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
  • A61K31/28—Compounds containing heavy metals
  • A61K31/282—Platinum compounds
• • 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
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
• • 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
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
  • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
  • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
• • 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
  • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
  • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
  • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
• • 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
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • 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
  • A61K47/56—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
  • A61K47/59—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
  • A61K47/60—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
  • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

• the invention relates generally to anti-cancer agent delivery vehicles capable of improved loading.
• a micelle is an aggregate of amphophilic or surfactant molecules dispersed in a liquid colloid.
• Each of the amphiphilic/surfactant molecules has a hydrophilic "head" end and a hydrophobic "tail” end.
• the tails of the micelle may include hydrocarbon groups, and the heads of the micelle may include charged (anionic or cationic) groups or polar groups.
• an aggregate of the micelle molecules typically form a normal micelle with the hydrophilic head ends extending outward and in contact with the surrounding solvent, sequestering the hydrophobic tail ends in the micelle centre, thereby forming the hydrophobic core.
• the polymeric micelles are generally formed from the self-assembly of amphiphilic block copolymers in an aqueous environment. It is known that polymeric micelles allow for enhanced accumulation of anti-cancer drug (i.e. enhanced drug loading) at tumor sites due to the enhanced permeability and retention (EPR) effect resulting from the leakiness of tumor vasculature (H. Maeda, Adv. Enzyme Regul.
• the outer hydrophilic shell of the micelles prevents the adhesion of proteins and reduces the uptake of micelles by the reticuloendothelial system (RES), thereby prolonging the blood circulation of micelles in the body (A. Lavasanifar et al, Adv. Drug. Deliv. Rev. 2002, 54, 169-190).
• RES reticuloendothelial system
• a conjugate of a delivery agent containing a chemical moiety and at least one flavonoid wherein the flavonoid exists in a monomeric form or dimeric form before
• the conjugate comprises two or more flavonoids, and each flavonoid does not undergo association with the other flavonoid.
• the flavonoids are chemically inert to each other and do not form oligomers during conjugation.
• the flavonoids may be identical or different.
• the flavonoid is a catechin-based flavonoid. More preferably, the flavonoid is (-)-epicatechin, (-)-epigallocatechin, (+)-catechin, (-)-epicatechin gallate, or (-)-epigallocatechin gallate. More preferably, the flavonoid is (-)- epigallocatechin gallate.
• the flavonoid may contain carboxyl-terminated group, amine-terminated group, succinimide-terminated group, or any other group suitable for conjugating with the chemical moiety.
• the chemical moiety is a polymer.
• the chemical moiety may be one selected from the group consisting of a polymer having a free aldehyde group or a functional group capable of being converted to an aldehyde group, a polymer having a free carboxyl group or a functional group capable of being converted to a carboxyl group, a polymer having a free amine group or a functional group capable of being converted to an amine group, a polymer having a free succinimide group or a functional group capable of being converted to a succinimide group, and a mixture thereof.
• the polymer is an aldehyde-terminated poly(ethylene glycol), aldehyde-derivatized hyaluronic acid, hyaluronic acid aminoacetylaldehyde diethylacetal conjugate, aldehyde-derivatized hyaluronic acid-tyramine, hyaluronic acid aminoacetylaldehyde diethylacetal conjugate- tyramine, cyclotriphosphazene core phenoxymethyl(methylhydrazono) dendrimer, thiophosphoryl core phenoxymethyl(methylhydrazono) dendrimer, carboxyl- terminated poly( ethylene glycol), amine-terminated poly( ethylene glycol), or succinimide-terminated poly(ethylene glycol). More preferably, the polymer is aldehyde-terminated poly(ethylene glycol).
• the delivery agent is conjugated at the C6 and/or the C8 position of the A ring of the flavonoid. While the C6 and C8 positions are most preferred, it will be apparent to a person skilled in the art that other conjugation positions of the flavonoid (e.g. at other rings) are also possible.
• a delivery vehicle comprising the conjugate of the first aspect of the invention.
• an anticancer agent delivery vehicle comprising an anti-cancer agent and a conjugate of the first aspect of the invention.
• the anti-cancer agent may be a protein, a nucleic acid, a small molecule, a drug, a peptide, an antibody, a hormone, an enzyme, a growth factor, a cytokine, single stranded DNA, double stranded DNA, single stranded RNA, double stranded RNA, a short hairpin RNA, an siRNA, an antibiotic, a chemotherapeutic agent or an angiogenesis inhibitor.
• the anti-cancer agent may be herceptin (trastuzumab) or TNP470, doxorubicin, cisplatin, paclitaxel, daunorubicin, or a mixture thereof.
• a pharmaceutical composition comprising the anti-cancer delivery vehicle of the thirdaspect of the invention.
• the pharmaceutical further comprises a pharmaceutical acceptable carrier.
