WO1999052936A2 - Analogues du peptide d'arginine utilises en tant qu'antagonistes du facteur de croissance des fibroblastes - Google Patents

Analogues du peptide d'arginine utilises en tant qu'antagonistes du facteur de croissance des fibroblastes Download PDF

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WO1999052936A2
WO1999052936A2 PCT/JP1999/001844 JP9901844W WO9952936A2 WO 1999052936 A2 WO1999052936 A2 WO 1999052936A2 JP 9901844 W JP9901844 W JP 9901844W WO 9952936 A2 WO9952936 A2 WO 9952936A2
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hydrogen
independently selected
fgf
alkyl
substituents
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PCT/JP1999/001844
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WO1999052936A3 (fr
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Ventkatachalapathi Yalamoori
Ramnarayan Kalyanaraman
Laura Schove
Vitukudi Narayanaiyengar Balaji
Ming Fai Chan
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Eisai Co., Ltd.
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Priority to AU31662/99A priority Critical patent/AU3166299A/en
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Publication of WO1999052936A3 publication Critical patent/WO1999052936A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/15—Oximes (>C=N—O—); Hydrazines (>N—N<); Hydrazones (>N—N=) ; Imines (C—N=C)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C281/00—Derivatives of carbonic acid containing functional groups covered by groups C07C269/00 - C07C279/00 in which at least one nitrogen atom of these functional groups is further bound to another nitrogen atom not being part of a nitro or nitroso group
    • C07C281/16—Compounds containing any of the groups, e.g. aminoguanidine
    • C07C281/18—Compounds containing any of the groups, e.g. aminoguanidine the other nitrogen atom being further doubly-bound to a carbon atom, e.g. guanylhydrazones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to compositions containing arginine amides, arginine peptides and analogs thereof, and methods using the compositions for treatment or prevention of fibroblast growth factor (FGF)-mediated diseases.
  • FGF fibroblast growth factor
  • compositions containing arginine amides, arginine peptides and analogs thereof and use of the compositions as FGF antagonists are provided.
  • Fibroblast growth factors are a family of polypeptide mitogens and are ubiquitous in mammals. FGFs and their corresponding receptors, FGFRs, are widely distributed in tissues throughout the body, i.e., the central and peripheral nervous system, retina, kidneys, and myocardium (see, e.g., Johnson et al. Adv. Cancer Res. 1993, 60, 1 ), and are expressed during embryogenesis (Kimelman et al. Science 1988, 242, 1053) . FGFs exhibit potent mitogenic activity in these areas (see, e.g., Gospodarowicz Nature 1974, 249, 123), are also mitogenic for mesenchymal, neuronal, and epithelial cells (see.
  • FGFs Fibroblast growth factors
  • the FGFs consist of a family of peptides, of which ten have been identified (FGF-1 through 10) .
  • the first two peptides of this family to be isolated and characterized were FGF-1 and FGF-2, more commonly referred to as acid FGF (aFGF) and basis FGF (bFGF), respectively, for their acidic and basic isoelectric points, respectively.
  • aFGF and bFGF were initially isolated from the bovine pituitary (Gospodarowicz J. Biol. Chem. 1975, 250, 251 5), then from bovine brain (Gospodarowicz et al. J. Biol. Chem. 1978, 253, 3736) and later isolated from human brain (Gimenez-Gallego et al.
  • aFGF and bFGF have common biological properties, including the ability to bind to one or more FGF receptors. They also exhibit 55% homology in their amino acid sequences and are highly conserved among species (i.e., human and bovine bFGF exhibit 98.7% identity (see, e.g., U.S. Patent No. 5,288,855; U.S. Patent No. 5, 1 55,214)) . Eight other FGFs have been identified based on these structures (FGF-3 through 1 0) : int-2 (FGF-3) (Moore et al. EMBO J .
  • keratinocyte growth factor (KGF)(FGF-7) (Finch et al. Science 1989, 245, 752), FGF-8, FGF-9 and FGF-10 (PCT International Publication Number WO 95/24,414) .
  • KGF keratinocyte growth factor
  • FGF-7 FGF-7
  • FGF-8 FGF-9
  • FGF-10 PCT International Publication Number WO 95/24,414.
  • the structures of aFGF and bFGF have also been determined through single-crystal x-ray diffraction (Eriksson Proc. Nat. Acad. Sci. USA 1991 , 88, 3441 ; Zhang et al. Proc. Nat. Acad . Sci. USA 1991 , 88, 3446; Zhu et al. Science 1991 , 25J_, 90) .
  • Basic FGF is a 1 6kD, acid- and thermally-sensitive peptide. It is an angiogenic factor causing the migration, proliferation and differentiation of endothelial cells to form blood vessels (see, e.g., Montesano et al. Proc. Nat. Acad. Sci. USA 1986, 83, 7279; Folkman et al. Science 1987, 235, 442) . This effect indicates possible therapeutic uses of bFGF for wound healing (Folkman Science 1987, 235, 442; Buntrock et al. Exp. Pathol.
  • FGFs have also been reported to be useful as hypotensive agents for reducing high blood pressure and preventing myocardial infarction and cerebral hemorrhages (Saltis et al. Atherosclerosis 1995, 1 1 8, 77; PCT International Publication No. WO 92/08,473), for the treatment of ulcers (U.S. Patent No. 5,401 ,721 ; U.S. Patent No.
  • Basic FGF may, however, be harmful in some cases in that cell proliferation and angiogenesis are important aspects of tumor growth and tumor development, rheumatoid arthritis, restenosis, In-Stent restenosis, proliferative diabetic retinopathies and diabetes (see, e.g., Folkman Adv. Cancer Res. 1985, 43, 1 75; Melnyk et al. Arthritis Rheum. 1990, 33, 493; Sivalingam Arch. Qphthalmol. 1990, 108, 869) .
  • Basic FGF also functions as an oncogene in melanoma. FGF activities are mediated by high and low affinity receptors. There are many diverse forms of aFGF and bFGF receptors (Hanneken et al.
  • FGF receptor genes have been identified of which at least two produce multiple mRNA transcripts through alternative splicing of the primary transcript. This splicing creates a large number of forms of the receptors, potentially ninety-six receptor isoforms, and leads to response of the cell to many FGF family members, Le ⁇ ., one gene gives FGFR-2 and KGF receptors, and alternate FGFR-1 splicing gives a 50 fold decrease in bFGF binding with unchanged aFGF binding. Receptor expression is also altered by injury and pathological conditions (restenosis, tumors and proliferative diseases) .
  • receptor mRNA and protein are present in melanoma cells (see, e.g., Becker et al. Oncogene 1992, 7, 2303), the receptor message is not usually found in palmar fascia, but is found in the proliferative hand disease Dupuytren's contracture (see, e.g.,
  • compositions containing arginine amides, arginine peptides and analogs thereof, or pharmaceutically acceptable derivatives, including, but not limited to, salts, esters, acids, bases, solvates, hydrates and prodrugs thereof, are provided.
  • Methods for modulating the interaction of an FGF peptide with FGF receptors using such compositions are also provided.
  • Methods of treating or preventing FGF- mediated diseases are also provided.
  • compositions containing arginine amides, peptides or analogs, and methods for inhibiting the binding of an FGF peptide to FGF receptors using such compositions are provided.
  • the arginine amides, peptides and analogs are arginine amide derivatives, or C-amido di- or tripeptides, or are aminoimino hydrazones.
  • the pharmaceutical compositions provided herein are those that are particularly active as bFGF antagonists, as evidenced by jn vitro assays described herein.
  • the pharmaceutical compositions contain arginine amides, peptides or analogs that have formula I:
  • R ⁇ R 2 , R 3 , R 4 , R 5 and R 6 are each independently selected from (i) or (ii) as follows:
  • R ⁇ R 3 and R 5 are each independently selected from alkyl, alkenyl, alkynyl, aryl, cycloalkyl, aralkyl and heteroaralkyl, and are unsubstituted or substituted with one or more substituents each independently selected from Z, and
  • R 2 , R 4 and R 6 are each independently selected from hydrogen or alkyl; or
  • R 1 and R 2 , or R 3 and R 4 , or R 5 and R 6 form alkylene or alkenylene which is unsubstituted or substituted with one or more substituents each independently selected from Z, and the others are selected as in (i);
  • A is hydrogen, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxy- carbonyl, aralkoxycarbonyl, heteroaralkoxycarbonyl, alkylcarbonyl, aryl- carbonyl, heteroarylcarbonyl, aralkylcarbonyl or heteroaralkylcarbonyl;
  • D is alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, aralkyl or heteroaralkyl, and is unsubstituted or substituted with one or more substituents each independently selected from Z;
  • Z is halo, hydroxy, amino, azido, guanidino, nitro, alkyl, aryl, heteroaryl, alkoxy, aryloxy, heteroaryloxy, aryialkoxy, heteroarylalkoxy, alkoxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, or any two Z substituents may form alkylene, alkenylene, alkylenedioxy, alkylenethioxyoxy or alkylenedithioxy, and is unsubstituted or substituted with one or more substituents each independently selected from Y; and
  • Y is halo, amino, alkylamino, dialkylamino, arylamino, diarylamino, acylamino, azido, nitro, alkyl, aryl, heteroaryl, alkoxy, aryloxy or heteroaryloxy.
  • the arginine amides, peptides and analogs of formula I are arginine amide derivatives or di- and tripeptide analogs which are carboxamides at the C-terminus.
  • the amino acids are of either the D- or L-configuration, or are racemic.
  • compositions contain arginine amides, peptides or analogs that have formula II:
  • Ar 1 is higher alkylene, which, as defined herein, contains about 7- 30 carbon atoms, or is monocyclic or polycyclic arylene, alkenylarylene, arylalkenylene, heteroarylene, alkenylheteroarylene or heteroarylalkenylene, and is unsubstituted or substituted with one or more substituents each independently selected from Z;
  • Z is halo, hydroxy, amino, azido, guanidino, nitro, alkyl, aryl, heteroaryl, alkoxy, aryloxy, heteroaryloxy, arylalkoxy, heteroarylalkoxy, alkoxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, or any two Z substituents may form alkylene, alkenylene, alkylenedioxy, alkylenethioxyoxy or alkylenedithioxy, and is unsubstituted or substituted with one or more substituents each independently selected from Y;
  • Y is halo, amino, alkylamino, dialkylamino, arylamino, diarylamino, acylamino, azido, nitro, alkyl, aryl, heteroaryl, alkoxy, aryloxy or heteroaryloxy;
  • R 7 , R 8 , R 9 , R 10 , R 1 1 and R 12 are each independently selected from (i) or (ii) as follows:
  • R 7 is selected from hydrogen and alkyl
  • R 8 and R 9 are hydrogen
  • R 10 is hydrogen, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyi, aralkyl or heteroaralkyl
  • R 1 1 is hydrogen
  • R 12 is hydrogen or Z, or
  • any two of R 7 , R 8 , R 9 , R 10 , R 1 1 and R 12 together form alkylene, alkenylene, carbonylalkylene or carbonylalkenylene, and the remainder are selected as in (i); and m is 0 or 1 when Ar 1 is arylalkenylene or heteroarylalkenylene, or is 1 .
  • those that inhibit an FGF- mediated activity by about 50% at concentrations of less than about 500 ⁇ M are preferred. More preferred are those that inhibit an FGF- mediated activity by about 50% at concentrations of less than about 300 ⁇ M, more preferably less than about 1 50 ⁇ M, and most preferably less than about 30 ⁇ M.
  • compositions formulated for administration by an appropriate route and means containing effective concentrations of one or more of the compounds described herein, or pharmaceutically acceptable salts, esters, acids, bases, solvates, hydrates or prodrugs thereof, that deliver amounts effective for the treatment of FGF-mediated disorders, and other conditions that are in some manner mediated by an FGF peptide or whose symptoms can be ameliorated by administration of a bFGF-specific FGF antagonist, are also provided.
  • the effective amounts and concentrations are effective for ameliorating any of the symptoms of any of the disorders.
  • any pharmaceutically-acceptable derivatives including salts, esters, acids, bases, solvates, hydrates and prodrugs of the arginine amides, peptides or analogs.
