectable label.
• labels include but are not limited to, radioactive isotopes, paramagnetic isotopes, compounds which can be imaged by positron emission tomography (PET), fluorescent or colored compounds, compounds which can be imaged by magnetic resonance, chemiluminescent compounds, bioluminescent compounds, etc.
• Suitable detectable labels include, but are not limited to, radioactive, fluorescent, fluorogenic or chromogenic labels.
• Useful radiolabels (radionuclides) which are detected simply by gamma counter, scintillation counter or autoradiography include, but are not limited to, 3 H, 125 I, 131 I, 35 S and 14 C.
• the metals are preferably detectable metal atoms, including radionuclides, and are complexed to proteins and other molecules (See, e.g., U.S. Pat. Nos. 5,627,286, 5,618,513, 5,567,408, 5,443,816 and 5,561,220).
• Common fluorescent labels include, but are not limited to, fluorescein, rhodamine, dansyl, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine (Haugland, Handbook of Fluorescent Probes and Research Chemicals , Sixth Ed., Molecular Probes, Eugene, Or., 1996) may be used to label compounds of structural formula (I). Fluorescein, fluorescein derivatives and fluorescein-like molecules such as Oregon GreenTM and its derivatives, Rhodamine GreenTM and Rhodol GreenTM, are coupled to amine groups using the isothiocyanate, succinimidyl ester or dichlorotriazinyl-reactive groups.
• fluorophores may also be coupled to thiols using maleimide, iodoacetamide, and aziridine-reactive groups.
• the long wavelength rhodamines which are basically Rhodamine GreenTM derivatives with substituents on the nitrogens are preferred labeling reagents. This group includes the tetramethylrhodamines, X-rhodamines and Texas RedTM derivatives.
• Other preferred fluorophores are those excited by ultraviolet light. Examples include, but are not limited to, cascade blue, coumarin derivatives, naphthalenes (of which dansyl chloride is a member), pyrenes and pyridyloxazole derivatives.
• Inorganic materials such as semiconductor nanocrystals (Bruchez, et al., 1998, Science 281:2013-2016) and quantum dots, e.g., zinc-sulfide-capped Cd selenide (Chan, et al., Science 1998, 281:2016-2018) may also be used as diagnostic labels.
• Peptides can also be labeled with fluorescence-emitting metals such as 152 Eu or others of the lanthanide series. These metals can be attached to compounds of structural formula (I) through acyl chelating groups such as
• DTPA diethylenetriaminepentaacetic acid
• EDTA ethylene-diamine-tetraacetic acid
• Radionuclides may be attached to peptides either directly or indirectly using an acyl chelating group such as DTPA and EDTA for in vivo diagnosis.
• an acyl chelating group such as DTPA and EDTA
• the chemistry of chelation is well known in the art and varying ranges of chelating agent to peptide may be used to provide the labeled peptide.
• the labeled peptide must retain the biological activity of the native peptide.
• radionuclide having diagnostic or therapeutic value can be used as the radiolabel in the present invention.
• the radionuclide is a y -emitting or beta -emitting radionuclide, for example, one selected from the lanthanide or actinide series of the elements.
• Positron-emitting radionuclides e.g 68 Ga or 64 Cu, may also be used.
• Suitable gamma -emitting radionuclides include those which are useful in diagnostic imaging applications.
• the gamma -emitting radionuclides preferably have a half-life of from 1 hour to 40 days, preferably from 12 hours to 3 days. Examples of suitable gamma -emitting radionuclides include 67 Ga, 111 In, 99m Tc, 169Yb and 186 Re. Most preferably, the radionuclide is 99m Tc.
• radionuclides are 67 Cu, 67 Ga, 68 Ga, 72 As, 89 Zr, 90 Y, 97 Ru, 99 Tc, 111 In, 123 I, 125 I, 131 I, 169 Yb, 186 Re, and 201 Tl. Though limited work have been done with positron-emitting radiometals as labels, certain proteins, such as transferrin and human serum albumin, have been labeled with 68 Ga.
• a number of metals (not radioisotopes) useful for magnetic resonance imaging include gadolinium, manganese, copper, iron, gold and europium. Gadolinium is most preferred.
• the amount of labeled peptide needed for detectability in diagnostic use will vary depending on considerations such as age, condition, sex, and extent of disease in the patient, contraindications, if any, and other variables, and is to be adjusted by the individual physician or diagnostician. Dosage can vary from 0.01 mg/kg to 100 mg/kg.
• Peptides may also be detected by coupling to a phosphorescent or a chemiluminescent compound, as is well known to the skilled artisan.
• Preferred chemiluminescent compounds include but are not limited to, luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
• bioluminescent compounds may be used to detect antibodies and/or conjugates thereof and include, but are not limited to, luciferin, luciferase and aequorin.
• Colorimetric detection based on chromogenic compounds which have, or result in, chromophores with high extinction coefficients may also be used to detect compounds of structural formula (I).
• the compounds of the invention may be obtained via conventional synthetic methods.
• Starting materials useful for preparing compounds of the invention and intermediates thereof are commercially available or can be prepared by well-known synthetic methods.
• Peptides may be prepared using solid-phase synthesis such as that generally described by Merrifield, J. Amer. Chem. Soc. 1963, 85:2149-54 using automated equipment, which may be purchased from chemical suppliers (e.g., Applied Biosystems, Foster City, Calif.) or manual equipment.
• Solid-phase peptide synthesis may be initiated from the C-terminus of the peptide by coupling a protected ⁇ -amino acid (either Boc or FMOC protected), to a suitable resin.
• a starting material can be prepared by attaching an ⁇ -amino-protected amino acid by an ester linkage to a chloromethylated resin, hydroxymethyl resin, BHA resin, MBHA resin or a Rink resin.
• transwells are coated with type I collagen (50 ⁇ g/mL) by adding 200 ⁇ L of the collagen solution per transwell, then incubating overnight at 37° C.
• the transwells are assembled in a 24-well plate and a chemoattractant (e.g., FGF-2) is added to the bottom chamber in a total volume of 0.8 mL media.
• ECs such as human umbilical vein endothelial cells (HUVEC), which have been detached from monolayer culture using typsin, are diluted to a final concentration of about 10 6 cells/niL with serum-free media and 0.2 mL of this cell suspension is added to the upper chamber of each transwell.
• a chemoattractant e.g., FGF-2
• Inhibitors to be tested may be added to both the upper and lower chambers and the migration is allowed to proceed for 5 hrs in a humidified atmosphere at 37° C.
• the transwells are removed from the plate stained using DiffQuik®.
• Cells which did not migrate are removed from the upper chamber by scraping with a cotton swab and the membranes are detached, mounted on slides, and counted under a high-power field (400x) to determine the number of cells migrated.
• Matrigel® is a reconstituted basement membrane containing type IV collagen, laminin, heparan sulfate proteoglycans such as perlecan, which bind to and localize bFGF, vitronectin as well as transforming growth factor- ⁇ (TGF ⁇ ), urokinase-type plasminogen activator (uPA), tissue plasminogens activator (tpA) and the serpin known as plasminogen activator inhibitor type 1 (PAI- 1) (Chambers et al., Canc. Res. 1995, 55:1578-1585).
• TGF ⁇ transforming growth factor- ⁇
• uPA urokinase-type plasminogen activator
• tpA tissue plasminogens activator
• PAI- 1 plasminogen activator inhibitor type 1
• Such assays employ transwell tissue culture inserts.
• Invasive cells are defined as cells which are able to traverse through the Matrigel® and upper aspect of a polycarbonate membrane and adhere to the bottom of the membrane.
• Transwells (Costar) containing polycarbonate membranes (8.0 ⁇ m pore size) are coated with Matrigel® (Collaborative Research), which has been diluted in sterile PBS to a final concentration of 75 ⁇ g/mL (60 ⁇ L of diluted Matrigel® per insert), and placed in the wells of a 24-well plate.
• the membranes are dried overnight in a biological safety cabinet, then rehydrated by adding 100 ⁇ L of DMEM containing antibiotics for 1 hour on a shaker table.
• the DMEM is removed from each insert by aspiration and 0.8 mL of DMEM/10% FBS/antibiotics is added to each well of the 24-well plate such that it surrounds the outside of the transwell (“lower chamber”).
• Fresh DMEM/antibiotics 100 ⁇ L
• human Glu-plasminogen 5 ⁇ g/mL
• any inhibitors to be tested are added to the top, inside of the transwell (“upper chamber”).
• the cells which are to be tested are trypsinized and resuspended in DMEM/antibiotics, then added to the top chamber of the transwell at a final concentration of 800,000 cells/mL.
• the final volume of the upper chamber is adjusted to 200 ⁇ L.
• the assembled plate is then incubated in a humid 5% CO 2 atmosphere for 72 hours. After incubation, the cells are fixed and stained using DiffQuik® (Giemsa stain) and the upper chamber is then scraped using a cotton swab to remove the Matrigel® and any cells which did not invade through the membrane.
• the membranes are detached from the transwell using an X-acto® blade, mounted on slides using Permount® and cover-slips, then counted under a high-powered (400x) field. An average of the cells invaded is determined from 5-10 fields counted and plotted as a function of inhibitor concentration.
• Endothelial cells for example, human umbilical vein endothelial cells (HUVEC) or human microvascular endothelial cells (HMVEC) which can be prepared or obtained commercially, are mixed at a concentration of 2 ⁇ 10 5 cells/mL with fibrinogen (5 mg/mL in phosphate buffered saline (PBS) in a 1:1 (v/v) ratio.