• the subject may be a vertebrate preferably a mammal. In one embodiment, the subject is a human.
• the anti-cancer agent delivery vehicle is used to formulate for injection, surgical implantation or topical administration.
• a method of conjugating a delivery agent containing a chemical moiety to at least one flavonoid comprising reacting the delivery agent with the flavonoid, wherein the flavonoid exists in a monomeric form or dimeric form before conjugation and remains in the monomeric form or dimeric form after conjugation.
• a method of delivering an anti-cancer agent to a cell In one embodiment, the cell is in-vitro.
• the cell is in-vivo and the method comprises
• the subject may be a vertebrate preferably a mammal. In one embodiment, the subject is a human.
• the administering of the anti-cancer agent delivery vehicle Preferably, the administering of the anti-cancer agent delivery vehicle
• FIG. 1 shows a scheme illustrating the self-assembly of poly ethylene glycol/(-)- epigallocatechin gallate (PEG-EGCG) conjugates loaded with doxorubicin;
• FIG. 2 demonstrates the formation of PEG-EGCG conjugates loaded with doxorubicin at varying initial PEG-EGCG concentrations.
• the numeral reference below each bottle sample represents the initial PEG-EGCG concentration in mg/ml;
• FIG. 3 shows the electrospray ionization time-of-flight mass spectra of (a) PEG and (b) PEG-EGCG, respectively;
• FIG. 4 shows a scheme illustrating the self-assembly of PEG-EGCG conjugates loaded with cisplatin and/or herceptin
• FIG. 5 shows the in-vivo HER2-overexpressed human breast cancer cell (BT- 474) xenograft to Athymic Nude-Foxn1 nu (n-6) with PEG-EGCG conjugates loaded with herceptin.
• FIG. 6 shows confocal images of Cy3-labeled negative control siRNA (red) counterstained with a nuclei stain, DAPI (Blue) 24 hours after siRNA transfection. siRNA concentration was 50nM. The overlay images show that the siRNA was localized only in the cytoplasm and not in the nuclei.
• A No PEG-EGCG;
• B 1.5pg/ml PEG-EGCG;
• C 3.0 g/ml PEG-EGCG;
• D 6.0 g/ml and
• E PEG alone.
• FIG. 7 shows delivery of 200nM siRNA to pGL3 DNA transfected cells using PEG-EGCG. Ratio of firefly to renilla luciferase activities were normalized against cell population transfected only with pGL3 DNA. Control refers to pGL3 DNA transfected cells treated with siRNA alone.
• siPORTTM is a polyamine-based transfection agent used as a positive control. Luciferase activities were measured 48 hours after transfection without change of medium. Data are means of 3 experiments; S.D. is shown.
• FIG. 8 shows fluorescence observation of GFP downregulation 48 hours after GFP-22 siRNA transfection.
• the microscopic images on the right shows the downregulation of GFP compared to the control microscopic images on the left.
• siPORTTM is a polyamine-based transfection agent used as a positive control.
• A No siRNA transfection;
• B siPORTTM + GFP-22 siRNA;
• C PEG-EGCG + control siRNA;
• D PEG-EGCG + GFP-22 siRNA.
• Figure 8 E & F show flow cytometric analysis of cells 48 hours after siRNA transfection. 1000 to 1200 cell events were counted.
• E PEG-EGCG + control siRNA;
• F PEG-EGCG + GFP-22 siRNA.
• the present invention relates generally to anti-cancer agent delivery vehicles capable of improved drug loading.
• WO 2006/124000 conjugates of a delivery agent containing a free aldehyde and a flavonoid where the delivery agent is conjugated at the C6 and/or the C8 position of the A ring of the flavonoid have been provided.
• WO 2009/054813 delivery vehicles comprising an anticancer agent and a conjugate of a delivery agent containing a free aldehyde and a flavonoid, where the delivery agent is conjugated at the C6 and/or the C8 position of the A ring of the flavonoid have been provided.
• the contents of both WO 2006/124000 and WO 2009/054813 are herein incorporated by way of reference.
• the inventors have found that the combination of a delivery vehicle containing a chemical moiety and at least one flavonoid, and a bioactive anti- cancer agent has a synergistic anti-cancer effect, greater than the combined effects of each of the delivery vehicle and bioactive anti-cancer agent when used alone.
• delivery vehicles loaded with an anti-cancer agent provide an effective way of delivering anti-cancer agents to a cell, taking advantage of the synergistic effect between the anti-cancer activity of the flavonoid portion of the delivery vehicle and the anti-cancer effect of the anti-cancer agent. It is believed that the synergistic effect and therefore a higher anti-cancer agent loading is due to the flavonoid remaining in its original monomeric form or dimeric form after conjugation with the chemical moiety.