  • Pharmaceutically-acceptable salts include, but are not limited to, amine salts, such as but not limited to N,N'- dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N- methylglucamine, procaine, N-benzylphenethylamine, 1 -para- chlorobenzyl-2-pyrrolidin-1 '-ylmethylbenzimidazole, diethylamine and other alkylamines, piperazine and tris(hydroxymethyl)aminomethane; alkali metal salts, such as but not limited to lithium, potassium and sodium; alkali earth metal salts, such as but not limited to barium, calcium and magnesium
  • FGF-mediated diseases including, but not limited to, diabetes, cancer, including, but not limited to, melanoma and tumor growth and development, restenosis, In-Stent restenosis, rheumatoid arthritis, ophthalmic disorders, including, but not limited to, corneal clouding following excimer laser surgery, closure of trabecuiectomies, hyperproliferation of lens epithelial cells following cataract surgery, the recurrence of pterygii and diabetic retinopathy, and other proliferative diseases, including, but not limited to, Dupuytren's contracture, conditions that are in some manner mediated by an FGF peptide that binds to FGF receptors, or that are ameliorated by administration of an FGF receptor bFGF antagonist are provided.
  • diseases including, but not limited to, diabetes, cancer, including, but not limited to, melanoma and tumor growth and development, restenosis, In-Stent restenosis, rheumatoid arthritis,
  • Methods for inhibiting binding of an FGF peptide to an FGF receptor are provided. These methods are practiced by contacting the receptor with one or more of the compositions provided herein simultaneously, prior to, or subsequent to contacting the receptor with an FGF peptide.
  • methods of treating FGF-mediated disorders by administering effective amounts of the arginine amides, peptides or analogs, or salts, acids, bases, solvates, hydrates, prodrugs or other suitable derivatives thereof are provided.
  • methods for treating FGF-mediated disorders including, but not limited to, diabetes, cancer, including, but not limited to, melanoma and tumor growth and development, restenosis, In-Stent restenosis, rheumatoid arthritis, ophthalmic disorders, including, but not limited to, corneal clouding following excimer laser surgery, closure of trabeculectomies, hyperproliferation of lens epithelial cells following cataract surgery, the recurrence of pterygii and diabetic retinopathy, and other proliferative diseases, including, but not limited to, Dupuytren's contracture, and other proliferative diseases in which FGF receptor bFGF-mediated physiological responses are implicated, by administering effective amounts of one or more
  • the methods are effected by contacting FGF receptors with one or more of the arginine amides, peptides or analogs prior to, simultaneously with, or subsequent to contacting the receptors with an FGF peptide.
  • effective amounts of compositions containing therapeutically effective concentrations of the compounds formulated for oral, intravenous, local and topical application for the treatment of FGF- mediated disorders including, but not limited to, diabetes, cancer, including, but not limited to, melanoma and tumor growth and development, restenosis, In-Stent restenosis, rheumatoid arthritis, ophthalmic disorders, including, but not limited to, corneal clouding following excimer laser surgery, closure of trabeculectomies, hyperproliferation of lens epithelial cells following cataract surgery, the recurrence of pterygii and diabetic retinopathy, and other proliferative diseases, including, but not limited to, Dupuytren's contracture, and other diseases in which FGF-
  • fibroblast growth factor (FGF) peptides include peptides that have substantially the amino acid sequence of any one of FGF-1 through 10 and that act as potent endogenous proliferative peptides.
  • an FGF-mediated condition is a condition that is caused by abnormal FGF activity or one in which compounds that inhibit FGF activity have therapeutic use.
  • diseases include, but are not limited to diabetes, cancer, including, but not limited to, melanoma and tumor growth and development, restenosis, In-Stent restenosis, rheumatoid arthritis, ophthalmic disorders, including, but not limited to, corneal clouding following excimer laser surgery, closure of trabeculectomies, hyperproliferation of lens epithelial cells following cataract surgery, the recurrence of pterygii and diabetic retinopathy, and other proliferative diseases, including, but not limited to, Dupuytren's contracture, and other diseases in which FGF-mediated physiological responses are implicated.
  • an effective amount of a compound for treating a particular disease is an amount that is sufficient to ameliorate, or in some manner reduce the symptoms associated with the disease. Such amount may be administered as a single dosage or may be administered according to a regimen, whereby it is effective. The amount may cure the disease but, typically, is administered in order to ameliorate the symptoms of the disease. Typically, repeated administration is required to achieve the desired amelioration of symptoms.
  • an FGF antagonist is a compound, such as a drug or an antibody, that inhibits FGF-stimulated proliferation and other FGF- mediated physiological responses.
  • the antagonist may act by interfering with the interaction of the FGF with an FGF-specific receptor or by interfering with the physiological response to or bioactivity of an FGF isopeptide, such as proliferation.
  • an FGF antagonist interferes with FGF-stimulated proliferation or other response or interferes with the interaction of an FGF peptide with an FGF-specific receptor, such as bFGF receptors, as assessed by assays known to those of skill in the art.
  • the effectiveness of potential FGF antagonists can be assessed using methods known to those of skill in the art. For example, the effectiveness may be measured by inhibition of binding of 125 l-bFGF to a human extracellular-domain FGFR1 -TPA fusion protein immobilized on a solid phase (hsRRA assay) (for the extracellular form of human FGFR, see U.S. Patent 5,288,855) . Effectiveness may also be measured through the use of a membrane-bound competitive binding assay, quantifying inhibition of binding of 125 l-bFGF to FGF receptors on cultured smooth muscle cells (SMCs) .
  • SMCs smooth muscle cells
  • Effectiveness may also be measured by determination of inhibition of 3 H-thymidine incorporation into DNA, which is promoted by bFGF stimulation of SMC proliferation (see, generally; Nachtigal et al. In Vitro Cellular and Developmental Biology 1989, 25, 892) .
  • the biological activity or bioactivity of an FGF peptide includes any activity induced, potentiated or influenced by FGF in vivo. It also includes the ability to bind to particular receptors and to induce a functional response, such as proliferation. It may be assessed by in vivo assays or by in. vitro assays, such as those exemplified herein.
  • the relevant activity includes, but is not limited to, proliferation. Any assay known to those of skill in the art to measure or detect such activity may be used to assess such activity (see, e.g., Nachtigal et al. In Vitro Cellular and Developmental Biology 1989, 25, 892; and the Examples herein) .
  • the IC 50 refers to an amount, concentration or dosage of a particular test compound that achieves a 50% inhibition of a maximal response, such as binding of FGF to tissue receptors, in an assay that measures such response.
  • EC 50 refers to a dosage, concentration or amount of a particular test compound that elicits a dose-dependent response at 50% of maximal expression of a particular response that is induced, provoked or potentiated by the particular test compound.
  • pharmaceutically acceptable derivatives of a compound include salts, esters, acids, bases, solvates, hydrates or prodrugs thereof that may be readily prepared by those of skill in this art using known methods for such derivatization and that produce compounds that may be administered to animals or humans without substantial toxic effects and that either are pharmaceutically active or are prodrugs.
  • acidic groups can be esterified or neutralized.
  • pharmaceutically-acceptable salts, esters, hydrates, solvates or other derivatives of the compounds include any such salts, esters and other derivatives that may be prepared by those of skill in this art using known methods for such derivatization and that produce compounds that may be administered to animals or humans without substantial toxic effects and that either are pharmaceutically active or are prodrugs.
  • Pharmaceutically- acceptable salts include, but are not limited to, salts of alkali metals and alkaline earth metals, including but not limited to sodium salts, potassium salts, lithium salts, calcium salts and magnesium salts; transition metal salts, such as zinc salts, copper salts and aluminum salts; polycationic counter ion salts, such as but not limited ammonium and substituted ammonium salts and organic amine salts, such as hydroxyalkylamines and alkylamines; salts of mineral acids, such as but not limited to hydrochlorides and sulfates, salts of organic acids, such as but not limited acetates, lactates, malates, tartrates, citrates, ascorbates, succinates, butyrate, valerate and fumarates. Also contemplated herein are the corresponding esters.
  • Preferred pharmaceutically-acceptable salts include, but are not limited to, N,N'-dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine, N-benzylphenethylamine, 1 -para-chlorobenzyl-2-pyrrolidin-1 '-ylmethylbenzimidazole, diethylamine and other alkyiamines, piperazine, tris(hydroxymethyl)aminomethane, aluminum, calcium, lithium, magnesium, potassium, sodium hydrogen phosphate, disodium phosphate, sodium, zinc, barium, gold, silver and bismuth salts.
  • Sodium salts, particularly the sodium salt of each of the compound, are most preferred herein.
  • treatment means any manner in which the symptoms of a conditions, disorder or disease are ameliorated or otherwise beneficially altered. Treatment also encompasses any pharmaceutical use of the compositions herein, such as use as contraceptive agents.
  • amelioration of the symptoms of a particular disorder by administration of a particular pharmaceutical composition refers to any lessening, whether permanent or temporary, lasting or transient that can be attributed to or associated with administration of the composition.
  • substantially pure means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis and high performance liquid chromatography (HPLC), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance.
  • TLC thin layer chromatography
  • HPLC high performance liquid chromatography
  • Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art.
  • a substantially chemically pure compound may, however, be a mixture of stereoisomers. In such instances, further purification might increase the specific activity of the compound.
  • biological activity refers to the [n vivo activities of a compound or physiological responses that result upon in vivo administration of a compound, composition or other mixture. Biological activity, thus, encompasses therapeutic effects and pharmaceutical activity of such compounds, compositions and mixtures.
  • a prodrug is a compound that, upon in vivo administration, is metabolized or otherwise converted to the biologically, pharmaceutically or therapeutically active form of the compound.
  • the pharmaceutically active compound is modified such that the active compound will be regenerated by metabolic processes.
  • the prodrug may be designed to alter the metabolic stability or the transport characteristics of a drug, to mask side effects or toxicity, to improve the flavor of a drug or to alter other characteristics or properties of a drug.
  • alkyl, alkenyl and alkynyl carbon chains if not specified contain from 1 to 20 carbons, preferably 1 to 1 6 carbons, and are straight or branched.
  • Alkenyl carbon chains of from 1 to 20 carbons preferably contain 1 to 8 double bonds, and the alkenyl carbon chains of 1 to 1 6 carbons preferably contain 1 to 5 double bonds.
  • Alkynyl carbon chains of from 1 to 20 carbons preferably contain 1 to 8 triple bonds, and the alkynyl carbon chains of 1 to 1 6 carbons preferably contain 1 to 5 triple bonds.
  • the alkyl, alkenyl and alkynyl groups may be optionally substituted, with one or more groups, preferably alkyl group substituents that may be the same or different.
  • lower alkyl, lower alkenyl, lower alkynyl, lower alkylene, lower alkenylene and lower alkynylene refer to carbon chains having up to about 6 carbons.
  • higher alkyl, higher alkenyl, higher alkynyl, higher alkylene, higher alkenylene and higher alkynylene refer to carbon chains having about 7-30 carbons.
  • an alkyl group substituent includes halo, haloalkyl, preferably halo lower alkyl, aryl, hydroxy, alkoxy, aryloxy, alkyloxy, alkylthio, arylthio, aralkyloxy, aralkylthio, carboxy, alkoxycarbonyl, oxo and cycloalkyl.
  • aryl refers to cyciic groups containing from 3 to 1 9 carbon atoms containing 1 -5 rings, including 1 -5 fused rings.
  • Aryl groups include, but are not limited to groups, such as phenyl, substituted phenyl, naphthyl, substituted naphthyl, in which the substituent is lower alkyl, halogen, or lower alkoxy.
  • an "aryl group substituent” includes alkyl, cycloalkyl, cycloalkylalkyl, aryl, heteroaryl optionally substituted with 1 or more, preferably 1 to 3, substituents selected from halo, halo alkyl and alkyl, arylalkyl, heteroarylalkyl, alkenyl containing 1 to 2 double bonds, alkynyl containing 1 to 2 triple bonds, halo, hydroxy, haloalkyl and polyhaloalkyl, preferably halo lower alkyl, especially trifluoromethyl, formyl, alkylcarbonyl, arylcarbonyl that is optionally substituted with 1 or more, preferably 1 to 3, substituents selected from halo, halo alkyl and alkyl, heteroarylcarbonyl, carboxy, alkoxycarbonyl, aryloxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,
  • cycloalkyl refers to a saturated mono- or multi- cyclic ring system, preferably of 3 to 10 carbon atoms, more preferably 3 to 6 carbon atoms; cycloalkenyl and cycloalkynyl refer to mono- or multicyclic ring systems that respectively include at least one double bond and at least one triple bond . Cycloalkenyl and cycloalkynyl groups may preferably contain 3 to 10 carbon atoms, with cycloalkenyl groups more preferably containing 4 to 7 carbon atoms and cycloalkynyl groups more preferably containing 8 to 10 carbon atoms.
