EP1765403A2 - Ciblage actif et passif combines d'agents biologiquement actifs - Google Patents

Ciblage actif et passif combines d'agents biologiquement actifs

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Publication number
EP1765403A2
EP1765403A2 EP05780057A EP05780057A EP1765403A2 EP 1765403 A2 EP1765403 A2 EP 1765403A2 EP 05780057 A EP05780057 A EP 05780057A EP 05780057 A EP05780057 A EP 05780057A EP 1765403 A2 EP1765403 A2 EP 1765403A2
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EP
European Patent Office
Prior art keywords
gly
seq
phe
leu
conjugate
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EP05780057A
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German (de)
English (en)
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EP1765403A4 (fr
Inventor
Vaikunth Cuchelkar
Pavla Kopechova
C. Matthew Peterson
Jindrich Kopecek
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University of Utah Research Foundation UURF
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University of Utah Research Foundation UURF
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Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
    • 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
    • A61K47/56—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
    • A61K47/58—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 by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
    • 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
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • 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
    • A61K47/68—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6869—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from a cell of the reproductive system: ovaria, uterus, testes, prostate
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal

Definitions

  • This invention relates to biotechnology, more particularly to targeted delivery of biologically active agents such as drugs, prodrugs, proteinaceous molecules, genes, and/or nucleic acid sequences.
  • BACKGROUND [0004] Low molecular weight therapeutic agents diffuse throughout a cell and are not concentrated at a specific subcellular location. Targeting these agents to the subcellular site where they are most effective increases their efficacy. In addition, if such drugs are administered intravenously, they are systemically distributed to all tissues of the body. The action of these drugs at these unintended sites of distribution results in observable systemic side effects. It is thus preferred to localize the drug to the sites in the body where the action is desired. [0005] Targeting of, for example, anticancer drugs in the case of tumors can be achieved by "passive targeting" and "active targeting”. Passive targeting is achieved by incorporating anticancer drugs into polymers.
  • Active targeting is achieved by incorporating cellular targeting moieties that are specific to recognition molecules on the surface of the cells.
  • Polymers localize preferentially in solid tumors when compared to normal tissue. This occurs due to a phenomenon called the Enhanced Permeability and Retention ("EPR") effect, which is attributed to morphological changes in tumor tissue, where the leaky vasculature produced due to neoangiogenesis results in the leakage of vascular contents into the extracellular tissue.
  • EPR Enhanced Permeability and Retention
  • the lymphatics may be blocked, which results in the accumulation of macromolecular agents in the extracellular tissue surrounding tumor cells (Matsumura Y, Maeda H, Cancer Res 4(12 Pt 1) (1986) 6387-6392).
  • This phenomenon can be used to target tumor cells by attaching drugs to the polymers. Since polymers localize around tumor cells, the drugs attached to the polymers are also available at higher concentrations around the tumor. Drugs attached to polymers are taken inside cells by endocytosis. However, since the drugs remain attached to the polymer backbone, they may not be as effective as free drugs. This may be overcome by the use of biodegradable sequences to link the drug to the polymer backbone. The sequences that are chosen are such that they can be degraded inside the cell under specific conditions. [0007] Polymer-based therapeutics have greater hydrodynamic volume in comparison to the free drug, which translates into a longer intravascular half-life. Polymer-based therapeutics also enhance the solubility and the bioavailability of insoluble drugs.
  • Macromolecular therapeutics for anticancer drugs also can overcome certain cases of drug resistance.
  • Other advantages afforded by polymer-based therapeutics include increased maximum tolerated dose, decreased non-specific toxicity, enhanced induction of apoptosis, and activation of alternate signaling pathways (Kopecek.et al., Advances in Polymer Science 122 (Biopolymers II) (1995) 55 - 123).
  • cancer cells often have surface molecules that are either absent in normal tissue or over-expressed in comparison to the normal tissue. These may include growth factor receptors and/or certain antigens. Attaching recognition molecules to polymers that bind these molecules results in a high concentration of the polymers in the local environment of the tumor.
  • Such targeting moieties include antibodies and ligands for cell surface receptors. Receptor mediated endocytosis initiated by the binding of some of these recognition molecules to their receptors can result in an increased intracellular concentration and correspondingly an enhanced effect.
  • PCT International Publication WO 00/11018A1 Conjugates Of DNA Interacting Groups With Steroid Hormones For Use As Nucleic Acid Transfection Agents", published March 2, 2000, (the contents of which are incorporated by this reference) discloses compounds comprising a steroid hormone (e.g., an androgen, estrogen, or glucocorticoid) linked to a DNA-interacting molecule that target nucleic acids to the cell nucleus (e.g., an intercalating agent, cross-linking agent, or psoralen) and a method of introducing nucleic acids into the nucleus of cells with the help of such compounds. Similar work is disclosed in Rebuffat et al. "Selective enhancement of gene transfer by steroid
  • the invention includes a polymeric delivery system for biologically active agents with concurrent nuclear targeting. Biologically active agents are modified by attaching a steroid hormone thereto.
