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WO1998011920A2 - Procede de capture d'un agent bioactif dans une preparation de nanoerythrosome - Google Patents

Procede de capture d'un agent bioactif dans une preparation de nanoerythrosome Download PDF

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Publication number
WO1998011920A2
WO1998011920A2 PCT/CA1997/000699 CA9700699W WO9811920A2 WO 1998011920 A2 WO1998011920 A2 WO 1998011920A2 CA 9700699 W CA9700699 W CA 9700699W WO 9811920 A2 WO9811920 A2 WO 9811920A2
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WO
WIPO (PCT)
Prior art keywords
neryt
nanoerythrosomes
nanoerythrosome
bioactive agent
present
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PCT/CA1997/000699
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English (en)
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WO1998011920A3 (fr
Inventor
René GAUDREAULT
Claude Gicquaud
Patrick Poyet
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Diagnocure Inc.
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Application filed by Diagnocure Inc. filed Critical Diagnocure Inc.
Priority to AU43733/97A priority Critical patent/AU4373397A/en
Publication of WO1998011920A2 publication Critical patent/WO1998011920A2/fr
Publication of WO1998011920A3 publication Critical patent/WO1998011920A3/fr

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    • 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
    • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
    • 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
    • A61K47/6849—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 receptor, a cell surface antigen or a cell surface determinant

Definitions

  • the present invention relates to nanoErythrosomes, a Drug Delivery System (DDS). More specifically, the present invention relates to a simple and efficient method of capture of bioactive agents by a nEryt preparation. Moreover, the present invention relates to the use of threreby prepared nanoErythrosome compositions in diagnostic and therapeutic methods. The invention further relates to the bioassays using the nanoErythrosome compositions of the present invention to diagnose or prognose a predetermined condition in mammal, as well as to kits containing these nanoErythrosome compositions of the present invention.
  • DDS Drug Delivery System
  • NanoErythrosomes are small vesicles that are produced from erythrocytes (red blood cells) which are treated in a low- salt solution to remove their haemoglobin content. Subsequently, these haemoglobin-free erythrocytes (ghosts) are extruded (intruding filtration under vacuum) to form small vesicles having a mean diameter of about 100 nm (USP 5,653,999).
  • the nanoErythrosome can in essence be considered lipoproteosomes (vesicles constituted of both lipids and proteins) by analogy with liposomes. They nevertheless represent totally distinct scientific universes when compared to the almost exclusively lipid-based liposomes.
  • the nanoErythrosomes are buoyant vesicles which remain in suspension for prolonged periods of time because of their high surface to volume ratio (approximately 80-fold higher than the parent red blood cell).
  • the nanoErythrosome can carry various types of molecules such as drugs or peptides that will be transported through the bloodstream to various targets. They can be coupled to antibodies or peptide ligands for selective delivery to cells, for example. Further, the nanoErythrosomes can encapsulate a biologically relevant substance and/or alternatively a biologically relevant molecule can be coupled thereto.
  • NanoErythrosomes are thus very versatile bioactive drug carriers or drug delivery systems (DDS). NanoErythrosomes have been shown to have advantages over other DDS and could constitute a breakthrough in biopharmacology.
  • nanoErythrosomes comprise: 1) they significantly modify the metabolism of the drug they transport by preventing their elimination via the liver or kidney; 2) they can encapsulate or bind numerous biologically relevant molecules or bioactive substances; 3) they can be used as a progressive drug- releasing system for several types of molecules, such as antineoplastics, phototherapeutic agents and peptides, thus maintaining an optimal concentration of the drug in the bloodstream for extended period of time; and 4) they can reduce the need for high dose instantaneous administration of large quantities of drugs into the bloodstream, thereby reducing deleterious effects such as nausea and vomiting which are caused by high blood concentrations of toxic drugs (USP 5,653,999).
  • nanoErythrosomes albeit to a reduced level as cortipared to other carriers can still, elicit on immune response in a mammal. Moreover, they are destroyed by gastric juices and hence could not be efficiently used in an unmodified from for per os administration.
  • NanoErythrosomes by nature, are constituted of both lipids and proteins.
  • proteins and lipids such as phosphatidylserine in the nEryt could lead, if used in non-autologous applications (a mammal is receiving nanoErythrosomes from another non compatible mammal, as opposed to autologous administration, whereby the blood donor and recipient are the same or at least the donor and recipient are compatible) to immunological reaction of the recipient mammal.
  • Those undesirable protein-protein interactions could seriously limit the potential for therapeutic, diagnostic and commercial applications of technologies based on nanoErythrosomes.
  • nanoErythrosomes could lead to deleterious immune responses.
  • it is used for diagnostic applications they they could be responsible for high background. There thus remain a need to find strategies that will abrogate those undesirable reactions.
  • there remains a need to provide a nEryt composition which is more stable at low pH conditions (such as those of the stomach).
  • This invention concerns a rapid and efficient method of capture of bioactive agents by nanoErythrosomes (nEryt), and to the nEryt-bioactive agent compositions as a diagnostic or therapeutic tool. More specifically, the present invention relates to nanoErythrosomes which have been coupled to a ligand, such as an antibody or portion thereof, in order to enable a targeting of the nanoErythrosome to a specific receptor for this ligand, such as an antigen (recognized by an antibody).
  • a ligand such as an antibody or portion thereof
  • the invention relates to a nEryt composition which comprises a nanoErythrosome coupled to an antibody which recognizes the cancer antigen gp54 TROP-2, the antibody-coupled- nanoErythrosome encapsulating a bioactive agent.