  • ring systems of the cycloalkyl, cycloalkenyl and cycloalkynyl groups may be composed of one ring or two or more rings which may be joined together in a fused, bridged or spiro-connected fashion, and may be optionally substituted with one or more alkyl group substituents.
  • heteroaryl refers to a monocyclic or multicyclic ring system, preferably of about 5 to about 1 5 members where one or more, more preferably 1 to 3 of the atoms in the ring system is a heteroatom, that is, an element other than carbon, for example, nitrogen, oxygen and sulfur atoms.
  • heteroaryl may be optionally substituted with one or more, preferably 1 to 3, aryl group substituents.
  • exemplary heteroaryl groups include, for example, furyl, thienyl, pyridyl, pyrrolyl, N- methylpyrrolyl, quinolinyl and isoquinoiinyi, with pyridyl and quinolinyl being preferred.
  • heterocyclic refers to a monocyclic or multicyclic ring system, preferably of 3 to 10 members, more preferably 4 to 7 members, even more preferably 5 to 6 members, where one or more, preferably 1 to 3 of the atoms in the ring system is a heteroatom, that is, an element other than carbon, for example, nitrogen, oxygen and sulfur atoms.
  • the heterocycle may be optionally substituted with one or more, preferably 1 to 3 aryl group substituents.
  • Preferred substituents of the heterocyclic group include hydroxy, alkoxy containing 1 to 4 carbon atoms, halo lower alkyl, including trihalomethyl, such as trifluoromethyl, and halogen.
  • heterocycle may include reference to heteroaryl.
  • exemplary heterocycles include, for example, pyrrolidinyl, piperidinyl, alkylpiperidinyl, morpholinyl, oxadiazolyl or triazolyl.
  • alkyl refers to saturated carbon chains that contain one or more carbons; the chains may be straight or branched or include cyclic portions or be cyclic.
  • alicyclic refers to aryl groups that are cyclic.
  • halogen or “halide” refers to F, CI, Br or I.
  • pseudohalides are compounds that behave substantially similar to halides. Such compounds can be used in the same manner and treated in the same manner as halides (X " , in which X is a halogen, such as CI or Br) .
  • Pseudohalides include, but are not limited to cyanide, cyanate, thiocyanate, selenocyanate, trifluoromethyl and azide.
  • haloalkyl refers to a lower alkyl radical in which one or more of the hydrogen atoms are replaced by halogen including, but not limited to, chloromethyl, trifluoromethyl, 1 -chloro-2-fluoroethyl and the like.
  • haloalkoxy refers to RO- in which R is a haloalkyl group.
  • sulfinyl refers to -S(O)-.
  • sulfonyl refers to -S(O) 2 -.
  • aminocarbonyl refers to -C(O)NH 2 .
  • alkylaminocarbonyl refers to -C(O)NHR in which R is hydrogen or alkyl, preferably lower alkyl.
  • dialkylaminocarbonyl refers to -C(O)NR R in which R and R are independently selected from hydrogen or alkyl, preferably lower alkyl;
  • carboxamide refers to groups of formula -NR COR.
  • diarylaminocarbonyl refers to -C(O)NRR' in which R and R' are independently selected from aryl, preferably lower aryl, more preferably phenyl.
  • arylalkylaminocarbonyl refers to -C(O)NRR' in which one of R and R' is aryl, preferably lower aryl, more preferably phenyl, and the other of R and R' is alkyl, preferably lower alkyl.
  • arylaminocarbonyl refers to -C(O)NHR in which R is aryl, preferably lower aryl, more preferably phenyl.
  • alkoxycarbonyl refers to -C(O)OR in which R is alkyl, preferably lower alkyl.
  • aryloxycarbonyl refers to -C(O)OR in which R is aryl, preferably lower aryl, more preferably phenyl.
  • alkoxy and alkylthio refer to RO- and RS-, in which R is alkyl, preferably lower alkyl.
  • aryloxy and “arylthio” refer to RO- and RS-, in which R is aryl, preferably lower aryl, more preferably phenyl.
  • alkylene refers to a straight, branched or cyclic, preferably straight or branched, bivalent aliphatic hydrocarbon group, preferably having from 1 to about 20 carbon atoms, more preferably 1 to 1 2 carbons, even more preferably lower alkylene. The alkylene group is optionally substituted with one or more "alkyl group substituents.
  • alkylene groups include methylene (-CH 2 -), ethylene (-CH 2 CH 2 -), propylene ( — (CH 2 ) 3 -), cyclohexylene (-C 6 H 10 -), methylenedioxy (-O-CH 2 - O-) and ethylenedioxy (-O-(CH 2 ) 2 -O-) .
  • lower alkylene refers to alkylene groups having 1 to 6 carbons. Preferred alkylene groups are lower alkylene, with alkylene of 1 to 3 carbon atoms being particularly preferred.
  • alkenylene refers to a straight, branched or cyclic, preferably straight or branched, bivalent aliphatic hydrocarbon group, preferably having from 1 to about 20 carbon atoms and at least one double bond, more preferably 1 to 1 2 carbons, even more preferably lower alkenylene.
  • the alkenylene group is optionally substituted with one or more "alkyl group substituents.” There may be optionally inserted along the alkenylene group one or more oxygen, sulphur or substituted or unsubstituted nitrogen atoms, where the nitrogen substituent is alkyl as previously described.
  • the term "lower alkenylene” refers to alkenylene groups having 2 to 6 carbons. Preferred alkenylene groups are lower alkenylene, with alkenylene of 3 to 4 carbon atoms being particularly preferred.
  • alkynylene refers to a straight, branched or cyclic, preferably straight or branched, bivalent aliphatic hydrocarbon group, preferably having from 1 to about 20 carbon atoms and at least one triple bond, more preferably 1 to 1 2 carbons, even more preferably lower alkynylene.
  • the alkynylene group is optionally substituted with one or more "alkyl group substituents. " There may be optionally inserted along the alkynylene group one or more oxygen, sulphur or substituted or unsubstituted nitrogen atoms, where the nitrogen substituent is alkyl as previously described.
  • Exemplary alkynylene groups include — C ⁇ C- C ⁇ C- , -C ⁇ C- and -C ⁇ C-CH 2 -.
  • the term "lower alkynylene” refers to alkynylene groups having 2 to 6 carbons.
  • Preferred alkynylene groups are lower alkynylene, with alkynylene of 3 to 4 carbon atoms being particularly preferred.
  • arylene refers to a monocyclic or polycyclic, preferably monocyclic, bivalent aromatic group, preferably having from 1 to about 20 carbon atoms and at least one aromatic ring, more preferably 1 to 1 2 carbons, even more preferably lower arylene.
  • the arylene group is optionally substituted with one or more "alkyl group substituents.” There may be optionally inserted around the arylene group one or more oxygen, sulphur or substituted or unsubstituted nitrogen atoms, where the nitrogen substituent is alkyl as previously described.
  • Exemplary arylene groups include 1 ,2-, 1 ,3- and 1 ,4- phenylene.
  • the term "lower arylene” refers to arylene groups having 5 or 6 carbons. Preferred arylene groups are lower arylene.
  • heteroarylene refers to a bivalent monocyclic or multicyclic ring system, preferably of about 5 to about 1 5 members where one or more, more preferably 1 to 3 of the atoms in the ring system is a heteroatom, that is, an element other than carbon, for example, nitrogen, oxygen and sulfur atoms.
  • the heteroarylene group may be optionally substituted with one or more, preferably 1 to 3, aryl group substituents.
  • Exemplary heteroarylene groups include, for example, 1 ,4-imidazolylene.
  • alkylenedioxy refers to -O-alkylene-O-; alkylenethioxyoxy refers to -S-alkylene-O-; and “alkylenedithioxy” refers to -S-alkylene-S-.
  • arylalkylidene refers to an alkylidene group in which either R' or R" is and aryl group.
  • amido refers to a bivalent group, either -C(O)NH- or -HNC(O)-.
  • Thioamido refers to a bivalent group, either -C(S)CH- or - HNC(S)-.
  • Oxyamido refers to a bivalent group, either -OC(O)NH- or - HNC(O)O-.
  • Thiaamido refers to a bivalent group, either -SC(O)NH- or - HNC(O)S-.
  • Dithiaamido refers to a bivalent group, either -SC(S)NH- or -HNC(S)S-.
  • Ureido refers to the bivalent group -HNCONH-.
  • Thioureido refers to the bivalent group -HNCSNH-.
  • amino acid refers to ⁇ -amino acids which are racemic, or of either the D- or L-configuration.
  • the pharmaceutical compositions contain arginine amides or C-amido arginine di- or tripeptide analogs.
  • the amino acids are of either the D- or L-configuration, or are racemic.
  • the pharmaceutical compositions contain compounds of formulae I:
  • R ⁇ R 2 , R 3 , R 4 , R 5 and R 6 are each independently selected from (i) or (ii) as follows:
  • R ⁇ R 3 and R 5 are each independently selected from alkyl, alkenyl, alkynyl, aryl, cycloalkyl, aralkyl and heteroaralkyl, and are unsubstituted or substituted with one or more substituents each independently selected from Z, and
  • R 2 , R 4 and R 6 are each independently selected from hydrogen or alkyl; or (ii) one or more of R 1 and R 2 , or R 3 and R 4 , or R 5 and R 6 form alkylene or alkenylene which is unsubstituted or substituted with one or more substituents each independently selected from Z, and the others are selected as in (i);
  • A is hydrogen, arylalkoxycarbonyl or arylcarbonyl; and D is aryl, heteroaryl, aralkyl or heteroaralkyl, and is unsubstituted or substituted with one or more substituents each independently selected from Z;
  • Z is halo, hydroxy, amino, azido, guanidino, nitro, alkyl, aryl, heteroaryl, alkoxy, aryloxy, heteroaryloxy, arylalkoxy, heteroarylalkoxy, alkoxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, or any
  • A is hydrogen, benzyloxycarbonyl (Cbz), 9-fluorenylmethoxycarbonyl (Fmoc) or benzoyl (Bz) .
  • D is benzyl (Bn), 4-nitrophenyl, 1 -methoxy-3- naphthyl (4MbNA) or 4-methyl-7-cumarinyl (AMC) .
  • A is hydrogen, benzyloxycarbonyl (Cbz), 9- fluorenylmethoxycarbonyl (Fmoc) or benzoyl (Bz) and D is benzyl (Bn), 4- nitrophenyl, 1 -methoxy-3-naphthyl (4MbNA) or 4-methyl-7-cumarinyl (AMC) .
  • R ⁇ R 2 , R 3 , R 4 , R 5 and R 6 are selected as in (i) or (ii) as follows:
  • R 1 , R 3 and R 5 are each independently selected from alkyl and aralkyl, and are unsubstituted or substituted with one or more substituents each independently selected from Z, and R 2 , R 4 and R 6 are each hydrogen; or
  • R 1 and R 2 , or R 3 and R 4 , or R 5 and R 6 form alkylene, and the others are selected as in (i) .
  • R ⁇ R 2 , R 3 , R 4 , R 5 and R 6 are selected as in (i) or (ii) as follows:
  • R ⁇ R 3 and R 5 are each independently selected from benzyl, 3- guanidinylpropyl, 4-hydroxyphenylmethyl, methyl, 2-methylpropyl, isopropyl and 4-aminobutyl, and R 2 , R 4 and R 6 are each hydrogen; or
  • R 3 and R 4 , or R 5 and R 6 form propylene, and the others are selected as in (i) .
  • the guanidinyl group is further substituted with 2,2,5,7,8-pentamethyl-6-chromylsulfonyl (Pmc) .
  • the peptides contain amino acids selected from phenylalanine, arginine, tyrosine, alanine, valine, leucine, lysine and proline.