  • Such biologically active agent-steroid derivatives are further targeted to the tumor tissue by attaching them to a polymer (for the EPR effect) with a biodegradable sequence.
  • the biodegradable sequences selected are ones that can be degraded by enzymes present inside the cell (especially the lysosomes) to link the drugs to the polymer (Duncan et al., Makromolecular Chemie 184 1997 - 2008 (1983)).
  • An example of such a biodegradable sequence is Gly-Phe-Leu-Gly (SEQ ID NO:l) that is degraded by Cathepsin B in lysosomes.
  • the invention also includes a "double-targeted polymeric delivery system".
  • the biologically active agent will be modified by attaching a steroid hormone as the nuclear targeting signal.
  • the biologically active agent-steroid derivative will be attached to the polymer by a biodegradable sequence.
  • the double-targeted system also includes attaching a cellular targeting moiety such as an antibody (e.g., polyclonal antibody, monoclonal antibody, phage display antibody, ribosome display, or antibody fragment) to the polymer.
  • a cellular targeting moiety such as an antibody (e.g., polyclonal antibody, monoclonal antibody, phage display antibody, ribosome display, or antibody fragment) to the polymer.
  • the attachment of the cellular targeting moiety like the antibody is expected to result in enhanced uptake by tumor cells. This effect, when combined with the potential to deliver the therapeutic agent in high concentrations to the nucleus due to nuclear targeting using the steroid hormone, will result in an unexpectedly high biological activity using this approach.
  • the use of the double targeting system will ensure that only the cells that express the surface recognition moiety will be targeted.
  • Use of subcellular targeting moieties will further enable a reduction of the dose of the drug that will need to be administered. This will ensure that only the right cells will be killed with a very small amount of drug.
  • This delivery system has great potential in the delivery of therapeutic agents for the treatment of cancer.
  • the use of cellular signaling pathways ensures that this strategy will work much more effectively in cells, which express the particular steroid hormone receptor — this affords another element of specificity to the whole approach.
  • Potential applications of the nuclear-targeted polymeric conjugates are numerous. Certain anticancer drugs are most effective in the nucleus.
  • Anticancer drugs that act on the DNA to elicit their cytotoxic effect would be greatly benefited by using this approach.
  • Agents used in the photodynamic therapy of cancer would also see an increase in the therapeutic effect following targeting to the nucleus since the nucleus is hypersensitive to photodynamic damage.
  • Another field in which this invention would prove useful would be in gene therapy. Gene therapy requires that genes be delivered to the nucleus effectively; this invention fulfils that need.
  • Other agents that can be targeted similarly are nucleic acid sequences (e.g., DNA or RNA) of epitopes for vaccine production.
  • drugs or genes as therapeutic agents
  • antibodies as targeting moieties
  • Gly-Phe-Leu-Gly SEQ ID NO:l
  • biodegradable linker sequences as mentioned below are merely illustrative of the numerous agents that could be used.
  • FIG. 1 depicts a general example of this system; wherein "P” is an inert polymeric backbone of the delivery system and connector "C” is linked to the polymer backbone
  • FIG. 2 depicts some examples of hormone-drug conjugates and their polymeric delivery systems.
  • A an example is given demonstrating a norethisterone (NET) - targeted system for the delivery of doxorubicin (Dox).
  • NET norethisterone
  • the connector used is Tyrosine (Tyr).
  • the connector is linked to the poly-L-lysine (PLL) polymeric backbone via a degradable glycylleucylglycyl (GLG) spacer.
  • the polymer also bears a LHRH moiety to target tissues overexpressing the LHRH receptor.
  • TTNPB 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-l-propenyl]benzoic acid
  • the connector used is glutamic acid (Glu).
  • FIG. 3 graphically depicts a scheme for the synthesis of a double-targeted polymeric delivery system for mesochlorin.
  • FIG. 4 graphically illustrates the synthesis of Cort-ONp
  • FIG. 5 graphically illustrates the synthesis of Lys-Mce 6 intermediate
  • FIG. 6 graphically illustrates the synthesis of Cort-Lys- Mce 6 [0023]
  • FIG. 3 graphically depicts a scheme for the synthesis of a double-targeted polymeric delivery system for mesochlorin.