  • the antibody coupled antibody encapsulates a fluorochrome.
  • the antibody coupled nEryt encapsulates an anti-cancer molecule.
  • the invention also relates to a method of preparing such ligand-coupled nanoErythrosomes and/or ligand-coupled nanoErythrosomes encapsulating a biologically relevant substance.
  • the invention relates to a method of preparing antibody-coupled-nEryt compositions encapsulating or not a biologically relevant or bioactive substance.
  • the invention further relates to a complex comprising a nanoErythrosome coupled to an antibody or portion thereof, wherein the antibody or portions thereof targets the nanoErythrosome to an antigen recognized by the antibody or portion thereof.
  • the invention relates to a method for encapsulating a biologically relevant substance into a nanoErythrosome comprising a cycle of heat shock.
  • the encapsulation or capture method comprises cycles of freeze thawing on ice, dry ice or other freezing mixture (for example, ice-sodium chloride (-20°C approx.), acetone-dry ice (-78°C), preferably in liquid nitrogen.
  • the present invention also relates to a nanoErythrosome composition having a significantly reduced immunogenic potential. More particularly, the invention relates to nEryt- PEG compositions.
  • the present invention relates to a nEryt composition which can be administered per os. More particularly, the invention relates to a PEG-nEryt composition which can be administered per os.
  • the invention is not so limited, since a person of ordinary skill will recognize that the method of the present invention can be adapted to the coupling of numerous types of ligands including antibodies or portions thereof.
  • the term "ligand" should thus be interpreted in a broad sense as defining a substance having high affinity for a receptor. Numerous types of ligands are well known to the person of ordinary skill.
  • a multitude of different biological relevant substances can be encapsulated in the nanoErythrosome in accordance with the present invention, and/or coupled to the nanoErythrosomes in accordance with the present invention.
  • Z is the group that will be responsible for the conjugation of PEGs to the proteins present in the nEryt membrane.
  • the composition of that reactive group is very important for the control of the kinetic of PEGs conjugation to available reactive groups, such as amino groups, such as those of lysine residues.
  • reactive groups targeting two families of nucleophiles present on proteins were used for that purpose i) NH 2 groups (i.e lysine residues), exemplified by activated ⁇
  • ⁇ N - esters such as succinimide ( ⁇ -*Q ) esters; and ii) SH groups (i.e
  • cysteine residues or iminothiolane derivative of lysine exemplified by
  • nEryt membrane can be modified according to known methods by the person of ordinary skill to provide and/or modify reactive groups which can serve as partners conjugating PEG or other substance.
  • PEG to nEryt include: cyanogen bromide (BrCN) , des amino acid esters (Zaiipsky et al., 1984, J. Macromol. Sci. Chem. A21 : 8339; Mutter et al., 1979, The Peptides; Gross et al., J., eds., 2, p. 285, Academic Press, New York), hydrazine derivates, Rubinstein, 1978, U.S. Patent 4,101 ,380; Davis et al., 1979, U.S. Patent 4,179,337 and Persson et al., 1988, J. Chromatog.
  • succinimidyl carbonate derivates Miron et al., 1993, Bioconjugate Chem. 4: 568; Zaiipsky et al., 1993, Bioconjugate Chem. 4: 296 and Zaiipsky et al., 1991, Polymeric Drugs and Drug delivery Systems; Dunn et al., eds) ACS, Washington, DC); oxycarbonylimiidazole derivates, Allen et al., 1991 , Biochem. Biophys. Acta 1066: 29; and Tondelli et al., 1985, J. Controlled Release 1: 25); nitrophenyl carbonate derivates, Satore et al., 1991 , Appl. Biochem.
  • the ⁇ Q" portion of the molecule is a linking arm allowing the conjugation of PEG to proteins.
  • n 1 to 8 carbon atoms, preferably 2 to 5 carbon atoms.
  • the use of a particular n can be readily adapted by the skilled artisan, depending on the particular nEryt-PEG composition desired and use thereof. In general, when n is more than 8, the molecule tends to be too liposoluble.
  • the YQ group is a critical group since there are very few ways to directly conjugate the OH group of polyethyleneglycols to proteins. One exception, is cyanuryl chloride derivatives that has been used in preliminary experiments.
  • X can be an oxygen (ether), a sulfur (thioether) atom or a -O-C(O) (ester), a -N-C(O) (amide) and a -S-C(O) (thioester).
  • Thioalkoxy, alkoxy bonds and amides are biologically stable, however, ester and thioester linkages are much more labile since they are hydrolyzed in biological media with a kinetic that is more or less rapid. The hydrolytic kinetic is related to the lenght of the YQ portion. However, they may prove very useful in the design of nEryt used as a slow releasing device.
  • a stable bond or more labile bond between the nEryt and the biologically relevant molecule or bioactive agent includes a situation in which after having targeted the nEryt composition to a specific location, phagocytosis of the nEryt rendered immunogenic by way of breakage of the bond linking same to the PEG moiety and possibly encapsulating bioactive agent by macrophages is desirable.
  • the (CH 2 -CH 2 -O) m moiety of the molecule is the polyethyleneglycol itself, m could be anything between 1 and 500.
  • PEGs having molecular weights from 350 to 10,000 can be used, preferably PEGs of between 1 ,000-10,000, more preferably 2,000-5,000 molecular weight.
  • (CH 2 ) n -W-R] group faces the media surrounding the nEryt.
  • (CH 2 ) n -W-R should preferably be an inert group such as OCH 3 (WR) in order to abrogate of the immune responses, in applications where nEryt is involved as a simple carrier (DNA vaccines) or for absorption through mucosas (lung, intestine).