• a conjugate of a delivery agent containing a chemical moiety and at least one flavonoid wherein the flavonoid exists in a monomeric form or dimeric form before conjugation and remains in the monomeric form or dimeric form after conjugation to allow for hydrophobic interactions and/or ⁇ - ⁇ stacking interections between the flavonoid and other chemical entities such as anti-cancer drugs allowing greater drug loading to the monomeric or dimeric conjugate formed.
• the conjugate comprises two or more flavonoids, and each flavonoid does not undergo association with the other flavonoid/s.
• the flavonoids are chemically inert to one another and do not form oligomers during conjugation.
• the flavonoids may be identical or different from one another.
• the flavonoid may be any flavonoid from the general class of molecules derived from a core phenylbenzyl pyrone structure, and includes flavones, isoflavones, flavonols, flavanones, flavan-3-ols, catechins, anthocyanidins and chalcones.
• the flavonoid is a catechin or a catech in-based flavonoid.
• a catechin, or a catechin-based flavonoid is any flavonoid that belongs to the class generally known as catechins (or flavan-3-ol derivatives), and includes catechin and catechin derivatives, including epicatechin, epigallocatechin, catechin, epicatechin gallate and epigallocatechin gallate, and including all possible stereoisomers of catechins or catechin-based flavonoids.
• the catechin-based flavonoid is (-)-epicatechin, (-)- epigallocatechin, (+)-catechin, (-)-epicatechin gallate, or (-)-epigallocatechin gallate.
• (-)-epigallocatechin gallate (EGCG) is thought to have the highest activity among the catechin-based flavonoids, possibly due to the trihydroxy B ring and gallate ester moiety at the C3 position of this flavonoid.
• EGCG is particularly chosen to form the conjugate because of its high activity and by conjugation with, say, PEG (to be discussed in subsequent paragraphs), the resultant PEG-EGCG conjugate forms a stable composition that is metabolised or degraded more slowly, and which thus may have a longer half-life in the body.
• the EGCG moiety is capable of forming interactions, such as hydrophobic interaction, ⁇ - ⁇ stacking interaction or other physical or chemical bond, with the drug molecule to thereby encapsulate or confine the drug molecule within the inner core of the delivery vehicle.
• the flavonoid may be EGCG containing carboxyl- terminated group, amine-terminated group, succinimide-terminated group, or any other group suitable for conjugating with the chemical moiety.
• the delivery agent contains a chemical moiety selected from the group consisting of a polymer having a free aldehyde group or a functional group capable of being converted to an aldehyde group, a polymer having a free carboxyl group or a functional group capable of being converted to a carboxyl group, a polymer having a free amine group or a functional group capable of being converted to an amine group, a polymer having a free succinimide group or a functional group capable of being converted to a succinimide group, and a mixture thereof.
• the polymer is a carboxyl-terminated
• poly(ethylene glycol) or succinimide-terminated poly(ethylene glycol) which can react with amine-terminated EGCG to form the conjugate.
• the polymer is an amine-terminated poly(ethylene glycol) which can react with carboxyl-terminated EGCG or succinimide-terminated EGCG to form the conjugate.
• the delivery agent is capable of being formed into a delivery vehicle, thus allowing for the incorporation of a conjugated flavonoid into the delivery vehicle without compromising the biological or pharmacological properties of the flavonoid.
• the delivery agent should be biocompatible, and may be biodegradable in some embodiments.
• the flavonoid is a catechin-based flavonoid and in which the delivery agent is an aldehyde- terminated polymer.
• the aldehyde is an aldehyde- terminated polymer.
• condensation reaction between an aldehyde-containing chemical group and a flavonoid is applicable to conjugation of any delivery agent having a functional group described above, including following acid treatment of the delivery agent, to any flavonoid.
• the reaction may involve conjugation of a polymer containing a free aldehyde group or a group that is able to be converted to a free aldehyde group, for example, in the presence of an acid to a catechin-based flavonoid.
• the catech in-based flavonoid may be used in its monomeric form or dimeric form and it is important to retain the monomeric form or dimeric form of the flavonoid during conjugation so that the conjugation does not result in the formation of oligomers of the flavonoid.
• conjugation of a polymer to a flavonoid results in augmentation of the flavonoid's biological or
• the polymer may be any polymer having a free aldehyde group prior to conjugation with the catechin-based flavonoid, or having a group that is converted to an aldehyde group in the presence of an acid, for example an acetal group.
• the polymer should be non-toxic, biocompatible and suitable for pharmacological use.
• the polymer may also have other desirable properties. For example, the polymer may have low
• immunogenicity and it may be biodegradable or non-biodegradable depending on the desired biological application of the composition, for example, for controlled release of catechin-based flavonoids and the anti-cancer agent at a particular site in a body.
• the polymer may be chosen based on its particular characteristics and its ability to form certain types of delivery vehicles.
• the polymer may be an aldehyde-terminated poly(ethylene glycol).
