EP2173359A2 - Heparin conjugates and methods - Google Patents

Heparin conjugates and methods

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Publication number
EP2173359A2
EP2173359A2 EP08778410A EP08778410A EP2173359A2 EP 2173359 A2 EP2173359 A2 EP 2173359A2 EP 08778410 A EP08778410 A EP 08778410A EP 08778410 A EP08778410 A EP 08778410A EP 2173359 A2 EP2173359 A2 EP 2173359A2
Authority
EP
European Patent Office
Prior art keywords
acid
heparin
residue
composition
bile acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP08778410A
Other languages
German (de)
French (fr)
Inventor
Youngro Byun
E Sak Lee
Ok-Cheol Jeon
Sang Yoon Kim
Rang-Woon Park
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Mediplex Corp
Mediplex Corp Korea
Original Assignee
Mediplex Corp
Mediplex Corp Korea
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Mediplex Corp, Mediplex Corp Korea filed Critical Mediplex Corp
Publication of EP2173359A2 publication Critical patent/EP2173359A2/en
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/727—Heparin; Heparan
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
    • C08B37/0081—Reaction with amino acids, peptides, or proteins
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug
    • A61K47/551—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug one of the codrug's components being a vitamin, e.g. niacinamide, vitamin B3, cobalamin, vitamin B12, folate, vitamin A or retinoic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/554—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/66—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • C08L5/10—Heparin; Derivatives thereof

Definitions

  • This invention relates to heparin conjugates and methods of making and using thereof. More particularly, this invention relates to heparin-bile acid conjugates, heparin-bile acid conjugates further including a targeting moiety, heparin-bile acid conjugates wherein the heparin is bonded to the bile acid through the 3-carbon of the bile acid, and heparin conjugates wherein heparin is covalently bonded to sulfonated moieties. Methods of using these conjugates for treating cancer are also described.
  • Heparin has anti-tumoral and anti-inflammatory activities as well as its well known anti-coagulant activity.
  • R. Sasisekharan et al. Roles of heparan-sulphate glycosa- minoglycans in cancer, 2 Nat. Rev. Cancer 521-528 (2002).
  • growth factors which are key regulators for cell mitogenic activity.
  • Growth factors usually bind with growth factor receptors and can modulate cell growth.
  • vascular endothelial growth factor (VEGF) is a key protein in physiological angiogenesis (or neo-vascularization), or formation of new blood vessels.
  • VEGF vascular endothelial growth factor
  • Angiogenesis is a complex multi-step process involving endothelial cell activation, controlled proteolytic degradation of the extracellular matrix (ECM), proliferation and migration of endothelial cells, and formation of capillary vessel lumina.
  • ECM extracellular matrix
  • LMWH low molecular weight heparin
  • MW 4500-6000 Da
  • in vitro heparin fragments of less than 18 saccharide residues reduce activity of VEGF, and fragments of less than 10 saccharide residues inhibit activity of bFGF.
  • G.C. Jayson et al. Heparin Oligosaccharides: Inhibitors of the Biological Activity of bFGF on Caco-2 Cells, 75 Br. J. Cancer 9-16 (1997); S.
  • Be- vacizumab (Avastin®) is an FDA-approved, anti-angiogenic drug that is representative of such VEGF inhibitors.
  • Bevacizumab is a basic monoclonal antibody that binds the negatively charged receptor binding domain of VEGF and, therefore, can block the interaction between VEGF and VEGF receptors (Flkl, KDR).
  • L.M. Ellis Mechanisms of Action of Bevacizumab as a Component of Therapy for Metastatic Colorectal Cancer, 33 Semin. Oncol. S107 (2006); E. Bergsland & M.N. Dickler, Maximizing the Potential of Bevacizumab in Cancer Treatment, 9 Oncologist 36-42 (2004).
  • LMWH can bind the heparin binding domain of VEGF.
  • the sulfate groups of heparin can bind with positively charged amino acid residues, such as arginine, histidine, and lysine.
  • a model of complexes formed between the heparin binding domain of VEGF and heparin or heparan sulfate predicts that sulfate and carboxylate groups of heparin contact these basic amino acid residues in the heparin-binding cleft of the VEGF protein. CJ.
  • VEGF165-binding sites within heparan sulfate encompass two highly sulfated domains and can be liberated by K5 lyase, 281 J. Biol. Chem. 1731-1740 (2006).
  • K5 lyase 281 J. Biol. Chem. 1731-1740 (2006).
  • binding of heparin to VEGF results in an anti-angiogenic effect.
  • Treatment of VEGF with either UFH or LMWH had no effect on tumor- associated angiogenesis in an experimental model of colon cancer metastasis in rat liver. S. M.
  • VEGF comprises two main parts, a positively charged heparin binding domain
  • HBD Vascular Endothelial Growth Factor Determinants for Binding KDR and FLT-I Receptors. Generation of Receptor- selective VEGF Variants by Site-directed Mutagenesis, 271 J. Biol. Chem. 5638-5646 (1996). Because HBD and RBD are in separate domains, even though heparin binds with HBD, the RBD maintains its structure.
  • HBD-deleted VEGF can bind to the VEGF receptor, but mitogenic activity is absent.
  • the HBD is critical to the cell growth activity of VEGF. RBD binding to VEGF cannot maintain cell growth.
  • the VEGF receptor is a monomer before binding with the RBD of VEGF.
  • This invention provides a composition represented by the formula:
  • B is a bile acid residue or a bile acid analog
  • H is a heparin residue
  • S 1 is a first spacer
  • S 2 is a second spacer
  • T is a targeting moiety
  • m is an integer of 0 to about 50
  • n is an integer of 0 to about 10, with the proviso that both m and n cannot be 0, and wherein B and S 1 are bonded to each other through a 3-carbon of B, and S 1 and S 2 can be the same or different.
  • m is about 1 to about 30.
  • m is about 1 to about 10.
  • n is 1 to about 10.
  • This invention also provides a method of making a heparin-spacer-bile acid or heparin-spacer-bile acid analog conjugate and a method of making a heparin- spacer-bile acid-spacer-targeting moiety or heparin-spacer-bile acid analog- spacer-targeting moiety.
  • This invention also provides a method of treating cancer comprising administering to an individual in need thereof an effective amount of a composition represented by the formula:
  • B is a bile acid residue or a bile acid analog
  • H is a heparin residue
  • S 1 is a first spacer
  • S 2 is a second spacer
  • T is a targeting moiety
  • m is an integer of 0 to about 50
  • n is an integer of 0 to about 10, with the proviso that both m and n cannot be 0, and wherein B and S 1 are bonded to each other through a 3-carbon of B, and S 1 and S 2 can be the same or different.
  • heparin-bile acid conjugates are available for treating cancer because they cna inhibit angiogenesis, metastasis and tumor growth.
  • FIG. 1 illustrates a scheme for synthesis of taurocholic acid carbonate derivative
  • FIG. 2 illustrates a scheme for synthesis of an ethylene amine derivative of taurocholic acid (Et-TCA) by reaction of CB-TCA with ethylene diamine such that the ethylene diamine replaces the 4-nitrophenyl group.
  • Et-TCA ethylene amine derivative of taurocholic acid
  • FIG. 3 illustrates a scheme for synthesis of a heparin-taurocholic acid conjugate by reaction of Et-TCA with heparin such that a carboxyl group on heparin bonds to the free amine group of Et-TCA, resulting in heparin conjugated to taurocholic acid through the 3-carbon thereof.
  • FIG. 4 illustrates a scheme for synthesis of activated deoxycholic acid (aDOCA) from deoxycholic acid (DOCA).
  • FIG. 5 illustrates a scheme for synthesis of JV-deoxycholylethylenediamine from aDOCA.
  • FIG. 6 illustrates a scheme for synthesis of a heparin-DOCA conjugate from N- deoxycholylethylenediamine and heparin.
  • FIG. 7 shows tumor volume as a function of days after inoculation of C3H/HeN mice with LMWH (5 mg/kg), HTlO (5 mg/kg), HT2 (5 mg/kg), and a control.
  • FIG. 8 shows tumor volume as a function of days after inoculation of C3H/HeN mice with LMWH (0.5 mg/kg), HTlO (0.5 mg/kg), HTlO (1 mg/kg), HTlO (5 mg/kg), be- vacizumab (5 mg/kg), and a control.
  • FIG. 9 shows tumor volume as a function of days after inoculation of C3H/HeN mice with HD (5 mg/kg), UFH (5 mg/kg), HL (5 mg/kg), and a saline control.
  • FIG. 10 shows tumor volume as a function of days after inoculation of C3H/HeN mice with HD (1 mg/kg), HD (5 mg/kg), HD (10 mg/kg), heparin (5 mg/kg), and a saline control.
  • FIG. 11 shows detection of microvessels and expression of the proliferating cell nuclear antigen (PCNA) in tumor tissues treated with a control, heparin, or HD, as detected by immunohistochemistry using either an anti-CD34 antibody or and anti- PCNA antibody.
  • PCNA proliferating cell nuclear antigen
  • FIG. 12-14 illustrates a scheme for synthesis of a cRGDyK-heparin-taurocholic acid conjugate.
  • FIG. 15 illustrates a scheme for synthesis of a highly sulfated heparin derivative, namely, a heparin-naphthalene trisulfonic acid conjugate.
  • An illustrative embodiment according to the present invention comprises a composition represented by the formula:
  • B is a bile acid residue or a bile acid analog
  • H is a heparin residue
  • S 1 is a first spacer
  • S 2 is a second spacer
  • T is a targeting moiety
  • m is an integer of 0 to about 50
  • n is an integer of 0 to about 10, with the proviso that both m and n cannot be 0, and wherein B and S 1 are bonded to each other through a 3-carbon of B, and S 1 and S 2 can be the same or different.