• fibrinogen 5 mg/mL in phosphate buffered saline (PBS) in a 1:1 (v/v) ratio.
• Thrombin is added (5 units/mL final concentration) and the mixture is immediately transferred to a 24-well plate (0.5 mL per well).
• the fibrin gel is allowed to form and then VEGF and bFGF are added to the wells (each at 5 ng/mL final concentration) along with the test compound.
• the cells are incubated at 37° C.
• results are expressed as the average of 5 different wells for each concentration of compound.
• cells typically, in the presence of angiogenic inhibitors, cells remain either rounded or form undifferentiated tubes (e.g. 0 or 1 branch). This assay is recognized in the art to be predictive of angiogenic (or anti-angiogenic) efficacy in vivo (Min et aL, Cancer Res. 1996, 56: 2428-2433).
• endothelial cell tube formation is observed when endothelial cells are cultured on Matrigel® (Schnaper et aL, J. Cell. PhysioL 1995, 165: 107-118). Endothelial cells (1 ⁇ 10 4 cells/well) are transferred onto Matrigel®-coated 24-well plates and tube formation is quantitated after 48 hrs. Inhibitors are tested by adding them either at the same time as the endothelial cells or at various time points thereafter. Tube formation can also be stimulated by adding (a) angiogenic growth factors such as bFGF or VEGF, (b) differentiation stimulating agents (e.g., PMA) or (c) a combination of these.
• angiogenic growth factors such as bFGF or VEGF
• differentiation stimulating agents e.g., PMA
• this assay models angiogenesis by presenting to the endothelial cells a particular type of basement membrane, namely the layer of matrix which migrating and differentiating endothelial cells might be expected to first encounter.
• the matrix components found in Matrigel® (and in basement membranes in situ) or proteolytic products thereof may also be stimulatory for endothelial cell tube formation which makes this model complementary to the fibrin gel angiogenesis model previously described (Blood et aL, Biochim. Biophys. Acta 1990, 1032: 89-118; Odedrat al., Pharmac. Ther. 1991, 49: 111-124).
• the ability of the compounds of the invention to inhibit the proliferation of EC's may be determined in a 96-well format.
• Type I collagen (gelatin) is used to coat the wells of the plate (0.1-1 mg/mL in PBS, 0.1 mL per well for 30 minutes at room temperature). After washing the plate (3x w/PBS), 3-6,000 cells are plated per well and allowed to attach for 4 hrs (37° C./5% CO 2 ) in Endothelial Growth Medium (EGM; Clonetics ) or M199 media containing 0.1-2% FBS.
• the media and any unattached cells are removed at the end of 4 hrs and fresh media containing bFGF (1-10 nglmL) or VEGF (1-10 ng/mL) is added to each well.
• Compounds to be tested are added last and the plate is allowed to incubate (37° C./5% CO 2 ) for 24-48 hrs.
• MTS Promega
• the absorbance at 490 nm, which is proportional to the cell number, is then measured to determine the differences in proliferation between control wells and those containing test compounds.
• a similar assay system can be set up with cultured adherent tumor cells. However, collagen may be omitted in this fonnat.
• Tumor cells e.g., 3,000-10,000/well
• Serum free medium is then added to the wells, and the cells are synchronized for 24 hrs.
• Medium containing 10% FBS is then added to each well to stimulate proliferation.
• Compounds to be tested are included in some of the wells. After 24 hrs, MTS is added to the plate and the assay developed and read as described above.
• the anti-proliferative and cytotoxic effects of compounds of the invention may be determined for various cell types including tumor cells, ECs, fibroblasts and macrophages. This is especially useful when testing a compound of the invention which has been conjugated to a therapeutic moiety such as a radiotherapeutic or a toxin.
• a conjugate of one of the compounds of the invention with Bolton-Hunter reagent which has been iodinated with 131 I would be expected to inhibit the proliferation of cells expressing an PHSCN binding site/receptor (most likely by inducing apoptosis).
• Anti-proliferative effects would be expected against tumor cells and stimulated endothelial cells but, under some circumstances not quiescent endothelial cells or normal human dermal fibroblasts. Any anti-proliferative or cytotoxic effects observed in the normal cells may represent non-specific toxicity of the conjugate.
• a typical assay would involve plating cells at a density of 5-10,000 cells per well in a 96-well plate.
• the compound to be tested is added at a concentration I Ox the IC 50 measured in a binding assay (this will vary depending on the conjugate) and allowed to incubate with the cells for 30 minutes.
• the cells are washed 3X with media, then fresh media containing [ 3 H]thymidine (1 ⁇ Ci/mL) is added to the cells and they are allowed to incubate at 37° C. in 5% CO 2 for 24 and 48 hours.
• Cells are lysed at the various time points using 1 M NaOH and counts per well determined using a ⁇ -counter.
• Proliferation may be measured non-radioactively using MTS reagent or CyQuant® to measure total cell number.
• MTS reagent or CyQuant® for cytotoxicity assays (measuring cell lysis), a Promega 96-well cytotoxicity kit is used. If there is evidence of anti-proliferative activity, induction of apoptosis may be measured using TumorTACS (Genzyme).
• the ability of the compounds of the invention to promote apoptosis of EC's may be determined by measuring activation of caspase-3.
• Type I collagen (gelatin) is used to coat a P100 plate and 5 ⁇ 10 5 ECs are seeded in EGM containing 10% FBS. After 24 hours (at 37° C. in5% CO 2 ) the medium is replaced by EGM containing 2% FBS, 10 ng/ml bFGF and the desired test compound. The cells are harvested after 6 hours, cell lysates prepared in 1% Triton and assayed using the EnzChek®Caspase-3 Assay Kit #1 (Molecular Probes) according to the manufactures instructions.
• Neovascularization is assessed at 5 and 7 days after implantation. On day 7, animals are anesthetized and infused with a dye such as colloidal carbon to stain the vessels. The animals are then euthanized, the corneas fixed with formalin, and the corneas flattened and photographed to assess the degree of neovascularization. Neovessels may be quantitated by imaging the total vessel area or length or simply by counting vessels.
• This assay is performed essentially as described by Passaniti et al., 1992, Lab Invest. 67:519-528. Ice-cold Matrigel® (e.g., 500 ⁇ L) (Collaborative Biomedical Products, Inc., Bedford, Mass.) is mixed with heparin (e.g., 50 ⁇ g/ml), FGF-2 (e.g., 400 ng/ml) and the compound to be tested. In some assays, bFGF may be substituted with tumor cells as the angiogenic stimulus.
• the Matrigel® mixture is injected subcutaneously into 4-8 week-old athymic nude mice at sites near the abdominal midline, preferably 3 injections per mouse.
• the injected Matrigel® forms a palpable solid gel. Injection sites are chosen such that each animal receives a positive control plug (such as FGF-2+heparin), a negative control plug (e.g., buffer+heparin) and a plug that includes the compound being tested for its effect on angiogenesis, e.g., (FGF-2+heparin+compound). All treatments are preferably run in triplicate. Animals are sacrificed by cervical dislocation at about 7 days post injection or another time that may be optimal for observing angiogenesis. The mouse skin is detached along the abdominal midline, and the Matrigel® plugs are recovered and scanned immediately at high resolution. Plugs are then dispersed in water and incubated at 37° C. overnight.
• a positive control plug such as FGF-2+heparin
• a negative control plug e.g., buffer+heparin
• All treatments are preferably run in triplicate. Animals are sacrificed by cervical dislocation at about 7 days post injection or another time that may be optimal for
• Hemoglobin (Hb) levels are determined using Drabkin's solution (e.g., obtained from Sigma) according to the manufacturers' instructions.
• the amount of Hb in the plug is an indirect measure of angiogenesis as it reflects the amount of blood in the sample.
• animals may be injected prior to sacrifice with a 0.1 ml buffer (preferably PBS) containing a high molecular weight dextran to which is conjugated a fluorophore.
• the amount of fluorescence in the dispersed plug determined fluorimetrically, also serves as a measure of angiogenesis in the plug.
• Staining with mAb anti-CD31 (CD31 is “platelet-endothelial cell adhesion molecule or PECAM”) may also be used to confirm neovessel formation and microvessel density in the plugs.
• This assay is performed essentially as described by Nguyen et al., Microvascular Res. 1994, 47:3140.
• a mesh containing either angiogenic factors (bFGF) or tumor cells plus inhibitors is placed onto the CAM of an 8-day old chick embryo and the CAM observed for 3-9 days after implantation of the sample.
• Angiogenesis is quantitated by determining the percentage of squares in the mesh which contain blood vessels.
• tumor cells for example 1 ⁇ 5 ⁇ 10 6 cells of the 3LL Lewis lung carcinoma or the rat prostate cell line MatLyLu, are mixed with Matrigel® and then injected into the flank of a mouse following the protocol described in Sec. B., above.
• a mass of tumor cells and a powerful angiogenic response can be observed in the plugs after about 5 to 7 days.
• the anti-tumor and anti-angiogenic action of a compound in an actual tumor environment can be evaluated by including it in the plug.
• Measurement is then made of tumor weight, Hb levels or fluorescence levels (of a dextran-fluorophore conjugate injected prior to sacrifice).
• the plugs are first homogenize with a tissue homogenizer.
• Nude mice are inoculated with MDA-MB-231 cells (human breast carcinoma) and MatrigelTM (1 ⁇ 10 6 cells in 0.2 mL) s.c. in the right flank of the animals.
