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WO2011051667A1 - Dendrimères ciblés - Google Patents

Dendrimères ciblés Download PDF

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Publication number
WO2011051667A1
WO2011051667A1 PCT/GB2010/001990 GB2010001990W WO2011051667A1 WO 2011051667 A1 WO2011051667 A1 WO 2011051667A1 GB 2010001990 W GB2010001990 W GB 2010001990W WO 2011051667 A1 WO2011051667 A1 WO 2011051667A1
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WIPO (PCT)
Prior art keywords
dendrimer
dab
molecule
composition according
dna
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PCT/GB2010/001990
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English (en)
Inventor
Christine Myriam Dufes
Original Assignee
University Of Strathclyde
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Priority to GB1209228.4A priority Critical patent/GB2487699B/en
Publication of WO2011051667A1 publication Critical patent/WO2011051667A1/fr

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal 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
    • A61K47/51Medicinal 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
    • A61K47/56Medicinal 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
    • A61K47/59Medicinal 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
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal 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
    • A61K47/51Medicinal 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
    • A61K47/54Medicinal 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
    • A61K47/543Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents

Definitions

  • the present invention relates to new compositions which are designed to be delivered in a targeted manner to desired cells in the body of a subject.
  • the cells to be targeted are cancer cells.
  • compositions as well as methods of treating diseases, such as cancer.
  • dendrimer nucleic acid compositions are described in WO03/033027, which describes compositions which normally target cells of the liver and spleen. However, there is no teaching of how to specifically target other cells or diseased cells, such as cancer cells.
  • Transferrin receptor is an attractive target for selective receptor-mediated gene delivery to tumours because it is overexpressed in a high percentage of human cancers, including ovarian, breast, colon cancers and glioblastoma cell lines (Calzolari et al., 2007).
  • Transferrin (Tf) has previously been used successfully as a tumour-targeting ligand for several drug delivery systems (Kircheis et al., 2001 ; Kircheis et al., 2002, Xu ei al., 2002; Dufes ef al., 2004; Nakase et al., 2005).
  • dendrisomes which are non-ionic surfactant vesicles which comprise dendrimers.
  • the transferrin molecule is conjugated to the vesicle surface and the requirement to make the vesicles may be undesirable.
  • the present invention is based on work by the present inventor into conjugating transferrin directly to a dendrimer and thereafter allowing nucleic acid to become associated with such a dendrimer/transferrin conjugate and using the resulting complex to target cancer cells.
  • compositions for targeted delivery of a bioactive molecule to cells in a body of a recipient comprising a cationic dendrimer and/or derivatives thereof to which one or more cell targeting molecule(s) have been conjugated thereto; and a bioactive molecule associated with said cationic dendrimer and/or derivative thereof.
  • cationic dendrimer refers to a dendrimer molecule which possesses a positive charge at physiological pH.
  • the dendrimer derivatives of the present invention may not in themselves be cationic as a result of the derivatisation.
  • the cationic dendrimers, or derivatives thereof, of the composition of the present invention may be derived from a core molecule comprising 2 to 10 carbon atoms, such as 3 or 4 carbon atoms, and in particular 4 carbon atoms with one or more functional groups which may, for example, be amine groups. It will be appreciated that, for example, the cationic dendrimers, or derivatives thereof, may be derived from a core molecule such as diaminoethane, diaminopropane or diaminobutane, and in particular, diaminobutane.
  • the groups attached to the core molecule may, for example, include propylamines
  • the dendrimers may be polypropylenimine dendrimers, or derivatives thereof, and may possess a diaminobutane core.
  • polypropylenimine dendrimer is intended to refer to dendrimers comprising a diaminobutane core with 1 , 2, 3, 4 or 5 generations of propylenimine molecules attached.
  • the term encompasses DAB 4, DAB 8, 16, 32 and 64, DSAM 4, DSAM 8, 16, 32 and 64, and QDAB 4, 8, 16, 32 and 64, HDAB 4, 8, 16, 32, 64 and bolamphiphilic polypropylenimine dendrimers BDAB 4, BDAB 8, BDAB 16, BDAB 32, BDAB 64.
  • DAB diaminobutane core with propylenimine groups attached thereto
  • DAB16 is a preferred polypropylenimine dendrimer.
  • the term "generation” refers to the number of iterative reaction steps that are necessary to produce the compound.