• the polymer may be an aldehyde-derivatized hyaluronic acid, hyaluronic acid aminoacetylaldehyde diethylacetal conjugate, aldehyde-derivatized hyaluronic acid-tyramine, hyaluronic acid aminoacetylaldehyde diethylacetal conjugate-tyramine, cyclotriphosphazene core phenoxymethyl(methylhydrazono) dendrimer, or thiophosphoryl core phenoxymethyl(methylhydrazono) dendrimer.
• the polymer may also be any biological polymer, modified to contain a free aldehyde group or a group that is convertible to an aldehyde in the presence of an acid, for example an aldehyde- modified protein, peptide, polysaccharide or nucleic acid.
• the polymer is an aldehyde-terminated poly(ethylene glycol) (PEG- CHO).
• PEG is particularly chosen to form the conjugate because PEG is a polymer widely used as a pharmacological ingredient, and possesses good hydrophilic, non-toxic, non-immunogenic and biocompatibility characteristics with low biodegradability.
• the free aldehyde group on the polymer allows for the conjugation of the polymer in a controlled manner to either the C6 or the C8 position of the A ring, or both, of the flavonoid structure, thus preventing disruption of the flavonoid structure, particularly the B and C rings of the flavonoid, and thus preserving the beneficial biological and pharmacological properties of the flavonoid. While the C6 and C8 positions are most preferred, it will be apparent to a person skilled in the art that other conjugation positions of the flavonoid (e.g. at other rings) are also possible.
• the polymer is conjugated to the catechin-based flavonoid via a reaction of the aldehyde group of the polymer with the C6 and/or the C8 position of the A ring of the catechin-based flavonoid.
• the conjugate is synthesized using acid catalysis of a condensation of the aldehyde group of the polymer with the catechin-based flavonoid, or using acid to convert a functional group on the polymer to a free aldehyde prior to condensation of the aldehyde group with the catechin-based flavonoid.
• the polymer and the catechin-based flavonoid may be separately dissolved in a suitable solvent.
• the polymer with the free aldehyde is added, for example by dropwise addition, to the solution containing the catechin-based flavonoid, in the presence of an acid.
• the reaction is allowed to go to completion.
• excess unreacted polymer or catechin-based flavonoid can be removed from the conjugated composition, for example by dialysis or by molecular sieving.
• a conjugate of a polymer containing a free aldehyde and a catechin-based flavonoid having the polymer conjugated at the C6 and/or the C8 position of the A ring of the flavonoid is also contemplated.
• Conjugation of the polymer also allows for the incorporation of catechin-based flavonoids into various compositions or vehicles.
• catechin-based flavonoids into various compositions or vehicles.
• selection of the particular polymer containing a free aldehyde group based on the physical properties of the polymer it is possible to incorporate flavonoids into a variety of different vehicle types, allowing for the delivery of high concentrations of flavonoids in different contexts to various targeted areas of the body.
• the conjugate resulting from the above-described reaction may be formed into a delivery vehicle, depending on the nature of the polymer portion of the conjugate.
• the delivery vehicle may be used to deliver the catechin-based flavonoid to a body, including a particular targeted site in a body, depending on the nature of the delivery vehicle.
• the conjugate, delivery vehicle, anti-cancer agent delivery vehicle or the pharmaceutical composition comprising any of these are of nanometer dimentions wherein the chemical moiety and the at least one flavonoid are of nanometer dimentions.
• the anti-cancer agent may be any agent that has an anti-cancer effect on a cell, including an anti-tumour effect, such as a cytotoxic, apoptotic, anti-mitotic anti- angiogenesis or inhibition of metastasis effect.
• the anti-cancer effect is intended to include inhibition or reduction of tumour cell growth, inhibition or reduction of carcinogenesis, killing of tumour cells, or inhibition or reduction of carcinogenic or tumourogenic properties of a cell, including a tumour cell.
• An anti-cancer agent includes a protein, a nucleic acid, a small molecule or a drug.
• An anti-cancer agent that is a protein may be a peptide, an antibody, a hormone, an enzyme, a growth factor, or a cytokine.
• An anti-cancer agent that is a nucleic acid may be single stranded or double stranded DNA or RNA, a short hairpin RNA, an siRNA, or may comprise a gene encoding an anti-cancer product. Also included in the scope of anti-cancer agent is a chemotherapeutic agent or an angiogenesis inhibitor.
• the anti-cancer agent may be an antibody, including a monoclonal antibody, directed against a tumour cell-surface marker, an :
• the anti-cancer agent may be Herceptin (trastuzumab) or TNP470 (/V-(2-Chloroacetyl)carbamic acid (3R,4S,5S,6 ⁇ )-5-Methoxy-4-[(2f?,3R)-2-methyl-3-(3-methyl-2-buten-1-yl)-2- oxiranyl]-1-oxaspiro[2.5]oct-6-yl ester) an analog of fumagillin, doxorubicin, cisplatin, paclitaxel, daunorubicin, or a mixture thereof.