• the tumors are staged to 200 mm 3 and then treatment with a test composition is initiated (100 ⁇ g/animal/day given q.d. IP).
• Tumor volumes are obtained every other day and the animals are sacrificed after 2 weeks of treatment.
• the tumors are excised, weighed and paraffin embedded. Histological sections of the tumors are analyzed by H and E, anti-CD31, Ki-67, TUNEL, and CD68 staining.
• the compounds of the invention are also tested for inhibition of late metastasis using an experimental metastasis model (Crowley et al., Proc. Natl. Acad. Sci. USA 1993, 90 5021-5025). Late metastasis involves the steps of attachment and extravasation of tumor cells, local invasion, seeding, proliferation and angiogenesis.
• Human prostatic carcinoma cells PC-3) transfected with a reporter gene, preferably the green fluorescent protein (GFP) gene, but as an alternative with a gene encoding the enzymes chloramphenicol acetyl-transferase (CAT), luciferase or LacZ, are inoculated into nude mice.
• a reporter gene preferably the green fluorescent protein (GFP) gene
• This approach permits utilization of either of these markers (fluorescence detection of GFP or histochemical calorimetric detection of enzymatic activity) to follow the fate of these cells.
• Cells are injected, preferably iv, and metastases identified after about 14 days, particularly in the lungs but also in regional lymph nodes, femurs and brain. This mimics the organ tropism of naturally occurring metastases in human prostate cancer.
• GFP-expressing PC-3 cells (1 ⁇ 10 6 cells per mouse) are injected iv into the tail veins of nude (nu/nu) mice. Animals are treated with a test composition at 100 ⁇ g/animal/day given q.d. IP.
• Single metastatic cells and foci are visualized and quantitated by fluorescence microscopy or light microscopic histochemistry or by grinding the tissue and quantitative colorimetric assay of the detectable label.
• the rat syngeneic breast cancer system employs Mat BIII rat breast cancer cells (Xing et al., Int. J. Cancer 1996, 67:423429).
• Tumor cells for example, about 10 6 suspended in 0.1 mL PBS, are inoculated into the mammary fat pads of female Fisher rats.
• a 14-day Alza osmotic mini-pump is implanted intraperitoneally to dispense the test compound.
• the compound is dissolved in PBS (e.g., 200 mM stock), sterile filtered and placed in the minipump to achieve a release rate of about 4 mg/kg/day.
• Control animals receive vehicle (PBS) alone or a vehicle control peptide in the minipump.
• This tumor line arose spontaneously as carcinoma of the lung in a C57BL/6 mouse (Malave et al., J. Nat'l. Canc. Inst. 1979, 62:83-88). It is propagated by passage in C57BL/6 mice by subcutaneous (sc) inoculation and is tested in semiallogeneic C57BL/6 ⁇ DBA/2 F 1 mice or in allogeneic C3H mice. Typically six animals per group for subcutaneously (sc) implant, or ten for intramuscular (im) implant are used. Tumor may be implanted sc as a 2-4 mm fragment, or im or sc as an inoculum of suspended cells of about 0.5-2 ⁇ 10 6 -cells.
• Treatment begins 24 hours after implant or is delayed until a tumor of specified size (usually approximately 400 mg) can be palpated.
• the test compound is administered ip daily for 11 days Animals are followed by weighing, palpation, and measurement of tumor size. Typical tumor weight in untreated control recipients on day 12 after im inoculation is 500-2500 mg. Typical median survival time is 18-28 days.
• a positive control compound, for example cyclophosphamide at 20 mg/kg/injection per day on days 1-11 is used. Results computed include mean animal weight, tumor size, tumor weight, survival time. For confirmed therapeutic activity, the test composition should be tested in two multi-dose assays.
• Test mice are male C57BL/6 mice, 2-3 months old. Following sc, im, or intra-footpad implantation, this tumor produces metastases, preferentially in the lungs.
• Single-cell suspensions are prepared from solid tumors by treating minced tumor tissue with a solution of 0.3% trypsin. Cells are washed 3 times with PBS (pH 7.4) and suspended in PBS. Viability of the 3LL cells prepared in this way is generally about 95-99% (by trypan blue dye exclusion). Viable tumor cells (3 ⁇ 10 4 -5 ⁇ 10 6 ) suspended in 0.05 ml PBS are injected subcutaneously, either in the dorsal region or into one hind foot pad of C57BL/6 mice.
• Visible tumors appear after 3-4 days after dorsal sc injection of 10 6 cells. The day of tumor appearance and the diameters of established tumors are measured by caliper every two days. The treatment is given as one to five doses of peptide or derivative, per week. In another embodiment, the peptide is delivered by osmotic minipump.
• mice are randomized into two groups: (1) primary tumor is completely excised; or (2) sham surgery is performed and the tumor is left intact. Although tumors from 500-3000 mm 3 inhibit growth of metastases, 1500 mm 3 is the largest size primary tumor that can be safely resected with high survival and without local regrowth. After 21 days, all mice are sacrificed and autopsied.
• Lungs are removed and weighed. Lungs are fixed in Bouin's solution and the number of visible metastases is recorded. The diameters of the metastases are also measured using a binocular stereoscope equipped with a micrometer-containing ocular under 8 ⁇ magnification. On the basis of the recorded diameters, it is possible to calculate the volume of each metastasis. To determine the total volume of metastases per lung, the mean number of visible metastases is multiplied by the mean volume of metastases. To further determine metastatic growth, it is possible to measure incorporation of 125 IdUrd into lung cells (Thakur et al., J. Lab. Clin. Med. 1977, 89:217-228).
• mice Ten days following tumor amputation, 25 ⁇ g of fluorodeoxyuridine is inoculated into the peritoneums of tumor-bearing (and, if used, tumor-resected mice). After 30 min, mice are given 1 ⁇ Ci of 125 IdUrd (iododeoxyuridine). One day later, lungs and spleens are removed and weighed, and a degree of 125 IdUrd incorporation is measured using a gamma counter.
• mice with footpad tumors when tumors reach about 8-10 mm in diameter, mice are randomized into two groups: (1) legs with tumors are amputated after ligation above the knee joints; or (2) mice are left intact as nonamputated tumor-bearing controls. (Amputation of a tumor-free leg in a tumor-bearing mouse has no known effect on subsequent metastasis, ruling out possible effects of anesthesia, stress or surgery). Mice are killed 10-14 days after amputation. Metastases are evaluated as described above.
• This assay is a surrogate marker of biological activity. Briefly, DU145 cell are harvested by brief incubation with trypsin, the peptide derivative to be tested (competitor) and standard are added and the suspension is agitated at 4° C. for 2 hours. The cells are pelleted, supernatant aspirated, PBS added and the process is repeated three times (washes). HPR-Streptavidin is added, allowed to bind followed by a second series of wash steps. The appropriate HRP substrate is added and color is developed within the previously defined linear part of the reaction.
• the assay is carried out in a 96 well assay and takes place for 48 hours. Briefly, HUVECs (Cascade Biologicals) are cultured overnight in M200 containing 2% FBS. The following day, 3,000 cells are plated in each well of a gelatin-coated 96-well plate. The cells are allowed to adhere and spread for 4-6 hours, at which time the medium is replaced with fresh medium containing 2% FBS, 1 ng/mL FGF-2, and varying concentrations of specific test compounds. Cells are then cultured for an additional 48 hours, and relative cell numbers in each well are determined using the Cell Titer Aqueous Cell Proliferation Assay (Promega). The result will be an IC 50 that can be compared to a standard compound. This is an assay that reflects biological function of the peptide derivatives against their target cells.
• peptide 2, 4, 8 and 10 ⁇ Ci for biodistribution and 10, 50, 100, and 200 ⁇ Ci for Gamma scintigraphy
• mice Four doses of peptide (2, 4, 8 and 10 ⁇ Ci for biodistribution and 10, 50, 100, and 200 ⁇ Ci for Gamma scintigraphy) are injected via tail vein into tumor (B16F10 melanoma cells) bearing (200-300 mm 3 ) C57Bl/6 mice.
• the lowest and highest dosages of peptides are tested first and second.
• the dosage of peptide derivative to be tested may be modified based on the initial results obtained.
• 1251 labeled peptide at a dose determined by the method of section 4.4.17 will be injected via tail vein into tumor bearing mice (200-300 mm 3 tumor). Three mice per time point will be anesthetized and imaged at 0.5 h, 1 h, 2 h, 4 h, 6 h, 24 h, and 48 h which will determine the optimal time for imaging. As a control, unlabelled peptide at a 100-fold molar excess will be co-injected with the 125 I labeled peptide to demonstrate equivalency of both species.
• nucleic acid is synonymous with “polynucleotide” and is intended to include a gene, a cDNA molecule, an mRNA molecule, as well as a fragment of any of these such as an oligonucleotide, and further, equivalents thereof (explained more fully below). Sizes of nucleic acids are stated either as kilobases (kb) or base pairs (bp).
• Protein size is stated as molecular mass in kilodaltons (kDa) or as length (number of amino acid residues). Protein size is estimated from PAGE, from sequencing, from presumptive amino acid sequences based on the coding nucleic acid sequence or from published amino acid sequences.
• DNA molecules encoding the amino acid sequence corresponding to the peptide multimers of the present invention, or active variants thereof, can be synthesized by the polymerase chain reaction (PCR) (see, for example, U.S. Pat. No. 4,683,202) using primers derived the sequence of the protein disclosed herein.