  • the cationic dendrimers of the composition of the present invention may be modified by covalently binding derivatising groups, such as hydrophobic, hydrophilic or amphiphilic groups to the surface of the dendrimer or by attaching two dendrimer molecules to either end of a hydrocarbon chain with a carbon length of 8, 12, 14, 16 or 18 carbons to give bolamphiphilic dendrimers (said modified dendrimers referred to herein as "derivatives").
  • the number of derivatising groups may vary from one derivatising group per dendrimer molecule up to, but not including derivatising all available surface or terminal groups on the dendrimer molecule, for example, derivatising all 1 to 15 surface groups of the DAB16 molecule.
  • the amphiphilic derivative comprises a hydrophilic and a hydrophobic segment.
  • the hydrophilic segment may be derived from a phosphoglycerate molecule, for example, glycerol 3-phosphate.
  • the hydrophobic segment is covalently bound to the hydrophilic segment, for example, via an ester linkage.
  • the hydrophobic segment is selected from any suitable hydrophobic group, for example, alkyl, alkenyl or alkynyl groups of 8-24 carbons in length.
  • the hydrophobic segment plus ester linkage can be defined as an acyl group.
  • the amphiphilic derivative is attached to the dendrimer by a linker molecule, such as polyethylene glycol (PEG) or a sugar unit such as muramic acid bound to the hydrophilic segment.
  • the length of the PEG linker molecule may for example be in the range of 1 to 120 ethylene glycol units, for example 50-100 and, for example, 70-80, for example, 77.
  • the linker molecule may be polyethylene glycol with a Mw of approximately 3,500.
  • the linker molecule may be an ester, amine or ether linkage for ordinary hydrophobic modifications or a sugar molecule such as muramic acid.
  • the derivative may be a phosphoglyceride such as a phosphatidyl ethanolamine, for example, distearoylphosphatidylethanolamine.
  • the number of amphiphilic derivatives per dendrimer molecule may range from 1 derivatising group per dendrimer molecule to derivatising all but one of the groups of the dendrimer (the generation of the dendrimer will determine the total number of surface groups available for derivatising), and may be, in particular, one group per dendrimer.
  • the hydrophobic derivative may be an alkyl, acyl, alkenyl, alkynyl or aryl group of 8-24 carbons in length. It is to be understood that the term "hydrophobic" can encompass acyl groups when the chain length of such acyl groups is 8 carbons or more and may, for example, be a hexadecanoyi group.
  • the number of hydrophobic groups per dendrimer molecule may range from 1 derivatising group per dendrimer molecule to derivatising all but one of the groups of the dendrimer (the generation of the dendrimer will determine the total number of surface groups available for derivatising), and may be, in particular, one group per dendrimer.
  • the dendrimers of the present invention include generations 1 , 2, 3, 4 and 5 of the hydrophobic- derivatised diaminobutane dendrimer referred to herein as HDAB 4, HDAB 8, HDAB 16, HDAB 32, HDAB 64.
  • the bolamphiphiles may consist of two molecules of any of the dendrimers DAB 4, DAB 8, DAB 16, DAB 32, DAB 64 linked to either end of an alkyl, acyl, alkenyl, alkynyl hydrophobic unit of 8 to 24 carbon chains in length or alternatively linked by an aryl group and may be a C12 bolamphiphile of DAB 4 or DAB 8.
  • the term "bolamphiphiles" is understood to refer to an amphiphilic molecule wherein the hydrophilic groups are separated by the hydrophobic groups.
  • the dendrimers of the present invention include C8-C16 alkyl bolamphiphiles of dendrimers of generations 1 ,2, 3,4 and 5 herein referred to as B8DAB 4, 8, 16, 32 or 64 ; B10DAB 4, 8, 16, 32 or 64; B12DAB 4, 8, 16, 32 or 64, B14DAB 4, 8, 16, 32 or 64 and B16DAB 4, 8, 16, 32 or 64.
  • the amino derivative may, for example, be a tertiary amine or quaternary ammonium derivative, and in particular a quaternary derivative comprising C1- C4 alkyl groups, such as 3 methyl groups, covalently bound to a nitrogen atom on the surface of the dendrimer.
  • the number of ammonium derivatives per dendrimer molecule may range from 1 derivatising group per dendrimer molecule to derivatising all but one groups of the dendrimer (the generation of the dendrimer will determine the total number of surface groups available for derivatising).