  • WR OCH 3
  • (CH 2 ) n -W-R must be a electropositively (i.e.-NH 2 ) or electronegatively (i.e.-COOH) charged group.
  • (CH 2 ) n -W-R could also be a group such as SH, 2-thiopyridyl or maleimide when the polyethyleneglycol derivative is required to bind a biologically relevant molecule such as antibodies to target a cell or a specific organ while abrogating the intrinsic immunogenicity of the nEryt.
  • n 1 to 7 carbon atoms.
  • WR is a member selected from the group consisting of: COOH, PO 4 , SO 3 H.
  • the nEryt can remain too immunogenic (which can be determined detrimental in therapeutic applications) or unprotected against various types of undesired interactions leading to non-specificity of the carrier for the target (undesirable in diagnostic interactions).
  • the percentage of reactive groups on the nEryt membrane (i.e. SH or NH 2 ) which is substituted by PEG can be readily adapted by the person of ordinary skill now cognizant of the present invention.
  • 2-30% of the reactive groups of the membrane of the nEryt will be substituted with PEG, more preferably 2-15%.
  • nEryt can be prepared from blood from various mammalian sources (mouse, rat, rabbit, ovine, bovine, echine, porcine, human, and the like).
  • the blood is collected on anticoagulant (heparine, EDTA, citrate, and the like).
  • anticoagulant heparine, EDTA, citrate, and the like.
  • the blood is centrifugated and the plasma and the buffy coat are discarded.
  • the packed erythrocytes are resuspended in phosphate buffer (PBS; 150 mM NaCI, 5.0 mM K 2 HPO 4 /KH 2 P0 4 , pH 7.4) to their initial volume of blood.
  • PBS phosphate buffer
  • the erythrocytes are washed four times and resuspended at the concentration of 2 X 10 9 cells/ml.
  • the erythrocytes in suspension can be depleted of their hemoglobin as previously described in UPS 5,653,999.
  • the present invention provides a more efficient, more quantitative, simpler and more easily scalable method of ghost preparation.
  • the erythrocytes in suspension are depleted of their hemoglobin by size-exclusion chromatography on a column containing the appropriate chromatographic gel Sepharose CL6B or Sephacryl S 400 high resolution using an hypotonic (2 to 5 mM) aqueous buffered solution at pH 8 to 11 without di and/or trivalent cations, a significant modification with respect to the previous protocol of Wood (1987, Methods in Enzymol. 149:271-280; and of USP 5,653,999). The recovery of the white ghosts from red blood cells is almost quantitative.
  • the next step of the production is providing a significant modification in the method of production of nanoErythrosomes as compared to that of USP 5,653,999.
  • the pH of the hypotonic suspension of white ghosts is brought to 7.4 is important to point out the essential role of hypotonic conditions and the absence of cations in the method of the present invention. Failure to comply with these requirement will totally compromise the production of nEryt.
  • the suspension is filtered through a filter of about 0.45 ⁇ m immediately followed by a filtration through a filter of about 0.22 m.
  • no extrusion is used.
  • no multiple extrusions through a 1 ⁇ m filter are required.
  • nEryt is also almost quantitative. As a maximum of 10% of nEryt are lost , preferably less than 5%. Using the method of USP 5,653,999, 20% or more of nEryt are lost. All manipulations are preferably performed under sterile conditions.
  • the diluted suspension (( 10 12 nEryt per ml) can be concentrated by several means commonly known by the person of ordinary skill i) either centrifugation of the aqueous solutions or on trehalose, sucrose gradients (i.e. 20,000g X 20 min.); ii) dialysis in cellulose acetate tubing having cut-off between 200,000 and 1X 10 6 molecular weight; iii) concentration using an Amicon system; iv) lyophiiization using trehalose, sucrose or raffinose solutions (ratio mg of sugar of nEryt [proteins] is between 1 to 100:1).
  • the terms "encapsulated”, “entrapped” and “capture” when relating to a bioactive agent are defined broadly as it is extremely difficult to assess where bioactive agent is specifically located following the "treatment". Indeed, it is difficult to assess whether the bioactive agent is inside the nEryt or deeply bonded to the membrane. Nevertheless, since experiments (i.e. with DNA) demonstrate that the DNA is protected from degradation (see below), it will be assumed that the bioactive agents are entrapped/captured by the nEryt.
  • nEryt are suspended in an hypotonic or isotonic solution, cation-free solution (di and/or trivalent) containing the highest concentration possible of the bioactive agent to be entrapped.
  • the capture of the molecule can take place at a low rate at room temperature. Preferably, it will be carried out more efficiently and quickly by quick freezing the nEryt-bioactive agent solution and allowed to warm up at rom temperature.
  • the nEryt-bioactive agent solution on the frozen ice, dry ice or other freezing mixture which are well known to the person of ordinary skill.
  • Non-limiting examples of freezing mixtures include ice-sodium chloride (approximately -20°C), acetone-dry ice (approx. -78°C) and the like.
  • the freezing mixture is liquid nitrogen.
  • the free bioactive agent can then be separated from captured ones through various processes adapted to their chemical nature (centrifugation-wash cycles, exclusion chromatography, dialysis, and the like) and adaptable by the person of ordinary skill.
  • nEryt having captured molecules can be concentrated (lyophilized) or used in latter processes such as conjugation of PEGs, ligands, antibodies to their membrane surface.
  • nEryt Various biologically important molecules such as ligands including antibodies, and polyethyleneglycols or chemical modifiers such as iminothiolane can now be conjugated to the membrane surface of nEryt.