• PCR polymerase chain reaction
• These cDNA sequences can then be assembled into a eukaryotic or prokaryotic expression vector and the resulting vector can be used to direct the synthesis of the fusion polypeptide or its fragment or derivative by appropriate host cells, for example COS or CHO cells.
• nucleic acid as used herein is intended to include such fragments or equivalents.
• the nucleic acid sequences of this invention can be DNA or RNA.
• Prokaryotic or eukaryotic host cells transformed or transfected to express the multimers are within the scope of the invention.
• the peptide multimer may be expressed in bacterial cells such as E. coli, insect cells ( baculovirus ), yeast, or mammalian cells such as Chinese hamster ovary cells (CHO) or human cells (which are preferred for human therapeutic use of the transfected cells).
• Other suitable host are known to those skilled in the art.
• yeast S. cerevisiae examples include pYepSec1 (Baldari et al., 1987, EMBO J. 6:229-234), pMFa (Kurjan et al. 1982 Cell 30:933-943), pJRY88 (Schultz et al., 1987, Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.).
• Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al., 1983, Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow et al., (1989) Virology 170:31-39).
• COS cells Gluzman 1981 Cell 23:175-182
• pCDM 8 Gluzman 1981 Cell 23:175-182
• CHO dhfr-negative CHO
• vectors such as pMT2PC (Kaufman et al., 1987, EMBO J. 6:187-195) for stable amplification/expression in mammalian cells.
• the NS0 myeloma cell line (a glutamine synthetase expression system.) is available from Celltech Ltd.
• Suitable vectors containing the desired coding and control sequences employs standard ligation and restriction techniques which are well understood in the art. Isolated plasmids, DNA sequences, or synthesized oligonucleotides are cleaved, tailored, and re-ligated in the form desired. The DNA sequences which form the vectors are available from a number of sources. Backbone vectors and control systems are generally found on available “host” vectors which are used for the bulk of the sequences in construction. For the pertinent coding sequence, initial construction may be, and usually is, a matter of retrieving the appropriate sequences from cDNA or genomic DNA libraries.
• the entire gene sequence for genes of sizeable length may be prepared by synthesizing individual overlapping complementary oligonucleotides and filling in single stranded nonoverlapping portions using DNA polymerase in the presence of the deoxyribonucleotide triphosphates.
• This approach has been used successfully in the construction of several genes of known sequence. See, for example, Edge, Nature 1981, 292:756; Nambair et al., Science 1984, 223:1299; and Jay, J. Biol. Chem. 1984, 259:6311.
• Synthetic oligonucleotides are prepared by either the phosphotriester method as described by references cited above or the phosphoramidite method as described by Beaucage et al., Tetrahedron Lett. 1981, 22:1859; and Matteucci et al., J. Am. Chem. Soc. 1981, 103:3185 and can be prepared using commercially available automated oligonucleotide synthesizers. Kinase treatment of single strands prior to annealing or for labeling is achieved using well-known methods.
• the components of the desired vectors can be excised and ligated using standard restriction and ligation procedures.
• Site-specific DNA cleavage is performed by treating with the suitable restriction enzyme (or enzymes) under conditions which are generally understood in the art, and the particulars of which are specified by the manufacturer of these commercially available restriction enzymes. See, e.g., New England Biolabs, Product Catalog.
• size separation of the cleaved fragments may be performed by polyacrylamide gel or agarose gel electrophoresis using standard techniques. A general description of size separations is found in Meth. Enzymol. (1980) 65:499-560.
• any of a number of methods are used to introduce mutations into the coding sequence to generate variants if these are to be produced recombinantly. These mutations include simple deletions or insertions, systematic deletions, insertions or substitutions of clusters of bases or substitutions of single bases. Modifications of the DNA sequence are created by site-directed mutagenesis, a well-known technique for which protocols and reagents are commercially available (Zoller et al., Nucleic Acids Res. 1982, 10:6487-6500 and Adelman et al., DNA 1983,2:183-193)). The isolated DNA is analyzed by restriction and/or sequenced by the dideoxy nucleotide method of Sanger, Proc. Natl. Acad. Sci. USA 1977, 74:5463) as further described by Messing, et al., Nucleic Acids Res. 1981, 9:309, or by the method of Maxam et al., Meth. Enzymol., supra.
• Vector DNA can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming host cells can be found in Sambrook et al. supra and other standard texts.
• a proteolytic cleavage site is introduced at the junction of the reporter group and the target protein to enable separation of the target protein from the reporter group subsequent to purification of the fusion protein.
• Proteolytic enzymes for such cleavage and their recognition sequences include Factor Xa, thrombin and enterokinase.
• a compound of the invention and/or a pharmaceutical composition thereof is administered to a patient, preferably a human, suffering from cancer or form a disease characterized by angiogenesis.
• the compounds of the invention and/or pharmaceutical compositions thereof may be used to treat or prevent cancer or undesired angiogenesis.
• cancers include any vascularized tumor, preferably, a solid tumor, including but not limited to, carcinomas of the lung, breast, ovary, stomach, pancreas, larynx, esophagus, testes, liver, parotid, bilary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, prostrate, thyroid, squamous cell carcinomas, adenocarcinomas, small cell carcinomas, melanomas, gliomas, neuroblastomas, sarcomas (e.g., angiosarcomas, chondrosarcomas )).
• a solid tumor including but not limited to, carcinomas of the lung, breast, ovary, stomach, pancreas, larynx, esophagus, testes, liver, parotid, bilary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, prostrate
• Disease characterized by undesired angiogenesis include, but are not limited to, arthritis, diabetes, arteriosclerosis, arteriovenous, malformations, corneal graft neovascularization, delayed wound healing, diabetic retinopathy, age related macular degeneration, granulations, burns, hemophilic joints, rheumatoid arthritis, hypertrophic scars, neovascular glaucoma, nonunion fractures, Osier Weber Syndrome, psoriasis, pyogenic, granuloma, retrolental fibroplasia, pterygium, scleroderma, trachoma, vascular adhesions, ocular neovascularization, parasitic diseases, hypertrophy following surgery, inhibition of hair growth, macular degeneration (including both wet and dry type), rheumatoid arthritis and osteoarthritis.
• Also contemplated are methods for treating a patient having a disease or condition associated with undesired cell migration, invasion or proliferation comprising administering to the subject an therapeutically effective amount of a compound of the invention and/or a pharmaceutical composition thereof.
• the patient has a tumor, and angiogenesis inhibition results in reduction in size or growth rate of the tumor or destruction of the tumor.
• the subject is a human.
• diseases or conditions against which the above methods may be effective include primary growth of a solid tumor, leukemia or lymphoma, tumor invasion, metastasis or growth of tumor metastases; benign hyperplasia; atherosclerosis, myocardial angiogenesis; post-balloon angioplasty vascular restinosis, neointima formation following vascular trauma, vascular graft restinosis, coronary collateral formation, deep venous thrombosis, ischemic limb angiogenesis; telangiectasia, pyogenic granuloma, corneal disease, rubeosis, neovascular glaucoma, diabetic and other retinopathy, retrolental fibroplasias, diabetic neovascularization, macular degeneration, endometriosis, arthritis, fibrosis associated with a chronic inflammatory condition, traumatic spinal cord injury including ischemia, scarring or fibrosis, lung fibrosis, chemotherapy-induced fibrosis
• a preferred disease or condition to be treated by the above methods are tumor growth, invasion or metastasis, particularly brain tumors.
• brain tumors are astrocytoma, anaplastic astrocytoma, glioblastoma, glioblastoma multiformae, pilocytic astrocytoma, pleiomorphic xanthoastrocytoma, subependymal giant cell astrocytoma, fibrillary astrocytoma, gemistocytic astrocytoma, protoplasmic astrocytoma, oligodendroglioma, anaplastic oligodendroglioma, ependymoma, anaplastic ependymoma, myxopapillary ependymoma, subependymoma, mixed oligoastrocytoma and malignant oligoastrocytoma.
• the above methods may also be used to treat uterine diseases such as endometriosis and pathogenic ocular neovascularization associated with, or a cause of, proliferative diabetic retinopathy, neovascular age-related macular degeneration, retinopathy of prematurity, sickle cell retinopathy or retinal vein occlusion.
• uterine diseases such as endometriosis and pathogenic ocular neovascularization associated with, or a cause of, proliferative diabetic retinopathy, neovascular age-related macular degeneration, retinopathy of prematurity, sickle cell retinopathy or retinal vein occlusion.
• compounds of the invention and/or pharmaceutical compositions thereof are administered to a patient, preferably a human, as a preventative measure against various diseases or disorders characterized by undesired angiogenesis including cancer.
• the compounds of the invention and/or pharmaceutical compositions thereof may be administered as a preventative measure to a patient having a predisposition for a disease characterized by undesired angiogenesis.
• the compounds and/or pharmaceutical compositions of the invention may be used for the prevention of one disease or disorder and concurrently treating another (e.g., preventing arthritis while treating cancer).
• a compound of the invention and/or a pharmaceutical composition thereof is administered to a patient, preferably a human, in a diagnostically effective amount to detect or image a disease such as those listed in Section 5.6 above.
• compounds of the invention and/or pharmaceutical compositions thereof may be used to detect or image diseases or conditions associated with undesired cell migration, invasion or proliferation such as those listed above in Section 5.6 by administering to a subject an diagnostically effective amount of a compound of the invention and/or a pharmaceutical composition thereof.
• Compounds of the invention may be diagnostically labeled and used, for example, to detect cell migration, cell invasion and cell proliferation.
• the disposition of a compound of the invention during and after binding may be followed in vitro or in vivo by using an appropriate method to detect the label.