  • the dendrimers in the present invention may also be derivatised with hydrophilic groups such as sugars, mono and oligohydroxy C1-C6 alkyl, mono and oligohydroxy C2-C6 acyl, C1- C2 alkoxy alkyl optionally having one or more hydroxy groups substituted on the alkoxy or alkylen groups, amino acids, peptides of 1-200 amino acids in length and oligo or poly- (oxa C1-C3 alkylen) such as polyoxyethylene comprising 1-120 ethylene oxide units.
  • hydrophilic groups such as sugars, mono and oligohydroxy C1-C6 alkyl, mono and oligohydroxy C2-C6 acyl, C1- C2 alkoxy alkyl optionally having one or more hydroxy groups substituted on the alkoxy or alkylen groups, amino acids, peptides of 1-200 amino acids in length and oligo or poly- (oxa C1-C3 alkylen) such as polyoxyethylene comprising
  • the recipient may be a mammal, such as a human.
  • the targeting molecule(s) may be any suitable molecule which can be covalently conjugated to the dendrimer.
  • the targeting molecule may be a ligand for a receptor present on the surface of a targeted cell.
  • One such ligand is transferrin which is capable of binding to transferrin which is capable of binding to transferrin receptor which is present on the surface and overexpressed by a variety of human cancers, such as ovarian, breast, colon and glioblastoma cancer cells.
  • lactoferrin lactoferricin
  • gambogic acid as tumour-targeting ligands.
  • Lactoferrin (Lf) also called lactotransferrin
  • lactoferricin (Lcin) are iron-binding members of the transferrin family, able to bind the same receptors than transferrin. Lf is found predominantly in the secreted fluids of mammals such as milk, whereas Lcin is released from Lf through proteolysis by pepsin.
  • Lf has been shown to inhibit the proliferation of human nasopharyngeal carcinoma, human breast carcinoma as well as head and neck cancer cell lines through induction of cell cycle arrest and modulation of the mitogen-activated protein kinase (MAPK) signalling pathway in vitro (Zhou ef a/., 2008).
  • the inhibition of tumour cell growth by LF may also be related to the ability of this protein to induce apoptosis of cancer cells by activating the Fas signalling pathway in cancerous cells.
  • LF has recently been shown to activate the NF- ⁇ signalling cascade in HeLa cervical carcinoma cells, which resulted in upregulation of the tumour suppressor protein p53 (Oh ef ai, 2004; Ward ef a/., 2005).
  • Lcin has been shown to exert antitumour effects against a number of cancer cell lines.
  • Lcin is a potent inducer of apoptosis in colon and ovarian carcinoma, neuroblastoma and melanoma (Gifford er a/., 2005; Eliassen er a/., 2006; Furlong et al., 2006).
  • Lcin-induced apoptosis results from reactive oxygen-species and caspase 2-dependent disruption of mitochondrial membrane integrity and the subsequent sequential activation of caspase 9 and caspase-3.
  • c-Jun N-terminal kinase/stress-activated protein kinase may also play an important role in Lcin-induced apoptosis of human cancer cells.
  • Lcin has also been reported to exert potent in vivo anti-tumour activity in mouse models of cancer. Direct injection of Lcin into solid Meth A tumours causes tumour cell lysis and a significant reduction in tumour size (Eliassen et al., 2002).
  • subcutaneous administration of Lcin inhibits tumour metastasis by highly metastatic murine L5178Y- L25 lymphoma cells and B16F10 melanoma cells (Yoo ef al., 1997).
  • Gambogic acid is the main active component of gamboge, a resin exuded from Garcinia hanburryi tree in Southeast Asia. Like Lf and Lcin, this drug binds the transferrin receptors (Pandley et al., 2007).
  • GA can inhibit the proliferation of various types of cancer cells, such as human lung carcinoma and hepatoma (Guo et al., 2004; Zhao et al., 2004; Liu ef al., 2005). It prevents tumour metastasis and angiogenesis, potentiates apoptosis through its interaction with the transferrin receptor (Kasibhatla et al., 2005; Pandley ef al., 2007). GA also enhances the apoptosis induced by TNF, which is of the highest importance for our current gene delivery studies involving this factor (Pandley et al., 2007).
  • targeting is meant that the composition is preferentially taken up by cells to which the targeting molecule is directed, but not necessarily exclusively by such cells.
  • more than one targeting molecule may be conjugated to each dendrimer molecule.