  • FIG. 4 graphically illustrates the synthesis of Cort-ONp
  • FIG. 5 graphically illustrates the synthesis of Lys-Mce 6 intermediate
  • FIG. 6 graphically illustrates the synthesis of Cort-Lys- Mce 6 [0023]
  • FIG. 8 demonstrates the biorecognition of Cort-Lys-Mce 6 derivative using the GFP-GR system. Cortisol and Cort-Lys-Mce 6 derivative is able to shuttle GFP tagged GR from the cytoplasm to the nucleus. Mce 6 by itself cannot cause redistribution of the GFP tagged GR.
  • FIG. 9 depicts the subcellular localization of free Mce 6 using confocal microscopy after a 24 hour incubation period at 37° C with (A) Mce 6 (red) and (B) DAPI (nuclear marker). DIG image is window (C).
  • FIG. 10 depicts the subcellular localization of Cort-Lys- Mce 6 derivative using confocal microscopy after 24 hours incubation at 37° C with (A) Mce 6 (red) and (B) DAPI (nuclear marker). DIC image is window (C).
  • Combined image (D) shows colocalization of Mce 6 and DAPI indicating presence of Cort-Lys- Mce 6 in the nucleus DETAILED DESCRIPTION OF THE INVENTION
  • Macromolecular therapeutic agents also afford other advantages in contrast to low molecular weight drugs: increased half life, increased maximum tolerated dose, decreased non-specific toxicity, targetability (by the attachment of ligands to the polymer that are specific to receptors on the cell surface), increased solubility and bioavailability, enhanced induction of apoptosis, and activation of alternate signaling pathways.
  • targetability by the attachment of ligands to the polymer that are specific to receptors on the cell surface
  • increased solubility and bioavailability enhanced induction of apoptosis, and activation of alternate signaling pathways.
  • antibodies and ligands for cell surface receptors growth factor molecules and hormones for example
  • Attaching such targeting moieties can also lead to internalization of the polymer conjugate by receptor-mediated endocytosis and an enhanced intracellular concentration.
  • the polymer-drug conjugates are designed such that they are stable in the blood stream, but release the drug in tumor tissue. This can be done by incorporating degradable spacers to link the drug to the polymer backbone. Biodegradable spacers like glycylphenylalanylleucylglycine (GFLG) (SEQ ID NO:l) are stable in the blood stream, but are broken down by proteases like cathepsin B in the lysosomes. The strategy of using the GFLG spacer to link the drug to the polymer enables us to exploit the advantages afforded by polymers as well as releasing the active drug inside the cell to obtain maximum therapeutic effect.
  • GFLG glycylphenylalanylleucylglycine
  • biodegradable spacers can be used depending on specific requirements.
  • Such a polymeric delivery system can be used to efficiently and specifically deliver therapeutic agents to the tumor tissue.
  • the distribution of low molecular weight therapeutic agents inside the cell depends on the mode of entry into the cell, its physico-chemical properties and its subsequent redistribution. Since the mode of entry is mainly via diffusion, free drug is not concentrated at a specific subcellular location. Targeting the free drug to the subcellular site where is it most effective increases its therapeutic efficacy.
  • photodynamic therapy is a therapeutic modality for cancer, which involves the administration of photosensitizers to the patient followed by their activation by illumination with light of a specific wavelength.
  • the photosensitizers are excited to their singlet state - however, this state is unstable and the excited photosensitizer decays rapidly and gives off excess energy. This energy is transferred to molecular oxygen in the environment, forming reactive oxygen species, the most important of which is singlet oxygen ( 1 O 2 ).
  • Singlet oxygen is highly reactive and nonspecifically reacts with biomolecules present in the surrounding leading to cellular damage.
  • singlet oxygen has a short half- life (about 4 ⁇ s) in the aqueous conditions and very limited diffusion capability. This results in cellular damage induced by singlet oxygen, localized to about 100 nm around the site of its generation.
  • the nucleus is known to be hypersensitive to photodynamic therapy induced damage (Peng et al., Ultrastructural Pathology 20(2) (1996) 109 - 129). However, most photosensitizers do not target this subcellular compartment. Singlet oxygen produces single strand breaks and base modifications in the DNA. Inactivation of enzymes in DNA repair produced by singlet oxygen further hampers the ability of the cell to repair the damage produced. Accumulation of cellular damage induced by the singlet oxygen eventually leads to cell death.
  • cytosolic steroid hormone receptors SHR
  • This genomic action is mediated by the formation of the steroid hormone-receptor complex in the cytoplasm followed by the shuttling of this complex from the cytoplasm to the nucleus through the nuclear pore complex (NPC) (Mangelsdorf et al., Cell, 83(6):835 - 839 (1995)).
  • NPC nuclear pore complex
  • This movement of the steroid hormone- receptor complex across the nuclear membrane occurs via nuclear localization signals (NLS) present on the receptor, which are exposed following binding of the steroid hormone.