  • m is about 1 to about 30.
  • m is about 1 to about 10.
  • n is 1 to about 10.
  • the bile acid residue may be selected, for example, from the group consisting of residues of cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid, ursocholic acid, ursodeoxycholic acid, isoursodeoxycholic acid, lagodeoxycholic acid, glycocholic acid, taurocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, dehydrocholic acid, hyocholic acid, and hyodeoxycholic acid.
  • the heparin can comprise heparin of any type, such as unfractionated heparin, high molecular weight heparin, low molecular weight heparin, heparin fragments, recombinant heparin, heparin analogs, and sulfonated polysaccharides containing heparin activity, and the like.
  • the spacer can comprise an alkyl chain, polyethylene glycol, an ethylene diamine residue, and the like.
  • the targeting moiety may comprise a folic acid residue, a cRGD residue, and the like.
  • An illustrative composition according to the present invention comprises a composition wherein B is a taurocholate residue, H is a low molecular weight heparin residue, S 1 and S 2 are an ethylene diamine residue, and T is a cRGD residue.
  • Another illustrative composition according to the present invention comprises a composition wherein B is a lithocholate residue, H is a low molecular weight heparin residue, S 1 and S 2 are an ethylene diamine residue, and T is folate residue.
  • Another illustrative embodiment according to the present invention comprises a composition comprising at least one sulfonated moiety, such as naphthalene trisulfonate, covalently bonded heparin.
  • Other illustrative embodiments comprise compositions wherein analogs of naphthalene trisulfonate or sulfonated naphthalenes are substituted for naphthalene trisulfonate.
  • Still another illustrative embodiment according to the present invention comprises a composition comprising heparin bonded to the 3-carbon of a bile acid or bile acid analog.
  • the bile acid analog comprises a sulfonyl group.
  • the composition may further comprise a spacer between heparin and the bile acid or bile acid analog, such as an ethylene diamine residue, an alkyl chain, polyethylene glycol, and the like.
  • the composition may also further comprise a targeting moiety coupled to the heparin through a second spacer.
  • Yet another illustrative embodiment of the invention comprises a method of treating cancer comprising administering to an individual in need thereof an effective amount of a composition represented by the formula:
  • B is a bile acid residue or a bile acid analog
  • H is a heparin residue
  • S 1 is a first spacer
  • S 2 is a second spacer
  • T is a targeting moiety
  • m is an integer of 0 to about 50
  • n is an integer of 0 to about 10, with the proviso that both m and n cannot be 0, and wherein B and S 1 are bonded to each other through a 3-carbon of B, and S 1 and S 2 can be the same or different.
  • Still another illustrative embodiment of the invention comprises a method of treating cancer comprising administering to an individual in need thereof an effective amount of a composition comprising heparin bonded to the 3-carbon of a bile acid or bile acid analog.
  • Another illustrative embodiment of the invention comprises a method of making a heparin-spacer-bile acid or heparin-spacer-bile acid analog conjugate.
  • the method comprises activating the 3-carbon of a bile acid or bile acid analog to result in an activated bile acid or activated bile analog, bonding a first spacer to the activated bile acid or activated bile acid analog to result in a spacer-bile acid or spacer-bile acid analog, activating a heparin to result in an activated heparin, and then bonding the activated heparin to the spacer-bile acid or spacer-bile acid analog to result in the heparin-spacer-bile acid or heparin-spacer-bile acid analog conjugate.
  • Still another illustrative embodiment of the invention comprises a method of making a heparin-spacer-bile acid-spacer-targeting moiety or heparin-spacer-bile acid analog- spacer-targeting moiety conjugate.
  • the method comprises activating the targeting moiety to result in an activated targeting moiety, bonding a second spacer to the activated targeting moiety to result in a second spacer-targeting moiety, and then bonding the second spacer-targeting moiety to a heparin-spacer-bile acid conjugate or heparin-spacer-bile acid analog conjugate to result in the heparin-spacer-bile acid- spacer-targeting moiety conjugate or heparin-spacer-bile acid analog-spacer-targeting moiety conjugate.
  • Bile acids means natural and synthetic derivatives of the steroid, cholanic acid, including, without limitation, cholic acid, deoxycholic acid, chen- odeoxycholic acid, lithocholic acid, ursocholic acid, ursodeoxycholic acid, isours- odeoxycholic acid, lagodeoxycholic acid, glycocholic acid, taurocholic acid, gly- codeoxycholic acid, glycochenodeoxycholic acid, dehydrocholic acid, hyocholic acid, hyodeoxycholic acid, and mixtures thereof, and the like.
  • Bile acid analogs can also be used according to the present invention. Examples of such bile acid analogs include bile acids bearing at least one sulfonyl group.
  • ⁇ means an amount of a heparin conjugate that is nontoxic but sufficient to provide the desired effect and performance at a reasonable benefit/risk ratio attending any cancer treatment.
  • administering means delivering the composition to the individual being treated such that the composition is capable of being circulated systemically to the parts of the body where cancer cells are located.
  • the composition is preferably administered to the individual by systemic administration, typically by subcutaneous, intramuscular, or intravenous administration, or intraperitoneal administration.
  • injectables for such use can be prepared in conventional forms, either as a liquid solution or suspension or in a solid form suitable for preparation as a solution or suspension in a liquid prior to injection, or as an emulsion.
  • Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, and the like; and if desired, minor amounts of auxiliary substances such as wetting or emulsifying agents, buffers, and the like can be added.
  • An illustrative embodiment according to the present invention comprises a composition represented by the formula : [63] (B-SVH-(S 2 -T) n
  • B is a bile acid residue or a bile acid analog
  • H is a heparin residue
  • S 1 is a first spacer
  • S 2 is a second spacer
  • T is a targeting moiety
  • m is an integer of 0 to about 50
  • n is an integer of 0 to about 10, with the proviso that both m and n cannot be 0, and wherein B and S 1 are bonded to each other through a 3-carbon of B, and S 1 and S 2 can be the same or different.
  • n is about 1 to about 30, and even more typically m is about 1 to about
  • n is about 1 to about 10.
  • the heparin can comprise heparin of any type, such as unfractionated heparin, high molecular weight heparin, low molecular weight heparin, heparin fragment, recombinant heparin, heparin analogs, heparin sulfate, and sulfonated polysaccharides containing heparin activity, and the like.
  • the spacers, S 1 and S 2 are independently selected from the group consisting of alkyl chains, polyethylene glycol, an ethylene diamine residue, and the like.
  • the targeting moiety may comprise a folic acid residue, a cRGD residue, and the like.
  • An illustrative composition according to the present invention comprises a composition wherein B is a taurocholate residue, H is a low molecular weight heparin residue, S 1 and S 2 are ethylene diamine residues, and T is a cRGD residue.
  • Another illustrative composition according to the present invention comprise a composition wherein B is lithocholate residue, H is a low molecular weight heparin residue, S 1 and S 2 are ethylene diamine residues, and T is a folate residue.
  • Another illustrative embodiment according to the present invention comprises a composition comprising at least one sulfonated moiety, such as naphthalene trisulfonate, covalently bonded to heparin.
  • sulfonated moieties that can be substituted for naphthalene trisulfonate include analogs of naphthalene trisulfonate, other sulfonated naphthalenes, and the like.
  • Still another illustrative embodiment according to the present invention comprises a composition comprising heparin bonded to the 3-carbon of a bile acid or bile acid analog.
  • the bile acid analog comprises a sulfonyl group.
  • the composition may further comprise a first spacer between heparin and the bile acid or bile acid analog, such as an ethylene diamine residue, an alkyl chain, polyethylene glycol, and the like.
  • the composition may also further comprise a targeting moiety coupled to the heparin through a second spacer.
  • Yet another illustrative embodiment of the invention comprises a method of treating cancer comprising administering to an individual in need thereof an effective amount of a composition represented by the formula:
  • B is a bile acid residue or a bile acid analog
  • H is a heparin residue
  • S 1 is a first spacer
  • S 2 is a second spacer
  • T is a targeting moiety
  • m is an integer of 0 to about 50
  • n is an integer of 0 to about 10, with the proviso that both m and n cannot be 0, and wherein B and S 1 are bonded to each other through a 3-carbon of B, and S 1 and S 2 can be the same or different.
  • Still another illustrative embodiment of the invention comprises a method of treating cancer comprising administering to an individual in need thereof an effective amount of a composition comprising heparin bonded to the 3-carbon of a bile acid or bile acid analog.
  • Another illustrative embodiment of the invention comprises a method of making a heparin-spacer-bile acid or heparin-spacer-bile acid analog conjugate.
  • the method comprises first activating the 3-carbon of a bile acid or bile acid analog to result in an activated bile acid or activated bile acid analog.
  • a first spacer is bonded to the activated bile acid or activated bile acid analog to result in a spacer-bile acid or spacer- bile acid analog.
  • Heparin is activated by reacting the heparin with an activating agent to result in an activated heparin.
  • the activated heparin is bonded to the spacer- bile acid or spacer-bile acid analog to result in the heparin-spacer-bile acid or heparin- spacer-bile acid analog conjugate.
  • Still another illustrative embodiment of the invention comprises a method of making a heparin-spacer-bile acid-spacer-targeting moiety or heparin-spacer-bile acid analog- spacer-targeting moiety conjugate.
  • the method comprises reacting a targeting moiety with an activating agent to result in an activated targeting moiety.
  • a second spacer is bonded to the activated targeting moiety to result in a second spacer-targeting moiety.