  • the dendrimer molecule is DAB16, which comprises 16 reactive amino groups, preferably 2, 4, 6, 8, or more of the amino groups react with and conjugate a transferrin molecule.
  • the targeting molecule such as transferrin may be conjugated to the dendrimer by any suitable means.
  • a preferred method was dimethylsuberimidate (DMSI) as cross- linking method, which may be carried out in a single-step.
  • DMSI dimethylsuberimidate
  • the compositions of the present invention may further comprise a molecule, such as a polymer to which amino acids selected from Arg, Lys and Leu have been conjugated.
  • bioactive molecules and “biologically active molecules” are intended to encompass polynucleotides, peptides/polypeptides and/or pharmaceutical agents.
  • polynucleotides generally refers to DNA unless otherwise indicated but may include RNA, cDNA, oligonucleotides, plasmids etc.
  • the term may also be used interchangeably herein with the terms “polynucleotide”, “gene”, “genetic material” and “genetic sequence”.
  • genes intended for expression are common to the field of gene therapy and include, but are not limited to, sense DNA or RNA for expressing a product in the target organ, or antisense DNA or RNA for reducing or eliminating expression of a native or introduced gene in the target organ.
  • the polynucleotides may be capable of expressing a functional gene which may encode a therapeutic protein, or may express part of a gene, so as to express a peptide which may compete with a native gene or protein eg tumour necrosis factor a (TNFa).
  • TNFa tumour necrosis factor a
  • Other exemplary bioactive molecules include interleukin-12 , tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), or p73.
  • Systemic 11-12 is able of suppressing tumour growth, metastasis and angiogenesis in many types of cancer in vivo (Salem et a/., 2006; Albini ef a/., 2007; Diez er a/., 2009).
  • IL-12 has been associated with significant dose-dependent toxicity, thus limiting its therapeutic application. The advantages and limitations of this cytokine thus make it particularly suitable as a therapeutic DNA in a tumour-targeting delivery system.
  • TRAIL a member of the TNF superfamily, specifically inhibits the growth of a variety of human tumours in vivo, including breast and colon carcinomas, gliomas, myelomas, while leaving normal non-transformed cells mostly unaffected (Yoo et al., 2006; Corazza et al., 2009; Holoch and Griffith, 2009).
  • TRAIL expression plasmid as a therapeutic agent would therefore provide an attractive approach to cancer gene therapy.
  • P73 a member of the p53 family of transcription factors, shares a high degree of sequence homology with p53 and can bind to p53-responsive elements, activating the transcription of p53 target genes, such as those inducing cell cycle arrest and promoting apoptosis.
  • p73 is upregulated, inducing cell cycle arrest and apoptosis in a p53-independent manner (Melino, 2003; Flores er al., 2005; Bell et al., 2007; Bell and Ryan, 2007, Bell and Ryan, 2008).
  • the polynucleotide may also encode and/or be capable of expressing a so-called RNAi or si NA molecule well known in the art.
  • peptide refers to a chain of 4 to 600 amino acids long, such as 4 to 200 amino acids long and therefore encompasses polypeptides and proteins, and includes enzymes and polypeptide hormones. Furthermore, peptides modified by, for example, glycosylation, are also included in the present invention, as is a protein comprising two or more polypeptide chains each of length of 4 to 600 amino acids long cross-linked by, for example, disulphide bonds.
  • pharmaceutical agent is intended to include any natural or synthetic compound administered to a recipient in order to induce a physiological or pharmacological effect. Examples of such agents are anti- tumour drugs, antibiotics, hormones, anti-inflammatory agents, antiparasitic agents, DNA vaccines, etc.
  • the cationic dendrimers comprising the conjugated targeting molecule may be admixed with the bioactive agents in preparing the compositions of the present invention for delivery.
  • the term "admixed" generally refers to the bioactive agent being associated with but not covalently bound to the dendrimer. The term is however also intended to encompass covalently binding the bioactive agent to the dendrimer via any suitable reactive group on the dendrimer and the agent.
  • the bioactive agent is a polynucleotide molecule
  • the molecule is usually associated with, that is, not covalently bound to, the dendrimer to allow the polynucleotide to be expressed.
  • expression of a covalently bound polynucleotide molecule can occur, and therefore, these covalently bound polynucleotide molecules are intended to be encompassed by the present invention.
  • compositions of the present invention may be administered to a subject alone, or may be administered in conjunction with other therapeutic agents.