  • AR androgen receptor
  • PR progesterone receptor
  • GR glucocorticoid receptor
  • PR progesterone receptor
  • GR glucocorticoid receptor
  • Nuclear targeting causes damage at the genomic level and induces apoptosis in the cancer cells leading to a relatively decreased inflammatory response.
  • the decreased inflammatory response is one of the desirable clinical features to be achieved using this strategy. It is expected that nuclear targeting will cause a similar increase in the efficacy of other therapeutic agents that are known to act at the level of the nucleus.
  • GR cortisol might be modified without impairment of binding to the receptor (Williams AP, Sigler PB, Nature, 393(6683):392-396 (1998)).
  • Lysine was used to connect the cortisol and the Mce 6 .
  • Cortisol was bound to the alpha-NH group, while Mce 6 was bound to the -COOH group of the lysine.
  • Cort-Lys-Mce 6 was able to localize to the nucleus and also demonstrate an enhanced cytotoxicity.
  • the free epsilon-NH group on the Cort-Lys-Mce 6 was then used to attach the Cort-Lys-Mce 6 to a PHPMA backbone bearing pendant ONp groups with biodegradable GFLG spacers.
  • PGFLG-Cort-Lys-Mce 6 had an enhanced cytotoxicity as compared to PGFLG-Mce 6 due to the nuclear targeting.
  • the remaining ONp groups will be used to bind OV-TL16 antibody (which is specific to OA antigen on ovarian carcinoma cells).
  • anticancer drugs like topoisomerase I inhibitors (for example, camptothecin, 9- aminocamptothecin, irinotecan, topotecan), anthracyclines (for example, doxorubicin, daunorubicin).
  • this strategy can also be used to deliver genes/ antisense oligonucleotides (ODNs)/ peptide nucleic acids (PNAs)/ small interfering RNAs (siRNAs) to the nucleus - this would involve replacement of the drug with the desired gene/ ODN/ PNA/ siRNA.
  • Nucleic acid sequences of epitopes for vaccine production can also be targeted similarly and result in an increased efficacy and specificity.
  • cytosolic receptors include the steroid hormone receptors (AR, PR, and ER), other nuclear receptors [Peroxisome Proliferation Activated Receptors (PPAR), Liver X receptors (LXR)] and various orphan receptors [Retinoid X receptors (RXR), Benzoate X receptor (BXR), Constitutive Androstane Receptor ⁇ (CAR ⁇ ), Pregnane X receptor (PXR), Steroid and Xenobiotic receptor (SXR), Farnesoid X receptors (FXR)].
  • AR steroid hormone receptors
  • PR Peroxisome Proliferation Activated Receptors
  • LXR Liver X receptors
  • RXR Retinoid X receptors
  • BXR Benzoate X receptor
  • CAR ⁇ Constitutive Androstane Receptor ⁇
  • PXR Pregnane X receptor
  • SXR Steroid and Xenobiotic receptor
  • Ligands that are specific for these cytosolic receptors can be used in the place of cortisol as the subcellular targeting moiety.
  • the cytosolic receptor can be so selected as to afford some selectivity in the type of cells that are targeted.
  • Receptors that are specifically present in certain tissues can be used to provide nuclear targeting in such tissues only and thereby focus their effect.
  • Besides lysine other molecules can serve to connect the three constituents of the system (drug, hormone, and polymer backbone).
  • the connector preferably has three functional groups present that can react with the aforementioned constituents.
  • polymeric carriers can serve as the backbone for the delivery of this system.
  • Some of the polymeric carriers (other than HPMA copolymers) that are suitable include Poly (L-glutamic acid) (PGA), Poly (L-lysine) (PLL), PEG (Polyethylene glycol) and PEG- block copolymers (for example, polyethylene glycol-co-aspartic acid).
  • polymeric carriers that could be used include polymers formed from monomeric units selected from the group including but not limited to N-(2-hydroxypropyl)methacrylamide, N-(2- hydroxyethyl)methacrylamide, N-isopropylacrylamide, acrylamide, N,N dimethylacrylamide, N- vinylpyrrolidone, vinyl alcohol, 2-methacryloxyethyl glucoside, acrylic acid, methacrylic acid, vinylphosphonic acid, styrenesulfonic acid, maleic acid, 2- methacryloxyethyltrimethylammonium chloride, methacrylamidopropyltrimethylammonium chloride, methacryloylchofine methyl sulfate, N-methylolacrylamide, 2-hydroxy-3- methacryloxypropyltrimethylammonium chloride, 2-methacryloxyethyltrimethylammonium bromide, 2-vinyl-l-methylpyridinium bromide, 4-vinyl-l-methylpyridinium
  • these polymer backbones can be used to attach the hormone-drug derivative via biodegradable spacers.