  • Presently preferred compounds of formula I include (Fmoc)-Phe- Arg-NHBn, (Fmoc)-Phe-D-Arg-NHBn, (Fmoc)-D-Phe-Arg-NHBn, (Fmoc)-D- Phe-D-Arg-NHBn, (Fmoc)-Tyr-Arg-NHBn, (Fmoc)-Ala-Arg-NHBn, (Fmoc)- Leu-Arg-NHBn, (Fmoc)-Arg-Phe-NHBn, (Fmoc)-Arg(Pmc)-Phe-NHBn, (Fmoc)-D-Arg-Phe-NHBn, (Fmoc)-Arg-D-Phe-NHBn, (Fmoc)-Arg-D-Phe-NHBn, (Fmoc)-Arg-D-Phe-NHBn, (Fmoc)-D-Arg-D-Phe
  • compositions contain aminoimino hydrazones of formula II:
  • R or pharmaceutically acceptable derivatives thereof including salts, esters, acids, bases, sovates, hydrates and prodrugs thereof, where:
  • Ar 1 is higher alkylene, which, as defined herein, contains about 7- 30 carbon atoms, or is monocyclic or polycyclic arylene, alkenylarylene, arylalkenylene, heteroarylene, alkenylheteroarylene or heteroarylalkenylene, and is unsubstituted or substituted with one or more substituents each independently selected from Z;
  • Z is halo, hydroxy, amino, azido, guanidino, nitro, alkyl, aryl, heteroaryl, alkoxy, aryloxy, heteroaryloxy, arylalkoxy, heteroarylalkoxy, alkoxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, or any two Z substituents may form alkylene, alkenylene, alkylenedioxy, alkylenethioxyoxy or alkylenedithioxy, and is unsubstituted or substituted with one or more substituents each independently selected from Y;
  • Y is halo, amino, alkylamino, dialkylamino, arylamino, diarylamino, acylamino, azido, nitro, alkyl, aryl, heteroaryl, alkoxy, aryloxy or heteroaryloxy;
  • R 7 , R 8 , R 9 , R 10 , R 1 1 and R 12 are each independently selected from (i) or (ii) as follows:
  • R 7 is selected from hydrogen and alkyl, R 8 and R 9 are hydrogen, R 10 is hydrogen, arylsulfonyl or aralkyl, R 1 1 is hydrogen, and R 12 is hydrogen or Z, or
  • any two of R 7 , R 8 , R 9 , R 10 , R 1 1 and R 12 together form alkylene or carbonylalkylene, and the remainder are selected as in (i); and m is 0 or 1 when Ar 1 is arylalkenylene or heteroarylalkenylene, or is 1 .
  • R 7 , R 8 , R 9 , R 0 , R 1 1 and R 12 are each independently selected from (i) or (ii) as follows: (i) R 7 is selected from hydrogen and alkyl, R 8 and R 9 are hydrogen, R 10 is hydrogen, arylsulfonyl or aralkyl, R 1 1 is hydrogen, and R 12 is hydrogen or Z, or
  • R 8 , R 9 and R 10 are selected as in (i), and (a) R 1 1 is selected as in (i), and R 7 and R 12 together form alkylene or carbonylalkylene, or (b) R 7 is selected as in (i), and R 1 1 and R 12 together form alkylene or carbonylalkylene.
  • Ar 1 and R 12 taken together are higher alkyl, preferably C 7 H 15 to C 20 H 41 , more preferably C 8 H 17 to C 15 H 31 , most preferably C g H 19 .
  • Ar 1 and R 12 taken together are monocyclic or bicyclic aryl, alkenylaryl, arylalkenylene or heteroarylalkenylene, and is unsubstituted or substituted with one or more substituents selected from Z.
  • Ar 1 and R 12 taken together are more preferably phenyl, 2-phenylalkenyl, 2-thienylalkylene or naphthyl, most preferably phenyl, 2-phenylethenyl, 2-thienylethylene and naphthyl, and are substituted with one or more groups selected from chloro, methoxy, biphenyl, benzoyloxy, 4-nitrophenyloxycarbonyl, 4-acetylamino- phenyloxycarbonyl, ethylene, methylene, fluoro, 2-phenylethyloxy, 3,4- dichlorobenzyioxy, 4-chlorobenzyloxy, phenyloxy, pentyl, benzyloxy and 2,4-dichlorobenzyloxy.
  • R 7 , R 8 , R 9 , R 10 , R 1 1 and R 12 are each independently selected from (i), (ii) or (iii) as follows:
  • R 7 is hydrogen or methyl, or forms ethylene or propylene with R 12 , R 8 and R 9 are each hydrogen, or together form ethylene or propylene
  • R 10 is hydrogen, 4-chlorophenylsulfonyl or benzyl
  • R 1 1 is hydrogen and R 12 forms ethylene or propylene with R 7 , or is hydrogen or Z
  • R 8 , R 9 , R 1 1 and R 12 are selected as in (i), and R 7 and R 10 together form methylene; or
  • R 8 , R 9 , R 10 and R 12 are selected as in (i), and R 7 and R 1 1 together form methylenecarbonyl.
  • Ar 1 and R 12 taken together are selected from (2-phenylethyloxy)phenyl, 2,4-dichlorophenylethen-1 - yl, 3-(3,4-dichlorobenzyloxy)phenyl, 2-(4-chlorobenzyloxy)phenyl, 4- phenyloxyphenyl, 1 -pentyl-2-phenylethenyl, 3,4-dibenzyloxyphenyl, 2- (2,4-dichlorobenzyloxy)phenyl, 2-(3,4-dichlorobenzyloxy)phenyl, 1 - naphthyl, phenyl, 2,6-dichlorophenyl, 2-methoxyphenyl, (4-(3- phenyl)phenyl)phenyl, 3-benzoyloxy
  • Presently preferred compounds of formula II include 3-(2-phenyl- ethoxy)acetophenone aminoimino hydrazone, 2,4-dichlorobenzylidene- acetone aminoimino hydrazone, 3-(3,4-dichlorobenzyloxy)benzaldehyde aminoimino hydrazone, 2-(4-chlorobenzyloxy)benzaldehyde aminoimino hydrazone, 4-phenyloxybenzaldehyde aminoimino hydrazone, 2- pentylcinnamaldehyde aminoimino hydrazone, decanal aminoimino hydrazone, 3,4-dibenzyloxybenzaldehyde aminoimino hydrazone, 2-(2,4- dichlorobenzyloxy)benzaldehyde aminoimino hydrazone, 2-(3,4-dichloro- benzyloxy)benzaldehyde aminoimino hydrazone, 1 -
  • Arginine amide, peptide and analog derivatives Also of interest are any pharmaceutically-acceptable derivatives, including salts, esters, acids, bases, solvates, hydrates and prodrugs of the arginine amides, peptides and analogs. Such derivatives may be readily prepared by methods known to those of ordinary skill in the art.
  • Pharmaceutically-acceptable salts include, but are not limited to, amine salts, such as but not limited to N,N'-dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine, N- benzylphenethyiamine, 1 -para-chlorobenzyl-2-pyrrolidin-1 '-ylmethyl- benzimidazole, diethylamine and other alkylamines, piperazine and tris(hydroxymethyl)aminomethane; alkali metal salts, such as but not limited to lithium, potassium and sodium; alkali earth metal salts, such as but not limited to barium, calcium and magnesium; transition metal salts, such as but not limited to zinc; and other metal salts, such as but not limited to sodium hydrogen phosphate and disodium phosphate; and also including, but not limited to, salts of mineral acids, such as
  • Arginine amides and di- and tripeptides of formulae I may be prepared according to methods known to those of skill in the art. Such methods may include solid phase synthesis. See, e.g., Merrifield J. Am. Chem. Soc. 1963, 85, 2149-21 54.
  • the C-terminal amino acid may be N-protected as the corresponding Fmoc or Cbz derivative by reaction with the appropriate chloroformate, e.g., 9-fluorenylmethyl chloroformate or benzyl chloroformate.
  • the C-terminal amino acid may then be derivatized as the corresponding amide, such as a benzylamide, by reaction of the N-protected amino acid with, e.g., benzylamine, either HOBt (1 -hydroxybenzotriazole) or HOAt (1 -hydroxy-7-azabenzotriazole), and either DCC (dicyclohexylcarbodiimide) or DIC (diisopropylcarbo- diimide). Removal of the nitrogen protecting group by, e.g., treatment with piperidine or catalytic hydrogenation, provides the free amino acid amide. Coupling of this amino acid amide with a N-protected amino acid, prepared as described above, provides a dipeptide.
  • benzylamine either HOBt (1 -hydroxybenzotriazole) or HOAt (1 -hydroxy-7-azabenzotriazole
  • DCC dicyclohexylcarbodiimide
  • DIC diisopropylcar
  • Coupling may be achieved by reaction in the presence of, e.g., HOBt or HOAt, and DCC or DIC. Removal of the amino protecting group provides the desired dipeptides. Further coupling with a N-protected amino acid, followed by deprotection, affords the desired tripeptides. 2. Aminoimino hydrazones
  • Aminoimino hydrazones of formula II may be prepared according to the method described below or by other methods known to those of ordinary skill in the art. See, generally, Buckingham Q. Rev., Chem. Soc. 1969, 23, 37-56; Newkome et al. Pro. Svnth. 1976, 50, 102; McMurry J. Am. Chem. Soc. 1968, 90, 6821 . Additionally, many of the compounds of formula II are commercially available from sources known to those of skill in the art (e.g. , Aldrich Chemical Co., Milwaukee, Wl) .
  • aminoimino hydrazones of formula II may be prepared by reacting aminoguanidine (neutral or as a salt, such as, but not limited to, the hydrochloride, bicarbonate or nitrate salt) with the appropriate aldehyde or ketone under conditions allowing for the removal of water.
  • Such conditions include, but are not limited to, azeotropic removal of water, use of a dehydrating agent, such as, but not limited to, magnesium sulfate, molecular sieves or phosphorous pentoxide, or an organic/aqueous biphasic system where the organic solvent (i.e., dichloromethane or hexane) is highly immiscible with water.
  • compounds of formula II may be prepared by heating an aqueous alcoholic solution of the aldehyde or ketone with aminoguanidine (neutral or as a salt, such as, but not limited to, the hydrochloride, bicarbonate or nitrate salt) . Evaporation of the solvent, followed by recrystallization of the crude product from, e.g., methanol or ethyl acetate/hexanes provides the desired compounds.
  • aminoguanidine neutral or as a salt, such as, but not limited to, the hydrochloride, bicarbonate or nitrate salt
  • Certain compounds of formula II may be synthesized by substituting 2-hydrazino-2-imidazoiine hydrobromide, 1 -benzyl-2- hydrazino-2-imidazoline or 2-hydrazino-1 ,4,5,6-tetrahydropyrimidine for aminoguanidine.
  • Other compounds of formula II are prepared by condensing 2-hydrazino-2-imidazoline hydrobromide with benzoylacetic acid or 3,4-difluoro- ⁇ -bromoacetophenone.
  • compositions provided herein contain therapeutically effective amounts of one or more of the arginine amides, peptides or analogs of formula I or II that are useful in the prevention or treatment of FGF-mediated diseases.
  • the compositions contain arginine amides, C-amido di- and tripeptide arginine peptide analogs, or aminoi- ino hydrazones.
  • the compounds are preferably formulated into suitable pharmaceutical preparations such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release formulations or elixirs, for oral administration or in sterile solutions or suspensions for parenteral administration, as well as transdermal patch preparation and dry powder inhalers.
  • suitable pharmaceutical preparations such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release formulations or elixirs, for oral administration or in sterile solutions or suspensions for parenteral administration, as well as transdermal patch preparation and dry powder inhalers.
  • suitable pharmaceutical preparations such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release formulations or elixirs, for oral administration or in sterile solutions or suspensions for parenteral administration, as well as transdermal patch preparation and dry powder inhalers.
  • the compounds described above are formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, e.g
  • the compounds may be derivatized as the corresponding salts, esters, acids, bases, solvates, hydrates or prodrugs prior to formulation, as described above.
  • concentrations of the compounds in the compositions are effective for delivery of an amount, upon administration, that ameliorates the symptoms of the FGF-mediated disease.
  • the compositions are formulated for single dosage administration.
  • the weight fraction of compound is dissolved, suspended, dispersed or otherwise mixed in a selected vehicle at an effective concentration such that the treated condition is relieved or ameliorated.
  • Pharmaceutical carriers or vehicles suitable for administration of the compounds provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration.
  • the compounds may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients.
  • Liposomal suspensions including tissue-targeted liposomes, may also be suitable as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art. For example, liposome formulations may be prepared as described in U.S. Patent No. 4,522,81 1 .
  • the active compound is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated.
  • the therapeutically effective concentration may be determined empirically by testing the compounds in known in vitro and ]n vivo systems (see, e.g., Moscatelli et al. J. Cell. Phvsiol. 1987, 1 31 , 1 23- 1 30; Gospodarowicz et al. Proc. Natl. Acad. Sci. U.S.A. 1984, 8_1, 6963-6967; Thomas et al. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 357; European Patent Application No.