• the anti-cancer agents may include tyrosine kinase inhibitors, or cisplatin, platinum, carboplatin, gemcitabine, paclitaxel, docetaxel, etoposide, vinorelbine, topotecan, or irinotecan.
• Enzymes inducing apoptosis may include TRAIL-R1 , TRAIL-R2 or FasL.
• Nucleic acid anti-cancer agents may include plasmid DNA (encoding therapeutic proteins) or antisense oligonucleotides (ODNs) or small interfering RNA (siRNA).
• ODN is a short single stranded DNA, which is
• siRNA is a short double or single stranded RNA, which assembles into endoribonuclease- containing complexes upon entering cells, also known as RNA-induced silencing complex (RISC). These siRNA molecules then unwind inside the RISC, making the complex activated to recognize and splice its mRNA target strands in the cells, and thus down-regulating the expression of the target protein.
• RISC RNA-induced silencing complex
• micro-RNA capable of inducing apoptosis may be initiated by a specific micro-RNA.
• the micro-RNA may include: MiR-15, MiR-16, MiR-99a/let7c/MiR-125b2 or other suitable pro-apoptotic micro-RNA.
• the biological activity of the anti-cancer agent is temporarily partially or completely masked while incorporated into the present delivery vehicles, making them less available while the anti-cancer agent is assembled in the delivery vehicle, meaning that the anti-cancer agent is not able to exert anti-cancer activity or interact with other molecules in a bioactive manner while contained in the delivery vehicle, and is also protected from activity of other molecules.
• the biological properties of the anti-cancer agent are once again available, and the anti-cancer agent is able to exert an anti-cancer effect once delivered to the cell.
• an anti-cancer agent is loaded into the conjugate or delivery agent.
• the anti-cancer agent may be loaded or encapsulated in the conjugate or anti-cancer delivery vehicle through hydrophobic interactions and/or ⁇ - ⁇ stacking interactions with the flavonoid.
• the drug loading of the anti-cancer agent in the delivery agent of the invention is preferably more than other known loading of drugs in delivery agents.
• Drug loading is defined as the weight percentage of the anti-cancer agent in the delivery vehicle, while drug loading efficiency is defined as the weight percentage of the entrapped anti-cancer agent compared to the free anti-cancer agent introduced initially, as described in the examples.
• the drug loading may be increased by 1.5 to 5 times the reported range of drug loading.
• the drug loading is increased by 1 .75 to 4.25 times the reported range of drug loading.
• a polypeptide and a nucleic acid is not only able to be successfully loaded into the delivery agent but can also be successfully delivered in vivo and in vitro.
• the ratio of drug to conjugate can range from 6:1 to 1 :6.
• ratios of doxorubicin to PEG/EGCG were successful at ratios of 6:1 , 4:1 , 2:1 1 :1 , and 1 :2.
• the amount of anti-cancer agent is greater than the amount of conjugate provided the anti-cancer agent is able to form an interaction with the flavonoid.
• (-)-Epigallocatechin gallate (EGCG), a main ingredient of green tea, exhibits numerous biological and pharmacological effects.
• conjugates of poly(ethylene glycol) with EGCG were synthesized using aldehyde- mediated condensation by an acid catalyst.
• Example 1 Formation of Delivery Vehicle Comprising Doxorubicin and PEG-
• FIG 1 shows a scheme illustrating the self-assembly of PEG-EGCG conjugates loaded with Dox to form the delivery vehicle.
• the delivery vehicle is in the form of a micelle. It is based on the self-assembly of PEG-EGCG conjugates into micelles in aqueous environment, stemming from the hydrophilic nature of the PEG chain of the PEG-EGCG conjugate. Due to the similarity in the multi-ring structures of the EGCG moiety and Dox, the EGCG moiety is designed to encapsulate Dox through the hydrophobic interactions and ⁇ - ⁇ stacking interactions.
• Dox was successfully encapsulated within the delivery vehicles by simple mixing and dialysis.
• the delivery vehicles demonstrate exceptionally high Dox loading in comparison to other delivery vehicles systems (e.g. polymeric micellar systems) known in the prior art.
• Dynamic light scattering (DLS) measurements also showed that the dimensions of the delivery vehicles are ideal for passive tumor targeting via EPR effect.
• the delivery vehicle was prepared by dialysis of a mixture of PEG-EGCG conjugates and Dox dissolved in organic solvent dimethylformamide (DMF) against distilled water.
• Different concentrations of PEG-EGCG conjugates (0, 0.5, 1 , 3, 6, 12, 24 mg/ml, respectively) were used to encapsulate a fixed concentration of Dox at 12 mg/ml.
• the dialysis the gradual replacement of the organic solvent with water triggered the self-assembly of the delivery vehicles and the entrapment of Dox within the assembled structures.