  • monoclonal antibodies such as 48-127, and M344 can be conjugated to nEryt.
  • the antibodies can be conjugated as described above using -NH 2 groups present on lysine residues, or -SH groups of cysteine residues on the nEryt membrane.
  • the conjugation can take place through numerous techniques using heterobifunctional linking arms such as, for example,
  • nEryt permits a modification of the proteins present in the membrane, for example, i) iminothiolane can add SH groups to the membrane. That reaction allows the conjugation of PEG or antibodies using electrophilic groups such as maleimides (ex: SMCC and the heterobifunctional PEGs), ii) addition, for example, of anhydrides such as succinic, cis aconitic, citraconic to nEryt will add -COOH groups that introduce negative charges on the surface.
  • electrophilic groups such as maleimides (ex: SMCC and the heterobifunctional PEGs)
  • anhydrides such as succinic, cis aconitic, citraconic to nEryt will add -COOH groups that introduce negative charges on the surface.
  • P0 4 2" SO NH or -substituted with polyanionic or polycationic molecules such as spermine, heparine, etc (see general formula of PEG).
  • polyethyleneglycol derivatives of the present invention can be represented by the general formula:
  • YQ is a member selected from the group consisting of:
  • R' lower alkyl of 1 to 5 carbon atoms
  • cyanuric chloride cyanogen halides
  • Br or Cl cyanogen halides
  • activating agents* such as mesyl, tosyl groups.
  • Z is a member selected from the group consisting, if
  • PEG to nEryt include: cyanogen bromide (BrCN) , des amino acid esters (Zaiipsky et al., 1984,
  • nitrophenyl carbonate derivates Satore et al., 1991, Appl. Biochem. Biotech. 27: 45; tresylate derivates, Klibanov et al., 1991 , Biochem. Biophys. Acta 1062:142. Delgado et al., 1990, Biotech. Appl. Biochem. 12:119); maleimide derivates, Kogan, 1992, Synthetic Commun. 22:2417 and Romani et al., 1984, Chemistry of Peptides and Proteins, Volter et al., eds, 2, p 29, Walter de Gruyter, Berlin).
  • R is a polyamine deriving from spermine, spermidine or putresceine or a lower alkyl chain of 1 to 6 carbon atoms bearing one or two COOH, SO 3 H or P0 4 groups
  • WR is a member selected from the group consisting of: COOH, P0 4 , SO 3 H.
  • Ya in the general formula presented above can be a number or reactive agents such as cyanuric chloride derivatives, aldehyde, succinide, benzinidazole, symetric disulfide, heterobidunctional PEG and the
  • biologically relevant substance and “bioactive agent” are used in a broad sense purposely so as to comprise, without being limited thereto, drugs, molecules, peptides, proteins, nucleic acid sequences, and fluorophores or other labelling molecules, and PEGs.
  • PEG is intended to include PEG derivatives.
  • the nEryt of the invention may be coupled to bioactive agents to form carriers for such agents.
  • the vesicles may be coupled to bioactive agents such as drugs, to provide a carrier for administration of the bioactive agent so that the bioactive agent may be efficiently delivered to the location in the body where the bioactive agent is required.
  • the vesicles are natural materials, biodegradable, non- immunogenic or having moderate immunogenic potential, non-toxic and non-pyrogenic, fully compatible with blood, and adapted for autologous administration.
  • autologous administration should be interpreted as meaning that the nanoErythrosomes administered to a mammal have been prepared from red blood cells obtained from compatible red blood cells or blood supply (including an administration to and from the same mammal; i.e. the same patient).
  • nEryt Although it is preferable to reduce the immunogenic potential of the nEryt when contemplating a no ⁇ -autologous administration of nanoErythrosomes, an administration of nEryt without treatment to reduce their immunoreactivity, to immunosuppressed or non-immunosuppressed mammals is also contemplated in certain situations.
  • the coupling is achieved with a coupling agent having a first group reactive with a reactive site of the vesicle and a second group reactive with a reactive group on the bioactive agent.
  • a coupling agent having a first group reactive with a reactive site of the vesicle and a second group reactive with a reactive group on the bioactive agent.
  • Numerous methods of coupling a bioactive agent to the nanoErythrosome exist and are well known in the art. These include, but are not limited thereto, to the use of well known crosslinking reagents, such as bifunctional reagents of homobifunctional or heterobifunctional type are well known in the art.
  • bioactive agent is designed to include, but is not limited to photosensitive compounds, drugs, antibiotics, antineoplastic agents, anti-inflammatory agents, proteins or parts thereof, enzymes, nucleotide sequences, nucleic acids or parts thereof, oligonucleotides, antisense, genes, vectors, expression vectors, radioactive isotopes, amino acid analogs or nucleoside analogs, as well as other medically or veterinarilly useful agents such as contrast materials (e.g. dyes) and diagnostic materials as well as growth factors, hormones such as corticosteroids or the like.
  • bioactive agent should be taken in a broad sense so as to also include a combination of
  • the term pharmaceutical should be understood as including veterinary, since the nanoErythrosomes of the present invention are suited for numerous types of treatment, prophylaxy or diagnosis in mammals.
  • the nanoErythrosomes of the present invention can also serve as a diagnostic tool.
  • bioactive agents could be coupled to the nanoErythrosomes of the invention, for example antibodies, in order to target a specific tissue or cell type.
  • the detection of the target can be assessed according to known methods, including for example the use of a label, radioactive or not, or a dye entrapped in the nanoErythrosomes.