  • the second spacer-targeting moiety is bonded to the previously described heparin-spacer-bile acid conjugate or heparin-spacer-bile acid analog conjugate to result in the heparin-spacer-bile acid-spacer-targeting moiety conjugate or heparin-spacer-bile acid analog-spacer-targeting moiety conjugate.
  • TCA-LMWH Taurocholic acid
  • TCA-TCA taurocholic acid ni- trophenyl carbonate
  • DMF JV,./V-dimethylformamide
  • NPC 4-nitrophenyl chloroformate
  • Et-TCA an amine derivative of taurocholic acid
  • FIG. 2 An amine derivative of taurocholic acid (Et-TCA) was synthesized (FIG. 2).
  • CB-TCA 0.5 g was dissolved in 5 ml of DMF.
  • 4-methyl-morpholine (0.144 g; Sigma) was added, followed by agitation for 1 h at 5O 0 C.
  • This solution was slowly added to an excess of ethylenediamine, followed by agitation for 16 h at room tem ⁇ perature.
  • the feed mole ratio of CT-TCA, 4-methylmorpholine, and ethylenediamine was 1:2:100. Since Et-TCA was water soluble, but ethylenediamine, 4-methylmorpholine, and NPC were not, acetone recrystallization was used to obtain pure Et-TCA, which was then dried under partial vacuum.
  • heparin taurocholate HT
  • Table 1 The coupling ratio and anticoagulant activity of each preparation were determined. Coupling ratio is expressed as the number of taurocholate moieties per heparin. Anticoagulant activity is expressed as the percent of activity of dialyzed LMWH, as determined with an FXa kit from Sigma.
  • Heparin 100 mg was dissolved in 3 ml of formamide by gentle heating. Different amounts of EDC were mixed with heparin solutions at room temperature, followed by the addition of different amounts of LITHO-NH 2 dissolved in DMF with slight heating. The reaction was performed at room temperature for 24 h. The product was precipitated in excess cold acetone, and precipitates were stored at O 0 C, washed 5 times with cold acetone to remove unreacted LITHO-NH 2 , followed by drying under reduced pressure. The resulting precipitates were collected by lyophilization to give heparin- lithocholic acid derivatives.
  • DOCA (Sigma) was mixed with DCC (7.4 g) and NHS (4.5 g) in 100 ml of tetrahydrofuran (THF). The mixture was reacted for 12 h at room temperature under a nitrogen atmosphere, then the precipitated dicyclohexylurea was removed by filtration. The filtrate was precipitated in n- hexane. The succinimido DOCA precipitate was filtered off and washed thoroughly with n-hexane, followed by vacuum drying at room temperature.
  • aDOCA was converted to JV-deoxycholylethylenediamine according to the procedure illustrated in FIG. 5.
  • JV-Deoxycholylethylenediamine (DOCA-NH 2 ) was synthesized by introducing ethylenediamine to the activated (with a succinimido group) DOCA.
  • Succinimido DOCA (1 g) was dissolved in DMF (5 ml), and the solution was slowly added dropwise into ethylenediamine (13.4 ml) solution. After reaction for 6 h, the mixture was precipitated in distilled water.
  • the white powder DOCA-NH 2 was obtained after washing 3 times with distilled water and drying at reduced pressure.
  • heparin was conjugated to JV-deoxycholylethylenediamine according to the procedure illustrated in FIG. 6.
  • Heparin 0.1 g was dissolved in formamide (5 ml) with gentle heating.
  • Different amounts of EDC were mixed with heparin solutions at room temperature, followed by addition of different amounts of DOCA- NH 2 dissolved in DMF.
  • the resulting solutions were stirred at room temperature under a nitrogen atmosphere for 24 h.
  • precipitates were carefully washed 3 times with acetone to remove excess DOCA- NH 2 , followed by drying at reduced pressure.
  • the dried HD conjugates were dissolved in water. Lyo- philization of the HD conjugates provided a white powder.
  • heparin activity means the anticoagulation ability of heparin.
  • COATEST HEPARIN FXa assay kit from Chromogenix was used for determining heparin activity of heparin conjugates. Results were recorded at 405 nm.
  • the size of self-aggregated dispersions was determined using dynamic light scattering (Spectra Physics Laser Model 127-35) operated at 633 nm and 25+0.1 0 C. Scattered light was measured at an angle of 90° and collected with a BI-9000At auto- correlator. The concentration of HD conjugates was kept constant at 1 mg/ml. The hy- drodynamic diameter of self-aggregates was calculated by the Stokes-Einstein equation. The polydispersity factor, represented as 2 /T 2 was evaluated from the cumulant method, where 2 is the second cumulant of the decay function and F is the average characteristic line width. The zeta potentials of the nanoparticles were measured using an ELS- 8000 electrophoretic light scattering spectrophotometer (Otsuka Electronics Co., Ltd., Japan).
  • the ultra-violet circular dichroism (CD) spectrum of proteins can predict important characteristics of their secondary structure. CD spectra can be readily used to estimate the fraction of a molecule that is in the alpha-helix conformation, the beta-sheet conformation, the beta-turn conformation, or some other (e.g., random coil) conformation. Jasco J-715 Circular Dichroism (Jasco, Japan) was used for making the determination.
  • Table 2 shows the characterization of heparin derivatives prepared according to Examples 1-4.
  • Example 6 [HO] Tumor Growth Inhibition [111] The anti-tumor activity of LMWH, HTlO, HT2, and control were compared in a tumor volume inhibition study. Seven-week-old male C3H/HeN mice (Orient Bio) were used for all animal experiments. Subcutaneous tumors were established by inoculating 1 x 10 6 SCC cells in the backs of the mice by subcutaneous injection. Care and maintenance of animals were performed in adherence to institutional guidelines of the Institutional Animal Care and Use Committee (IACUC).
  • IACUC Institutional Animal Care and Use Committee
  • mice When the tumors had grown to about 50-70 mm 3 , the mice were given intravenous injections of 0.1 ml of saline containing HTlO (5 mg/kg), HT2 (5 mg/kg), or LMWH (5 mg/kg), or saline alone (control), every three days. On the 15th day, the mice were sacrificed and their tumors removed. All treatment groups contained 7 or 8 mice. Tumor tissues were isolated from three representative treated and untreated tumor-bearing mice. Detection of micro vessels and expression of the proliferating cell nuclear antigen (PCNA) marker in tumor tissues were carried out by immunohistochemistry using a specific anti-CD34 antibody and anti-PCNA antibody (Dako, Carpinteria, California), respectively. FIG. 7 shows that HTlO resulted in about 69% tumor volume inhibition compared to the control treatment.
  • PCNA proliferating cell nuclear antigen
  • Example 6 The procedure of Example 6 was followed, except that HTlO (5 mg/kg), HTlO (1 mg/kg), HTlO (0.5 mg /kg), LMWH (0.5 mg/kg), and control were intravenously administered daily, and bevacizumab (5 mg/kg) was intraperitoneally administered twice weekly.
  • HTlO is a nontoxic heparin derivative, and its anticoagulation activity is less than 13% that of LMWH, thus daily administration was not problematic.
  • FIG. 8 shows that HTlO administered in doses of 1 mg/kg and 5 mg/kg was similar in its tumor growth inhibition effect to bevacizumab.
  • HTlO (5 mg/kg) inhibited tumor growth about 71% compared with the control, while HTlO (1 mg/kg) inhibited tumor growth about 63%, and bevacizumab inhibited tumor growth about 74%.
  • HTlO nor LMWH administered at 0.5 mg/kg were effective in inhibiting tumor growth.
  • Example 6 The procedure of Example 6 was followed except that UFH (5 mg/kg), HL (5 mg/ kg), HD (5 mg/kg), and a control were administered. As shown in FIG. 9, HD and HL exhibited about 55-60% tumor volume inhibition compared with the control.
  • HD was selected for a study on the in vivo dose effect of this heparin conjugate.
  • the procedure of Example 6 was carried out, except that treatments were HD (10 mg/kg), HD (5mg/kg), HD (1 mg/kg), heparin (5 mg/kg), ad control.
  • Detection of micro vessels and expression of PCNA was carried out using immunohistochemistry and anti-CD34 antibody and anti-PCNA antibody (Dako). Results are shown in FIGS. 10 and 11.
  • FIGS. 12-14 show synthesis of a cRGD-HT conjugate.
  • 50 mg of cRGDyK peptide was dissolved in DMF (10 ml) and then methyl-N-succinimidyl adipate (MSA, 32 mg) was added. After stirring for 12 h at room temperature, phenyl ester was added to the reaction mixture and stirring was continued for 6 h to protect the carboxyl group of the cRGDyK peptide.
  • the reaction mixture was precipitated with a 3-fold excess of water to remove any unreacted reagents, and the precipitate was washed with water and then dried in vacuo.
  • heparin in an amount indicated in column A was dissolved in the corresponding amount of formamide from column B, and this solution was agitated on an ice bath.
  • EDAC in the corresponding amount from column C was added to the heparin solution.
  • trisodium 8-amino-l,3,6-naphthalene trisulfonate (ANTS) in the corresponding amount from column D dissolved in the corresponding amount of formamide from column E was mixed with the activated heparin solution. After stirring for 12 h at room temperature, the reaction mixture was dialyzed against water using a 1000 MWCO membrane for 2 days and was then lyophilized.
  • the amount of naphthalene trisulfonate in the resulting heparin derivative was determined by HPLC, and the anti-coagulation activity of this highly sulfated heparin derivative was evaluated using an anti-FXa chromogenic assay.