• precipitates were obtained during the dialysis when PEG-EGCG concentrations of 0, 0.5 and 1 mg/ml were used, indicating ineffective delivery vehicle formation.
• PEG-CHO Aldehyde-terminated PEG (PEG-CHO, Mw 5000) was purchased from NOF Co., Japan.
• EGCG was purchased from Kurita Ltd., Japan.
• Doxorubicin hydrochloride Dox HCI was obtained from Boryung Pharmaceuticals Inc., Korea.
• Delivery Vehicle Dox HCI (12 mg, 21 ⁇ ) was dissolved in 1 ml of DMF.
• Triethylamine (29 ⁇ , 210 pmol) was then added to the solution to neutralize the hydrochloride.
• PEG-EGCG conjugates (1 ml) with different concentrations dissolved in DMF were added to the solution, and the resulting mixtures were vortexed for 20 min.
• Each sample solution was then transferred to dialysis tubes with a MWCO of 3500 Da. The tubes were dialyzed against distilled water for 2 days, with the water replaced every 12 h. The sample solutions were lyophilized to obtain solid micelles.
• Determination of Drug Loading The absorbance of the sample solutions was measured at 480 nm using a Hitachi U2810 UV-Vis spectrophotometer after the dried samples were dissolved in DMF. The amount of Dox in each sample was estimated by comparing to the Dox standards. Drug loading was calculated from the amount of Dox in the delivery vehicles, and the weight of the dried delivery vehicles samples.
• Determination of Delivery Vehicle Size The size of the delivery vehicle samples was determined by DLS using a Brookhaven 90Plus Particle Size Analyzer. Each sample solution was prepared using distilled water to give 10 ⁇ g/m ⁇ of Dox before conducting the DLS measurements at 25°C.
• Results characteristics of the delivery vehicle The characteristics of the delivery vehicle of Example 1 are summarized in Table 1 below.
• Drug loading is defined as the weight percentage of Dox in the delivery vehicle, while drug loading efficiency is defined as the weight percentage of the entrapped Dox compared to the free Dox introduced initially.
• the samples with initial PEG-EGCG concentrations of 3 and 6 rrig/ml showed an extraordinarily high drug loading of 85 and 68 wt%, respectively.
• the highest Dox loading achieved (85 wt%) corresponded to more than 4 times the average Dox loading (10-20 wt%) in the block copolymeric delivery vehicles developed by other groups (H. M. Aliabadi et al, Expert Opinion on Drug Delivery, 2006, 3, 139-162).
• the size of the delivery vehicles ranged from 78.5 to 192.4 nm (see Table 1 ).
• the size increased as the drug loading increased, but decreased with increasing initial PEG-EGCG concentration.
• the increase in size should be correlated to the encapsulation efficiency since the EGCG moiety played a key role in
• the encapsulating Dox could be controlled by the initial PEG- EGCG concentration introduced.
• the nanometer dimensions of the delivery vehicles (less than 200 nm) conferred the ability of targeting tumor tissue via EPR effect. At the same time, they enabled prolonged blood residence time, as they helped to avoid opsonization and renal clearance of delivery vehicles.
• the electrospray ionization time-of-f light (ESI-TOF MS) mass spectra of (a) PEG and (b) PEG-EGCG, respectively, are shown in FIG. 3.
• the peaks of PEG-EGCG shifted higher with molecular weight of two EGCG (e.g.
• the drug molecule is cisplatin.
• FIG. 4 shows a scheme illustrating the self-assembly of PEG-EGCG conjugates loaded with cisplatin to form the delivery vehicle.
• the delivery vehicle may be in a form of a chelate-complex.
• the interaction between cisplatin and the PEG-EGCG conjugates is probably due to the chelation of Pt (platinum) by the OH-moieties of the EGCG.
• the chelate-complex is formed as a conseguence of the chelation where the PEG-EGCG conjugates cluster around the central cisplatin, thereby confining cisplatin therein.
• the cisplatin/PEG-EGCG complex was prepared by dialysis of a mixture of PEG-EGCG and cisplatin dissolved in (i) water or (ii) organic solvent dimethylformamide (DMF) against distilled water.
• Different concentrations of PEG- EGCG conjugates (1.2, 2.4, 6, and/or 2 mg/ml) were used against a fixed concentration of cisplatin at 1.2 mg/ml.
• the PEG-EGCG conjugates cluster around the central cisplatin to confine cisplatin within the cluster.
• Aldehyde-terminated PEG (PEG-CHO, Mw 5000) was purchased from NOF Co., Japan.
• EGCG was purchased from Kurita Ltd., Japan.
• Cisplatin [c/s-dichlorodiammineplatinum(ll); CDDP] was purchased from Sigma-Aldrich Pte. Ltd., Singapore.