  • the composition may be allowed to associate with drug containing vesicles, such as niosomes.
  • the nucleic acid associated with the dendrimer/targeting agent may encode a protein such as tumour necrosis factor and the vesicles may contain an anticancer agent such as doxorubicin.
  • anti-cancer natural products may be encapsulated in the present delivery systems and include tocotrienol; epigallocatchin; sulphoraphane; quercetin; curcumin; evodiamine; emodin.
  • the present invention provides a pharmaceutical formulation comprising a composition of the present invention, and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.1 M and preferably 0.05M phosphate buffer or 0.9% (w/v) saline.
  • such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solutions are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, cheating agents, inert gases and the like.
  • composition or pharmaceutical formulation of the present invention may include an agent which assists in forming a colloidal suspension, for example, 5% dextrose solution.
  • agents which may be included are viscosity enhancing polymers such as alginates and polyethyleneglycol polymers, buffering agents and mixtures of aqueous and non- aqueous solvents in emulsions.
  • the present invention also provides the use of the composition or pharmaceutical formulation of the present invention for the delivery of bioactive molecules to a target location in the body of a recipient.
  • the formulations of the present invention are formulated for administration parenterally.
  • compositions suitable for parenteral administration include sterile solutions or suspensions of a composition in aqueous or oleaginous vehicles.
  • Injectable preparations may be adapted for bolus injection or continuous infusion. Such preparations are conveniently presented in unit dose or multi-dose containers, which are sealed after introduction of the formulation until required for use.
  • a composition may be in powder form that is constituted with a suitable vehicle, such as sterile, pyrogen-free water, before use.
  • a composition may also be formulated as long-acting depot preparations, which may be administered by intramuscular injection or by implantation, e.g. subcutaneously or intramuscularly.
  • Depot preparations may include, for example, suitable polymeric or hydrophobic materials, or ion-exchange resins. Such long-acting formulations are particularly convenient for prophylactic use.
  • the present invention provides a method of delivering a bioactive molecule to target cells in the body of a recipient, which method comprises preparing a composition comprising a cationic dendrimer, or derivative thereof to which one or more cell targeting molecule(s) have been conjugated thereto, and a bioactive molecule associated with said cationic dendrimer and/or derivative thereof, and subsequently administering the composition to said recipient.
  • the dendrimers of the present invention display suitable properties for the delivery of bioactive molecules in vivo, they are also useful for transfecting mammalian cells in vitro.
  • the present invention also provides a composition of the present invention for transfecting mammalian cells with a bioactive molecule in vitro.
  • the mammalian cells may, for example, be human cells.
  • the present invention also provides a method of preparing a composition as described above, said method comprising admixing a cationic dendrimer, and/or derivatives thereof, to which one or more cell targeting molecule(s) have been conjugated thereto, with a bioactive molecule.
  • the targeting molecule is conjugated to the dendrimer via an amide linkage, most preferably using dimethylsuberimidate (DMSI)
  • Figure 1 shows 1 H NMR spectra of generation 3 polypropylenimine dendrimer conjugated to transferrin (DAB-Tf) (A) (B: 1 H NMR spectra of the unmodified dendrimer); and (C schematic representation of transferrin conjugated to DAB).
  • Figure 3 shows confocal microscopy imaging of the cellular uptake of Cy3- labelled DNA (6 g / dish) either complexed with DAB-Tf (A, D), DAB (B, E) or free in solution (C, F) after incubation for 24 hours with T98G (top) and A431 cells (bottom) (Blue: nuclei stained with DAPI (excitation: 405 nm laser line , bandwidth: 415-491 nm ), green: Cy3-labelled DNA (excitation: 453 nm laser line, bandwidth: 550-620 nm) (Magnification: x 40).
  • tumour response to treatments on the last day of the experiment stratified according to change in tumour volume: progressive disease (increase >1 .2-fold, dark grey), stable disease (0.7-1.2, white), partial response (0-0.7, medium grey with stripes) and complete response (0, light grey) over the duration of the experiment (40 days).
  • Figure 7 shows transfection efficacy of DAB-Lf, DAB-Lcin, DAB-Tf poiyplexes relative to native DAB-DNA and DOTAP-DNA in A431 cells.
  • * P ⁇ 0.05 vs the transfection resulting from DAB-DNA treatment.