  • Various moieties can be used to target certain tissues and cancers specifically.
  • Antibodies that target specific antigens on the surface of cancer cells can be used to localize the polymeric conjugate to tumor tissue actively.
  • Some such antigen-antibody combinations include OA3-OVTL16, Pgp-UIC2 and EGFR-anti-EGFR antibody.
  • various other ligands that are specific for receptors on the surface of the cells can be used.
  • Some such ligand-receptor combinations include LHRH- LHRH receptor and insulin-insulin receptor.
  • binding of the conjugates to the cells can result in an enhanced uptake due to receptor-mediated endocytosis. This results in an increase in the concentration of the drug inside the cell and further increases the effect.
  • the examples of the cellular targeting moieties mentioned are examples of the general idea and do not limit the targeting molecules that can be used.
  • Various kinds of biodegradable spacers can be used in the polymeric delivery system.
  • peptide sequences made from L-amino acids (for example, Gly-Phe-Leu- Gly (SEQ ID NO:l), Gly-Leu-Gly (SEQ ID NO:2), Gly-Val-Gly (SEQ ID NO:3), Gly-Phe-Ala
  • spacer The type of spacer that will be used will depend on the individual circumstance and the expected mechanism of action.
  • protein transduction domains can be used to achieve increased concentration in the cells is another method to increase intracellular concentrations - however, it is to be realized that if this approach is followed, the conjugate will bypass the traditional endocytic pathway and hence the use of lysosomally degradable sequences may not be the most optimal approach.
  • S-S disulfide
  • C is the connector that is linked to the water-soluble inert polymer backbone (P) via a spacer (SI), which may be biodegradable or non- biodegradable.
  • DI spacer
  • D is the therapeutic agent (anticancer drug/ genes/ ODN / PNA / siRNA) bonded to one arm of the connector C.
  • N is the nuclear targeting moiety (ligand specific for nuclear receptor) bonded to the other arm of the connector C.
  • T is the optional tissue targeting moiety covalently bound to the polymer backbone (P) via biodegradable or non-degradable spacer (S3);
  • L is an optional bio-assay label covalently bonded to the polymer backbone (P) via a non- degradable spacer (S2) which can be the same or different than SI or S2 when they are non- degradable;
  • X is an optional biodegradable cross-linkage between two polymer chains (P).
  • the first example considered is that of a norethisterone (NET) targeted conjugate of the anthracycline anti-cancer drug, doxorubicin (Dox) and the polymeric delivery system for the delivery of the NET-Dox conjugate.
  • the connector used in this case is tyrosine
  • NET is a progesterone analog that will interact with the progesterone receptor (PR) and facilitate transport of the NET-Tyr-Dox into the nucleus.
  • the connector Tyr is linked to the polymer backbone via a biodegradable glycylleucylglycyl (GLG) spacer.
  • LCG biodegradable glycylleucylglycyl
  • the system in consideration also actively targets tissues overexpressing the LHRH receptor by using an LHRH moiety.
  • the LHRH is connected to the poly-L-lysine (PLL) polymer backbone via a non-degradable glycylglycyl (GG) spacer.
  • the monomeric structure with the NET-Tyr-Dox attached by the GLG spacer can range from 0.01 - 80 mole % of the polymer backbone.
  • the content of the LHRH connected to the PLL backbone by a GG spacer can range from 0.01 to 20 mole % of the polymer backbone.
  • the second example uses glutarnic acid (Glu) as a connector.
  • Glu glutarnic acid
  • TTNPB 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-l- ⁇ ropenyl]benzoic acid
  • FXR Farnesoid X receptor
  • the drug used in this case is a Topoisomerase I inhibitor, 9-aminocamptothecin (9-AC).
  • the TTNPB-Glu-9-AC conjugate is linked covalently to the polymer backbone via a nondegradable GG spacer.
  • the polymer also bears an OV-TL 16 antibody to target ovarian cancer tissue specifically (which overexpresses the OA3 antigen that is recognized by OV-TL16).
  • the OV- TL16 antibody is linked to the polymer backbone (which is a copolymer of polyethylene glycol and aspartic acid) (PEG-co-Asp) via a nondegradable GG spacer.
  • the monomeric structure with the TTNPB-Glu-9-AC conjugate attached by the GG spacer can range from 0.01 - 90 mole % of the polymer backbone.
  • a higher drug loading content can be achieved in this case because the polymer backbone is extremely hydrophilic.
  • the content of the OV-TL16 antibody connected to the PEG-co-Asp backbone can range from 0.01 to 50 mole % of the polymer backbone.