  • One of numerous examples of the diagnostic use of the nanoErythrosomes of the invention is to target a tumoral antigen, through an antibody specific to this antigen, in order to detect, quantify or analyse the presence of metastases.
  • bioactive agent and whether it is entrapped in the nanoErythrosome or conjugated thereto will depend on the desired application, the purpose of delivery, the route of delivery, the target, and other parameters relating to the use of the nanoErythrosomes.
  • the nanoErythrosomes may be administered by a number of routes: in man and animals these include but are not limited to injection (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, intraauricular, intramammary, intraurethrally, etc.), topical application (e.g., on afflicted areas), and by absorption through epithelial or mucocutaneous linings (e.g., ocular epithelia, oral mucosa, rectal and vaginal epithelial linings, the respiratory tract linings, nasopharyngeal mucosa, intestinal mucosa, etc.).
  • injection e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, intraauricular, intramammary, intraurethrally, etc.
  • topical application e.g., on afflicted areas
  • epithelial or mucocutaneous linings e.g., ocular epithelia, oral
  • NanoErythrosomes can be administered alone but will generally be administered in admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
  • Such preparations may be injected parenterally, for example, intraperitoneally, intra-arterially or intravenously.
  • the preparations may also be administered via oral, subcutaneous, intramuscular and, of course, intraorgan routes.
  • parenteral administration they can be used, for example, in the form of a sterile aqueous solution which may contain other solutes, for example, enough salts or glucose to make the solution isotonic.
  • nanoErythrosome formulation by way of an aerosol is also contemplated as a method of administration.
  • the prescribing medical professional will ultimately determine the appropriate dosage for a given subject, and this can be expected to vary according to the agent, weight, and response of the animal as well as the nature and severity of the disease.
  • the same principle can be applied for a diagnostic use of the nanoErythrosomes.
  • the dosage of the bioactive agent in a nanoErythrosome formulation can, according to the present invention, be lower than that employed for the free bioactive agent. In some cases, however, it may be necessary to administer equal or higher doses. It is also contemplated that periodic treatments or different cycles of treatment might be beneficial.
  • the route of delivery of nanoErythrosomes can also affect their distribution in the body. Passive delivery of nanoErythrosomes involves the use of various routes of administration, e.g., intravenous, subcutaneous and topical. Each route produces differences in localization of the nanoErythrosomes. Targeting of the nanoErythrosomes and bioactive agent to selected target areas is also contemplated.
  • Nucleic acid sequences of the invention can be utilized with naturally occurring sugar-phosphate backbones as well as modified backbones including phosphorothioates, dithionates, alkyl phosphonates and nucleotides and the like. Modified sugar-phosphate backbones are generally taught by Miller, 1988, Ann. Reports Med. Chem. 23:295 and Moran et al., 1987, Nucleic acid molecule. Acids Res., 1_4:5019.
  • the nucleic acid sequence can be radioactively labeled so as to specifically permit radiotherapy.
  • the term "gene" is well known in the art and relates to a nucleic acid sequence defining a single protein or polypeptide.
  • a "structural gene” defines a DNA sequence which is transcribed into RNA and translated into a protein having a specific amino acid sequence thereby giving rise the a specific polypeptide or protein. It will readily recognized by the person of ordinary skill, that the nucleic acid sequence of the present invention can be incorporated into anyone of numerous established kit formats which are well known in the art.
  • the term "vector” is commonly known in the art and defines a plasmid DNA, phage DNA, viral DNA and the like, which can serve as a DNA vehicle into which DNA of the present invention can be cloned. Numerous types of vectors exist and are well known in the art.
  • expression defines the process by which a structural gene is transcribed into mRNA (transcription), the mRNA is then being translated (translation) into one polypeptide (or protein) or more.
  • expression vector defines a vector or vehicle as described above but designed to enable the expression of an inserted sequence following transformation into a host.
  • the cloned gene (inserted sequence) is usually placed under the control of control element sequences such as promoter sequences.
  • control element sequences such as promoter sequences.
  • the placing of a cloned gene under such control sequences is often referred to as being operably linked to control elements or sequences.
  • Expression control sequences will vary depending on whether the vector is designed to express the operably linked gene in a prokaryotic or eukaryotic host or both (shuttle vectors) and can additionally contain transcriptional elements such as enhancer elements, termination sequences, tissue-specificity elements, and/or translational initiation and termination sites.
  • the present invention relates to a kit comprising a nanoErythrosome of the present invention comprising at least one container means containing a nanoErythrosome composition in accordance with the present invention.
  • the kit comprises other containers comprising one or more of the following reagents, wash reagents, detection reagents, and the like.
  • antibody includes monoclonal and polyclonal antibodies as well as fragments thereof, single chain antibodies, antibody fragments which contain the idiotype of a specific molecule and antibody fragments.
  • the term antibody as used herein also includes F(AB') 2 fragment, the F(ab') fragments, F(ab) fragment and Fv fragments.
  • the nanoErythrosome compositions have broad therapeutic applications.
  • One non-limiting example of such application includes gene therapy by which a nucleic acid segment can be transferred to a patient.
  • a nanoErythrosome composition of the present invention permits a specific targeting of an organ, tissue or cell by way of a specific ligand directed against this organ, tissue or cell. It will be clear that the nanoErythrosome compositions of the present invention can deliver agonists, and antagonists of gene products.
  • antisense molecules using the nanoErythrosome compositions of the present invention is also contemplated.
  • the specific design of the antisense and modification thereof to increase its stability, are well known in the art.
  • Suitable pharmaceutical carriers, excipients, and therapeutic or diagnostic compositions in general are described in Remington's Pharmaceutical Sciences, 1980, 16th, Ed., a standard reference in this particular field.