  • Figure 8. shows confocal microscopy imaging of the cellular uptake of Cy3- labelled DNA (6 pg/dish) either complexed with DAB-Lf, DAB-Lcin, DAB-Tf or free in solution after incubation for 24 hours with A431 cells (Blue: nuclei stained with DAPI (excitation: 405 nm laser line, bandwidth: 415-491 nm), green: Cy3-labelled DNA (excitation: 453 nm laser line, bandwidth: 550-620 nm) (Magnification: x 40).
  • Quanti-iTTM PicoGreen ® dsDNA reagent and tissue culture media were obtained from Invitrogen (Paisley, UK). Vectashield ® mounting medium with 4',6-diamidino-2-phenylindole (DAPI) was obtained from Vector Laboratories (Peterborough, UK). Passive lysis buffer was purchased from Promega (Southampton, UK). Label IT ® Cy3 Nucleic Acid Labelling kit was from Cambridge Biosciences (Cambridge, UK). Polypropylenimine dendrimer generation 3 (PPI G3; DAB-Am16), transferrin and all other chemicals were purchased from Sigma Aldrich (Poole, UK).
  • A431 epidermoid carcinoma and T98G glioblastoma were obtained from the European Collection of Cell Cultures (Salisbury, UK).
  • the expression plasmids encoding Tumour necrosis factor (TNF)a (pORF9-mTNFa) and ⁇ -galactosidase (pCMVsport ⁇ -galactosidase) were obtained respectively from InvivoGen (San Diego, CA) and Invitrogen (Paisley, UK).
  • the expression plasmid encoding p73 (pcDNA3-HA-TA-p73alpha) was obtained from a kind gift from Professor Kevin Ryan (Beatson Institute, Glasgow, Scotland and has been described (De Laurenzi et a/., 1998). They were purified using an Endotoxin- free Giga Plasmid Kit (Qiagen, Hilden, Germany).
  • Polypropylenimine dendrimer generation 3 was conjugated to transferrin (Tf) by using dimethylsuberimidate (DMSI) as a cross-linking agent in a similar manner to that reported for transferrin-bearing vesicles [9].
  • DMSI dimethylsuberimidate
  • Polypropylenimine dendrimer DAB 24 mg was added to transferrin (6 mg) and DMSI (12 mg) in triethanolamine HCI buffer (pH 7.4, 2mL). The coupling reaction was allowed to take place at room temperature for 2h whilst stirring. The final product was purified by size exclusion chromatography using a Sephadex G75 column and freeze-dried. DAB-Tf was then dissolved in D 2 0 at a concentration of 5 mg/mL The grafting of transferrin to DAB was assessed by 1 H NMR spectroscopy, using a Jeol Oxford NMR AS 400 spectrometer.
  • the degree of DNA accessibility following complexation with DAB-Tf was assessed by PicoGreen ® assay, performed according to the supplier's protocol.
  • the fluorescence of PicoGreen ® significantly increases on intercalation with double stranded DNA.
  • the electrostatic interaction between the anionic DNA and cationic group of the polymer on formation of the Tf-bearing DAB-DNA polyplex condenses the DNA and reduces the number of PicoGreen ® binding sites, ultimately reducing the fluorescence intensity for the PicoGreen ® solution.
  • PicoGreen ® reagent was diluted 200-fold in Tris-EDTA (TE) buffer (10 mM Tris, 1m EDTA, pH 7.5) on the day of the experiment.
  • Tris-EDTA (TE) buffer 10 mM Tris, 1m EDTA, pH 7.5
  • the DNA concentration in the cuvette (10 g/mL) was kept constant throughout the experiment.
  • A431 and T98G cell lines overexpressing Tf receptors were grown as monolayers in DMEM medium supplemented with 10% (v/v) fetal bovine serum, 1 % (v/v) L-glutamine and 0.5% (v/v) penicillin-streptomycin. Cells were cultured at 37°C in a 5% carbon dioxide atmosphere.
  • Transfection efficacy of the DNA carried by the transferrin-bearing dendrimer was assessed with a plasmid coding for ⁇ -galactosidase (pCMV gal), using a ⁇ -galactosidase transfection assay.
  • pCMV gal plasmid coding for ⁇ -galactosidase
  • A431 and T98G cells were seeded in quintuplicate at a density of 2 000 cells/well in 96-well plates. After 72h incubation, the cells were treated with DAB-Tf complexed to plasmid DNA encoding ⁇ -galactosidase, at the polymer: DNA weight ratios used for the DNA condensation experiment.