  • Cortisol was acylated with twice molar excess of 4-nitrophenyl chloroformate in methylene chloride and thrice molar excess of 4-methyl morpholine to form Cort-ONp (Cortisol-C17-4-nitrophenyl ester) (carbonic acid 2-(l l,17-dihydroxy-10,13- dimethyl-3- oxo-2,3,6,7,8,9,10,11,12,13,14,15, 16,17-tetradecahydro-lH- cyclopenta[a]phenanthren-17-yl)-2-oxo-ethyl ester 4-nitro-phenyl ester) (FIG. 4).
  • Cort-ONp Cortisol-C17-4-nitrophenyl ester
  • Mce 6 was reacted with N-alpha-Fmoc, N- ⁇ -Boc-Lysine N- hydroxysuccinimide ester (6-tert-Butoxycarbonylamino-2-(9H-fluoren-9-ylmethoxy- carbonylamino)-hexanoic acid 2,5-dioxo-pyrrolidin-l-yl ester) in dimethylformamide (DMF) followed by the deprotection of the Fmoc group using 20 % piperidine in DMF for 30 minutes (FIG.
  • DMF dimethylformamide
  • Lys- Mce 6 intermediate (alpha-NH , N- ⁇ -Boc-Lysyl-Mesochlorin ethyl amide or 20- ⁇ [2-(2-Amino-6-tert-butoxycarbonylamino-hexanoylamino)-ethylcarbamoyl]- methyl ⁇ - 18-(2-carboxy-ethyl)-7, 12-diethyl-3 ,8, 13 , 17-tetramethyl- 17, 18,21 ,23 -tetrahydro- p ⁇ hine-2-carboxylic acid) (FIG. 5).
  • the reactive ONp group on Cort-ONp was aminolysed with the alpha-NH 2 of the lysine of the Lys-Mce 6 intermediate in DMF with DIPEA (Diisopropylethylamine).
  • the reaction mixture was purified by column chromatography using Sephadex LH-20 beads equilibrated in methanol. The major fraction was collected and verified to be the product by mass spectrometry.
  • the N- ⁇ -Boc group of lysine of the product was deprotected with 50% trifluoroacetic acid (TFA) in methylene chloride to obtain the Cort-Lys- Mce 6 derivative (FIG. 6).
  • the product was purified using preparative HPLC/RPC in acetonitrile/H 2 O.
  • Cort-Lys-Mce 6 was covalently bound to an ⁇ -(2-hydroxypropyl)- methacrylamide (“HPMA”) copolymer backbone via a biodegradable glycylphenylalanylleucylglycine (“GFLG”) spacer. Characterization of the HPMA copolymer- bound Cort-Lys-Mce 6 (P-GFLG-Cort-Lys-Mce 6 ) revealed 1.3 mol % drug-terminated side chains as determined by UN spectrometry. The weight average molecular weight, M w , of the conjugate was determined to be approximately 22 kDa by size-exclusion chromatography.
  • the polydispersity (M w / M n ) was approximately 1.55.
  • UN spectrophotometry revealed 3.5 mol % drug-terminated side chains.
  • the weight average molecular weight, M w , of the conjugate was determined to be approximately 27 kDa.
  • the polydispersity (M w / M n ) was approximately 1.35.
  • Example V Biorecognition of Cort-Lys-Mce ⁇ [0053] To demonstrate the ability of Cort-Lys-Mce 6 to bind the glucocorticoid receptor (GR), the green fluorescent protein tagged-GR (GFP-GR) system was utilized. This system involves the generation of GFP labeled GR in the cell.
  • GFP-GR green fluorescent protein tagged-GR
  • the cells are then treated with the drug and the change in the localization of the fluorescently tagged GR is followed.
  • Cells were transfected with GFP-GR plasmids as previously. Briefly, 5 x 10 6 cells were transfected with 2 ⁇ g of the GFP-GR plasmid plus carrier DNA using an Electrosquare porator ECM 830 system (BTX, San Diego, CA). The electroporation was performed using 3 pulses each of 135 V for 10 milliseconds, and 3 pulses. Cells were allowed to recover on ice for 5 minutes and were then diluted with phenol red-free DMEM with 10 % charcoal stripped FBS and plated on a clear cover glass (Corning no.
  • 150000 cells were seeded on previously sterilized glass coverslips in 35 mm dishes 24 hours before incubation with the drug.
  • the cells were incubated with the drug (free Mce 6 , Cort-Lys-Mce 6 or HPMA copolymer-bound Mce 6 conjugates) for 24 hours and the cells were washed twice with DPBS (Dulbeco's Phosphate Buffered Saline) after incubation.
  • the cells were then fixed with 3 % paraformaldehyde for 20 minutes at room temperature.