  • cytotoxic agents and drugs can be used in accordance with the present invention.
  • Non-limiting examples of cytotoxic agents include toxins such as diphtheria toxin.
  • radionuclides can also be coupled to the nanoErythrosome compositions of the present invention to exert the cytotoxic effect by local irradiation of cells and hence to radiotherapy. Radionuclides which can be used in radiotherapy are well known in the art and can be readily adapted to a particular clinical or diagnostic situation.
  • Figure 1 shows a summary of the methodologies used to produce nanoErythrosomes to be used in diagnostic and/or therapeutic applications
  • FIG. 1 shows phase contrast microscopy A and C and fluorescence microscopy B-D of T24 (AB) and EFFRON cells (CD).
  • Figure 1 shows a summary of the methodologies used to produce nanoErythrosomes that are involved in diagnostic or therapeutic applications.
  • nanoErythrosomes The technology of the production of the nanoErythrosomes has been taken a step further by exploring administration thereof through parenteral or anteral routes.
  • parenteral route nanoErythrosomes were used successfully to show that they could identify cancer cells of the bladder.
  • nanoErythrosomes were concentrated by centrifugation and mixed in a concentrated solution of dextran-fluorocsein isothyocyanate (Dextran-FITC; MW 4000 to 2 X 10 6 ). After one round of freeze-thawing in liquid nitrogen, nanoErythrosomes which had encapsulated the dextran-FITC were purified either by filtration through size-exclusion, gel using Sephadex or Sepharose (i.e.
  • NanoErythrosome membranes were modified by a first reaction with SPDP.
  • the chosen antibody in this case monoclonal antibody 48-127, which recognizes the cancer antigen TROP-2, an antigen present on a large number of bladder cancer cells, was first modified with SPDP followed by treatment with a reducing agent such as DTT (dithiothreitol) liberate the free SH group.
  • a reducing agent such as DTT (dithiothreitol) liberate the free SH group.
  • DTT dithiothreitol
  • the modified nanoErythrosomes from above and the monoclonal antibody 48-127, also modified were mixed together in a physiological buffer in order to favour the formation of a composition termed the nanoErythrosome - Dextran - FITC - 48127.
  • Biological assays using this complex clearly demonstrated that the cells carrying the TROP-2 antigen recognized by 48-127 were very selectively decorated by the complex while non-TROP-2 carrying cells were not.
  • Pseudomonas toxin is being encapsulated in nanoErythrosomes in order to show that the nanoEiythrosome-Pseudomo.-ias toxin - 48127 complex can be efficiently used as a cytotoxic delivery system of high specificity.
  • These latter two types of complexes may be shown to be useful for intravesicular treatment of various bladder cancers.
  • Such complexes could overcome the main problem encountered with currently used treatment methodologies which is the very short period of contact between the drugs and the targeted bladder cells (this is caused by the rapid accumulation in the bladder of the urine produced by the kidneys).
  • nanoErythrosomes can encapsulate, using the method of the present invention, molecules having molecular weights ranging from 1000 (phthalocyanine octacarboxylic) to 2 X 10 6 (Dextran).
  • the NanoErythrosome is thus a very versatile drug delivery system.
  • dextran and phthalocyanine molecules have been shown to be stably encapsulated by the nanoErythrosomes for periods longer than six months.
  • nanoErythrosomes could eventually permit a per os administration of biologically relevant substances such as drugs, enzymes, proteins (i.e. insulin), immunomodulating compounds, peptides or other substances sensitive to the gastro-intestinal system.
  • the strategy used to obtain such a modified delivery system consists in encapsulating under the thermal shock treatment method described above, a biologically relevant substance inside the nanoErythrosomes.
  • derivatives of the Polyethyleneglycol PEG-cyanuryl chloride or the like
  • Such a Polyethyleneglycol derivative is known to abrogate antigenicity and to increase the biological half life of proteins and enzymes. Such a system could thus permit the use of non-autologous and non-homologous nanoErythrosomes.
  • the invention is not limited to PEG-cyanuryl chloride since numerous other PEG derivatives can be used. Such derivatives are well known to a person of ordinary skill. Non- limiting examples of such PEG derivatives comprise PEG derivatives having activated esters. It follows, that a person of ordinary skill could adapt the teachings of the present invention to other types of PEG derivatives.
  • the nanoErythrosomes are conjugated to PEG-5000 - cyanuryl chloride.
  • PEG derivatives between about 350 and 10,000 molecular weight can be used in accordance with the present invention, it should be understood that it is preferable to use a PEG derivative having a molecular weight between about 1 ,000 and 7,000 and more preferably between about 2,000 and 10,000.
  • nanoErythrosomes The stability of PEG-modified nanoErythrosomes is currently tested using Dextran-FITC encapsulated nanoErythrosomes. Experiments are under way to verify the encapsulation and stability of insulin-1125 and evaluate the biopharmaceutical parameters of such nanoErythrosomes in animals following different types of administration (iv, ip and per os). In addition, the capacity of nanoErythrosome - PEG containing insulin or hormone such as LHRH to induce a pharmacological response when administered peros also will be tested.
  • Blood (2 X 30 ml) from mammal donors (echine, bovine, porcine, human, etc) is centrifuged at 500 X g (1500 rpm) for 10 min at 4°C. Plasma and buffy coat are removed by aspiration. The volume removed is replaced by the same volume of Phosphate Buffer Saline (PBS) sodium phosphate 5 mM, sodium chloride 150mM) at pH 7.4 and at 4°C. The mixture is gently homogeneized by several inversion of the tubes. Blood is then recentrifuged again at 500 X g for 10 min at 4°C. The sequence described herein is repeated 3 times.
