WO1994021281A1 - Composition polymere-polypeptide et procede d'administration de ladite composition - Google Patents

Composition polymere-polypeptide et procede d'administration de ladite composition Download PDF

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Publication number
WO1994021281A1
WO1994021281A1 PCT/US1994/003102 US9403102W WO9421281A1 WO 1994021281 A1 WO1994021281 A1 WO 1994021281A1 US 9403102 W US9403102 W US 9403102W WO 9421281 A1 WO9421281 A1 WO 9421281A1
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Prior art keywords
polypeptide
peg
liposome
composition
attached
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PCT/US1994/003102
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English (en)
Inventor
Samuel Zalipsky
Francis Martin
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Liposome Technology, Inc.
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Priority to AU63683/94A priority Critical patent/AU6368394A/en
Publication of WO1994021281A1 publication Critical patent/WO1994021281A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/35—Allergens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K47/6905—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 the form being a colloid or an emulsion
    • A61K47/6911—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 the form being a colloid or an emulsion the form being a liposome
    • 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
    • A61K47/6905—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 the form being a colloid or an emulsion
    • A61K47/6911—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 the form being a colloid or an emulsion the form being a liposome
    • A61K47/6913—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 the form being a colloid or an emulsion the form being a liposome the liposome being modified on its surface by an antibody
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers

Definitions

  • the present invention relates to an improvement in a method for treating a subject by parenteral administration of an immunogenic polypeptide.
  • This invention also relates to a composition utilized for parenteral administration of the polypeptide.
  • Zalipsky, S., et al. (1987) Int. J. Peptide Res. 30:740.
  • Zalipsky, S., et al. (1990) J. Bioactive Compat. Polym. 5:227.
  • a variety of medical conditions can be treated by parenteral administration of antibodies or enzyme polypeptides.
  • monoclonal or polyclonal antibodies against different epitopes of a patient's CD4+ cells are administered alone, or in combination with other immunosuppressive compounds, for treatment of rheumatoid arthritis and other autoimmune diseases, or for the suppression of graft/host reactions or transplantation rejection.
  • anti-tumor antibodies may be coupled to toxins or radionucleotides, or to chemotherapeutic drugs for targeting these compounds to tumor cells (Sehon, 1992) .
  • streptokinase is commonly administered for the treatment or prevention of blood clotting and thrombus formation.
  • Tissue plasminogen activator is also used to prevent thrombus formation (Cherng) .
  • these therapeutic polypeptides are derived from nonhuman sources, or otherwise may include regions which are able to provoke an immune response. Repeated administration of such polypeptides can result in undesired side effects, such as serum sickness, anaphylactic symptoms, and/or liver complications.
  • a chimeric antibody constructed to have human constant regions and mouse variable regions has been proposed (Greiner) .
  • the antibody may still provoke an immune response against the variable regions.
  • the polypeptide is derivatized with multiple polyethylene glycol chains, by covalent attachment of the chains to reactive sites, typically amines, on the polypeptide.
  • the derivatized polypeptide when administered parenterally, appears to reduce or eliminate the immune response that ordinarily is directed against the free polypeptide.
  • the immune system After an initial treatment with the derivatized polypeptide, the immune system is less responsive to the polypeptide in free form, allowing the polypeptide to be administered in free form with reduced immune-response complications (Lee, 1977) .
  • This invention relates to an improvement in a method for treating a subject by parenteral admininistration of an immunogenic polypeptide.
  • the improvement includes pretreating a subject by parenteral administration of a polymer-polypeptide composition, composed of the polypeptide and polyethylene glycol (PEG) chains which surround, but are not covalently attached to, the polypeptide.
  • PEG polyethylene glycol
  • the polypeptide is attached to a particle support surface and is surrounded by a layer of PEG chains attached to the particle surface.
  • the PEG chains have a molecular weight between about 1,000-10,000 daltons, and the particle to which PEG chains and polypeptides are attached is a liposome.
  • the polypeptide is: (a) a xenogeneic antibody, or a xenogeneic antibody F ⁇ fragment, attached to the liposome surface by high-affinity, specific antibody binding to an antigen substrate covalently attached to the liposome surface, (b) an allergen attached to the liposome surface by biotin/avidin high-affinity coupling, or (c) an enzyme attached to the liposome surface by high affinity, irreversible binding to an irreversible enzyme inhibitor.
  • the invention includes a composition of the type described above.