• Diagnostically labeled compounds may be utilized in vivo for diagnosis and prognosis, for example, to image occult metastatic foci or for other types of in situ evaluations.
• compounds of the invention may include bound linker moieties, which are well known to those of skill in the art
• In situ detection of the labeled compound may be accomplished by removing a histological specimen from a subject and examining it by microscopy under appropriate conditions to detect the label.
• histological methods such as staining procedures
• the type of detection instrument available is a major factor in selecting a radionuclide.
• the radionuclide chosen must have a type of decay which is detectable by a particular instrument.
• any conventional method for visualizing diagnostic imaging can be utilized in accordance with this invention.
• Another factor in selecting a radionuclide for in vivo diagnosis is that its half-life be long enough so that the label is still detectable at the time of maximum uptake by the target tissue, but short enough so that deleterious irradiation of the host is minimized.
• a radionuclide used for in vivo imaging does not emit particles, but produces a large number of photons in a 140-200 keV range, which may be readily detected by conventional gamma cameras.
• In vivo imaging may be used to detect occult metastases which are not observable by other methods.
• Compounds of the present invention may be used in diagnostic, prognostic or research procedures in conjunction with any appropriate cell, tissue, organ or biological sample of a desired animal species.
• biological sample any fluid or other material derived from the body of a normal or diseased subject, such as blood, serum, plasma, lymph, urine, saliva, tears, cerebrospinal fluid, milk, amniotic fluid, bile, ascites fluid, pus and the like.
• a organ or tissue extract and a culture fluid in which any cells or tissue preparation from the subject has been incubated is also included within the meaning of this term.
• an effective amount means an amount sufficient to be detected using the appropriate detection system e.g., magnetic resonance imaging detector, gamma camera, etc.
• the minimum detectable amount will depend on the ratio of labeled compound specifically bound to a tumor (signal) to the amount of labeled compound either bound non-specifically or found free in plasma or in extracellular fluid.
• the amount of a composition to be administered depends on the precise compound selected, the disease or condition, the route of administration, and the judgment of the skilled imaging professional. Generally, the amount of a compound needed for detectability in diagnostic use will vary depending on considerations such as age, condition, sex, and extent of disease in the patient, contraindications, if any, and other variables, and is to be adjusted by the individual physician or diagnostician. Dosage can vary from 0.01 mg/kg to 100 mg/kg.
• the compounds the invention and/or pharmaceutical compositions thereof may be advantageously used in human medicine. As previously described in Section 4.6 above, compounds of the invention and/or pharmaceutical compositions thereof are useful for the treatment or prevention of various diseases or disorders such as cancer.
• compounds of the invention and/or pharmaceutical compositions thereof When used to treat or prevent the above disease or disorders, compounds of the invention and/or pharmaceutical compositions thereof may be administered or applied singly, or in combination with other agents.
• the compounds of the invention and/or pharmaceutical compositions thereof may also be administered or applied singly, in combination with other pharmaceutically active agents (e.g., other anti-cancer agents, other anti-angiogenic agents such as chelators as zinc, penicillamine, thiomolybdate etc.), including other compounds of the invention.
• the current invention provides methods of treatment and prophylaxis by administration to a patient of a therapeutically effective amount of a compound and/or pharmaceutical composition of the invention.
• the patient may be an animal, is more preferably, a mammal and most preferably, a human.
• the present compounds and/or pharmaceutical compositions of the invention may be administered orally.
• the compounds and/or pharmaceutical compositions of the invention may also be administered by any other convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.). Administration can be systemic or local.
• Various delivery systems e.g., encapsulation in liposomes, microparticles, microcapsules, capsules, etc.
• Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectally, by inhalation, or topically, particularly to the ears, nose, eyes, or skin.
• the preferred mode of administration is left to the discretion of the practitioner, and will depend in-part upon the site of the medical condition. In most instances, administration will result in the release of the compounds and/or pharmaceutical compositions of the invention into the bloodstream.
• This may be achieved, for example, and not by way of limitation, by local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
• administration can be by direct injection at the site (or former site) of cancer or arthritis.
• Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir.
• a compound and/or pharmaceutical composition of the invention may also be administered directly to the lung by inhalation.
• a compound and/or pharmaceutical composition of the invention may be conveniently delivered to the lung by a number of different devices.
• a Metered Dose Inhaler (“MDI”), which utilizes canisters that contain a suitable low boiling propellant, (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or any other suitable gas) may be used to deliver compounds of the invention directly to the lung.
• a suitable low boiling propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or any other suitable gas
• a Dry Powder Inhaler (“DPI”) device may be used to administer a compound and/or pharmaceutical composition of the invention to the lung.
• DPI devices typically use a mechanism such as a burst of gas to create a cloud of dry powder inside a container, which may then be inhaled by the patient.
• DPI devices are also well known in the art.
• a popular variation is the multiple dose DPI (“MDDPI”) system, which allows for the delivery of more than one therapeutic dose.
• MDDPI devices are available from companies such as AstraZeneca, GlaxoWellcome, IVAX, Schering Plough, SkyePharma and Vectura.
• capsules and cartridges of gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of a compound of the invention and a suitable powder base such as lactose or starch for these systems.
• liquid spray device Another type of device that may be used to deliver a compound and/or pharmaceutical composition of the invention to the lung is a liquid spray device supplied, for example, by Aradigm Corporation, Hayward, Calif.
• Liquid spray systems use extremely small nozzle holes to aerosolize liquid drug formulations that may then be directly inhaled into the lung.
• a nebulizer is used to deliver a compound and/or pharmaceutical composition of the invention to the lung.
• Nebulizers create aerosols from liquid drug formulations by using, for example, ultrasonic energy to form fine particles that may be readily inhaled (see e.g., Verschoyle et al., British J. Cancer, 1999, 80, Suppl. 2, 96, which is herein incorporated by reference).
• Examples of nebulizers include devices supplied by Batelle Pulmonary Therapeutics (Columbus, Ohio) (See, Armer et al., U.S. Pat. No.5,954,047; van der Linden et al., U.S. Pat. No.5,950,619; van der Linden et al., U.S. Pat. No. 5,970,974).
• an electrohydrodynamic (“EHD”) aerosol device is used to deliver a compound and/or pharmaceutical composition of the invention to the lung.
• EHD aerosol devices use electrical energy to aerosolize liquid drug solutions or suspensions (see e.g., Noakes et al., U.S. Pat. No. 4,765,539).
• the electrochemical properties of the formulation may be important parameters to optimize when delivering a compound and/or pharmaceutical composition of the invention to the lung with an EHD aerosol device and such optimization is routinely performed by one of skill in the art.
• EHD aerosol devices may more efficiently deliver drugs to the lung than existing pulmonary delivery technologies.
• the compounds and/or pharmaceutical compositions of the invention can be delivered in a vesicle, in particular a liposome (see Langer, 1990, Science, 249:1527-1533; Treat et al., in “Liposomes in the Therapy of Infectious Disease and Cancer,” Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989); see generally “Liposomes in the Therapy of Infectious Disease and Cancer,” Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989)).
• a liposome see generally “Liposomes in the Therapy of Infectious Disease and Cancer,” Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989)).
• the compounds and/or pharmaceutical compositions of the invention can be delivered via sustained release systems, preferably, oral sustained release systems.
• a pump may be used (Langer, supra; Sefton, 1987, CRC Crit. Ref Biomed. Eng. 14:201; Saudek et al., 1989, N. Engl. J Med. 321:574).
• polymeric materials can be used (see “Medical Applications of Controlled Release,” Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); “Controlled Drug Bioavailability,” Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Langer t al., 1983, J Macromol. Sci. Rev. Macromol Chem. 23:61; Levy et al., 1985, Science 228: 190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105).
• polymeric materials are used for oral sustained release delivery.
• Preferred polymers include sodium carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose and hydroxyethylcellulose (most preferred, hydroxypropyl methylcellulose).
• Other preferred cellulose ethers have been described (Alderman, Int. J. Pharm. Tech. & Prod. Mfr., 1984, 5(3) 1-9). Factors affecting drug release are well known to the skilled artisan and have been described in the art (Bamba et al., Int. J. Pharm., 1979, 2, 307).
• enteric-coated preparations can be used for oral sustained release administration.
• Preferred coating materials include polymers with a pH-dependent solubility (i.e., pH-controlled release), polymers with a slow or pH-dependent rate of swelling, dissolution or erosion (i.e., time-controlled release), polymers that are degraded by enzymes (i.e., enzyme-controlled release) and polymers that form firm layers that are destroyed by an increase in pressure (i.e., pressure-controlled release).
• osmotic delivery systems are used for oral sustained release administration (Verma et al., Drug Dev. Ind. Pharm. 2000, 26:695-708).
• OROSTM osmotic devices are used for oral sustained release delivery devices (Theeuwes et al., U.S. Pat. No. 3,845,770; Theeuwes et al., U.S. Pat. No.3,916,899).
• a controlled-release system can be placed in proximity of the target of the compounds and/or pharmaceutical composition of the invention, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in “Medical Applications of Controlled Release,” supra, vol. 2, pp. 115-138 (1984)).
• Other controlled-release systems discussed in Langer, 1990, Science 249:1527-1533 may also be used.
• the present pharmaceutical compositions contain a therapeutically or diagnostically effective amount of one or more compounds of the invention, preferably, in purified form, together with a suitable amount of a pharmaceutically acceptable vehicle, so as to provide the form for proper administration to a patient.