  • Folate -heparin-lithocholate was prepared as follows. Folic acid (1 mmol) dissolved in 20 ml DMSO was reacted with DCC (1.2 mmol) and NHS (2 mmol) at 5O 0 C for 6 h. Folate has two " ⁇ - and ⁇ -carboxylic acids, but the ⁇ -carboxylic acid is more selectively activated due to its higher reactivity. The resulting folate-NHS was reacted with ethylene diamine (13 mmol) and pyridine (500 mg) at room temperature overnight.
  • the folylethylamine (folate-NH 2 ) was precipitated by the addition of excess acetonitrile, and the precipitate was filtered and washed with diethyl ether before trying under vacuum to get yellow powder.
  • This product was added to HL (100 mg), dissolved in 20 ml of formamide, and activated by EDAC (3.38 mg) with 5 ⁇ l of N, N - diisopropylethylamine (DIEA) for 12 h.
  • the unreacted folate-NH 2 was removed by dialysis (MWCO 2000).
  • the final product, FHL was obtained by lyophilization at a yield of 91%.
  • the folate content in FHL was determined by quantitative UV spectrophotometry at 365 nm.
  • the anti-coagulation activity of FHL was measured by Fxa chromogenic assay (COATEST®eparin, Milan, Italy).

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Abstract

Heparin conjugates and methods of making and use thereof are disclosed. An illustrative heparin conjugate includes a composition wherein a bile acid is bonded to heparin through the 3-carbon of the bile acid. A spacer may be interposed between the bile acid and the heparin. Another embodiment includes a taheting moiety bonded to the heparin-bile acid conjugate through a spacer. Another illustartive heparin conjugate includes heparin covalently bonded to a sulfonated moiety, such as naphthaalene trisulfonate residue. A method of treating cancer includes administering a heparin conjugate to an individual in need thereof.

Description

Description
HEPARIN CONJUGATES AND METHODS
Technical Field
[1] This invention relates to heparin conjugates and methods of making and using thereof. More particularly, this invention relates to heparin-bile acid conjugates, heparin-bile acid conjugates further including a targeting moiety, heparin-bile acid conjugates wherein the heparin is bonded to the bile acid through the 3-carbon of the bile acid, and heparin conjugates wherein heparin is covalently bonded to sulfonated moieties. Methods of using these conjugates for treating cancer are also described.
[2]
Background Art
[3] Heparin, due to its heterogeneous structure, can interact with many kinds of proteins.
Heparin has anti-tumoral and anti-inflammatory activities as well as its well known anti-coagulant activity. R. Sasisekharan et al., Roles of heparan-sulphate glycosa- minoglycans in cancer, 2 Nat. Rev. Cancer 521-528 (2002). Among the many sorts of proteins that interact with heparin are growth factors, which are key regulators for cell mitogenic activity. Growth factors usually bind with growth factor receptors and can modulate cell growth. In particular, vascular endothelial growth factor (VEGF) is a key protein in physiological angiogenesis (or neo-vascularization), or formation of new blood vessels. N. Ferrara et al., The biology of VEGF and its receptors, 9 Nat. Med. 669-676 (2003). Angiogenesis is a complex multi-step process involving endothelial cell activation, controlled proteolytic degradation of the extracellular matrix (ECM), proliferation and migration of endothelial cells, and formation of capillary vessel lumina. Diaz-Flores et al., 33 Anat. Histol. Embryol. 334-338 (2004).
[4] Binding of growth factors to heparins or heparan sulfates is thought to have a crucial role in the modulation of activity of the high-affinity receptors. S. Colin et al., In Vivo Involvement of Heparan Sulfate Proteoglycan in the Bioavailability, Internalization, and Catabolism of Exogenous Basic Fibroblast Growth Factor, 55 MoI. Pharmacol. 74-82 (1999); IJ. Mason, The Ins and Outs of Fibroblast Growth Factors, 78 Cell 547-552 (1994); S. Tessler et al., Heparin Modulates the Interaction of VEGF165 with Soluble and Cell Associated flk-1 Receptors, 269 J. Biol. 12456-12461 (1994). Unfrac- tionated heparin (UFH) or heparan sulfates promote basic fibroblast growth factor (bFGF) receptor dimerization and activation, thus enhancing cell growth. J. Sch- lessinger et al., Regulation of Growth Factor Activation by Proteoglycans: What Is the Role of the Low Affinity Receptors?, 83 Cell 357-360 (1995). Interestingly, it has been shown that low molecular weight heparin (LMWH; MW = 4500-6000 Da), in contrast to UFH, can hinder binding of growth factors to their high-affinity receptors as a result of its smaller size. Indeed, in vitro heparin fragments of less than 18 saccharide residues reduce activity of VEGF, and fragments of less than 10 saccharide residues inhibit activity of bFGF. G.C. Jayson et al., Heparin Oligosaccharides: Inhibitors of the Biological Activity of bFGF on Caco-2 Cells, 75 Br. J. Cancer 9-16 (1997); S. Soker et al., Variations in the Size and Sulfation of Heparin Modulate the Effect of Heparin on the Binding of VEGF165 to its Receptors, 203 Biochem. Biophys. Res. Comm. 1339-1347 (1994).
[5] Many anti-cancer drugs were designed as inhibitors of VEGF and its receptors. Be- vacizumab (Avastin®) is an FDA-approved, anti-angiogenic drug that is representative of such VEGF inhibitors. Bevacizumab is a basic monoclonal antibody that binds the negatively charged receptor binding domain of VEGF and, therefore, can block the interaction between VEGF and VEGF receptors (Flkl, KDR). L.M. Ellis, Mechanisms of Action of Bevacizumab as a Component of Therapy for Metastatic Colorectal Cancer, 33 Semin. Oncol. S107 (2006); E. Bergsland & M.N. Dickler, Maximizing the Potential of Bevacizumab in Cancer Treatment, 9 Oncologist 36-42 (2004).
[6] LMWH can bind the heparin binding domain of VEGF. The sulfate groups of heparin can bind with positively charged amino acid residues, such as arginine, histidine, and lysine. A model of complexes formed between the heparin binding domain of VEGF and heparin or heparan sulfate predicts that sulfate and carboxylate groups of heparin contact these basic amino acid residues in the heparin-binding cleft of the VEGF protein. CJ. Robinson et al., VEGF165-binding sites within heparan sulfate encompass two highly sulfated domains and can be liberated by K5 lyase, 281 J. Biol. Chem. 1731-1740 (2006). However, there is no evidence that binding of heparin to VEGF results in an anti-angiogenic effect. Treatment of VEGF with either UFH or LMWH had no effect on tumor- associated angiogenesis in an experimental model of colon cancer metastasis in rat liver. S. M. Smorenburg et al., In Vivo Treatment of Rats Unfractionated Heparin (UFH) or Low Molecular Weight Heparin (LMWH) Does Not Affect Experimentally Induced Colon Carcinoma Metastasis, 17 Clin. Exp. Metastasis 451-456 (1999).
[7] VEGF comprises two main parts, a positively charged heparin binding domain
(HBD; amino acid residues 111-165) and a negatively charged receptor binding domain (RBD; amino acid residues 8-109). B.A. Keyt et al., Identification of Vascular Endothelial Growth Factor Determinants for Binding KDR and FLT-I Receptors. Generation of Receptor- selective VEGF Variants by Site-directed Mutagenesis, 271 J. Biol. Chem. 5638-5646 (1996). Because HBD and RBD are in separate domains, even though heparin binds with HBD, the RBD maintains its structure.
[8] It has been reported that HBD-deleted VEGF can bind to the VEGF receptor, but mitogenic activity is absent. B. A. Keyt et al., The carboxyl-terminal domain (111-165) of vascular endothelial growth factor is critical for its mitogenic potency, 271 J. Biol. Chem. 7788-7795 (1996). Thus, it was concluded that the HBD is critical to the cell growth activity of VEGF. RBD binding to VEGF cannot maintain cell growth.
[9] In vitro experiments have suggested that the VEGF HBD-mediated interaction with neuropilin-1 (NP-I) increases the affinity of VEGF for KDR (VEGF receptor 2). Furthermore, the affinity of VEGF for the NP-I extracellular domain is greatly enhanced by the addition of heparin. These results suggest that heparin mediates a successful interaction between VEGF and the receptor. H. Jia et al., Characterization of a Bicyclic Peptide Neuropilin-1 (NP-I) Antagonist (EG3287) Reveals Importance of Vascular Endothelial Growth Factor Exon 8 for NP-I Binding and Role of NP-I in KDR Signaling, 281 J. Biol. Chem. 13493-13502 (2006).
[10] Finally, the VEGF receptor is a monomer before binding with the RBD of VEGF.
When a VEGF ligand binds with the monomer receptor, then receptor dimerization can be initiated. After dimerization, two domains of the receptor experience conformational changes, resulting in a coiled structure. After all these steps, angiogenic signaling occurs. C. Ruch et al., Structure of a VEGF-VEGF receptor complex determined by electron microscopy, 14 Nat. Struct. MoI. Biol. 249-250 (2007).
[H]
Disclosure of Invention
Technical Problem
[12] It is an object of the present invention to provide heparin conjugates.
[13] It is another object of the present invention to provide methods of making heparin conjugates.
[14] It is still another object of the present invention to provide use of heparin conjugates.