  • DAPI 6- diamidino-2-phenylindole
  • DAPI is represented by green fluorescence.
  • the nucleus will be stained green in color. Though the staining is stippled, we can delineate the nuclear boundary and hence the nucleus from the stained areas.
  • Mce 6 is stained red in color. Hence we can determine the subcellular distribution of Mce 6 in the cell by the red fluorescence.
  • window A displays the fluorescence only due to Mce 6
  • window B displays the fluorescence only due to DAPI.
  • These two windows separately indicate the subcellular localization of Mce 6 and DAPI respectively. These 2 windows are superimposed in window D to display a composite image.
  • Example VII Determination of cytotoxicity The IC 50 (concentration that inhibits growth by 50%) was determined utilizing a WST-8 assay. Four thousand cells were seeded into 96 well plates and incubated overnight in a humidified atmosphere containing 5% CO 2 . Varying concentrations of free Mce 6 and Cort-Lys-Mce 6 or HPMA copolymer-bound Mce 6 conjugates (P-GFLG-Cort-Lys-Mce 6 and P-GFLG-Mce 6 ) were added to each well. Incubation was carried out for 4 hours in the case of free Mce 6 and Cort-Lys-Mce 6 .
  • the incubation period was 10 hours in the case of the HPMA copolymer-bound Mce 6 conjugates. After the period of incubation, the media containing the drug was removed and 100 ⁇ L of fresh media was added and the cells were promptly illuminated for 30 min.
  • the light source was three ENH Tungsten halogen lamps (120 V/ 250 W) placed in parallel, attenuated by three band-pass interference filters (Melles Griot Co., Carlsbad, CA). The light energy was approximately 2 mW cm " as determined using a radiometer. [0061] After illumination, the cells were incubated in a humidified atmosphere containing 5% CO 2 for 24 hours. The media was replaced with 100 ⁇ L fresh media containing 10 ⁇ L of WST-8.
  • IC 50 values calculated for P-GFLG-Mce 6 and P-GFLG-Cort-Lys-Mce 6 after 10 hours of incubation with the drug are shown in Table 2. IC 50 values have been expressed in terms of Mce 6 equivalents. Thus, P-GFLG-Cort-Lys-Mce ⁇ is about 2.5 times more cytotoxic than P-GFLG-Mce 6 .
  • the invention provides nuclear targeting polymeric drug delivery systems based on HPMA copolymers wherein hormone analogs like cortisol are used as nuclear-targeting moieties. In vitro studies indicated that these systems are effective in achieving nuclear localization and increasing the efficacy of therapeutic agents.
  • Example VIII Cytotoxicity Experiment [0064] The details of the cytotoxicity experiment are as follows - SK-OV3 cells were plated at a density of 10,000 cells/ well in 96 well plates. 24 hours after plating, the cells were incubated with varying concentrations of the drugs (both nuclear-targeted polymer-bound anticancer drug and non-nuclear targeted polymer-bound drug). Specifically, these drugs were cortisol-targeted HPMA-copolymer bound mesochlorin (the nuclear targeted construct) and HPMA-copolymer bound mesochlorin (the non-nuclear targeted construct). The drugs were incubated with the cells for varying durations.
  • the drugs both nuclear-targeted polymer-bound anticancer drug and non-nuclear targeted polymer-bound drug.
  • these drugs were cortisol-targeted HPMA-copolymer bound mesochlorin (the nuclear targeted construct) and HPMA-copolymer bound mesochlorin (the non-n
  • the cells were washed, the medium was replaced and then irradiated for 30 minutes. 24 hours later, the cell viability was assessed using a modified MTT assay.
  • the drugs were incubated with the cells for 10 hours.
  • the cytotoxicity values that were obtained in this case were 110.00 ⁇ 7.07 micromolar for cortisol-targeted HPMA-copolymer bound mesochlorin (the nuclear targeted construct) and 3.38 ⁇ 1.33 micromolar for HPMA-copolymer bound mesochlorin (the non-nuclear targeted construct). This translates into approximately a 33 -fold increase in cytotoxicity following nuclear targeting.
  • Example IX Nuclear Localization [0065] The details of the experiment to prove nuclear localization of the nuclear targeted construct are as follows - 1471.1 cells were transfected with a plasmid (pCI-nGFP- C656G-Han) that causes the expression of Green Fluorescent Protein (GFP)-tagged Glucocorticoid Receptor (GR) using standard electroporation protocols. 24 hours after electroporation, the cells were plated in live cell chambers at a concentration of 200,000 cells/ chamber. The cells were then treated with 100 micromolar concentration of cortisol-targeted HPMA-copolymer bound mesochlorin (the nuclear targeted construct) and HPMA-copolymer bound mesochlorin (the non-nuclear targeted construct).