• a pharmaceutically acceptable vehicle When administered to a patient, the compounds of the invention and pharmaceutically acceptable vehicles are preferably sterile. Water is a preferred vehicle when the compounds of the invention are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid vehicles, particularly for injectable solutions.
• Suitable pharmaceutical vehicles also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
• excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
• the present pharmaceutical compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
• auxiliary, stabilizing, thickening, lubricating and coloring agents may be used.
• compositions comprising a compound of the invention may be manufactured by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
• Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries, which facilitate processing of compounds of the invention into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
• the present pharmaceutical compositions can take the form of solutions, suspensions, emulsion, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use.
• the pharmaceutically acceptable vehicle is a capsule (see e.g., Grosswald et al., U.S. Pat. No. 5,698,155).
• suitable pharmaceutical vehicles have been described in the art (see Remington's Pharmaceutical Sciences, Philadelphia College of Pharmacy and Science, 19th Edition, 1995).
• compounds of the invention may be formulated as solutions, gels, ointments, creams, suspensions, etc. as is well-known in the art.
• Systemic formulations include those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal, oral or pulmonary administration.
• Systemic formulations may be made in combination with a further active agent that improves mucociliary clearance of airway mucus or reduces mucous viscosity.
• active agents include, but are not limited to, sodium channel blockers, antibiotics, N-acetyl cysteine, homocysteine and phospholipids.
• the compounds of the invention are formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
• compounds of the invention for intravenous administration are solutions in sterile isotonic aqueous buffer.
• a compound of the invention may be formulated in aqueous solutions, preferably, in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.
• the solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
• the pharmaceutical compositions may also include a solubilizing agent.
• Pharmaceutical compositions for intravenous administration may optionally include a local anesthetic such as lignocaine to ease pain at the site of the injection.
• the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
• a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
• the compound of the invention When the compound of the invention is administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline.
• an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
• penetrants appropriate to the barrier to be permeated are used in the formulation.
• penetrants are generally known in the art.
• compositions for oral delivery may be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, for example.
• Orally administered pharmaceutical compositions may contain one or more optionally agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry coloring agents and preserving agents, to provide a pharmaceutically palatable preparation.
• the compositions when in tablet or pill form, the compositions may be coated to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action over an extended period of time.
• Selectively permeable membranes surrounding an osmotically active driving compound are also suitable for orally administered compounds of the invention.
• fluid from the environment surrounding the capsule is imbibed by the driving compound, which swells to displace the agent or agent composition through an aperture.
• delivery platforms can provide an essentially zero order delivery profile as opposed to the spiked profiles of immediate release formulations.
• a time delay material such as glycerol monostearate or glycerol stearate may also be used.
• Oral compositions can include standard vehicles such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Such vehicles are preferably of pharmaceutical grade.
• suitable carriers, excipients or diluents include water, saline, alkyleneglycols (e.g., propylene glycol), polyalkylene glycols (e.g., polyethylene glycol) oils, alcohols, slightly acidic buffers between pH 4 and pH 6 (e.g., acetate, citrate, ascorbate at between about 5.0 mM to about 50.0 mM), etc.
• alkyleneglycols e.g., propylene glycol
• polyalkylene glycols e.g., polyethylene glycol
• slightly acidic buffers between pH 4 and pH 6 e.g., acetate, citrate, ascorbate at between about 5.0 mM to about 50.0 mM
• flavoring agents, preservatives, coloring agents, bile salts, acylcamitines and the like may be added.
• the pharmaceutical compositions may take the form of tablets, lozenges, etc. formulated in conventional manner.
• Liquid drug formulations suitable for use with nebulizers and liquid spray devices and EHD aerosol devices will typically include a compound of the invention with a pharmaceutically acceptable vehicle.
• the pharmaceutically acceptable vehicle is a liquid such as alcohol, water, polyethylene glycol or a perfluorocarbon.
• another material may be added to alter the aerosol properties of the solution or suspension of compounds of the invention.
• this material is liquid such as an alcohol, glycol, polyglycol or a fatty acid.
• Other methods of formulating liquid drug solutions or suspension suitable for use in aerosol devices are known to those of skill in the art (see, e.g., Biesalski, U.S. Pat. No. 5,112,598; Biesalski, U.S. Pat. No. 5,556,611).
• a compound of the invention may also be formulated in rectal or vaginal pharmaceutical compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
• a compound of the invention may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection.
• a compound of the invention may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
• a compound of the invention when it is acidic, it may be included in any of the above-described formulations as the free acid or a pharmaceutically acceptable salt.
• Pharmaceutically acceptable salts substantially retain the activity of the free acid, may be prepared by reaction with bases and tend to be more soluble in aqueous and other protic solvents than the corresponding free acid form.
• a compound of the invention, or pharmaceutical compositions thereof will generally be used in an amount effective to achieve the intended purpose.
• the amount of a compound of the invention that will be effective in the treatment, prevention or detection of a particular disorder or condition disclosed herein will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques known in the art as previously described. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges.
• the amount of a compound of the invention administered will, of course, be dependent on, among other factors, the subject being treated, the weight of the subject, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.
• the dosage may be delivered in a pharmaceutical composition by a single administration, by multiple applications or controlled release.
• the compounds of the invention are delivered by oral sustained release administration.
• the compounds of the invention are administered twice per day (more preferably, once per day). Dosing may be repeated intermittently, may be provided alone or in combination with other drugs and may continue as long as required for effective treatment of the disease state or disorder.
• Suitable dosage ranges for oral administration are dependent on the potency of the drug, but are generally about 0.001 mg to about 200 mg of a compound of the invention per kilogram body weight. Dosage ranges may be readily determined by methods known to the artisan of ordinary skill.
• Suitable dosage ranges for intravenous (i.v.) administration are about 0.01 mg to about 100 mg per kilogram body weight.
• Suitable dosage ranges for intranasal administration are generally about 0.01 mg/kg body weight to about 1 mg/kg body weight.
• Suppositories generally contain about 0.01 milligram to about 50 milligrams of a compound of the invention per kilogram body weight and comprise active ingredient in the range of about 0.5% to about 10% by weight.
• Recommended dosages for intradermal, intramuscular, intraperitoneal, subcutaneous, epidural, sublingual or intracerebral administration are in the range of about 0.001 mg to about 200 mg per kilogram of body weight.
• Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. Such animal models and systems are well-known in the art.
• the compounds of the invention are preferably assayed in vitro and in vivo, as described above, for the desired therapeutic, prophylactic or diagnostic activity, prior to use in humans.
• in vitro assays can be used to determine whether administration of a specific compound of the invention or a combination of compounds of the invention is preferred for treating, preventing or diagnosing cancer.
• the compounds of the invention may also be demonstrated to be effective and safe using animal model systems.
• a therapeutically effective dose of a compound of the invention described herein will provide therapeutic benefit without causing substantial toxicity.
• a diagnostically effective dose of a compound of the invention described herein will provide diagnostic benefit without causing substantial toxicity.
• Toxicity of compounds of the invention may be determined using standard pharmaceutical procedures and may be readily ascertained by the skilled artisan.
• the dose ratio between toxic and therapeutic effect is the therapeutic index.
• a compound of the invention will preferably exhibit particularly high therapeutic indices in treating disease and disorders.
• the dosage of a compound of the inventions described herein will preferably be within a range of circulating concentrations that include an effective therapeutic or diagnostic does dose with little or no toxicity.
• the compounds and/or pharmaceutical compositions of the invention can be used in combination therapy with at least one other therapeutic agent.
• the compound and/or pharmaceutical composition of the invention and the therapeutic agent can act additively or, more preferably, synergistically.
• a compound and/or pharmaceutical composition of the invention is administered concurrently with the administration of another therapeutic agent, which may be part of the same pharmaceutical composition or a different pharmaceutical composition.
• a pharmaceutical composition of the invention is administered prior or subsequent to administration of another therapeutic agent.
• the compounds and/or pharmaceutical compositions of the invention can be used in combination therapy with other chemotherapeutic agents (e.g., alkylating agents (e.g., nitrogen mustards (e.g., cyclcphosphamide, ifosfamide, mechlorethamine, melphalen, chlorambucil, hexamethylmelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas, triazines), antimetabolites (e.g., folic acid analogs, pyrimidine analogs (e.g., fluorouracil, floxuridine, cytosine arabinoside, etc.), purine analogs (e.g., mercaptopurine, thiogunaine, pentostatin, etc.), natural products (e.g., vinblastine, vincristine, etoposide, tertiposide, dact
• alkylating agents
• the current invention provides therapeutic kits comprising the compounds of the invention or pharmaceutical compositions of the invention.
• the therapeutic kits may also contain other compounds (e.g., chemotherapeutic agents, natural products, hormones or antagonists, anti-angiogenesis agents or inhibitors, apoptosis-inducing agents or chelators) or pharmaceutical compositions of these other compounds.
• Therapeutic kits may have a single containers which contains the compound of the invention or pharmaceutical compositions of the invention with or without other components (e.g., other compounds or pharmaceutical compositions of these other compounds) or may have distinct container for each component.
• therapeutic kits of the invention include a compound and/or a pharmaceutical composition of the invention packaged for use in combination with the co-administration of a second compound (preferably, a chemotherapeutic agent, a natural product, a hormone or antagonist, a anti-angiogenesis agent or inhibitor, a apoptosis-inducing agent or a chelator) and/or a pharmaceutical composition thereof.