[15]
Technical Solution
[16] This invention provides a composition represented by the formula:
[17] (B-S1)m-H-(S2-T)n
[18] wherein B is a bile acid residue or a bile acid analog, H is a heparin residue, S1 is a first spacer, S2 is a second spacer, T is a targeting moiety, m is an integer of 0 to about 50, and n is an integer of 0 to about 10, with the proviso that both m and n cannot be 0, and wherein B and S1 are bonded to each other through a 3-carbon of B, and S1 and S2 can be the same or different. In another illustrative embodiment of the invention, m is about 1 to about 30. In still another illustrative embodiment of the invention, m is about 1 to about 10. In yet another illustrative embodiment of the invention, n is 1 to about 10. [19] This invention also provides a method of making a heparin-spacer-bile acid or heparin-spacer-bile acid analog conjugate and a method of making a heparin- spacer-bile acid-spacer-targeting moiety or heparin-spacer-bile acid analog- spacer-targeting moiety.
[20] This invention also provides a method of treating cancer comprising administering to an individual in need thereof an effective amount of a composition represented by the formula:
[21] (B-SVH-(S2-T)n
[22] wherein B is a bile acid residue or a bile acid analog, H is a heparin residue, S1 is a first spacer, S2 is a second spacer, T is a targeting moiety, m is an integer of 0 to about 50, and n is an integer of 0 to about 10, with the proviso that both m and n cannot be 0, and wherein B and S1 are bonded to each other through a 3-carbon of B, and S1 and S2 can be the same or different.
[23]
Advantageous Effects
[24] According to the present invention, heparin-bile acid conjugates; heparin-bile acid conjugates further including a targeting moiety; heparin-bile acid conjugates wherein the heparin is bonded to the bile acid through the 3-carbon of the bile acid; and heparin conjugates wherein heparin is covalently bonded to sulfonated moieties are available for treating cancer because they cna inhibit angiogenesis, metastasis and tumor growth.
[25]
Brief Description of the Drawings
[26] FIG. 1 illustrates a scheme for synthesis of taurocholic acid carbonate derivative
(CB-TCA) by reaction of taurocholic acid sodium salt with 4-nitrophenyl chlo- roformate such that the 4-nitrophenyl formate group bonds to the 3 -hydroxy group of taurocholic acid.
[27] FIG. 2 illustrates a scheme for synthesis of an ethylene amine derivative of taurocholic acid (Et-TCA) by reaction of CB-TCA with ethylene diamine such that the ethylene diamine replaces the 4-nitrophenyl group.
[28] FIG. 3 illustrates a scheme for synthesis of a heparin-taurocholic acid conjugate by reaction of Et-TCA with heparin such that a carboxyl group on heparin bonds to the free amine group of Et-TCA, resulting in heparin conjugated to taurocholic acid through the 3-carbon thereof.
[29] FIG. 4 illustrates a scheme for synthesis of activated deoxycholic acid (aDOCA) from deoxycholic acid (DOCA).
[30] FIG. 5 illustrates a scheme for synthesis of JV-deoxycholylethylenediamine from aDOCA. [31] FIG. 6 illustrates a scheme for synthesis of a heparin-DOCA conjugate from N- deoxycholylethylenediamine and heparin.
[32] FIG. 7 shows tumor volume as a function of days after inoculation of C3H/HeN mice with LMWH (5 mg/kg), HTlO (5 mg/kg), HT2 (5 mg/kg), and a control.
[33] FIG. 8 shows tumor volume as a function of days after inoculation of C3H/HeN mice with LMWH (0.5 mg/kg), HTlO (0.5 mg/kg), HTlO (1 mg/kg), HTlO (5 mg/kg), be- vacizumab (5 mg/kg), and a control.
[34] FIG. 9 shows tumor volume as a function of days after inoculation of C3H/HeN mice with HD (5 mg/kg), UFH (5 mg/kg), HL (5 mg/kg), and a saline control.
[35] FIG. 10 shows tumor volume as a function of days after inoculation of C3H/HeN mice with HD (1 mg/kg), HD (5 mg/kg), HD (10 mg/kg), heparin (5 mg/kg), and a saline control.
[36] FIG. 11 shows detection of microvessels and expression of the proliferating cell nuclear antigen (PCNA) in tumor tissues treated with a control, heparin, or HD, as detected by immunohistochemistry using either an anti-CD34 antibody or and anti- PCNA antibody.
[37] FIG. 12-14 illustrates a scheme for synthesis of a cRGDyK-heparin-taurocholic acid conjugate.
[38] FIG. 15 illustrates a scheme for synthesis of a highly sulfated heparin derivative, namely, a heparin-naphthalene trisulfonic acid conjugate.
[39]
Best Mode for Carrying Out the Invention
[40] An illustrative embodiment according to the present invention comprises a composition represented by the formula:
[41] (B-S1)m-H-(S2-T)n
[42] wherein B is a bile acid residue or a bile acid analog, H is a heparin residue, S1 is a first spacer, S2 is a second spacer, T is a targeting moiety, m is an integer of 0 to about 50, and n is an integer of 0 to about 10, with the proviso that both m and n cannot be 0, and wherein B and S1 are bonded to each other through a 3-carbon of B, and S1 and S2 can be the same or different. In another illustrative embodiment of the invention, m is about 1 to about 30. In still another illustrative embodiment of the invention, m is about 1 to about 10. In yet another illustrative embodiment of the invention, n is 1 to about 10.
[43] The bile acid residue may be selected, for example, from the group consisting of residues of cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid, ursocholic acid, ursodeoxycholic acid, isoursodeoxycholic acid, lagodeoxycholic acid, glycocholic acid, taurocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, dehydrocholic acid, hyocholic acid, and hyodeoxycholic acid. The heparin can comprise heparin of any type, such as unfractionated heparin, high molecular weight heparin, low molecular weight heparin, heparin fragments, recombinant heparin, heparin analogs, and sulfonated polysaccharides containing heparin activity, and the like. The spacer can comprise an alkyl chain, polyethylene glycol, an ethylene diamine residue, and the like. The targeting moiety may comprise a folic acid residue, a cRGD residue, and the like. An illustrative composition according to the present invention comprises a composition wherein B is a taurocholate residue, H is a low molecular weight heparin residue, S1 and S2 are an ethylene diamine residue, and T is a cRGD residue. Another illustrative composition according to the present invention comprises a composition wherein B is a lithocholate residue, H is a low molecular weight heparin residue, S1 and S2 are an ethylene diamine residue, and T is folate residue.
[44] Another illustrative embodiment according to the present invention comprises a composition comprising at least one sulfonated moiety, such as naphthalene trisulfonate, covalently bonded heparin. Other illustrative embodiments comprise compositions wherein analogs of naphthalene trisulfonate or sulfonated naphthalenes are substituted for naphthalene trisulfonate.
[45] Still another illustrative embodiment according to the present invention comprises a composition comprising heparin bonded to the 3-carbon of a bile acid or bile acid analog. Illustratively, the bile acid analog comprises a sulfonyl group. The composition may further comprise a spacer between heparin and the bile acid or bile acid analog, such as an ethylene diamine residue, an alkyl chain, polyethylene glycol, and the like. The composition may also further comprise a targeting moiety coupled to the heparin through a second spacer.
[46] Yet another illustrative embodiment of the invention comprises a method of treating cancer comprising administering to an individual in need thereof an effective amount of a composition represented by the formula:
[47] (B-SVH-(S2-T)n
[48] wherein B is a bile acid residue or a bile acid analog, H is a heparin residue, S1 is a first spacer, S2 is a second spacer, T is a targeting moiety, m is an integer of 0 to about 50, and n is an integer of 0 to about 10, with the proviso that both m and n cannot be 0, and wherein B and S1 are bonded to each other through a 3-carbon of B, and S1 and S2 can be the same or different.
[49] Still another illustrative embodiment of the invention comprises a method of treating cancer comprising administering to an individual in need thereof an effective amount of a composition comprising heparin bonded to the 3-carbon of a bile acid or bile acid analog.
[50] Another illustrative embodiment of the invention comprises a method of making a heparin-spacer-bile acid or heparin-spacer-bile acid analog conjugate. The method comprises activating the 3-carbon of a bile acid or bile acid analog to result in an activated bile acid or activated bile analog, bonding a first spacer to the activated bile acid or activated bile acid analog to result in a spacer-bile acid or spacer-bile acid analog, activating a heparin to result in an activated heparin, and then bonding the activated heparin to the spacer-bile acid or spacer-bile acid analog to result in the heparin-spacer-bile acid or heparin-spacer-bile acid analog conjugate.
[51] Still another illustrative embodiment of the invention comprises a method of making a heparin-spacer-bile acid-spacer-targeting moiety or heparin-spacer-bile acid analog- spacer-targeting moiety conjugate. The method comprises activating the targeting moiety to result in an activated targeting moiety, bonding a second spacer to the activated targeting moiety to result in a second spacer-targeting moiety, and then bonding the second spacer-targeting moiety to a heparin-spacer-bile acid conjugate or heparin-spacer-bile acid analog conjugate to result in the heparin-spacer-bile acid- spacer-targeting moiety conjugate or heparin-spacer-bile acid analog-spacer-targeting moiety conjugate.
[52]
Mode for the Invention
[53] Before the present heparin-bile acid conjugates and methods are disclosed and described, it is to be understood that this invention is not limited to the particular configurations, process steps, and materials disclosed herein as such configurations, process steps, and materials may vary somewhat. It is also to be understood that the terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.
[54] The publications and other reference materials referred to herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference. The references discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.
[55] It must be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.
[56] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. [57] In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.
[58] As used herein, "comprising", "including", "containing", "characterized by" and grammatical equivalents thereof are inclusive or open-ended terms that do not exclude additional, unrecited elements or method steps. "Comprising" is to be interpreted as including the more restrictive terms "consisting of" and "consisting essentially of". As used herein, "consisting of" and grammatical equivalents thereof exclude any element, step, or ingredient not specified in the claim. As used herein, "consisting essentially of" and grammatical equivalents thereof limit the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic or characteristics of the claimed invention.