  • PBS Phosphate Buffer Saline
  • Example 2 Five ml of blood treated as described in Example 1 are added to 35 ml (in 50 ml NalgeneTM tubes) of hypotonic buffer (sodium phosphate 5mM) at pH 7.4. The erythrocytes suspension is centrifuged at 25,000 X g for 20 min at 4°C.
  • hypotonic buffer sodium phosphate 5mM
  • NanoErythrosomes can be prepared as described previously in USP 5,653,999. It is important to point-out that ghosts are extruded through polycarbonate filters having pores of approximately
  • the polycarbonate filter provide the advantage of not sticking to nanoErythrosomes.
  • the pore size is important for the preparation of vesicles. Ghosts are extruded from isotonic suspensions and under nitrogen pressure. The suspension is preferably extruded 4 times.
  • the nanoErythrosomes obtained according to method 3.1 have a mean diameter of 100 to 200nm.
  • Light scattering experiments using nEryt suspensions prepared according to method 3.2 three populations of nEryt a) 40-50nm; b) 100 nm; and c) a smaller subpopulation of 200nm.
  • the mean diameter of the nEryt suspension prepared in accordance with method 3.2 remain however at about 100-200 nm.
  • nEryt suspensions 350 ml at 0.2 mg of protein per ml are concentrated using an AmiconTM concentrator (model 8400, membrane having a molecular cut-off smaller than 500 000 (for example ZM 500 and YM 100) to a volume of 50 ml under a nitrogen pressure of 10 psi and a very gentle stirring.
  • AmiconTM concentrator model 8400, membrane having a molecular cut-off smaller than 500 000 (for example ZM 500 and YM 100) to a volume of 50 ml under a nitrogen pressure of 10 psi and a very gentle stirring.
  • a general protocol can be used to capture (entrap) bioactive agents such as proteins (i.e insulin), photodynamic agents (i.e porphyrine-like molecules), fluorophores (i.e dextran (4,000 to 2 000 000 mw)-fluorescein isothiocyanate (FITC)).
  • bioactive agents such as proteins (i.e insulin), photodynamic agents (i.e porphyrine-like molecules), fluorophores (i.e dextran (4,000 to 2 000 000 mw)-fluorescein isothiocyanate (FITC)).
  • nucleotides i.e DNA
  • glycoproteins i.e inulin
  • polysaccharides i.e inulin
  • vitamins drugs (i.e. bleomycin) and the like.
  • nEryt In eppendorf tubes, suspension of nEryt (1.5 ml, 1 to 2 mg/ml) are centrifugated (microfugeTM) at 16,000 X g for 10 min at 4°C. The supernatant is removed and discarded. The protein content of the pellet is determined according to well known methods (i.e. a commercially available kit). One ml of the concentrated solution of the substance to be capture (entrapped) is added to the pellet. The suspension is gently homogeneized by several inversion of the tubes and kept at 4°C room temperature for 10 min. Afterward, the suspension is frozen in liquid nitrogen for 2 min and unfrozen in a water bath at 25°C.
  • nEryt are freed from the unentrapped molecules by 3 to 5 cycles of dilution with 1 ml of cold (4°C) PBS (isotonic at pH 7.4), centrifugation at 16,000 X g for 8 min, removal of the supernatant and again dilution with PBS.
  • the nEryt are freed from the unentrapped molecules by using size exclusion chromatography with the proper Sephadex or Sephacryl column according to well known methods allowing the retardation of free molecules and the rapid elution of nEryt.
  • the captured (entrapped) material can evaluated by usual methodologies (adapted to the molecule being entrapped) after solubilization of nEryt using a suitable detergent. Stability
  • the capture results in the formation of a stable nanoErythrosome-bioactive agent composition as evidenced by the fact that suspensions of dextran-FITC-nEryt are stable for at least 7 months when kept at 4°C. Furthermore, entrapment of DNA was carried out. Briefly, two hundred nanograms of nEryt suspended in PBS ( 500 ⁇ l) are washed 3 times with the TKN buffer (to eliminate phosphates). DNA (30 to 320 ng) in TKN buffer (10 to 300 ⁇ l) is added to the suspension and gently homogeneized. The suspension is incubated at 4°C for 10 min and frozen in liquid nitrogen for 2 min followed by incubation at 25°C for 4 min. The suspension is treated as described previously using TKNM buffer.
  • nEryt-DNA was amplified using conventional protocols of PCR and confirmed the presence of the vector within the nEryt. Table 1
  • a typical preparation consists of adding 1 ml (1 mg of proteins) of a suspension of nEryt (or nEryt having a captured bioactive agent or a PEG coupled nEryt having a captured bioactive agent or a nEryt antibody on its surface) in either hypotonic PB at pH 7.4 or isotonic PBS.
  • nEryt a fresh solution of iminithiolane (IT) is prepared (2 M) in PBS-EDTA. The solution is kept on ice. nEryt are suspended in cold PBS-EDTA buffer and IT solution is added to obtain the ratio 100 ⁇ moles of IT per mg of nEryt (3 mg nEryt in 320 ⁇ l of PBS-EDTA, 151 ⁇ l of IT in 677 ⁇ l of PBS-EDTA). The mixture is kept at 4°C in the dark with gentle agitation for 90 min. After the incubation, the mixture is purified on Sephadex G-25 (PD-10).
  • IT iminithiolane
  • chromatography column is equilibrated with 20 ml of cold PBS-EDTA prior to use.