  • Naked DNA served as a negative control, formulations of DOTAP-DNA (polymer: DNA weight ratio 5:1 ) and DAB-DNA (polymer: DNA weight ratio 5:1 ) served as positive controls.
  • DNA concentration (1 pg/well) was kept constant for all the formulations tested.
  • cells were lysed with 1X passive lysis buffer (PLB) (50 pL/well) for 20 min. The cell lysates were subsequently analyzed for ⁇ -galactosidase expression [10].
  • PLB passive lysis buffer
  • the assay buffer (2 mM magnesium chloride, 100 mM mercaptoethanol, 1.33 mg/mL o-nitrophenol- -galactopyranoside, 200 mM sodium phosphate buffer, pH 7.3) was added in each well containing the lysates. After 2 hours incubation at 37 °C, the absorbance of the samples was read at 405 nm with a plate reader (Thermo Lab Systems, Multiscan Ascent, UK).
  • Imaging of the cellular uptake of the DNA carried by DAB-Tf was carried out using confocal microscopy. Labelling of plasmid DNA with the fluorescent probe Cy3 was performed using a Label IT ® Cy3 Nucleic Acid Labelling kit, as described by the manufacturer. A431 and T98G cells were grown on microscope slides (0.6 x 10 6 cells / 90-mm Petri dish) at 37 °C for 24 hours.
  • Anti-proliferative activity of transferrin-bearing DAB complexed with plasmid DNA encoding TNFa or p73 was assessed in A431 and T98G cancer cell lines.
  • Cells (2 x 10 3 cells per well in 96-well plates seeded 72 hours prior treatment) were incubated for 72 hours with the DNA formulations at final concentrations of 10 "3 to 10 000 pg/mL.
  • mice Female immunodeficient BALB/c mice were housed in groups of five at 19°C to 23°C with a 12-hour light-dark cycle. They were fed a conventional diet (Rat and Mouse Standard Expanded, B&K Universal, Grimston, United Kingdom) with mains water ad libitum. Experimental work was carried out in accordance with UK Home Office regulations and approved by the local ethics committee.
  • Progressive disease is defined as an increase in relative tumour volume higher than 1 ,2-fold, stable disease as a relative volume between 0,7 and 1 ,2 of starting volume, partial response as measureable tumour with a reduction of more than 30% (0-0,7) and complete response as the absence of any tumour.
  • Results were expressed as means ⁇ standard error of the mean (S.E.M). Statistical significance was determined by one-way analysis of variance (ANOVA) followed by the Bonferroni multiple comparison post-test (GraphPad Prism software). Differences were considered as significant when P ⁇ 0.05.
  • DAB-Tf The synthesis of DAB-Tf was confirmed by 1 H NMR spectrum, as follows (see Fig. 1 ).
  • the NMR spectrum of DAB had a characteristic broad triplet peak for the CH 2 adjacent to peripheral primary amino group at 52.65, which was slightly shifted to 2.80 ppm in the NMR spectrum of DAB-Tf.
  • an additional broad triplet at 63.70 was observed which is compatible to the methylene protons adjacent to an amide unit. This indicated that some of the peripheral amino groups had reacted with the carboxyl group of the DMSI to form an amide linkage with transferrin. Multiple small peaks between ⁇ 1.30 and 3.40 corresponded to protons for transferrin.
  • the polypropylenimine dendrimer DAB can efficiently form complexes with plasmid DNA through electrostatic interactions between its protonated primary amines and the negatively charged phosphodiester groups of the DNA.
  • the conjugation of transferrin may affect the dendrimer ability to complex DNA.
  • PicoGreen ® assay we examined the influence of the conjugation of transferrin on the dendrimer ability to form complexes with DNA, by using a PicoGreen ® assay. Transferrin-bearing DAB was able to condense more than 70% of the DNA, independently of the weight ratio tested (Fig. 2A). DNA condensation occurred almost instantaneously and was found to be stable over at least 24h.
  • Transferrin-bearing DAB polyplex displayed a z-average mean diameter of 287 nm (polydispersity index: 0,393).
  • the conjugation of transferrin to the periphery of DAB led to a slight increase of the polyplex size compared to the unmodified DAB polyplex, which had an average size of 196 nm (polydispersity index: 0,683).
  • the cut-off size for extravasation has been found to be 400 nm for most tumours [13], this delivery system has the required properties to access the receptor-expressing tumour cells and specifically deliver the therapeutic DNA.