  • EP 645 451 International Application Publication No. WO 92/1 2245; Moscatelli et al. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 2091 -2095; Phadke Biochem. Biophvs. Res. Comm. 1987, 1 42. 448-453; Togari et al. Biochem. Biophvs. Res. Comm. 1983, 1 14, 1 1 89-1 1 93; and Wagner et al. J. Cell Biol. 1986, 1 03, 1 363-1 367) and then extrapolated therefrom for dosages for humans.
  • the concentration of active compound in the drug composition will depend on absorption, inactivation and excretion rates of the active compound, the physicochemical characteristics of the compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art. For example, the amount that is delivered is sufficient to treat the symptoms of diabetes.
  • a therapeutically effective dosage should produce a serum concentration of active ingredient of from about 0.1 ng/ml to about 50-100 ⁇ g/ml.
  • the pharmaceutical compositions typically should provide a dosage of from about 0.001 mg to about 2000 mg of compound per kilogram of body weight per day.
  • Pharmaceutical dosage unit forms are prepared to provide from about 1 mg to about 1 000 mg and preferably from about 10 to about 500 mg of the essential active ingredient or a combination of essential ingredients per dosage unit form.
  • the active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.
  • Preferred pharmaceutically acceptable derivatives include acids, bases, salts, esters, hydrates, solvates and prodrug forms.
  • the derivative is selected such that its pharmacokinetic properties are superior to the corresponding neutral compound.
  • effective concentrations or amounts of one or more of the compounds described herein or pharmaceutically acceptable derivatives thereof are mixed with a suitable pharmaceutical carrier or vehicle for systemic, topical or local administration to form pharmaceutical compositions.
  • Compounds are included in an amount effective for ameliorating or treating the FGF-mediated disorder for which treatment is contemplated.
  • the concentration of active compound in the composition will depend on absorption, inactivation, excretion rates of the active compound, the dosage schedule, amount administered, particular formulation as well as other factors known to those of skill in the art.
  • compositions are intended to be administered by a suitable route, which includes orally, parenterally, rectally and topically and locally depending upon the disorder being treated.
  • a suitable route which includes orally, parenterally, rectally and topically and locally depending upon the disorder being treated.
  • capsules and tablets are presently preferred.
  • the compositions are in liquid, semi-liquid or solid form and are formulated in a manner suitable for each route of administration. Preferred modes of administration include parenteral and oral modes of administration.
  • Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include any of the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvent; antimicrobial agents, such as benzyl alcohol and methyl parabens; antioxidants, such as ascorbic acid and sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates and phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • a sterile diluent such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvent
  • antimicrobial agents such as benzyl alcohol and methyl parabens
  • antioxidants such as ascorbic acid and sodium bisul
  • Parenteral preparations can be enclosed in ampules, disposable syringes or single or multiple dose vials made of glass, plastic or other suitable material.
  • methods for solubilizing compounds may be used. Such methods are known to those of skill in this art, and include, but are not limited to, using cosolvents, such as dimethylsulfoxide (DMSO), using surfactants, such as TWEEN ® , or dissolution in aqueous sodium bicarbonate.
  • cosolvents such as dimethylsulfoxide (DMSO)
  • surfactants such as TWEEN ®
  • dissolution in aqueous sodium bicarbonate such as sodium bicarbonate
  • Derivatives of the compounds, such as prodrugs of the compounds may also be used in formulating effective pharmaceutical compositions.
  • the resulting mixture may be a solution, suspension, emulsion or the like.
  • the form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle.
  • the effective concentration is sufficient for ameliorating the symptoms of the disease, disorder or condition treated and may be empirically determined.
  • the pharmaceutical compositions are provided for administration to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral solutions or suspensions, and oil-water emulsions containing suitable quantities of the compounds or pharmaceutically acceptable derivatives thereof.
  • the pharmaceutically therapeutically active compounds and derivatives thereof are typically formulated and administered in unit-dosage forms or multiple-dosage forms.
  • Unit-dose forms as used herein refers to physically discrete units suitable for human and animal subjects and packaged individually as is known in the art. Each unit-dose contains a predetermined quantity of the therapeutically active compound sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carrier, vehicle or diluent.
  • unit-dose forms include ampoules and syringes and individually packaged tablets or capsules. Unit-dose forms may be administered in fractions or multiples thereof.
  • a multiple-dose form is a plurality of identical unit-dosage forms packaged in a single container to be administered in segregated unit-dose form. Examples of multiple-dose forms include vials, bottles of tablets or capsules or bottles of pints or gallons. Hence, multiple dose form is a multiple of unit-doses which are not segregated in packaging.
  • the composition can contain along with the active ingredient: a diluent such as lactose, sucrose, dicalcium phosphate, or carboxymethylcellulose; a lubricant, such as magnesium stearate, calcium stearate and talc; and a binder such as starch, natural gums, such as gum acaciagelatin, glucose, molasses, polvinylpyrrolidine, celluloses and derivatives thereof, povidone, crospovidones and other such binders known to those of skill in the art.
  • a diluent such as lactose, sucrose, dicalcium phosphate, or carboxymethylcellulose
  • a lubricant such as magnesium stearate, calcium stearate and talc
  • a binder such as starch, natural gums, such as gum acaciagelatin, glucose, molasses, polvinylpyrrolidine, celluloses and derivatives thereof, povidone, crospovidones and
  • Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, or otherwise mixing an active compound as defined above and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution or suspension.
  • a carrier such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution or suspension.
  • the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, or solubilizing agents, pH buffering agents and the like, for example, acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents.
  • auxiliary substances such as wetting agents, emulsifying agents, or solubilizing agents, pH buffering agents and the like, for example, acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents.
  • auxiliary substances such as wetting agents, emulsifying agents, or solubilizing agents, pH buffering agents and the like, for example, acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine ole
  • compositions containing active ingredient in the range of 0.005% to 100% with the balance made up from non-toxic carrier may be prepared.
  • a pharmaceutically acceptable non-toxic composition is formed by the incorporation of any of the normally employed excipients, such as, for example pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, talcum, cellulose derivatives, sodium crosscarmellose, glucose, sucrose, magnesium carbonate or sodium saccharin.
  • compositions include solutions, suspensions, tablets, capsules, powders and sustained release formulations, such as, but not limited to, implants and microencapsuiated delivery systems, and biodegradable, biocompatible polymers, such as collagen, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid and others. Methods for preparation of these compositions are known to those skilled in the art.
  • the contemplated compositions may contain 0.001 %-100% active ingredient, preferably 0.1 -85%, typically 75-95%.
  • the active compounds or pharmaceutically acceptable derivatives may be prepared with carriers that protect the compound against rapid elimination from the body, such as time release formulations or coatings.
  • compositions may include other active compounds to obtain desired combinations of properties.
  • the compounds of formulae I or II, or pharmaceutically acceptable derivatives thereof as described herein may also be advantageously administered for therapeutic or prophylactic purposes together with another pharmacological agent known in the general art to be of value in treating one or more of the diseases or medical conditions referred to hereinabove, such as beta-adrenergic blocker (for example atenolol), a calcium channel blocker (for example nifedipine), an angiotensin converting enzyme (ACE) inhibitor (for example lisinopril), a diuretic (for example furosemide or hydrochlorothiazide), an endothelin converting enzyme (ECE) inhibitor (for example phosphoramidon), a neutral endopeptidase (NEP) inhibitor, an HMGCoA reductase inhibitor, a nitric oxide donor, an anti-oxidant, a vasodilator, a dopamine agonist, a neuroprotect
  • the solid dosage forms are tablets, capsules, granules, and bulk powders.
  • Types of oral tablets include compressed, chewable lozenges and tablets which may be enteric-coated, sugar-coated or film-coated.
  • Capsules may be hard or soft gelatin capsules, while granules and powders may be provided in non-effervescent or effervescent form with the combination of other ingredients known to those skilled in the art.
  • the formulations are solid dosage forms, preferably capsules or tablets.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder; a diluent; a disintegrating agent; a lubricant; a glidant; a sweetening agent; and a flavoring agent.
  • binders include microcrystalline cellulose, gum tragacanth, glucose solution, acacia mucilage, gelatin solution, sucrose and starch paste.
  • Lubricants include talc, starch, magnesium or calcium stearate, lycopodium and stearic acid.
  • Diluents include, for example, lactose, sucrose, starch, kaolin, salt, mannitol and dicalcium phosphate.
  • Glidants include, but are not limited to, colloidal silicon dioxide.
  • Disintegrating agents include crosscarmellose sodium, sodium starch glycolate, alginic acid, corn starch, potato starch, bentonite, methylcellulose, agar and carboxymethylcellulose.
  • Coloring agents include, for example, any of the approved certified water soluble FD and C dyes, mixtures thereof; and water insoluble FD and C dyes suspended on alumina hydrate.
  • Sweetening agents include sucrose, lactose, mannitol and artificial sweetening agents such as saccharin, and any number of spray dried flavors.
  • Flavoring agents include natural flavors extracted from plants such as fruits and synthetic blends of compounds which produce a pleasant sensation, such as, but not limited to peppermint and methyl salicylate.
  • Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene laural ether.
  • Emetic-coatings include fatty acids, fats, waxes, shellac, ammoniated shellac and cellulose acetate phthalates.
  • Film coatings include hydroxyethylcellulose, sodium carboxymethylcellulose, polyethylene glycol 4000 and cellulose acetate phthalate.
  • the compound could be provided in a composition that protects it from the acidic environment of the stomach.
  • the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine.
  • the composition may also be formulated in combination with an antacid or other such ingredient.
  • the dosage unit form When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil.
  • dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents.
  • the compounds can also be administered as a component of an elixir, suspension, syrup, wafer, sprinkle, chewing gum or the like.
  • a syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.
  • the active materials can also be mixed with other active materials which do not impair the desired action, or with materials that supplement the desired action, such as antacids, H2 blockers, and diuretics.
  • the compound is used for treating asthma or hypertension, it may be used with other bronchodilators and antihypertensive agents, respectively.
  • the active ingredient is a compound or pharmaceutically acceptable derivative thereof as described herein. Higher concentrations, up to about 98% by weight of the active ingredient may be included.
  • Pharmaceutically acceptable carriers included in tablets are binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, and wetting agents.
  • Enteric-coated tablets because of the enteric-coating, resist the action of stomach acid and dissolve or disintegrate in the neutral or alkaline intestines.
  • Sugar-coated tablets are compressed tablets to which different layers of pharmaceutically acceptable substances are applied.
  • Film-coated tablets are compressed tablets which have been coated with a polymer or other suitable coating.
  • Multiple compressed tablets are compressed tablets made by more than one compression cycle utilizing the pharmaceutically acceptable substances previously mentioned.
  • Coloring agents may also be used in the above dosage forms. Flavoring and sweetening agents are used in compressed tablets, sugar-coated, multiple compressed and chewable tablets. Flavoring and sweetening agents are especially useful in the formation of chewable tablets and lozenges.
  • Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules.
  • Aqueous solutions include, for example, elixirs and syrups.
  • Emulsions are either oil-in-water or water-in-oil.
  • Elixirs are clear, sweetened, hydroalcoholic preparations.
  • Pharmaceutically acceptable carriers used in elixirs include solvents. Syrups are concentrated aqueous solutions of a sugar, for example, sucrose, and may contain a preservative.
  • An emulsion is a two-phase system in which one liquid is dispersed in the form of small globules throughout another liquid.
  • Pharmaceutically acceptable carriers used in emulsions are non-aqueous liquids, emulsifying agents and preservatives. Suspensions use pharmaceutically acceptable suspending agents and preservatives.
  • Pharmaceutically acceptable substances used in non-effervescent granules, to be reconstituted into a liquid oral dosage form include diluents, sweeteners and wetting agents.
  • Pharmaceutically acceptable substance used in effervescent granules, to be reconstituted into a liquid oral dosage form include organic adds and a source of carbon dioxide. Coloring and flavoring agents are used in all of the above dosage forms.
  • Solvents include glycerin, sorbitol, ethyl alcohol and syrup.
  • preservatives include glycerin, methyl and propylparaben, benzoic add, sodium benzoate and alcohol.
  • non-aqueous liquids utilized in emulsions include mineral oil and cottonseed oil.
  • emulsifying agents include gelatin, acacia, tragacanth, bentonite, and surfactants such as polyoxyethylene sorbitan monooleate.
  • Suspending agents include sodium carboxymethylcellulose, pectin, tragacanth, Veegum and acacia.
  • Diluents include lactose and sucrose.
  • Sweetening agents include sucrose, syrups, glycerin and artificial sweetening agents such as saccharin.
  • Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene lauryl ether.
  • Organic adds include citric and tartaric acid.
  • Sources of carbon dioxide include sodium bicarbonate and sodium carbonate.
  • Coloring agents include any of the approved certified water soluble FD and C dyes, and mixtures thereof.
  • Flavoring agents include natural flavors extracted from plants such fruits, and synthetic blends of compounds which produce a pleasant taste sensation.
  • the solution or suspension in for example propylene carbonate, vegetable oils or triglycerides, is preferably encapsulated in a gelatin capsule.
  • a gelatin capsule Such solutions, and the preparation and encapsulation thereof, are disclosed in U.S. Patent Nos 4,328,245; 4,409,239; and 4,410,545.
  • the solution e.g., for example, in a polyethylene glycol, may be diluted with a sufficient quantity of a pharmaceutically acceptable liquid carrier, e.g., water, to be easily measured for administration.
  • liquid or semi-solid oral formulations may be prepared by dissolving or dispersing the active compound or salt in vegetable oils, glycols, triglycerides, propylene glycol esters (e.g., propylene carbonate) and other such carriers, and encapsulating these solutions or suspensions in hard or soft gelatin capsule shells.
  • Other useful formulations include those set forth in U.S. Patent Nos. Re 28,81 9 and 4,358,603.
  • tablets and capsules formulations may be coated as known by those of skill in the art in order to modify or sustain dissolution of the active ingredient.
  • they may be coated with a conventional enterically digestible coating, such as phenylsalicylate, waxes and cellulose acetate phthalate.
  • enterically digestible coating such as phenylsalicylate, waxes and cellulose acetate phthalate.
  • injectables, solutions and emulsions Parenteral administration, generally characterized by injection, either subcutaneously, intramuscularly or intravenously is also contemplated herein. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol.
  • the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such agents, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins.
  • Implantation of a slow-release or sustained-release system such that a constant level of dosage is maintained (see, e.g ., U.S. Patent No. 3,71 0,795) is also contemplated herein.
  • the percentage of active compound contained in such parenteral compositions is highly dependent on the specific nature thereof, as well as the activity of the compound and the needs of the subject.
  • Parenteral administration of the compositions includes intravenous, subcutaneous and intramuscular administrations.
  • Preparations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use and sterile emulsions.
  • the solutions may be either aqueous or nonaqueous.
  • suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof.
  • PBS physiological saline or phosphate buffered saline
  • thickening and solubilizing agents such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof.
  • Pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents and other pharmaceutically acceptable substances.
  • aqueous vehicles examples include Sodium Chloride Injection, Ringers Injection, Isotonic Dextrose Injection, Sterile Water Injection, Dextrose and Lactated Ringers Injection.
  • Nonaqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil and peanut oil.
  • Antimicrobial agents in bacteriostatic or fungistatic concentrations must be added to parenteral preparations packaged in multiple-dose containers which include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride.
  • Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcelluose, hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifying agents include Polysorbate 80 (TWEEN ® 80). A sequestering or chelating agent of metal ions include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol and propylene glycol for water miscible vehicles and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.
  • the concentration of the pharmaceutically active compound is adjusted so that an injection provides an effective amount to produce the desired pharmacological effect.
  • the exact dose depends on the age, weight and condition of the patient or animal as is known in the art.
  • the unit-dose parenteral preparations are packaged in an ampoule, a vial or a syringe with a needle. All preparations for parenteral administration must be sterile, as is known and practiced in the art.
  • intravenous or intraarterial infusion of a sterile aqueous solution containing an active compound is an effective mode of administration.
  • Another embodiment is a sterile aqueous or oily solution or suspension containing an active material injected as necessary to produce the desired pharmacological effect.
  • Injectables are designed for local and systemic administration.
  • a therapeutically effective dosage is formulated to contain a concentration of at least about 0.1 % w/w up to about 90% w/w or more, preferably more than 1 % w/w of the active compound to the treated tissue(s).
  • the active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the tissue being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the age of the individual treated.
  • the compound may be suspended in micronized or other suitable form or may be derivatized to produce a more soluble active product or to produce a prodrug.
  • the form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle.
  • the effective concentration is sufficient for ameliorating the symptoms of the condition and may be empirically determined. 3.
  • Lyophilized powders Of interest herein are also lyophilized powders, which can be reconstituted for administration as solutions, emulsions and other mixtures. They may also be reconstituted and formulated as solids or gels.
  • the sterile, lyophilized powder is prepared by dissolving a compound of formula (I) or (II) in a buffer solution.
  • the buffer solution may contain an excipient which improves the stability or other pharmacological component of the powder or reconstituted solution, prepared from the powder. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides the desired formulation.
  • the lyophilized powder is prepared by dissolving an excipient, such as dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agent, in a suitable buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, typically, about neutral pH. Then, a selected compound of formula (I) or (II) is added to the resulting mixture, and stirred until it dissolves. The resulting mixture is sterile filtered or treated to remove particulates and to insure sterility, and apportioned into vials for lyophilization. Each vial will contain a single dosage (100-500 mg, preferably 250 mg) or multiple dosages of the compound.
  • the lyophilized powder can be stored under appropriate conditions, such as at about 4 °C to room temperature.
  • Reconstitution of this lyophilized powder with water for injection provides a formulation for use in parenteral administration.
  • For reconstitution about 1 -50 mg, preferably 5-35, more preferably about 9- 30 is added per mL of sterile water or other suitable carrier. The precise amount depends upon the indication treated and selected compound. Such amount can be empirically determined.
  • Topical administration Topical mixtures are prepared as described for the local and systemic administration.
  • the resulting mixture may be a solution, suspension, emulsions or the like and are formulated as creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, irrigations, sprays, suppositories, bandages, dermal patches or any other formulations suitable for topical administration.
  • the compounds or pharmaceutically acceptable derivatives thereof may be formulated as aerosols for topical application, such as by inhalation (see, e.g., U.S. Patent Nos. 4,044, 1 26, 4,414,209, and 4,364,923, which describe aerosols for delivery of a steroid useful for treatment inflammatory diseases, particularly asthma).
  • These formulations for administration to the respiratory tract can be in the form of an aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose.
  • the particles of the formulation will typically diameters of less than 50 microns, preferably less than 10 microns.
  • the compounds may be formulated for local or topical application, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intracisternal or intraspinal application.
  • Topical administration is contemplated for transdermal delivery and also for administration to the eyes or mucosa, or for inhalation therapies. Nasal solutions of the active compound alone or in combination with other pharmaceutically acceptable excipients can also be administered.
  • solutions particularly those intended for ophthalmic use, may be formulated as 0.01 % - 10% isotonic solutions, pH about 5-7, with appropriate salts.
  • compositions for ophthalmic administration are formulated in an ophthalmically acceptable carrier.
  • local administration either by topical administration or by injection is preferred.
  • Time release formulations are also desirable.
  • the compositions are formulated for single dosage administration, so that a single dose administers an effective amount.
  • Ophthalmologically effective concentrations or amounts of one or more of the compounds are mixed with a suitable pharmaceutical carrier or vehicle.
  • concentrations or amounts of the conjugates that are effective requires delivery of an amount, upon administration, that prevents or substantially reduces the effects of FGF-mediated ophthalmological conditions, including, but not limited to, diabetic retinopathy, corneal clouding following excimer laser surgery, closure of trabeculectomies, hyperproliferation of lens epithelial cells following cataract surgery and the recurrence of pterygii.
  • the compounds can also be mixed with other active materials, that do not impair the desired action, or with materials that supplement the desired action, including viscoelastic materials, such as hyaluronic acid, which is sold under the trademark HEALON (solution of a high molecular weight (MW of about 3 million) fraction of sodium hyaluronate; manufactured by Pharmacia, Inc. see, e.g., U.S. Patent Nos.
  • HEALON solution of a high molecular weight (MW of about 3 million) fraction of sodium hyaluronate
  • the viscoelastic materials are present generally in amounts ranging from about 0.5 to 5.0%, preferably 1 to 3% by weight of the conjugate material and serve to coat and protect the treated tissues.
  • the compositions may also include a dye, such as methylene blue or other inert dye, so that the composition can be seen when injected into the eye or contacted with the surgical site during surgery.
  • compositions for other routes of administration are also contemplated herein.
  • pharmaceutical dosage forms for rectal administration are rectal suppositories, capsules and tablets for systemic effect.
  • Rectal suppositories are used herein mean solid bodies for insertion into the rectum which melt or soften at body temperature releasing one or more pharmacologically or therapeutically active ingredients.
  • Pharmaceutically acceptable substances utilized in rectal suppositories are bases or vehicles and agents to raise the melting point.
  • bases examples include cocoa butter (theobroma oil), glycerin-gelatin, carbowax (polyoxyethylene glycol) and appropriate mixtures of mono-, di- and triglycerides of fatty acids. Combinations of the various bases may be used.
  • Agents to raise the melting point of suppositories include spermaceti and wax. Rectal suppositories may be prepared either by the compressed method or by molding. The typical weight of a rectal suppository is about 2 to 3 gm. Tablets and capsules for rectal administration are manufactured using the same pharmaceutically acceptable substance and by the same methods as for formulations for oral administration. 7.
  • compositions containing compounds or pharmaceutically acceptable derivatives may be packaged as articles of manufacture containing packaging material, a composition containing a compound or pharmaceutically acceptable derivative thereof provided herein, which is effective for antagonizing the effects of an FGF peptide, preferably bFGF, ameliorating the symptoms of an FGF-mediated disorder, or inhibiting binding of an FGF peptide to an FGF receptor with an IC 50 of less than about 500 ⁇ M, within the packaging material, and a label that indicates that the composition containing the compound or derivative thereof is used for antagonizing the effects of FGF, treating FGF-mediated disorders or inhibiting the binding of an FGF peptide to an FGF receptor.
  • FGF peptide preferably bFGF
  • Standard physiological, pharmacological and biochemical procedures are available for testing the compounds to identify those that possess biological activities of compounds that interfere with or inhibit or otherwise modulate the activity of FGF peptides.
  • Numerous assays are known to those of skill in the art for evaluating the ability of compounds to modulate the activity of one or more FGF peptides.
  • the properties of a potential antagonist may be assessed as a function of its ability to inhibit FGF activity including the ability ]n vitro to compete for binding to FGF receptors present on the surface of tissues or recombinant cell lines, cell-based competitive assays (see, e.g., Moscatelli et al. J. Cell. Phvsiol.
  • FGF isotype specific antagonists may be identified by the ability of a test compound to interfere with one or more FGF peptide binding to different tissues or cells expressing different endothelin receptor subtypes, or to interfere with the biological effects of an FGF peptide (see, e.g., International Patent Application Publication No. WO 95/24414).
  • the relative affinities of the compounds for FGF receptors have been and can be assessed. Those that possess the desired in vitro properties, such as specific inhibition of the binding of bFGF, are selected. The selected compounds that exhibit desirable activities may be therapeutically useful in the methods described herein and are tested for such uses employing the above-described assays from which the vivo effectiveness may be evaluated (Gospodarowicz et al. Endocrin. Rev. 1987, 8, 95-1 14; Buntrock et al. Exp. Pathol. 1982, 21, 62-67; International Patent Application Publication No WO 92/08473) .
  • compositions to prevent the undesired growth and proliferation of FGF- sensitive cells occurring in vascular disorders characterized by accelerated smooth muscle cell proliferation, such as rheumatoid arthritis, tumor angiogenesis, Kaposi's sarcoma, restenosis, In-Stent restenosis, certain ophthalmic disorders and dermatological disorders, such as psoriasis, are provided herein.
  • the medicament containing the compound is administered intravenously (IV), although treatment by localized administration may be tolerated in some instances.
  • IV intravenously
  • the medicament containing the compound is injected into the circulatory system of a subject in order to deliver a dose to the targeted cells.
  • Targeting may be effected by linking the compound to a targeting agent specific for FGF receptors, particularly bFGF receptors. Dosages may be determined empirically, but will typically be in the range of about 0.01 mg to about 100 mg of the compound per kilogram of body weight as a daily dosage. 1 . Restenosis and vascular injury
  • vascular injury particularly, restenosis or In- Stent restenosis by contacting the vascular wall with an effective amount of a composition containing compound(s) of formulae I or II are provided (see generally, Lindner et al. Proc. Natl. Acad. Sci. USA 1991 , 88, 3739; Kearney et al. Circulation 1997, 95, 1 998) .
  • Atherosclerosis also referred to as arteriosclerosis, results from the development of an intimal lesion and the subsequent narrowing of the vessel lumen.