[59] As used herein, "bile acids" means natural and synthetic derivatives of the steroid, cholanic acid, including, without limitation, cholic acid, deoxycholic acid, chen- odeoxycholic acid, lithocholic acid, ursocholic acid, ursodeoxycholic acid, isours- odeoxycholic acid, lagodeoxycholic acid, glycocholic acid, taurocholic acid, gly- codeoxycholic acid, glycochenodeoxycholic acid, dehydrocholic acid, hyocholic acid, hyodeoxycholic acid, and mixtures thereof, and the like. The carbon atoms in a bile acid are numbered according to the standard numbering system used for steroids, which is well known in the art. R.T. Morrison & R.N. Boyd, Organic chemistry 514(3d ed. 1973). Thus, the 3 -carbon of a bile acid is contained in the A ring thereof. Bile acid analogs can also be used according to the present invention. Examples of such bile acid analogs include bile acids bearing at least one sulfonyl group.
[60] As used herein, "effective amount" means an amount of a heparin conjugate that is nontoxic but sufficient to provide the desired effect and performance at a reasonable benefit/risk ratio attending any cancer treatment.
[61] As used herein, "administering" and similar terms mean delivering the composition to the individual being treated such that the composition is capable of being circulated systemically to the parts of the body where cancer cells are located. Thus, the composition is preferably administered to the individual by systemic administration, typically by subcutaneous, intramuscular, or intravenous administration, or intraperitoneal administration. Injectables for such use can be prepared in conventional forms, either as a liquid solution or suspension or in a solid form suitable for preparation as a solution or suspension in a liquid prior to injection, or as an emulsion. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, and the like; and if desired, minor amounts of auxiliary substances such as wetting or emulsifying agents, buffers, and the like can be added.
[62] An illustrative embodiment according to the present invention comprises a composition represented by the formula : [63] (B-SVH-(S2-T)n
[64] wherein B is a bile acid residue or a bile acid analog, H is a heparin residue, S1 is a first spacer, S2 is a second spacer, T is a targeting moiety, m is an integer of 0 to about 50, and n is an integer of 0 to about 10, with the proviso that both m and n cannot be 0, and wherein B and S1 are bonded to each other through a 3-carbon of B, and S1 and S2 can be the same or different.
[65] Typically, m is about 1 to about 30, and even more typically m is about 1 to about
10. In yet another illustrative embodiment of the invention, n is about 1 to about 10.
[66] The heparin can comprise heparin of any type, such as unfractionated heparin, high molecular weight heparin, low molecular weight heparin, heparin fragment, recombinant heparin, heparin analogs, heparin sulfate, and sulfonated polysaccharides containing heparin activity, and the like. The spacers, S1 and S2, are independently selected from the group consisting of alkyl chains, polyethylene glycol, an ethylene diamine residue, and the like. The targeting moiety may comprise a folic acid residue, a cRGD residue, and the like.
[67] An illustrative composition according to the present invention comprises a composition wherein B is a taurocholate residue, H is a low molecular weight heparin residue, S1 and S2 are ethylene diamine residues, and T is a cRGD residue. Another illustrative composition according to the present invention comprise a composition wherein B is lithocholate residue, H is a low molecular weight heparin residue, S1 and S2 are ethylene diamine residues, and T is a folate residue.
[68] Another illustrative embodiment according to the present invention comprises a composition comprising at least one sulfonated moiety, such as naphthalene trisulfonate, covalently bonded to heparin. Other sulfonated moieties that can be substituted for naphthalene trisulfonate include analogs of naphthalene trisulfonate, other sulfonated naphthalenes, and the like.
[69] Still another illustrative embodiment according to the present invention comprises a composition comprising heparin bonded to the 3-carbon of a bile acid or bile acid analog. Illustratively, the bile acid analog comprises a sulfonyl group. The composition may further comprise a first spacer between heparin and the bile acid or bile acid analog, such as an ethylene diamine residue, an alkyl chain, polyethylene glycol, and the like. The composition may also further comprise a targeting moiety coupled to the heparin through a second spacer.
[70] Yet another illustrative embodiment of the invention comprises a method of treating cancer comprising administering to an individual in need thereof an effective amount of a composition represented by the formula:
[71] (B-SVH-(S2-T)n
[72] wherein B is a bile acid residue or a bile acid analog, H is a heparin residue, S1 is a first spacer, S2 is a second spacer, T is a targeting moiety, m is an integer of 0 to about 50, and n is an integer of 0 to about 10, with the proviso that both m and n cannot be 0, and wherein B and S1 are bonded to each other through a 3-carbon of B, and S1 and S2 can be the same or different.
[73] Still another illustrative embodiment of the invention comprises a method of treating cancer comprising administering to an individual in need thereof an effective amount of a composition comprising heparin bonded to the 3-carbon of a bile acid or bile acid analog.
[74] Another illustrative embodiment of the invention comprises a method of making a heparin-spacer-bile acid or heparin-spacer-bile acid analog conjugate. The method comprises first activating the 3-carbon of a bile acid or bile acid analog to result in an activated bile acid or activated bile acid analog. Next, a first spacer is bonded to the activated bile acid or activated bile acid analog to result in a spacer-bile acid or spacer- bile acid analog. Heparin is activated by reacting the heparin with an activating agent to result in an activated heparin. Finally, the activated heparin is bonded to the spacer- bile acid or spacer-bile acid analog to result in the heparin-spacer-bile acid or heparin- spacer-bile acid analog conjugate.
[75] Still another illustrative embodiment of the invention comprises a method of making a heparin-spacer-bile acid-spacer-targeting moiety or heparin-spacer-bile acid analog- spacer-targeting moiety conjugate. The method comprises reacting a targeting moiety with an activating agent to result in an activated targeting moiety. Then, a second spacer is bonded to the activated targeting moiety to result in a second spacer-targeting moiety. Finally, the second spacer-targeting moiety is bonded to the previously described heparin-spacer-bile acid conjugate or heparin-spacer-bile acid analog conjugate to result in the heparin-spacer-bile acid-spacer-targeting moiety conjugate or heparin-spacer-bile acid analog-spacer-targeting moiety conjugate.
[76] As described above, the present invention is drawn to heparin conjugates and methods of making and using them. These conjugates and methods are illustrated in the following examples, which are not to be construed as limiting the claims.
[77]
[78] Example 1
[79] Synthesis of Heparin-Taurocholic Acid ("HT") Conjugates
[80] Taurocholic acid (TCA)-LMWH was synthesized by conjugating carboxyl groups of
LMWH with amine groups of TCA-amine derivatives. First, taurocholic acid ni- trophenyl carbonate was synthesized (CB-TCA; FIG. 1), which comprises an activated derivative of taurocholic acid. TCA sodium salt (0.5 g; Sigma Chemical Co., St. Louis, Missouri) was mixed with JV,./V-dimethylformamide (DMF; 4.6 ml; Sigma), and the mixture was agitated at O0C. Then, triethylamine (0.565 g; Sigma) and 4-nitrophenyl chloroformate (NPC; Sigma) were added to the mixture and reacted for 1 h at O0C followed by 6 h at room temperature. This resulted in the 4-nitrophenyl formate group bonding to the 3-hydroxyl group of TCA. The feed mole ratio of TCA, NPC, and tri- ethylamine was 1:5:6.
[81] Second, an amine derivative of taurocholic acid (Et-TCA) was synthesized (FIG. 2). CB-TCA (0.5 g) was dissolved in 5 ml of DMF. Then, 4-methyl-morpholine (0.144 g; Sigma) was added, followed by agitation for 1 h at 5O0C. This solution was slowly added to an excess of ethylenediamine, followed by agitation for 16 h at room tem¬ perature. The feed mole ratio of CT-TCA, 4-methylmorpholine, and ethylenediamine was 1:2:100. Since Et-TCA was water soluble, but ethylenediamine, 4-methylmorpholine, and NPC were not, acetone recrystallization was used to obtain pure Et-TCA, which was then dried under partial vacuum.
[82] Finally, Et-TCA was conjugated to heparin (FIG. 3). LMWH (Fraxiparin; average molecular weight, 4500 Da; Sanofi-Synthelabo Co., Gentilly, France) was dissolved in distilled water. The carboxyl group of LMWH was activated by adding l-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (EDC; Sigma) with chilling in an ice bath and agitation for 5 min. JV-Hydroxysuccinimide (NHS(HOSu); Sigma) was then added to this solution and agitated for 30 min. Finally, Et-TCA was added slowly, then the reaction was carried out at room temperature overnight. The reaction was then dialyzed overnight to remove unreacted EDC, NHS(HOSu), and Et- TCA. Several preparations of heparin taurocholate (HT) were made by controlling the feed mole ratio, as shown in Table 1. The coupling ratio and anticoagulant activity of each preparation were determined. Coupling ratio is expressed as the number of taurocholate moieties per heparin. Anticoagulant activity is expressed as the percent of activity of dialyzed LMWH, as determined with an FXa kit from Sigma.
[83]
[84] Example 2
[85] Synthesis of Heparin-Lithocholic Acid ("HL") Conjugates
[86] A solution of lithocholic acid (LITHO; 23. 5 mmol; Sigma) in 25 ml of methanol was acidified 2 with 0.3 ml of concentrated HCl. This mixture was stirred and heated to reflux at 750C for 6 h. The solution was then cooled to O0C until crystallization occurred. The product was thrice filtered and washed with cold methanol, followed by vacuum drying at room temperature. The resulting lithocholyl methyl ester (3.8 g) was reacted with 75 ml of ethylenediamine by reflux at 12O0C for 7 h. After cooling to room temperature, the mixture was precipitated in cold distilled water with vigorous stirring, stored at O0C for 1 day, filtered, and washed with cold distilled water. The white powder LITHO-NH2 was obtained after drying under partial vacuum.