  • the mixture is applied on the column and eluted with cold PBS-EDTA.
  • the first 2.5 ml is discarded (dead volume) and nEryt are collected in the following 2.5 ml fraction.
  • the samples are kept on ice prior to the next reaction.
  • nEryt-IT are unstable and should be prepared extemporaneously.
  • SMCC succinimidyl 4-(n-maleimidomethyl)cyclohexane-1-carboxylate
  • the solution is kept at room temperature.
  • a solution of 48-127 (1 mg) in PBS (700 ⁇ l) containing 4.7 mM EDTA is prepared.
  • One hundred and twenty five nanomoles of SMCC are added, the volume of the reaction is 712 ⁇ l.
  • the solution is incubated for 30 min at 30°C with agitation.
  • the mixture is separated on a PD-10 column previously equilibrated using 5 ml of PBS containing 0.5% of bovine serum albumin and washed with 20 ml of cold PBS-EDTA buffer.
  • the mixture is applied on the column and eluted with cold PBS-EDTA.
  • the first 2.5 ml is discarded (dead volume) and nEryt are collected in the following 2.5 ml fraction.
  • the samples are kept on ice prior to the next reaction.
  • nEryt-SMCC-48127 composition was treated as previously described (Example 6).
  • the resulting nEryt (FITC)-SMCC- 48127 composition (15 mg/ml) was incubated with T24 cells and EFFRON cells according to known methods. Briefly, T24 cells and EFFRON cells were used after
  • the cells and nanoErythrosome composition was incubated for 1h at 37°C in the presence of 5% CO 2 .
  • the slides were washed with MEM containing CAV serum (3 ml) and PBS (2 X 3 ml).
  • the slides were mounted on microscopic slid and rinced with 2 ml of PBS.
  • FIG. 2 shows the immunofluorescence results.
  • Fig. 2A and 2C show the phase contrast microscopy (400 X) of the T24 and EFFRON cells, respectively.
  • Fig. 2B and 2D show the same microscopy field as 2A and 2C under the fluorescent microscope.
  • SMCC nEryt-Dextran-F.TC-48127
  • the present invention thus teaches a simple, efficient, quantitative method for the encapsulation/capture/entrapment of a bioactive agent in a preparation of nanoErythrosomes.
  • the nanoErythrosomes of the present invention, entrapping or being linked to a bioactive agents can be further modified by pegilation (substitution of groups on a membrane of a nanoErythrosome by PEG -derivatives).
  • the nEryt entrapping a bioactive agent can also be further modified by conjugating an antibody thereto, thereby enabling of the targeting of the nEryt and its captured bioactive agent.
  • the nEryt compositions described are stable.
  • Heterobifunctional PEG bearing a monoclonal antibody were conjugated to nanoErythrosomes having an entrapped dextran-fluorochrome conjugate, and in vitro results showed that this PEG bearing monoclonal antibody-nanoErythrosome compositions were specifically decorating cells expressing the antigen recognized by the antibody.

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Abstract

L'invention porte sur des nanoérythrosomes, système de distribution de médicaments (DDS), et plus particulièrement sur un nouveau procédé de piégeage/capture d'un agent bioactif par des nanoérythrosomes. L'invention porte également sur des compositions de nanoérythrosomes utilisées à des fins diagnostiques et thérapeutiques. L'invention porte en outre sur des bioessais recourant aux compositions de nanoérythrosomes de la présente invention pour diagnostiquer ou pronostiquer certain états prédéterminés chez l'animal, et sur des trousses contenant lesdites compositions de nanoérythrosomes.
PCT/CA1997/000699 1996-09-19 1997-09-19 Procede de capture d'un agent bioactif dans une preparation de nanoerythrosome WO1998011920A2 (fr)

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DE10000838A1 (de) * 2000-01-12 2001-07-26 Schott Glas Alkalifreies Aluminoborosilicatglas und dessen Verwendungen
US7858329B2 (en) 2002-09-30 2010-12-28 Topotarget Germany Ag Antibody tools for the diagnostic use in the medical therapy with inhibitors of histone deacetylases

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US7432331B2 (en) 2002-12-31 2008-10-07 Nektar Therapeutics Al, Corporation Hydrolytically stable maleimide-terminated polymers
AU2003300133B2 (en) 2002-12-31 2008-11-13 Nektar Therapeutics Hydrolytically stable maleimide-terminated polymers
EP2015071A1 (fr) * 2007-07-13 2009-01-14 FUJIFILM Corporation Support, son procédé de production, bioréacteur et puce pour analyse par résonance à plasmons de surface
WO2011091154A2 (fr) * 2010-01-21 2011-07-28 The Regents Of The University Of California Utilisation d'érythrocytes humains dans la prévention et le traitement de la dissémination et de la croissance du cancer

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CA2150617C (fr) * 1995-02-07 2001-10-30 Universite Laval Nanoerythrosome comme porteur d'agent bioactif

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10000838A1 (de) * 2000-01-12 2001-07-26 Schott Glas Alkalifreies Aluminoborosilicatglas und dessen Verwendungen
US6852658B2 (en) 2000-01-12 2005-02-08 Schott Glas Flat panel liquid-crystal display, such as for a laptop computer
DE10000838B4 (de) * 2000-01-12 2005-03-17 Schott Ag Alkalifreies Aluminoborosilicatglas und dessen Verwendungen
US7858329B2 (en) 2002-09-30 2010-12-28 Topotarget Germany Ag Antibody tools for the diagnostic use in the medical therapy with inhibitors of histone deacetylases

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