  • atherosclerosis originally appears as a result of the buildup of plaque which lines the interior of blood vessels, particularly the arteries.
  • bypass surgery is sometimes employed to replace such clogged arteries, in recent years, a number of surgical procedures have been developed so as to interarterially remove such plaque, often by balloon catheterization or other such treatments in which the plaque is either compressed against or scraped away from the interior surface of the artery. This scraping of the interior wall removes endotheiial cells, which constitute the lining of the blood vessel.
  • the smooth muscle cells which are normally located exterior of the endotheiial cells (ECs) and form the blood vessel structure, begin to grow and multiply causing a narrowing of the vessel iumem.
  • the patient so treated finds a recurrence of such narrowing of the vessel lumen in a relatively short period thereafter as a result of this proliferation, generally referred to as restenosis, requiring a repetition of the surgical procedure to again remove the increasing blockage.
  • Proliferating SMCs express functional FGF receptors and are responsive to bFGF.
  • SMCs migrating smooth muscle cells
  • Basic FGF appears to play a pivotal role in the subsequent responses of the vascular wall.
  • Basic FGF is known to be synthesized by endotheiial and smooth muscle cells (SMCs) and is thought to be stored in the subendothelial matrix, and in some instances, this growth factor is released from cells after injury.
  • compounds that inhibit FGF-mediated proliferation of SMCs may be used in methods for treating restenosis by preventing the proliferation that causes the narrowing of the vessel lumem.
  • Treatment is effected by administering a therapeutically effective amount of a medicament containing the compound in a physiologically acceptable carrier or recipient, in a manner so that the compound reaches regions in a human or other mammal where the compound will inhibit the proliferation of the target cells.
  • a therapeutically effective amount of a medicament containing the compound in a physiologically acceptable carrier or recipient in a manner so that the compound reaches regions in a human or other mammal where the compound will inhibit the proliferation of the target cells.
  • intraarterial infusion will be among the preferred methods.
  • IV administration over a period of time is preferred.
  • the compounds for treating restenosis may be formulated for intravenous or local administration.
  • compounds may be conjugated to an agent that specifically targets proliferating SMCs, such as antibodies, hormones, ligands or the like to improve delivery and uptake of the compound.
  • the therapeutically effective concentration may be determined empirically by testing the compounds in known m vitro and in vivo systems (see, e.g., Moscatelli et al. J. Cell. Phvsiol. 1987, 1 31 , 123-130); mitogenic assays (Gospodarowicz et al. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 6963-6967; Thomas et al. Proc. Natl.
  • Rheumatoid arthritis is a systemic, chronic inflammatory disease, that is characterized by the destruction of the joint cartilage and inflammation of the synovium.
  • the hallmark feature of rheumatoid arthritis is the production circulating autoantibodies, also referred to as rheumatoid factors, which are reactive with the Fc portions of the patients own IgG molecules (e.g., see Abbas et al. , Cellular and Molecular Immunology, W.B. Saunders Co., Philadelphia, PA).
  • T-cells e.g., interleukin-1 , IFN-i and tumor necrosis factor (TNF)
  • TNF tumor necrosis factor
  • bFGF growth factors
  • cytokines and bFGF stimulate fibroblast and collagen proliferation resulting in angiogenesis, and prolonged exposure can result in hyperproliferation of epithelial cells that form fibrous tissue, referred to as fibrosis.
  • compounds that inhibit the FGF-mediated hyperproliferation of epithelial cells may be used to treat rheumatoid arthritis.
  • the compounds for treating rheumatoid arthritis may be formulated for oral administration or intravenous injection and an effective concentration may be administered. The effective concentration is sufficient for ameliorating the symptoms of the disease, disorder or condition treated and may be empirically determined.
  • Angiogenesis plays a critical role in embryonic development and in several physiologic and pathologic conditions, including wound healing, ovulation, diabetic retinopathy and malignancy. In particular, without the nutrients and oxygen provided via this neovascularization, solid tumors would be unable to grow beyond about 2 mm in diameter.
  • the compounds may be specifically targeted to tumorigenic tissues by linking the compound to an agent that specifically binds to the surface of the tumorigenic cell, e.g., an anti-tumor antigen antibody, or linking the compound to an agent that is preferentially interacts with or taken up by targeted tumor.
  • compounds may be encapsulated in tissue-targeted liposomal suspensions for targeted delivery of the compound.
  • the compounds for treating tumor angiogenesis may be formulated for topical application and administered to the skin, e.g., for treatment of melanoma, or may be formulated for intravenous administration for treatment of solid tumors, such as carcinomas.
  • the therapeutically effective concentration may be determined empirically by testing the compounds in known in vitro (e.g ., inhibition of angiogenesis in vitro (see, e.g., European Patent Application No. EP 645 451 )) and then extrapolated therefrom for dosages for humans. 4.
  • Ophthalmic Disorders e.g ., inhibition of angiogenesis in vitro (see, e.g., European Patent Application No. EP 645 451 )
  • compositions provided herein may be used in methods of treating ophthalmic disorders resulting from FGF-mediated hyper-proliferation of lens epithelial cells, fibroblasts or keratinocytes.
  • ophthalmic disorders that may be treated using the methods and compositions provided herein include, but are not limited to, corneal clouding following excimer laser surgery, closure of trabeculectomies, hyperproliferation of lens epithelial cells following cataract surgery, the recurrence of pterygii and diabetic retinopathy (see, Dell Drug Discovery Today 1996, 1, 221 ).
  • the compounds for treating ophthalmic disorders may be formulated for local or topical application and administered by topical application of an effective concentration to the skin and mucous membranes, such as in the eye.
  • the compositions may also include a dye, such as methylene blue or other inert dye, so that the composition can be seen when injected into the eye or contacted with the surgical site during surgery.
  • the effective concentration is sufficient for ameliorating the symptoms of the disease, disorder or condition treated and may be empirically determined.
  • Fmoc-Arg(Pmc)-OH (1 .324 g) was dissolved in N-methylpiperidone (NMP) .
  • HOBt (1 -hydroxybenzotriazole) (0.306 g) was added followed by DCC (dicyclohexylcarbodiimide) (0.41 2 g) .
  • DCC diclohexylcarbodiimide
  • the mixture was stirred for 25 min.
  • Benzylamine 0.1 96 mL was added slowly in six equal amounts. The mixture was stirred overnight.
  • the mixture was diluted with water and extracted into ethyl acetate (EtOAc) (3 x 25 mL) .
  • Fmoc-Arg(Pmc)-Benzylamide 1 .1 g was treated with 25% piperidine in DMF (dimethylformamide) . The mixture was stirred for 1 hr, when TLC showed the absence of starting material, the solution was concentrated to a small volume and diluted with ether. The white solid precipitated was filtered, washed with ether and dried under vacuum. Yield: 0.7 g. Fmoc-Phe-OH (0.774 g) was dissolved in NMP. HOBt (0.306 g) and DCC (0.41 2 g) were added and the mixture was stirred for 25 min. H-Arg(PMC)-Benzylamide (0.4 g) was added and the mixture was stirred overnight.
  • Fmoc-Phe-dArg-Benzylamide A. Fmoc-dArg(Pmc)-Benzylamide Fmoc-dArg(Pmc)-OH ( 1 .324 g) was dissolved in N- methylpiperidone (NMP) . HOAt (1 -hydroxy-7-azabenzotriazole) (0.272 g) was added followed by diisopropylcarbodiimide (DIC) (0.252 g) . The mixture was stirred for 25 min. Benzylamine (0.1 96 mL) was added slowly in six equal amounts. The mixture was stirred overnight. The mixture was diluted with water and extracted into EtOAc (3 x 25 mL) .
  • Fmoc-dArg(Pmc)-Benzylamide ( 1 .1 g) was treated with 25% piperidine in DMF. The mixture was stirred for 1 hr, when TLC showed the absence of starting material, the solution was concentrated to a small volume and diluted wtih ether. The white solid which precipitated was filtered, washed with ether and dried under vacuum. This product used in the next reaction.
  • EXAMPLE 8 The following compounds were prepared by minor modifications of the methods in Examples 1 -7: Fmoc-Ala-Arg-benzylamide, Fmoc-Leu-
  • Compounds of formulae I or II that exhibit FGF antagonist activity were and can be identified by testing their ability to compete with 125 l- bFGF for binding to one or more FGF receptor or FGF-binding fragment thereof.
  • a recombinant FGF receptor fusion protein was used in which the extracellular domain of a human FGF receptor,
  • FGFR1 tissue plasminogen activator (tPA) protein. This fusion protein retains the ability to bind
  • FGF such as bFGF (Zhu et al. J. Biol. Chem. 1995, 270, 21 869-21 874) .
  • FGFR1 human basic fibroblast growth factor receptor 1
  • This shorter form of FGFR1 is a 731 amino acid polypeptide that has a signal peptide, two extracellular immunoglobulin- like domains, a transmembrane domain and an intracellular tyrosine kinase domain.
  • PCR polymerase chain reactions
  • ATG initiation codon of FGFR1 (e.g., Itoh et al. Biochem. Biophvs. Res. Comm. 1990, 1 69, 680-685) and an oligonucleotide complementary to nt 221 8-2243 were used as primers to amplify a 2,243 bp PCR product encoding the entire FGRF1 coding region.
  • the full-length FGFR1 -encoding DNA was used as a template for a subsequent PCR reaction, performed as described above, to amplify a 869 bp DNA fragment encoding only the FGFR1 extracellular domain.
  • a Hindlll restriction endonuclease site was introduced upstream of the FGFR1 initiation codon and a Sail site was introduced downstream of the second immunoglobulin-like extracellular domain (Igll) to facilitate cloning of the amplified product.
  • the Hindlll site was introduced at nt -8 to -3 during the PCR reaction by synthesizing an oligonucleotide primer corresponding to nt - 1 2 to + 22 that introduced nucleotide changes at three positions in the FGFR1 sequence: nt -3 (G to T), nt -6 (A to G) and nt -8 (G to A) .
  • the Sail site was introduced at nt 849 to nt 854 by synthesizing an oligonucleotide primer complementary to nt 823 to 857 containing nucleotide substitutions at three positions in the FGFR1 sequence: nt 849 (C to G), nt 851 (G to C) and nt 854 (G to C) .
  • the 857 bp PCR fragment was incubated with Hindlll and Sail and purified by agarose gel electrophoresis according to the standard procedures (Sambrook et al. (1 989) Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory Press, New York). The DNA was isolated from gel by electroelution and recovered by precipitation with ethanol.
  • the resulting Hindlll to SaJI DNA fragment consists of nt -7 to nt 849 of the FGFR1 cDNA described by Itoh et al. and encodes amino acid residues 1 to 284 of the shorter form of the bFGF receptor.
  • tPA human tissue plasminogen activator
  • oligonucleotides complementary to sequences flanking the tPA coding region were synthesized and used as primers in PCR reactions to isolate a full-length cDNA encoding human tPA from a human placenta cDNA library (Clontech, Palo Alto, CA) .
  • An oligonucleotide corresponding to nt -6 to + 21 , relative to the A of the initiation codon of the of human tPA prepro polypeptide e.g., see Pennica et al.
  • oligonucleotide complementary to nt 1 558 to nt 1 584 were used to amplify a 1 591 bp DNA encoding the entire human tPA prepro polypeptide.
  • the full-length DNA was used as a template for a subsequent PCR reaction to amplify a 599 bp DNA encoding the a portion of the signal peptide-finger-growth factor-first Kringle domains of tPA, and which also to introduce an in-frame amber stop codon (i.e., UGA) at amino acid codon 180 of mature tPA sequence.
  • UGA in-frame amber stop codon
  • a Sail restriction endonuclease site and a mutation substituting a Pro for an Arg at position -6 were introduced upstream of the first Ser codon of mature tPA and a BamHI site was introduced downstream of newly introduced translational stop codon to allow for convenient subcloning of the amplified product.
  • the substitution of Pro for Arg at amino acid residue position -6 introduces a proteolytic cleavage site for thrombin in the linker sequence (i.e., Phe-Pro-Arg-Gly at positions -7 to -4) .
  • the Sail s 'te and the amino acid substitution were introduced at nt 76 to 81 and 91 and 92 (nt -30 to -25 and -1 5 and -1 4, respectively, relative to the first nucleotide of mature tPA) during the PCR reaction by synthesizing an oligonucleotide primer corresponding to nt 72 to nt 1 1 1 containing nucleotide substitutions at six positions in the tPA sequence: nt 76 (A to G), nt 79 (C to G), nt 81 (T to C), nt 91 (A to C) and nt 92 (G to C) .