[87] Heparin (100 mg) was dissolved in 3 ml of formamide by gentle heating. Different amounts of EDC were mixed with heparin solutions at room temperature, followed by the addition of different amounts of LITHO-NH2 dissolved in DMF with slight heating. The reaction was performed at room temperature for 24 h. The product was precipitated in excess cold acetone, and precipitates were stored at O0C, washed 5 times with cold acetone to remove unreacted LITHO-NH2, followed by drying under reduced pressure. The resulting precipitates were collected by lyophilization to give heparin- lithocholic acid derivatives.
[88]
[89] Example 3
[90] Synthesis of Folate-HL Derivatives
[91] Folic acid (1 mmol; Sigma) was dissolved in 20 ml of dimethylsulf oxide (DMSO) with gentle heating (above 5O0C). ,/V,./V-dicyclohexylcarbodiimide (DCC; Sigma) and HOSu (2 mmol) were then added to the solution and reacted in the dark for 6 h. After removing precipitants (dicyclohexyl urea) by filtration, the resulting folate-NHS was mixed with 13 mmolar equiv of ethylenediamine plus 500 μl of pyridine and allowed to react at room temperature overnight. The crude product was precipitated by addition of excess acetonitrile, filtered, and washed 10 times with diethyl ether before drying under reduced pressure. For further purification, gamma-linked folate-NH2 was separated from the unwanted folic acid by HPLC.
[92] HL was dissolved in 20 ml of formamide (Sigma) with EDC (3.38 mg) with 5 μl of
DIEA for 24 h. The gamma- linked folate-NH2 (16 mg) dissolved in 2 ml of DMF was then added to the reaction plus TEA (16.5 μl) for 20 h. The unreacted folate- NH2 was removed by dialysis (MWCO 2000). The final yellowish product, folate-HL (FHL), was obtained by lyophilization.
[93]
[94] Example 4 [95] Synthesis of Heparin-DOCA (HD) Conjugates
[96] First, deoxycholic acid (DOCA) was activated to result in activated deoxycholic acid
(aDOCA) according to the procedure illustrated in FIG. 4. DOCA (Sigma) was mixed with DCC (7.4 g) and NHS (4.5 g) in 100 ml of tetrahydrofuran (THF). The mixture was reacted for 12 h at room temperature under a nitrogen atmosphere, then the precipitated dicyclohexylurea was removed by filtration. The filtrate was precipitated in n- hexane. The succinimido DOCA precipitate was filtered off and washed thoroughly with n-hexane, followed by vacuum drying at room temperature.
[97] Next, aDOCA was converted to JV-deoxycholylethylenediamine according to the procedure illustrated in FIG. 5. JV-Deoxycholylethylenediamine (DOCA-NH2) was synthesized by introducing ethylenediamine to the activated (with a succinimido group) DOCA. Succinimido DOCA (1 g) was dissolved in DMF (5 ml), and the solution was slowly added dropwise into ethylenediamine (13.4 ml) solution. After reaction for 6 h, the mixture was precipitated in distilled water. The white powder DOCA-NH2 was obtained after washing 3 times with distilled water and drying at reduced pressure.
[98] Finally, heparin was conjugated to JV-deoxycholylethylenediamine according to the procedure illustrated in FIG. 6. Heparin (0.1 g) was dissolved in formamide (5 ml) with gentle heating. Different amounts of EDC were mixed with heparin solutions at room temperature, followed by addition of different amounts of DOCA- NH2 dissolved in DMF. The resulting solutions were stirred at room temperature under a nitrogen atmosphere for 24 h. After mixtures were precipitated in cold acetone, precipitates were carefully washed 3 times with acetone to remove excess DOCA- NH2, followed by drying at reduced pressure. The dried HD conjugates were dissolved in water. Lyo- philization of the HD conjugates provided a white powder.
[99]
[100] Example 5
[101] Analysis of Heparin Derivatives
[102] Heparin derivatives prepared according to the procedures of Examples 1-4 were analyzed using IR and ff-NMR (JEOLJNM-LA300 WB FT-NMR/FT-IR, Tokyo, Japan), which was operated in deuterium-substituted proper solvents. The NMR data show that taurocholic acid was successfully conjugated to LMWH, and the HPLC data show the purity of the synthesized materials that were used as intermediates in the synthesis reactions.
[103] As used herein, "heparin activity" means the anticoagulation ability of heparin. The COATEST HEPARIN FXa assay kit from Chromogenix was used for determining heparin activity of heparin conjugates. Results were recorded at 405 nm.
[104] The size of self-aggregated dispersions was determined using dynamic light scattering (Spectra Physics Laser Model 127-35) operated at 633 nm and 25+0.10C. Scattered light was measured at an angle of 90° and collected with a BI-9000At auto- correlator. The concentration of HD conjugates was kept constant at 1 mg/ml. The hy- drodynamic diameter of self-aggregates was calculated by the Stokes-Einstein equation. The polydispersity factor, represented as 2/T2 was evaluated from the cumulant method, where 2 is the second cumulant of the decay function and F is the average characteristic line width. The zeta potentials of the nanoparticles were measured using an ELS- 8000 electrophoretic light scattering spectrophotometer (Otsuka Electronics Co., Ltd., Japan).
[105] The ultra-violet circular dichroism (CD) spectrum of proteins can predict important characteristics of their secondary structure. CD spectra can be readily used to estimate the fraction of a molecule that is in the alpha-helix conformation, the beta-sheet conformation, the beta-turn conformation, or some other (e.g., random coil) conformation. Jasco J-715 Circular Dichroism (Jasco, Japan) was used for making the determination.
[106] Table 2 shows the characterization of heparin derivatives prepared according to Examples 1-4.
[107]
[108] [109] Example 6 [HO] Tumor Growth Inhibition [111] The anti-tumor activity of LMWH, HTlO, HT2, and control were compared in a tumor volume inhibition study. Seven-week-old male C3H/HeN mice (Orient Bio) were used for all animal experiments. Subcutaneous tumors were established by inoculating 1 x 106 SCC cells in the backs of the mice by subcutaneous injection. Care and maintenance of animals were performed in adherence to institutional guidelines of the Institutional Animal Care and Use Committee (IACUC). When the tumors had grown to about 50-70 mm3, the mice were given intravenous injections of 0.1 ml of saline containing HTlO (5 mg/kg), HT2 (5 mg/kg), or LMWH (5 mg/kg), or saline alone (control), every three days. On the 15th day, the mice were sacrificed and their tumors removed. All treatment groups contained 7 or 8 mice. Tumor tissues were isolated from three representative treated and untreated tumor-bearing mice. Detection of micro vessels and expression of the proliferating cell nuclear antigen (PCNA) marker in tumor tissues were carried out by immunohistochemistry using a specific anti-CD34 antibody and anti-PCNA antibody (Dako, Carpinteria, California), respectively. FIG. 7 shows that HTlO resulted in about 69% tumor volume inhibition compared to the control treatment.
[112]
[113] Example 7
[114] The procedure of Example 6 was followed, except that HTlO (5 mg/kg), HTlO (1 mg/kg), HTlO (0.5 mg /kg), LMWH (0.5 mg/kg), and control were intravenously administered daily, and bevacizumab (5 mg/kg) was intraperitoneally administered twice weekly. HTlO is a nontoxic heparin derivative, and its anticoagulation activity is less than 13% that of LMWH, thus daily administration was not problematic. FIG. 8 shows that HTlO administered in doses of 1 mg/kg and 5 mg/kg was similar in its tumor growth inhibition effect to bevacizumab. HTlO (5 mg/kg) inhibited tumor growth about 71% compared with the control, while HTlO (1 mg/kg) inhibited tumor growth about 63%, and bevacizumab inhibited tumor growth about 74%. Neither HTlO nor LMWH administered at 0.5 mg/kg were effective in inhibiting tumor growth.
[115]
[116] Example 8
[117] The procedure of Example 6 was followed except that UFH (5 mg/kg), HL (5 mg/ kg), HD (5 mg/kg), and a control were administered. As shown in FIG. 9, HD and HL exhibited about 55-60% tumor volume inhibition compared with the control.
[118]
[119] Example 9
[120] HD was selected for a study on the in vivo dose effect of this heparin conjugate. The procedure of Example 6 was carried out, except that treatments were HD (10 mg/kg), HD (5mg/kg), HD (1 mg/kg), heparin (5 mg/kg), ad control. Detection of micro vessels and expression of PCNA was carried out using immunohistochemistry and anti-CD34 antibody and anti-PCNA antibody (Dako). Results are shown in FIGS. 10 and 11.
[121]
[122] Example 10
[123] FIGS. 12-14 show synthesis of a cRGD-HT conjugate. First, 50 mg of cRGDyK peptide was dissolved in DMF (10 ml) and then methyl-N-succinimidyl adipate (MSA, 32 mg) was added. After stirring for 12 h at room temperature, phenyl ester was added to the reaction mixture and stirring was continued for 6 h to protect the carboxyl group of the cRGDyK peptide. Next, the reaction mixture was precipitated with a 3-fold excess of water to remove any unreacted reagents, and the precipitate was washed with water and then dried in vacuo.
[124] The resulting MSA-linked cRDG (4.2 mg) was dissolved in DMF (1 ml) and was mixed with end-aminated heparin (100 mg) dissolved in formamide (7 ml) and stirred in the presence of sodium cyanide for 24 h. The reaction mixture was then precipitated in cold acetone, and the precipitate was washed with acetone several times. After completely evaporating the acetone, the precipitate was dissolved in distilled water and then lyophilized.