  • Zeta -potential experiments demonstrated that transferrin-bearing and control DAB polyplexes were bearing a positive surface charge.
  • the zeta-potential of DAB- Tf polyplex was 1.03 mV, smaller than that of the unmodified DAB polyplex (6.42 mV). This decrease is most likely due to the presence of the negatively charged amino acids of transferrin and would lead to a minimization of the non-specific interactions of the polyplex with the negatively charged cellular membranes [14].
  • transferrin-bearing DAB has suitable physicochemical properties for being an efficient gene delivery system.
  • Transfection efficacy was determined by quantifying the expression of ⁇ -galactosidase encoded on the plasmid.
  • the conjugation of Tf to DAB led to an improved transfection compared to the unmodified DAB and DOTAP on both the tested cell lines.
  • the improved ⁇ -gal expression induced by Tf-bearing DAB compared to unmodified DAB most likely resulted from the transferrin-specific uptake by the cancer cells overexpressing Tf receptors.
  • the higher zeta-potential of the DAB polyplex would have otherwise resulted in a higher transfection efficacy of the unmodified dendrimer, due to the strong correlation between cellular uptake and positive charge density of polyplexes [14].
  • This improved ⁇ -gal expression was in accordance with that observed with other Tf-bearing gene delivery systems [15-16].
  • Tf-targeted DAB dendrimer complexed with TNFa plasmid induced a tumour regression within 24 hours compared to untreated tumours (Fig. 5).
  • the complexes comprising p 73 expressing DNA are able to kill the tumour cells, but complexes which are also targeted to the cells by way of transferrin, do so in a much more effective manner.
  • Eliassen LT Berge G, Sveinbjornsson B, Svendsen JS, Vorland LH, Redkal O, Evidence of a direct antitumor mechanism of action of bovine lactoferricin, Anticancer Research, 22 (2002) 2703-2710.
  • Eliassen LT Berge G, Leknessund A, Wikman M, Lindin I, Lokke C, Ponthan F, Johnsen Jl, Sveinbjornsson B, Kogner P, Flaegstad T, Rekdal O,
  • the antimicrobial peptide, Lactoferricin B is cytotoxic to neuroblastoma cells in vitro and inhibits xenograft growth in vivo, International Journal of Cancer, 1 19 (2006) 493-500.
  • Furlong SJ, ader JS, Hoskin DW, Lactoferricin-induced apoptosis in estrogen-non responsive MDA- B-435 breast cancer cells is enhanced by C6 ceramide or tamoxifen, Oncology reports, 15 (2006) 1385-1390.
  • Lactoferricin a lactoferrin-derived peptide with antimicrobial, antiviral, antitumor and immunological properties, Cell and Molecular Life Sciences, 62 (2005) 2588-2598.

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Abstract

La présente invention concerne une composition pour la distribution ciblée d'une molécule bioactive vers des cellules dans le corps d'un récepteur, ladite composition comportant un dendrimère polypropylénimine et/ou des dérivés de celui-ci au(x)quel(s) une ou des molécule(s) de ciblage cellulaire ont été conjuguées; et une molécule bioactive associée à ce dendrimère polypropylénimine et/ou un dérivé de celui-ci. Selon un mode de réalisation, les cellules à cibler sont des cellules cancéreuses. La molécule de ciblage peut être la transferrine, la lactoferrine, la lactoferricine ou l'acide gambogique. L'invention concerne également les utilisations de telles compositions, ainsi que des procédés de traitement de maladies, comme le cancer.
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WO2003033027A2 (fr) 2001-10-19 2003-04-24 University Of Strathclyde Dendrimeres utilises pour une administration ciblee
WO2006040579A1 (fr) * 2004-10-14 2006-04-20 The University Court Of The University Of Glasgow Polymeres bioactifs
WO2008010000A1 (fr) * 2006-07-21 2008-01-24 National Center For Scientific Research Transporteurs moléculaires dendritiques

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003033027A2 (fr) 2001-10-19 2003-04-24 University Of Strathclyde Dendrimeres utilises pour une administration ciblee
WO2006040579A1 (fr) * 2004-10-14 2006-04-20 The University Court Of The University Of Glasgow Polymeres bioactifs
WO2008010000A1 (fr) * 2006-07-21 2008-01-24 National Center For Scientific Research Transporteurs moléculaires dendritiques

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