• a second compound preferably, a chemotherapeutic agent, a natural product, a hormone or antagonist, a anti-angiogenesis agent or inhibitor, a apoptosis-inducing agent or a chelator
• the components of the kit may be pre-complexed or each component may be in a separate distinct container prior to administration to a patient.
• the components of the kit may be provided in one or more liquid solutions, preferably, an aqueous solution, more preferably, a sterile aqueous solution.
• the components of the kit may also be provided as solids, which may be converted into liquids by addition of suitable solvents, which are preferably provided in another distinct container.
• the container of a therapeutic kit may be a vial, test tube, flask, bottle, syringe, or any other means of enclosing a solid or liquid.
• the kit will contain a second vial or other container, which allows for separate dosing.
• the kit may also contain another container for a pharmaceutically acceptable liquid.
• a therapeutic kit will contain apparatus (e.g., one or more needles, syringes, eye droppers, pipette, etc.), which enables administration of the components of the kit.
• apparatus e.g., one or more needles, syringes, eye droppers, pipette, etc.
• Rink Amide AM resin (Novabiochem) was treated with 20% piperidine in DMF (1 mL per 100 mg of resin) for three minutes with nitrogen agitation or vibration and the reaction mixture was filtered. This step was repeated an additional two times. The resin was washed three times with DMF, three times with methanol and three times with dichloromethane.
• NMM N-Methylmorpholine
• the resin was treated with 20% piperidine in DMF (3 ⁇ 3 minutes each) as described above to remove the Fmoc group on the first amino acid.
• the next desired Fmoc protected, tritylated amino acid (3 eq), HBTU (3 eq), and HOBt (3 eq) were dissolved in DMF (1 mL per 100 mg of resin) and added to the above resin, followed by the addition of N-Methylmorpholine (NMM) (6 eq) and the mixture was agitated for 1 hour.
• NMM N-Methylmorpholine
• the reaction mixture was filtered and the resin was washed with DMF three times, MeOH three times and DCM three times. Subsequent amino acids were single coupled in a similar manner.
• peptides containing an N-terminus acetyl the following commercially available amino acids, Ac-Pro-OH (3 eq) or Ac-Tyr-OH (3 eq) were used and coupled with HBTU (3 eq), HOBT (3 eq) and NMM (6 eq) for an hour.
• the capping of the N-terminus was performed by coupling the terminal amine on the resin with the appropriate acid (3 eq), HBTU (3 eq), HOBT (3 eq) and NMM (6 eq).
• the crude peptide was dissolved in a minimum amount of methanol and water or in a few drops of glacial acetic acid and water and was purified by preparative reverse phase HPLC (Beckman) with a Phenomenex Synergi hydro-RP C18 column (250 mm ⁇ 21.2 mm).
• the peptide was eluted using a gradient from 3-100% B over 30 min with a flow rate of 20 mL/min, where solvent A was water containing 0.1% trifluoroacetic acid and solvent B was acetonitrile containing 0.1% trifluoroacetic acid. Detection was at 220 or 254 nm.
• the resin was treated with 2% hydrazine in DMF (1 mL per 100 mg of resin) for three minutes with nitrogen agitation or vibration and the reaction mixture was filtered. This step was repeated an additional five times.
• the resin was washed three times with DMF and three times with dichloromethane.
• the desired carboxylic acid (4 eq), HBTU (4 eq) and HOBt (4 eq) were dissolved in DMF (1 mL per 100 mg of resin) and added to the above resin, followed by the addition of N-methylmorpholine (8 eq) and the mixture was agitated for 1 hour.
• the reaction mixture was filtered and the resin was washed three times with DMF and three times with dichloromethane.
• the resin was treated with TFA/TIS/water (95:2.5:2.5, 1 mL per 100 mg of resin) and agitated with nitrogen or vibration for 1 hour.
• the reaction mixture was filtered, the resin was washed once with TFA/TIS/water, three times with dichloromethane and the filtrate was concentrated. The residue was triturated 4 times with diethyl ether.
• Example 2 The procedure of Example 2 was used to cleave and purify the peptide and afforded 282 mg (46%) of a white solid, and as a mixture of two compounds in a ratio of 83:17: 1 H NMR (300 MHz, DMSO-d6) ⁇ 8.97 (s, 1H), 8.46-8.27 (m, 2H), 8.25-8.15 (m, 1H), 7.98-7.87 (m, 1H), 7.40-7.35 (m, 2H), 6.93 (s, 1H), 4.79-4.60 (m, 1H), 4.56-4.47 (m, 2H), 4.39-4.25 (m, 2H), 3.66-3.60 (m, 3H, overlapping with water peak), 3.22-3.12 (m, 2H), 3.05-2.94 (m, 1H), 2.91-2.82 (m, 1H), 2.68-2.41 (m, 3H, overlapping with DMSO peak), 2.07 (s, 3H), 2.00 (s, 3H), 1.91-1.
• Triethylamine (17 ⁇ L, 0.12 mmol) was added to a solution of Ac-Pro-His-Ser-Cys(Me)-Asn-Gly-Gly-Lys-NH 2 (21 mg, 0.024 mmol) and 4-fluorobenzoyl succinimide (6.2 mg, 0.026 mmol) in 1 mL of DMF and the solution was stirred at room temperature for 3.5 hours.
• This compound was prepared from Rink amide AM resin (0.0584 mmol) according to the procedures of Example 1 and 2, with the exceptions that the coupling of Fmoc-Cys(trt)-OH was performed using 3 equivalents of amino acid, 3 equivalents of HBTU, 3 equivalents of HOBt, and 6 equivalents of NMM, while the couplings of Fmoc-Ser(trt)-OH, Fmoc-His(trt)-OH, Fmoc-Pro-OH and 5-(&6-)carboxyfluorescein were performed using 2 equivalents of the acid, 2 equivalents of HBTU, 2 equivalents of HOBt, and 4 equivalents of NMM.
• This compound was prepared from Ac-Pro-His(trt)-Ser(trt)-Cys(trt)-Asn(trt)-Gly-Gly-Lys(ivDde)-Rink amide AM resin (208 mg, loading of 0.32 mmol/g, 0.067 mmol) according to Procedure C.
• This compound was prepared from Ac-Pro-His-Ser-Cys(Me)-Asn-OH (62 mg, 0.10 mmol) and doxorubicin hydrochloride (37 mg, 0.063 mmol) according to the procedure of Example 4.
• This compound was prepared from Ac-Pro-His-Ser-Cys(Ac)-Asn-OH (52 mg, 0.081 mmol) and doxorubicin hydrochloride (33 mg, 0.057 mmol) according to the procedure of Example 3.
• This compound was prepared from Ac-Pro-His(trt)-Ser(trt)-Cys(trt)-Asn(trt)-Gly-Gly-Lys(ivDde)-Rink amide AM resin (317 mg, loading of 0.32 mmol/g, 0.101 mmol) according to the procedure of Example 3.
• This compound was prepared from Ac-Pro-His(trt)-Ser(trt)-Cys(trt)-Asn(trt)-Gly-Gly-Lys(ivDde)-Rink amide AM resin (312 mg, loading of 0.32 mmol/g, 0.0997 mmol) according to the procedure of Example 3.
• the product (42.3 mg of crude material) was purified according to the procedure of Example 2 and afforded 3.9 mg (11%) of PLG-107 as a fluffy, white solid: ES MS m/z (M+H) + 1104.0.
• This compound was prepared according to the procedures of Examples 1 and 2 with the following modification: capping of the N-terminus was carried out using N-succinimidyl-3-(4-hydroxyphenyl)propionate (4.0 eq) and TEA (12.0 eq). The title compound (43.0 mg, 38%) was isolated as a fine white powder.
• the NMR data indicated a mixture of two species in a ratio of about 80:20: 1 H NMR (300 MHz, DMSO-d 6 ) ⁇ 9.12 (bs, 1 H),8.87-8.95 (m, 1 H), 8.09-8.39 (m, 4 H), 7.97 (app d, 1 H), 7.39 (s, 1 H), 7.33 (bs, 1 H), 6.99-7.09 (m, 4 H), 6.90-6.92 (2 H), 6.61-6.66 (m, 2 H), 5.09 (bs, 1 H), 4.59-4.77 (m, 2 H), 4.27-4.46 (m, 5 H), 3-57-3.70 (m, 3 H), 3.12-3.19 (m, 1 H), 2.97-3.02 (m, 1 H), 2.66-2.80 (m, 4 H), 2.37-2.45 (m, 2 H), 1.63-2.30 (m, 5 H); MS m/z (C 30 H 41 N 9 O 9 S+H) + 704.4; Anal. calcd
• This compound was prepared according to the procedures of Examples 1 and 2 with the following modification: capping of the N-terminus was performed by doing a single coupling with 3-(4-hydroxyphenyl)propionic acid (3.0 eq). The title compound (23.9 mg, 23%) was isolated as a fine white powder.
• This compound was prepared according to the procedures of Examples 1 and 2 with the following modification: only single couplings were performed with Fmoc- ⁇ -Ala-OH and Acetyl-Tyr(3,5-diiodo)-OH. The title compound was isolated as a fine white powder (55.0 mg, 51%).