  • Estrogen receptor (ER) Estrogen
  • LXR Liver X receptors
  • Retinoid X receptors 9-cis retinoic acid
  • FXR Farnesoid X receptors 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8- tetramethyl-2-naphthalenyl)- 1 - propenyljbenzoic acid (TTNPB)

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Abstract

La présente invention concerne un conjugué comprenant un agent biologiquement actif (médicaments) divisé par une fraction de ciblage sous cellulaire qui cible un médicament spécifiquement sur le noyau. Le ciblage est effectué par l'attache d'une hormone stéroïde (ou d'un analogue) à ce médicament. Cette hormone stéroïde attachée au médicament se lie à son récepteur correspondant, la formation du complexe récepteur-ligand entraînant l'externalisation de ce complexe dans le noyau, ce qui entraîne la translocalisation nucléaire du médicament. Cette invention concerne aussi un conjugué (comprenant le complexe du médicament et de l'hormone stéroïde) liée à un polymère par des espaceurs permettant un ciblage passif simultané sur la cellule tumorale (permis par l'attache au polymère par l'effet EPR) et un ciblage nucléaire de ce conjugué (due à la présence du stéroïde). Un espaceur dégradable adapté permet de libérer le médicament libre dans la tumeur et renforce l'efficacité du ciblage nucléaire. Ce polymère peut aussi être lié à une molécule de ciblage cellulaire, cette molécule de ciblage dirigeant le polymère vers des cellules spécifiques. On peut ainsi cibler efficacement des médicaments sur le noyau de cellule tumorale. Avec quelques petites modifications, plusieurs agents thérapeutiques peuvent être ciblés grâce à cette invention.
EP05780057A 2004-05-10 2005-05-05 Ciblage actif et passif combines d'agents biologiquement actifs Withdrawn EP1765403A4 (fr)

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US5853713A (en) * 1994-02-28 1998-12-29 Sterling Winthrop Inc. Biologically compatible linear block copolymers of polyalkylene oxide and peptide units
WO1999029303A1 (fr) * 1997-12-12 1999-06-17 Samyang Corporation Micelles polymeres biodegradables melangees destinees a l'apport de genes
WO2001091798A2 (fr) * 2000-06-01 2001-12-06 Universite Catholique De Louvain Composes de promedicaments a activation tumorale et procedes de fabrication et d'utilisation de ces derniers
US20020099041A1 (en) * 2000-10-06 2002-07-25 Gallop Mark A. Bile-acid derived compounds for enhancing oral absorption and systemic bioavailability of drugs
WO2003046185A1 (fr) * 2001-11-28 2003-06-05 Genta Salus Llc Copolymere polycationique hydrosoluble et procede de passage de macromolecules polyanioniques a travers des barrieres biologiques
WO2003070982A1 (fr) * 2002-02-22 2003-08-28 Avaris Ab Complexe comportant au moins deux elements biospecifiques separes par un lieur d'acides nucleiques, par exemple pour l'identification de candidats d'administration de medicaments ; bibliotheques combinatoires de tels complexes
WO2004009136A2 (fr) * 2002-07-22 2004-01-29 Psimei Pharmaceuticals Plc Nouveaux composes anti-cancereux

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Publication number Priority date Publication date Assignee Title
US5853713A (en) * 1994-02-28 1998-12-29 Sterling Winthrop Inc. Biologically compatible linear block copolymers of polyalkylene oxide and peptide units
WO1999029303A1 (fr) * 1997-12-12 1999-06-17 Samyang Corporation Micelles polymeres biodegradables melangees destinees a l'apport de genes
WO2001091798A2 (fr) * 2000-06-01 2001-12-06 Universite Catholique De Louvain Composes de promedicaments a activation tumorale et procedes de fabrication et d'utilisation de ces derniers
US20020099041A1 (en) * 2000-10-06 2002-07-25 Gallop Mark A. Bile-acid derived compounds for enhancing oral absorption and systemic bioavailability of drugs
WO2003046185A1 (fr) * 2001-11-28 2003-06-05 Genta Salus Llc Copolymere polycationique hydrosoluble et procede de passage de macromolecules polyanioniques a travers des barrieres biologiques
WO2003070982A1 (fr) * 2002-02-22 2003-08-28 Avaris Ab Complexe comportant au moins deux elements biospecifiques separes par un lieur d'acides nucleiques, par exemple pour l'identification de candidats d'administration de medicaments ; bibliotheques combinatoires de tels complexes
WO2004009136A2 (fr) * 2002-07-22 2004-01-29 Psimei Pharmaceuticals Plc Nouveaux composes anti-cancereux

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