  • Figs. 1A-1E are representations of liposome polymer- polypeptide compositions, in which (a) a polypeptide is directly attached to a liposome surface (Fig. 1A) , (b) a polypeptide is attached to a liposome surface by a short polyethylene glycol (PEG) spacer arm (Fig. IB) , (c) a polypeptide is bound to a liposome surface by high-affinity binding to an antigen attached to the liposome surface (Fig. 1C) , (d) a polypeptide is bound to a liposome surface by high-affinity binding to an enzyme inhibitor attached to the liposome surface (Fig. ID) , and (e) a polypeptide is bound by biotin/avidin coupling to a liposome having surface-bound biotin;
  • Fig. 2 illustrates a step in the formation of a phosphatidylethanolamine (PE) whose polar head group is derivatized with a maleimide group
  • Fig. 3 illustrates a step in the formation of a PE whose polar head group is derivatized with a bromoacetamide group
  • Fig. 4 shows steps in forming of a PE derivatized by a short PEG spacer chain having a reactive maleimide group at its free end;
  • Fig. 5 shows steps in forming of a PE derivatized by a short PEG spacer chain having an aldehyde group at its free end;
  • Fig. 6 shows steps in forming a PE derivatized by a PEG spacer chain having a hydrazine group at its free end
  • Fig. 7 shows a reaction for covalent attachment of a polypeptide, via a sulhydryl group, to a maleimide group of a derivatized PE;
  • Fig. 8 shows a reaction for covalent coupling of a polypeptide to a derivatized PE having aldehyde groups via reductive amination
  • Figs. 9A-9C illustrate the preparation of a biotinylated PE (Fig. 9A) for use in preparing liposomes with surface-bound biotin, the preparation of a biotinylated IgG (Fig. 9B) , and binding of the biotinylated IgG molecule to a liposome surface containing biotinylated PE via high affinity interactions between surface-bound biotin, avidin, and biotin attached to IgG (Fig. 9C) ; and
  • Fig. 10 shows a plot of a time course of gallium-67 labelled liposomes composed of hydrazide PEG-DSPE, partially hydrogenated egg phosphatidylcholine (PHEPC) , and cholesterol (PEG-HZ fluid liposomes) , or hydrazide PEG-DSPE, hydrogenated serum phosphatidylcholine (HSPC) , and cholesterol (PEG-HZ rigid liposomes) in the bloodstream.
  • PHEPC partially hydrogenated egg phosphatidylcholine
  • HSPC hydrogenated serum phosphatidylcholine
  • HSPC hydrogenated serum phosphatidylcholine
  • Vehicle-forming lipid refers to any lipid capable of forming part of a stable micelle or liposome composition and typically including two hydrophobic acyl hydrocarbon chains or a steroid group and a polar group which contains a reactive chemical group, such as an a ine, acid, ester, aldehyde or alcohol.
  • Allergen refers to a polypeptide capable of eliciting an immune response, typically involving immunoglobulin E (IgE) , when a subject is exposed parenterally to the allergen.
  • IgE immunoglobulin E
  • Antigen refers to a polypeptide capable of eliciting an response, typically involving an IgG or IgM immune response, when a subject is exposed parenterally to the antigen.
  • Immunogen refers to either an allergen or an antigen.
  • polypeptide refers to both ⁇ to peptides, e.g., having less than 50 amino acid residues, and to larger polypeptides, e.g., having up to 200 amino acid residues or even larger.
  • Substrate refers to a compound attached to a liposome surface capable of binding, covalently or nocovalently, a polypeptide to a liposome surface.
  • High-affinity binding refers to substrate binding to a polypeptide with an affinity constant greater than 10 6 M *1 , and preferably greater than 10 9 K" 1 . Examples of such high-affinity binding between substrate and polypeptide, include binding of biotin to avidin, and binding of an antigen to its antibody.
  • irreversible binding refers to high-affinity substrate binding by a polypeptide, where the substrate- polypeptide complex is stable and is characterized by a lifetime of at least 48 hours due to slow dissociation of substrate from the polypeptide. Irreversible binding can occur by covalent attachment of a substrate to an enzyme, or by high affinity noncovalent binding of a substrate to an enzyme. Examples of such irreversible binding between substrate and enzyme, include covalent complex formation between organic fluorophosphates and acetylcholinesterase, and vanadate binding to nucleotide- hydrolyzing enzymes.
  • FIGS. 1A-1E illustrate various embodiments of a liposomal polymer-polypeptide composition for use in the invention.
  • the composition will be described in its general features with respect to to Fig. 1A.
  • the figure shows a portion of the outer bilayer 10 of a liposome 12 having an outer surface 14.
  • the liposome will include additional bilayers which are within the outer layer shown.
  • the outer bilayer itself is composed of confronting lipid layers 10a and 10b which are the interior and exterior lipid layers, respectively, of the billayer, each layer being composed of vesicle-forming lipids, such as phospholipids and cholesterol. Methods for forming liposomes suitable for use in the composition are described below.
  • the liposome is representative of a particle support forming part of the composition.
  • the composition includes molecules of a polypeptide, such as polypeptide 16 which is attached to the outer liposome surface by covalent coupling to one of the lipids, such as indicated at 18, forming the outer layer of the outer bilayer.
  • polypeptides suitable for use in the invention, and methods of their preparation are described below.
  • the polypeptide is attached by direct covalent coupling to the polar head group of a lipid, such as lipid 18, in the outer layer of the liposome's outer bilayer.