• the NMR data indicated a mixture of two species in a ratio of about 80:20: 1 H NMR (300 MHz, DMSO-d 6 ) ⁇ 9.35 (bs, 1 H), 8.96 (m, 1 H), 8.22-8.41 (m, 3 H), 7.97-8.14 (m, 4 H), 7.60-7.63 (m, 2 H), 7.39 (s, 1 H), 7.34 (bs, 1 H), 7.09 (app d, 2 H), 6.92 (s, 1 H), 4.60-4.78 (m, 2 H), 4.28-4.50 (m, 6 H), 3.59-3.71 (m, 2 H), 2.94-3.06 (m, 2 H), 2.72-2.83 (m, 3 H), 2.38-2.46 (m, 3 H), 1.76-2.09 (m, 7 H); MS m/z (C 35 H 47 I 2 N 11 O 11 S+H) + 1084.5; Anal. calcd for C 35 H 47 I 2 N 11 O 11 S: N, 14.22. Found: N
• This compound was prepared according to the procedures of Examples 3 and 2 with the following modifications: the amide formation on lysine was carried out with 3-(4-hydroxy-3,4-diiodophenyl)propionic acid (4 eq), HBTU (4 eq), HOBT (4 eq) and NMM (8 eq). The title compound was isolated (16.2 mg, 13%) as a fine white powder.
• This compound was prepared according to the procedures of Examples 1 and 2 with the following modification: capping of the N-terminus was carried out using 3-(4-hydroxy-3,5-diiodophenyl)propionic acid (4.0 eq), HBTU (4.0 eq), HOBT (4.0 eq) and NMM (8.0 eq). The title compound (23.3 mg, 32%) was isolated as a fine white powder.
• This compound was prepared according to the procedures of Examples 2 and 3 with the following modifications: the amide formation on lysine was carried out with 5-(and 6)-carboxyfluorescein (3 eq), HBTU (3 eq), HOBT (3 eq) and NMM (6 eq). The title compound was isolated (10.5 mg, 18%) as a fine white powder.
• This compound was prepared according to the procedures of Examples 1 and 2 with the following modification: capping of the N-terminus was carried out using 3-(4-hydroxy-3,5-diiodophenyl)propionic acid (3 eq), HBTU (3 eq), HOBT (3 eq) and NMM (6 eq). The title compound was isolated as a fine white powder (24.2 mg, 30%).
• This compound was prepared according to the procedures of Examples 2 and 3 with the following modifications: the amide formation on lysine was carried out with 3-(4-hydroxyphenyl)propionic acid (4 eq), HBTU (4 eq), HOBT (4 eq) and NMM (8 eq). The title compound was isolated (16.2 mg, 13%) as a fine white powder.
• This compound was prepared according to the procedures of Examples 2 and 3 with the following modification: capping of the N-terminus was carried out using 3-(4-hydroxy-3,5-diiodophenyl)propionic acid (3 eq.), HBTU (3 eq.), HOBT (3 eq.) and NMM (6 eq.). The title compound was isolated as a fine white powder (27.3 mg, 29%).
• This compound was prepared according to the procedures of Examples 1 and 2 with the following modification: capping of the N-terminus was carried out using 3-(4-hydroxy-3,5-diiodophenyl)propionic acid (3 eq), HBTU (3 eq), HOBT (3 eq) and NMM (6 eq). The title compound was isolated as a fine white powder (17.1 mg, 16%).
• the NMR data indicated a mixture of two species in a ratio of about 70:30: 1 H NMR (300 MHz, DMSO-d 6 ) ⁇ 9.11 (s, 1 H), 8.86, 8.92 (s, s, 1 H), 8.51, 8.05-8.16 (m, d, 6 H, J 8.5 Hz), 8.22-8.32 (m, 2 H) 7.87-7.98 (m, 2 H), 7.34-7.39 (m, 2 H), 7.11 (s, 1 H), 7.08 (s, 1 H), 6.98 (app d, 2 H), 6.92 (s, 1 H), 6.64 (app d, 2 H), 5.11 (bs, 1 H), 4.59-4.77 (m, 1 H), 4.31-4.50 (m, 4 H), 3.83-4.02 (m, 2 H), 3.71-3.79 (m, 9 H), 3.58-3.67 (m, 2 H), 3.48-3.53 (m, 2 H), 3.11-3.19 (m, 1 H), 2.91-3.04 (m
• This compound was prepared according to the procedures of Examples 1 and 2 with the following modification: capping of the N-terminus was carried out using 3-(4-hydroxyphenyl)propionic acid (3 eq), HBTU (3 eq), HOBT (3 eq) and NMM (6 eq). The title compound was isolated as a fine white powder (47.4 mg, 57%).
• the NMR data indicated a mixture of two species in a ratio of about 70:30: 1 H NMR (300 MHz, DMSO-d 6 ) ⁇ 8.98 (m, 1 H), 8.21-8.44 (m, 2 H), 7.98-8.14 (m, 2 H), 7.71-7.77 (m, 1 H), 7.40 (s, 1 H), 7.35 (m, 1 H), 7.09 (d, 2 H, J 9.8 Hz), 6.95-6.98 (m, 2 H), 6.92 (s, 1 H), 6.62-6.54 (m, 2 H), 4.62-4.79 (m, 4 H), 4.28-4.50 (m, 8 H), 3.47-3.70 (m, 4 H), 3.12-3.19 (m, 1 H), 2.90-3.03 (m, 3 H), 2.76-2.83 (m, 2 H), 2.65-2.70 (m, 2 H), 2.40-2.46 (m, 2 H), 2.25-2.30 (m, 3 H), 1.72-2.08 (m, 4 H), 1.14-1
• the NMR data indicated a mixture of two species in a ratio of about 80:20: 1 H NMR (300 MHz, DMSO-d 6 ) ⁇ 8.97 (m, 1 H), 8.21-8.44 (m, 2 H), 8.06-8.14 (m, 1 H), 7.97-7.99 (m, 2 H), 7.85-7.87 (m, 1 H), 7.40 (s, 1 H), 7.35 (s, 1 H), 7.09 (d, 2 H, J 9.7 Hz), 6.89-7.00 (m, 2 H), 6.92 (s, 1 H), 6.61-6.64 (m, 2 H), 4.59-4.66 (m, 2 H), 4.25-4.50 (m, 6 H), 3.59-3.71 (m, 2 H), 3.48-3.55 (m, 2 H), 3.12-3.19 (m, 1 H), 2.95-3.10 (m, 3 H), 2.76-2.83 (m, 3 H), 2.57-2.65 (m, 1 H), 2.38-2.46 (m, 2 H), 2.24-2.27
• the NMR data indicated a mixture of three species in a ratio of about 70:20:10 1 H NMR (300 MHz, D 2 O) ⁇ 8.69-8.90 (m, 1 H), 7.39-7.55 (m, 1 H), 4.53-4.64 (m, 3 H), 4.39-4.48 (m, 1 H), 3.88-3.96 (m, 5 H), 3.24-3.68 (m, 28 H), 3.00-3.02 (m, 3 H), 2.69-2.94 (m, 9 H), 2.24-2.43 (m, 2 H), 1.84-2.10 (m, 5 H); MS m/z (C 40 H 61 InN 14 O 15 S+H) + 11245.8; Anal. Calcd for C 40 H 61 InN 14 O 15 S: N, 17.43 Found: N, 12.42 (peptide content: 71%).
• This compound was prepared according to the procedures of Examples 1 and 2 with the following modifications: capping of the N-terminus was carried out using 3-(4-hydroxyphenyl)propionic acid (3 eq), HBTU (3 eq), HOBT (3 eq) and NMM (6 eq). The title compound was isolated (61.1 mg, 53%) as a fine white powder.
• This compound was prepared according to the procedures of Examples 1 and 2 with the following modification: capping of the N-terminus was carried out using 8-(4-fluorobenzylamino)suberic acid (3 eq), HBTU (3 eq), HOBT (3 eq) and NMM (6 eq). The title compound was isolated as a fine white powder (70.8 mg, 53%).
• the NMR data indicated a mixture of two species in a ratio of about 70:30: 1 H NMR (300 MHz, DMSO-d 6 ) ⁇ 8.97 (s, 1 H), 8.22-8.28 (m, 3 H), 8.11-8.14 (m, 1 H), 7.96-7.99 and 8.40-8.43 (m, 1 H), 7.74-7.80 (m, 1 H), 7.34-7.39 (m, 2 H), 7.24-7.29 (m, 2 H), 7.08-7.16 (m, 4 H), 6.92 (bs, 1 H), 4.29-4.50 (m, 7 H), 4.22 (d, 2 H, J 5.9 Hz), 3.63-3.69 (m, 2 H), 3.44-3.52 (m, 2 H), 3.16-3.26 (m, 3 H), 2.97-3.02 (m, 1 H), 2.76-2.83 (m, 2 H), 2.38-2.49 (m, 3 H), 1.99-2.13 (m, 5 H), 1.72-1.89 (m, 3 H), 1.4
• DTPA-PHSCN (20 mg, 0.018 mmol) was dissolved in 1 mL of 0.1M AcOH(aq) solution and InCl 3 (40 mg, 0.18 mmol) was dissolved in 2 mL of 0.02M HCl solution. The two solutions were combined and incubated for an hour at room temperature.
• This compound was prepared according to procedures of Examples 2 and 3 with the following modifications: the 2% hydrazine treatment for the deprotection of ivDde on lysine was repeated 10 times and the amide formation on lysine was carried out with protoporphyrin IX (2 eq), PYBOP (2 eq), and NMM (6 eq). The title compound was isolated (7.6 mg, 5.49 ⁇ M, 8.6%) as a fine dark red powder.; MS m/z (C 67 H 85 N 17 O 14 S+H) + 1385.6.
• This compound was prepared from Rink amide AM resin according to the procedures of Examples 1 and 2 sand was isolated as a white, fluffy solid; ES MS m/z (M+H) + 1066.5.

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