• PEG-EGCG Conjugate The PEG-EGCG conjugates were synthesised as described in Example 1.
• the polymer may be a carboxyl-terminated poly(ethylene glycol) which can react with amine-terminated EGCG to form the conjugate, the synthesis of which is apparent to persons skilled in the art.
• CDDP 1.2 mg, 4 pmol
• PEG-EGCG conjugates (1 ml) with different concentrations dissolved in water or DMF were added to the solution, and the resulting mixtures were vortexed for 20 min.
• Each sample solution was then transferred to dialysis tubes with a MWCO of 1000 Da.
• the tubes were dialyzed against distilled water for 2 days, with the water replaced every 12 h.
• the delivery vehicles were then harvested.
• Determination of Drug Loading The absorbance of the sample solutions was measured at 280 nm using a Hitachi U2810 UV-Vis spectrophotometer for the PEG-EGCG content.
• the amount of cisplatin in each sample was measured by PelkinElmer SCIEX ICP Mass Spectrometer Elan DRC II. Drug loading efficiency was calculated from the amount of cisplatin confined in the delivery vehicle, and the amount of the cisplatin added into the system initially. Drug loading was calculated from the amount of cisplatin in the delivery vehicle, and the weight of the delivery vehicle.
• Dynamic light scattering (DLS) measurements also showed that the dimensions of the delivery vehicle were in the range of 153 to 194 nm (see Table 2 and Table 3) which are ideal for passive tumor targeting via EPR effect.
• the nanometer dimensions of the delivery vehicle (less than 200 nm) conferred the ability of targeting tumor tissue via EPR effect. At the same time, they enabled prolonged blood residence time, as they helped to avoid opsonization and renal clearance of the delivery vehicle.
• PEG-EGCG Cisplatin hydrodynamic loading loading concentration concentration diameter efficiency (wt%) c (mg/ml) (mg/ml) (nm) a (wt%) b
• PEG-EGCG Cisplatin hydrodynamic loading loading concentration concentration diameter efficiency (wt%) c (mg/ml) (mg/ml) (nm) a (wt%) b
• Example 3 Test Results of Using Delivery Vehicle Comprising Herceptin and PEG-EGCG Conjugates
• the present inventors have also successfully encapsulated herceptin (Herceptin being purchased from Roche, Switzerland) within PEG-EGCG conjugates following the synthesis route outlined in Example 2 above.
• the conjugates have shown enhanced anti-cancer effect on BT-474 HER2/neu overexpressed breast cancer xenograft, as shown in FIG. 5.
• mice In-Vivo Anti-cancer Efficacy of Delivery Vehicle: Athymic (nu/nu) mice (5-6 weeks old) were purchased from the Harlan, UK and all animal experiments were performed in compliance with Institutional Animal Care and Use Committee
• Herceptin used was 2.5 mg/kg and PEG-EGCG was 100 ⁇ .
• Treatments were administered twice per week. Sixteen days later, the animals were injected with vehicle (PBS) ( ⁇ ), PEG-EGCG ( A ), herceptin ( ⁇ ), (herceptin + PEG-EGCG) micelle ( ⁇ ).
• the results were reported as mean ⁇ standard deviation.
• the enhanced anti-cancer effect on BT-474 HER2/neu overexpressed breast - cancer xenograft could be due to the accumulation of the delivery vehicles at the tumor site and hence reduced the tumor size significantly as compared to the animal groups treated with herceptin only.
• the mechanism for the encapsulation of herceptin within the delivery vehicle may be similar to that contemplated in
• Example 1 i.e. the EGCG moiety encapsulates herceptin through the hydrophobic interactions and ⁇ - ⁇ stacking interactions between the proline residues and the EGCG moiety.
• the present delivery vehicles overcome the challenge of low drug loading faced by the conventional drug delivery vehicles (such as polymeric micellar carriers) comprising of a variety of block copolymers as mentioned in previous paragraphs.
• the conventional drug delivery vehicles such as polymeric micellar carriers
• the present delivery vehicles provide strong drug interactions via the EGCG moiety to achieve high drug loading.
• the enhanced encapsulation or confinement capability of the present delivery vehicles may also be extended to other anti-cancer drugs without any modification of their structure and hence makes it a potential system for co-delivery of multi anti-cancer agents, such as herceptin and doxorubicin or paclitaxel and daunorubicin.
• Unique properties of the present delivery vehicles which make them particularly suitable for use in cancer therapy include (i) their nanometer-dimensions of between 10 and 200 nm; (ii) their amphiphilic structure that confers increased solubility in water; and (iii) their ability to encapsulate or confine anti-cancer agents within its inner core.
• the design of a delivery vehicle with a higher drug loading is important towards achieving an efficient cancer therapy. It is also expected that such a delivery vehicle would minimize the side effects caused by the potential unspecific drug delivery as the dose of the delivery vehicle may be reduced.