  • nt 652 to nt 657 and translational stop codon at amino acid codon 1 80 were introduced by synthesizing an oligonucleotide primer complementary to nt 623 to 661 containing nucleotide substitutions at three positions in the tPA sequence: nt 644 (C to A), nt 655 (A to T) and nt 657 (G to C) .
  • the amplified PCR fragment was incubated with Sail and BamHI and subjected to agarose gel electrophoresis according to the standard procedures (Sambrook et al. ( 1 989) Molecular Cloning, 2nd ed., Cold
  • the isolated Sail to BamHI fragment encoding the portion of human tPA was ligated into the Sail and BamHI sites of pUC1 8 to generate plasmid HTPA3/4-pUC1 8.
  • HTPA3/4-pUC1 8 was then digested with Hindlll arid Sail into which the isolated Hindlll to Sail FGFR1 - encoding fragment was inserted.
  • the plasmid carrying the FGFR1 -tPA chimeric DNA was digested with Hindlll and BamHI, subjected to agarose gel electrophoresis and the 1 ,426 bp DNA fragment was excised from the gel and isolated as described above.
  • the resulting DNA encodes a 472 amino acid peptide comprised of amino acids 1 -284 of human FGFR1 , a 10 amino acid linker sequence VDARFPRGAR, derived from the human tPA signal peptide, and amino acids 1 -1 78 from human tPA.
  • the resulting DNA encoding the FGFR1 -tPA fusion protein is shown in SEQ ID No: 1 and the deduced amino acid is shown in SEQ ID No: 2.
  • the DNA of SEQ ID No. 1 was digested with Hindlll to BamHI and the 1 ,434 bp fragment (nt 2-1435 of SEQ ID No: 1 ) was isolated and ligated into the mammalian expression vector pK4K for recombinant expression of the FGFR1 -tPA fusion protein (Niidome et al. Biochem. Biophvs. Res. Commun. 1994, 203, 1 821 -1 827) .
  • the plasmid pK4K is a pBR322-based vector that has unique Hindlll and BamHI sites for directional cloning of heterologous DNAs whose expression is under the control of the SV40 early promoter. This plasmid also contains the ⁇ - lactamase and DHFR genes for use as selectable markers in prokaryotes and eukaryotic organisms, respectively.
  • Baby hamster kidney cells BHK cells; Waechter, D.E., et al. Proc
  • FGFR1 -tPA fusion protein Upon expression, the recombinant FGFR1 -tPA fusion protein is secreted into the surrounding culture medium. Recombinant FGFR1 -tPA fusion protein expression in BHK cells was monitored by sandwich enzyme-linked immunosorbent assays (sandwich ELISAs) . A mouse IgG monocional antibody specific for human tPA, designated 14-6, was used as the capture antibody and a polyclonal, rabbit anti-lgG antibody conjugated to horseradish peroxidase was used as the secondary-labeled antibody. 5. Purification of FGFR1 -tPA chimeric protein
  • the recombinant FGFR1 -tPA fusion protein was purified from conditioned medium of BHK-expressing cells by affinity chromatography. Transfected cells were grown as described above and the condition medium was harvested. The osmolarity of the conditioned medium was adjusted to a final concentration of 0.5 M NaCl by the addition of solid NaCl. The sample was applied onto a column of Cellulofine (Seikagaku Kogyo, Tokyo, Japan) conjugated with anti-tPA 14-6 monoclonal antibody previously equilibrated in column buffer (50 mM Tris-HCI, pH 7.5, and 0.5 M NaCl) .
  • column buffer 50 mM Tris-HCI, pH 7.5, and 0.5 M NaCl
  • the soluble, recombinant FGFR1 -tPA fusion protein was immobilized to a solid support by attachment to the surface of the wells of an enzyme-linked immunosorbent assay plate (High binding plates, COSTAR) .
  • An enzyme-linked immunosorbent assay plate High binding plates, COSTAR
  • a 0.1 ml aliquot of a 1 0 ⁇ g/ml solution of rFGFR1 -tPA in PBS was added and the plate was incubated for approximately 1 6 hr at 4 °C. Unbound fusion protein was removed by washing three times with an equal volume of cold PBS.
  • blocking buffer 25 mM HEPES, pH 7.5, 100 mM NaCl and 0.5% gelatin
  • binding buffer 25 mM HEPES, pH 7.5, 100 mM NaCl and 0.3% gelatin
  • binding buffer 25 mM HEPES, pH 7.5, 100 mM NaCl and 0.3% gelatin
  • 0.1 ml of binding buffer supplemented with 2 ⁇ g/ml heparin and a range of 1 -20ng/ml of labeled 125 l-bFGF (800-1 200Ci/mmol; Amersham, Arlington Heights, IL) and incubated in the absence or presence of 2.5 ⁇ g/ml unlabeled bFGF or a test compound for 3 hr at ambient temperature.
  • the buffer was removed by aspiration and the wells were washed twice each with PBS and a solution of 25 mM HEPES, pH 7.5, containing 2 M NaCl.
  • Bound bFGF was dissociated from the immobilized fusion protein by the addition of two aliquots of a solution of 25 mM sodium acetate, pH 4.0, containing 2 M NaCl. The two sodium acetate washes were combined and the amount of radioactivity present was determined using a gamma counter. The amount of bound radiolabeled bFGF in each well was calculated and the specificity of bFGF binding was analyzed according to Scatchard (Scatchard Ann. N.Y. Acad. Sci. 1949, 5 _, 660) .
  • the rabbit aortic smooth muscle cell line, Rb-1 expresses high and low affinity FGF receptors (e.g., see Nachtigal et al. In Vitro Cell. & Develop. Biol.1 989, 25, 892-897) .
  • Compounds of formula I or II that have FGF antagonist activity were and can be identified by their ability to compete with 125 l-bFGF for binding to the FGF receptors expressed on cell surface of such cells (see e.g., see, Moscatelli et al. J. Cell. Phvsiol. 1987, 131, 1 23-1 30) .
  • Rb-1 cells were grown in 24-well plates to near-confluence in
  • DMEM Dulbecco's modified Eagle's medium
  • penicillin 100 unit/ml
  • streptomycin (1 00 ug/ml)
  • the culture medium was removed by aspiration and the cells were incubated in binding buffer (serum-free DMEM supplemented with 20 mM HEPES (pH 7.5) and 0.1 % BSA) containing 0.2 ng/ml recombinant human 125 l-bFGF (800-1 200Ci/mmol; Amersham, Arlington Heights, IL) and varying concentrations of test compound, for 2 hr at ambient temperature.
  • binding buffer serum-free DMEM supplemented with 20 mM HEPES (pH 7.5) and 0.1 % BSA
  • binding buffer serum-free DMEM supplemented with 20 mM HEPES (pH 7.5) and 0.1 % BSA
  • 0.2 ng/ml recombinant human 125 l-bFGF 800-1 200Ci/m
  • the cells were washed twice with cold phosphate-buffered saline (PBS) and the bFGF bound to low affinity heparin sulfate proteoglycan (HSPG) receptors was dissociated by the addition to each well of a 1 ml solution of 25 mM HEPES (pH 7.5) containing 2 M NaCl.
  • the bFGF bound to high affinity FGF receptors was dissociated by the addition to each well of a 1 ml solution of 25 mM sodium acetate (pH 4.0) containing 2 M NaCl.
  • a 1 ml aliquot from each well was transferred to a polypropylene tube and the amount of radioactivity present in the high affinity samples was determined using a gamma counter.
  • EGF EGF-binding protein
  • SMCs such as rat aortic SMCs, incorporate tritiated thymidine into DNA upon stimulation with bFGF or PDGF.
  • the cells were washed twice with serum-free medium (DMEM supplemented with 0.1 % BSA, 5 ⁇ g/ml transferrin, penicillin (100 unit/ml) and streptomycin (100 ug/ml)) and cultured for an additional three days in serum-free DMEM medium.
  • serum-free medium DMEM supplemented with 0.1 % BSA, 5 ⁇ g/ml transferrin, penicillin (100 unit/ml) and streptomycin (100 ug/ml)
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE: (ix) FEATURE:
  • AAA ACG CAG ATG ATA TAC CAG CAA CAT CAG TCA TGG CTG CGC CCT GTG 962 Lys Thr Gin Met lie Tyr Gin Gin His Gin Ser Trp Leu Arg Pro Val 305 310 315
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • Gin Met lie Tyr Gin Gin His Gin Ser Trp Leu Arg Pro Val Leu Arg 305 310 315 320

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Abstract

L'invention concerne des compositions pharmaceutiques contenant des amides, des peptides d'arginine ou leurs analogues ainsi que des procédés d'utilisation de ces compositions pharmaceutiques pour moduler l'activité de la famille FGF des peptides. L'invention se rapporte à des procédés pour inhiber la liaison d'un peptide FGF à un récepteur de FGF par la mise en contact du récepteur avec des amides, des peptides d'arginine ou leurs analogues correspondant aux formules I et II. L'invention concerne aussi des procédés pour traiter des troubles induits par FGF par l'administration de quantités efficaces d'un ou de plusieurs de ces composés ou de leurs dérivés pharmaceutiquement acceptables qui inhibent l'activité d'un ou de plusieurs peptides FGF.
PCT/JP1999/001844 1998-04-09 1999-04-07 Analogues du peptide d'arginine utilises en tant qu'antagonistes du facteur de croissance des fibroblastes WO1999052936A2 (fr)

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AU31662/99A AU3166299A (en) 1998-04-09 1999-04-07 Arginine peptide analogs useful as fibroblast growth factor antagonists

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Cited By (5)

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US7939505B2 (en) 2007-05-04 2011-05-10 Marina Biotech, Inc. Amino acid lipids and uses thereof
US8148429B2 (en) 2000-08-07 2012-04-03 Anamar Ab Use of benzylideneaminoguanidines and hydroxyguanidines as melanocortin receptor ligands
JP2017521494A (ja) * 2014-07-02 2017-08-03 インフレクティス・バイオサイエンス プロテオパチーの処置のためのベンジリデングアニジン誘導体の新規な治療的使用
CN111233973A (zh) * 2020-02-21 2020-06-05 重庆医药高等专科学校 一种精氨酸衍生物Pro-Phe-Arg-AMC的合成方法及用途
US10973920B2 (en) 2014-06-30 2021-04-13 Glykos Finland Oy Saccharide derivative of a toxic payload and antibody conjugates thereof

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US9227927B2 (en) 2000-08-07 2016-01-05 Anamar Ab Method of treating inflammation
US8148429B2 (en) 2000-08-07 2012-04-03 Anamar Ab Use of benzylideneaminoguanidines and hydroxyguanidines as melanocortin receptor ligands
US8309609B2 (en) 2000-08-07 2012-11-13 Anamar Ab Use of benzylideneaminoguanidines and hydroxyguanidines as melanocortin receptor ligands
US8410174B2 (en) 2000-08-07 2013-04-02 Anamar Ab Method for treating arthritis
US8501824B2 (en) 2007-05-04 2013-08-06 Marina Biotech, Inc. Amino acid lipids and uses thereof
US8877729B2 (en) 2007-05-04 2014-11-04 Marina Biotech, Inc. Amino acid lipids and uses thereof
US7939505B2 (en) 2007-05-04 2011-05-10 Marina Biotech, Inc. Amino acid lipids and uses thereof
US9339461B2 (en) 2007-05-04 2016-05-17 Marina Biotech, Inc. Arginine-based lipids for delivery of therapeutics
US9731016B2 (en) 2007-05-04 2017-08-15 Marina Biotech, Inc. Tyrosine-based lipids for delivery of therapeutics
US10973920B2 (en) 2014-06-30 2021-04-13 Glykos Finland Oy Saccharide derivative of a toxic payload and antibody conjugates thereof
JP2017521494A (ja) * 2014-07-02 2017-08-03 インフレクティス・バイオサイエンス プロテオパチーの処置のためのベンジリデングアニジン誘導体の新規な治療的使用
JP6994939B2 (ja) 2014-07-02 2022-02-04 インフレクティス・バイオサイエンス プロテオパチーの処置のためのベンジリデングアニジン誘導体の新規な治療的使用
CN111233973A (zh) * 2020-02-21 2020-06-05 重庆医药高等专科学校 一种精氨酸衍生物Pro-Phe-Arg-AMC的合成方法及用途

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