[125] Next, the cRDG-coupled heparin (50 mg) was dissolved in 10 ml of distilled water and l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC; 10.6 mg) was then added. The, Et-TCA (47.4 mg), prepared according to the procedure of Example 1 was added to the heparin solution. The pH was adjusted to 5.0 with 1 N HCl. After stirring for 12 h at room temperature, hydrogen peroxide was added to the reaction mixture, which was incubated for 6 h. The reaction mixture was then dialyzed using 2000 MWCO dialysis membrane and lyophilized.
[126]
[127] Example 11
[128] Highly sulfated heparin derivatives were prepared according to the scheme illustrated in FIG. 15 and Table 3.
[129] First, heparin (Fraxiparine) in an amount indicated in column A was dissolved in the corresponding amount of formamide from column B, and this solution was agitated on an ice bath. Next, EDAC in the corresponding amount from column C was added to the heparin solution. Then, trisodium 8-amino-l,3,6-naphthalene trisulfonate (ANTS) in the corresponding amount from column D dissolved in the corresponding amount of formamide from column E was mixed with the activated heparin solution. After stirring for 12 h at room temperature, the reaction mixture was dialyzed against water using a 1000 MWCO membrane for 2 days and was then lyophilized. The amount of naphthalene trisulfonate in the resulting heparin derivative was determined by HPLC, and the anti-coagulation activity of this highly sulfated heparin derivative was evaluated using an anti-FXa chromogenic assay.
[130]
[131]
[132] [133] Example 12 [134] Folate -heparin-lithocholate (FHL) was prepared as follows. Folic acid (1 mmol) dissolved in 20 ml DMSO was reacted with DCC (1.2 mmol) and NHS (2 mmol) at 5O0C for 6 h. Folate has two "α- and γ-carboxylic acids, but the γ-carboxylic acid is more selectively activated due to its higher reactivity. The resulting folate-NHS was reacted with ethylene diamine (13 mmol) and pyridine (500 mg) at room temperature overnight. The folylethylamine (folate-NH2) was precipitated by the addition of excess acetonitrile, and the precipitate was filtered and washed with diethyl ether before trying under vacuum to get yellow powder. This product was added to HL (100 mg), dissolved in 20 ml of formamide, and activated by EDAC (3.38 mg) with 5 μl of N, N - diisopropylethylamine (DIEA) for 12 h. The unreacted folate-NH2 was removed by dialysis (MWCO 2000). The final product, FHL, was obtained by lyophilization at a yield of 91%. The folate content in FHL was determined by quantitative UV spectrophotometry at 365 nm. The anti-coagulation activity of FHL was measured by Fxa chromogenic assay (COATEST®eparin, Milan, Italy).

Claims

Claims
[I] A composition represented by the formula: (B-SX-H-(Si-T)n wherein B is a bile acid residue or a bile acid analog, H is a heparin residue, S1 is a first spacer, S2 is a second spacer, T is a targeting moiety, m is an integer of 0 to about 50, and n is an integer of 0 to about 10, with the proviso that both m and n cannot be 0, and wherein B and S1 are bonded to each other through a 3-carbon of B, and S1 and S2 can be the same or different.
[2] The composition of claim 1, wherein B is a bile acid residue selected from the group consisting of residues of cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid, ursocholic acid, ursodeoxycholic acid, isoursodeoxycholic acid, lagodeoxycholic acid, glycocholic acid, taurocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, dehydrocholic acid, hyocholic acid, and hyo- deoxycholic acid.
[3] The composition of claim 1, wherein B is a deoxycholic acid residue.
[4] The composition of claim 1, wherein B is a lithocholic acid residue.
[5] The composition of claim 1, wherein B is a taurocholic acid residue.
[6] The composition of claim 1, wherein B is a bile acid analog comprising a sulfonyl group.
[7] The composition of claim 1, wherein said heparin residue is a member selected from the group consisting of unfractionated heparin, high molecular weight heparin, low molecular weight heparin, heparin fragments, recombinant heparin, heparin analogs, heparan sulfate, and sulfonated polysaccharides containing heparin activity.
[8] The composition of claim 1, wherein H is low molecular weight heparin.
[9] The composition of claim 1, wherein S1 and S2 are independently selected from the group consisting of alkyl chains, polyethylene glycol, and an ethylene diamine residue.
[10] The composition of claim 1, wherein T is a folic acid residue.
[I I] The composition of claim 1, wherein T is a cRGD residue.
[12] The composition of claim 1, wherein B is a taurocholate residue, S1 is an ethylene diamine residue, H is low molecular weight heparin, S2 is an ethylene diamine residue, and T is a cRGD residue.
[13] The composition of claim 1, wherein B is a lithocholate residue, S1 is an ethylene diamine residue, H is low molecular weight heparin, S2 is an ethylene diamine residue, and T is a folate residue.
[14] A composition comprising at least one sulfonated moiety covalently bonded to a heparin.
[15] The composition of claim 14, wherein the sulfonated moiety comprises naphthalene trisulfonate, analogs of naphthalene trisulfonate, or sulfonated naphthalene.
[16] The composition of claim 14, wherein the heparin is a member selected from the group consisting of unfractionated heparin, high molecular weight heparin, low molecular weight heparin, heparin fragments, recombinant heparin, heparin analogs, heparin sulfate, and sulfonated polysaccharides containing heparin activity.
[17] A composition comprising a heparin bonded to the 3-carbon of a bile acid or a bile acid analog.
[18] The composition of claim 17, further comprising a spacer between the heparin and the bile acid or bile acid analog.
[19] The composition of claim 18, wherein the spacer is a member selected from the group consisting of alkyl chains, polyethylene glycol, and an ethylene diamine residue.
[20] The composition of claim 17, wherein the heparin is a member selected from the group consisting of unfractionated heparin, high molecular weight heparin, low molecular weight heparin, heparin fragments, recombinant heparin, heparin analogs, heparin sulfate, and sulfonated polysaccharides containing heparin activity.
[21] The composition of claim 17, wherein the bile acid is a member selected from the group consisting of cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid, ursocholic acid, ursodeoxycholic acid, isoursodeoxycholic acid, lagodeoxycholic acid, glycocholic acid, taurocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, dehydrocholic acid, hyocholic acid, and hyo- deoxycholic acid.
[22] The composition of claim 17, wherein the bile acid analog comprises a sulfonyl group.
[23] A method of treating cancer comprising administering to an individual in need thereof an effective amount of a composition represented by the formula: (B-S%-H-(S2-T)n wherein B is a bile acid residue or a bile acid analog, H is a heparin residue, S1 is a first spacer, S2 is a second spacer, T is a targeting moiety, m is an integer of 0 to about 50, and n is an integer of 0 to about 10, with the proviso that both m and n cannot be 0, and wherein B and S1 are bonded to each other through a 3-carbon of B, and S1 and S2 can be the same or different.
[24] The method of claim 23, wherein B is a bile acid residue selected from the group consisting of cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid, ursocholic acid, ursodeoxycholic acid, isoursodeoxycholic acid, Ia- godeoxycholic acid, glycocholic acid, taurocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, dehydrocholic acid, hyocholic acid, and hyo- deoxycholic acid.
[25] The method of claim 23, wherein B is a deoxycholic acid residue.
[26] The method of claim 23, wherein B is a lithocholic acid residue.
[27] The method of claim 23, wherein B is a bile acid analog comprising a bile acid to which is bonded a sulfonyl group.
[28] The method of claim 23, wherein said heparin residue is a member selected from the group consisting of unfractionated heparin, high molecular weight heparin, low molecular weight heparin, heparin fragments, recombinant heparin, heparin analogs, heparan sulfate and sulfonated polysaccharides containing heparin activity.
[29] The method of claim 23, wherein H is low molecular weight heparin.
[30] The method of claim 23, wherein S1 and S2 are independently selected from the group consisting of alkyl chains, polyethylene glycol, and an ethylene diamine residue.
[31] The method of claim 23, wherein T is a folic acid residue.
[32] The method of claim 23, wherein T is a cRGD residue.
[33] The method of claim 23, wherein B is a taurocholate residue, S1 is an ethylene diamine residue, H is low molecular weight heparin, S2 is an ethylene diamine residue, and T is a cRGD residue.
[34] The method of claim 23, wherein B is a lithocholate residue, S1 is an ethylene diamine residue, H is low molecular weight heparin, S2 is an ethylene diamine residue, and T is a folate residue.
[35] A method of treating cancer comprising administering to an individual in need thereof an effective amount of a composition comprising heparin bonded to the
3-carbon of a bile acid or bile acid analog.
[36] The method of claim 35, further comprising a spacer between the heparin and the bile acid or bile acid analog.
[37] The method of claim 36, wherein the spacer is a member selected from the group consisting of alkyl chains, polyethylene glycol, and an ethylene diamine residue.
[38] The method of claim 35, wherein the heparin is a member selected from the group consisting of unfractionated heparin, high molecular weight heparin, low molecular weight heparin, heparin fragments, recombinant heparin, heparin analogs, heparan sulfate, and sulfonated polysaccharides containing heparin activity.
[39] The method of claim 35, wherein the bile acid is a member selected from the group consisting of cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid, ursocholic acid, ursodeoxycholic acid, isoursodeoxycholic acid, lagodeoxycholic acid, glycocholic acid, taurocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, dehydrocholic acid, hyocholic acid, and hyo- deoxycholic acid.
[40] The method of claim 35, wherein the bile acid analog comprises a sulfonyl group.
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