• having carriers with a higher anti-cancer drug loading reduces the frequency of injections, thereby improving the compliance of patient and reducing trauma caused to the patient.
• the present delivery vehicles are designed to provide for such unique properties.
• Example 4 In vitro imaging of siRNA delivery and subcellular localization Cells were seeded in Lab-TekTM 8-well chambered coverglasses (Nunc, USA) at a density of 1 x 10 4 cells/coverglass. Two days after plating, the culture medium was removed and PEG-EGCG/siRNA transfection complexes were added to the chambered coverglasses. Complexes were prepared as described above by adding varying concentrations of PEG-EGCG to 50nM Silencer® CyTM3-Labeled Negative Control #1 siRNA (Ambion, USA).
• DPBS Dulbecco's Phosphate-Buffered Saline
• PFA paraformaldehyde
• the cells were stained with 4', 6- diamidino-2-phenylin (DAPI, Invitrogen, USA) before being examined by confocal microscopy. Confocal laser scanning microscopy was carried out using an
• the images reveal a cytoplasmic sublocalization for the siRNA, as seen by overlaying the images of the DAPI stained nucleus and the Cy-3 labelled siRNA.
• siRNA was delivered without PEG-EGCG as carriers ( Figure 6A)
• no fluorescence was observed, demonstrating that siRNA alone was not able to enter the cells.
• PEG alone was used to complex siRNA ( Figure 6E)
• no fluorescence was detected, indicating that the red fluorescence, observed in cells transfected with PEG-EGCG/siRNA complexes, was not due to the PEG polymer.
• the Renilla luciferase serves a dual purpose. Firstly, it is a transfection control that can be used to normalize the activity of the firefly luciferase. Secondly, its activity can be monitored to detect any unspecific effect of the siRNA/PEG-EGCG complexes.
• Transfections were performed with pGL3 luciferase siRNA (Promega.USA) and Allstars negative control siRNA (QIAGEN, Singapore). After 48 hours, the transfection medium was removed and the cells rinsed with DPBS. Cells were lysed in 50 ⁇ of passive lysis buffer (Promega, USA) and placed on an orbital shaker (Thermoscientific, USA) at room temperature for 15 minutes. Luciferase assay reagent II (LARII) and Stop & Glo® reagent (Promega, USA) used for measuring gene expression were prepared and added to cell lysates following the manufacturer's protocol. Luciferase activity was measured using a Lumat LB9507 luminometer (Berthold, Germany).
• Transfection efficiency was evaluated as the ratio of the activity of the firefly luciferase (expressed in relative light units (RLU) over the Renilla luciferase's activity.
• RLU relative light units
• siRNA siRNA on luciferase activity was also investigated.
• the ratio of both luciferase was expressed as the relative light units of respectively firefly and Renilla (RLU1/RLU2).
• RLU1/RLU2 The ratio obtained in cell population transfected with both luciferases but without siRNA was used to normalize the ratio obtained from various siRNA transfection conditions.
• a functional siRNA is expected to reduce the levels of the firefly luciferase protein and consequently its activity.
• the lowered activity will translate in a decrease of the normalized ratio of firefly to Renilla luciferase activity.
• This normalization minimizes experimental variability caused by differences in transfection efficiency or cell viability, thereby allowing more reliable interpretation of the experimental data.
• To examine the transfection efficiency of the two plasmid DNA and optimize their ratio cells were transfected with ratios of Renilla to firefly luciferase DNA ranging from 1 : 10 to 1 :400. Indeed, it has been reported that the two promoters used to drive the Renilla and firefly luciferase (SV40 and the cytomegalovirus promoters, respectively) could influence each other. The amount of firefly luciferase was maintained constant. Their activities were measured and expressed as a relative ratio.
• Figure 8A-D showed positive downregulation when GFP-22 siRNA was delivered with siPORTTM .
• the image was used as a positive control in this study.
• Cell transfected with pEGFP-1 plasmid vector. (Clontech.USA) were transfected with GFP-22 and Allstars negative control siRNA (QIAGEN, Singapore) 4 hours later using complexes described above. 48 hours later, fluorescence images were taken using Olympus 1x71 fluorescence microscope (Olympus, Singapore). The images were acquired using Image-Pro Plus software (Media-Cybernetics, USA). Confirming gene silencing using flow cytometry
• transfection medium was removed and 50ul of accutase enzyme cell detachment medium (PAA Laboratories GmbH, Austria) was added per wells. Cells were incubated for 5 minutes; after cells were detached, 150ul of DPBS was added per well and the contents of the well were transferred into 1.5ml tubes and spun down at 3000rpm for 3 minutes. The supernatant was removed and cells were resuspended in cell fluorescence buffer from the Agilent Cell Fluorescent LabChip® Kit (Agilent Technologies, Singapore) at a density of 2.5 x 10 6 cells/ml.

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