  • other methods of attaching polypeptides to the outer surfaces of liposomes are described.
  • the polypeptide molecules attached to the liposome outer surface are surrounded by, but not covalently linked to, hydrophilic polymer chains, such as chains 20, which are also carried on the liposome's outer surface.
  • the polymer chains are preferably polyethylene glycol (PEG) chains having molecular weights between about 1,000 and 10,000 daltons, correspondig to polymer chain lengths of about 22 to 220 ethylene oxide units.
  • PEG polyethylene glycol
  • the polymer chains are covalently attached to the polar hea groups of vesicle-forming lipids in the outer layer of th liposome's outer bilayer. Methods of forming PEG-derivatize lipids will be described below.
  • th polymer chains form a layer 22 whose thickness is sufficient to cover at least a portion of the polypeptide.
  • the PE chains are selected in length so that the layer formed by the polymers will cover all or most of the polypeptide attached to the liposome surface.
  • a polymer layer composed of PEG chains having a molecular weight of about 2,000 daltons has an apparent layer thickness of about 50 angstroms (Needham) . This polymer layer would therefore be effective in covering a globular polypeptide having rough dimensions of less than about 50 A diameter.
  • the polypeptide in the composition is attached to the liposome outer surface by a short polymer chain.
  • the figure shows a liposome bilayer portion 24 with a layer 26 of PEG chains, as above.
  • the polypeptide, such as polypeptide 28 is attached to the liposome outer surface by a spacer chain, such as chain 30.
  • the spacer chain is preferably a short hydrophilic chain, such as a 100-500 dalton PEG chain, which is itself coupled to the polar head group of a lipid, such as lipid 32, in the outer layer 34 of the liposome bilayer.
  • the short spacer chain contains a derivatizable group 36 at its free end.
  • the present embodiment allows the polypeptide in the polymer layer to be positioned at a selected depth in the layer, as shown, to increase or decrease the extent to which the polypeptide is buried in the polymer layer.
  • a small polypeptide e.g., one having a globular size of less than 20 A, may be advantageously placed adjacent the outer surface of the layer by a 100-500 dalton PEG spacer chain, as shown.
  • the polpeptide is surrounded by, but not covalently linked to, the PEG chains which form the polymer layer on the liposomes.
  • the polypeptide in the composition is an antibody or antibody fragment attached to the liposome outer surface by specific, high-affinity binding to an antigen, or antigenic substrate, carried on the liposome outer surface.
  • the figure shows a liposome bilayer portion 38 with a layer 40 of PEG chains, as above.
  • a polypeptide, such as polypeptide 42 is attached to the liposome outer surface by high-affinity binding to an antigen, such as antigen 44, covalently attached to to the polar head group of a lipid, such as lipid 46, in the outer layer of the liposome bilayer.
  • an antigen such as antigen 44
  • the polpeptide is surrounded by, but not covalently linked to, the PEG chains which form the polymer layer on the liposome.
  • An important feature of this embodiment of the invention is that polypeptides exhibit high affinity for the antigenic substrates attached to the liposome surface. These sites on the liposome surface which exhibit a high affinity for the polypeptide will likely facilitate polypeptide binding to the liposome surface.
  • the polypeptide in the composition is an enzyme which is attached to the liposome outer surface by specific, high-affinity binding to an enzyme inhibitor carried on an outer liposome surface.
  • the figure shows a liposome bilayer portion 48 with a layer 50 of PEG chains, as above.
  • the liposome contains, on or close to its surface 52, numerous enzyme inhibitor molecules, such as enzyme inhibitor 54.
  • a polypeptide such as polypeptide 56, binds with high affinity to the enzyme inhibitor molecule, and once bound, the polypeptide will remain substantially bound to the liposome surface, with a very slow dissociation rate if any.
  • the enzyme is surrounded by, but not covalently linked to, the PEG chains which form the polymer layer on the liposome surface.
  • An important feature of this composition is that enzymes having a high affinity for the enzyme inhibitors may be attached to the liposome surface with a polymer layer by covalent or noncovalent inhibitor substrate binding.
  • the polypeptide in the composition is a biotinylated polypeptide attached to the liposome outer surface by specific, high- affinity binding to avidin carried on the liposome outer surface.
  • the avidin is bound noncovalently to the liposome outer surface by high-affinity interactions with biotin which has been used to derivatize lipid head groups on the liposome surface.
  • the figure shows a liposome bilayer portion 58 with a layer 60 of PEG chains, as above.
  • the liposome outer surface contains a number of lipid polar head groups, such as lipid polar head group 62, which have been derivatized by biotin.
  • lipid polar head group 62 which have been derivatized by biotin.
  • an avidin molecule such as avidin molecule 64
  • Each avidin molecule contains four high-affinity biotin binding sites, such as biotin binding site 68. To one of these sites is attached the liposome bound biotin as previously indicated.
  • biotinylated polypeptide such as biotinylated polypeptide 70, which is derivatized by a biotin molecule, such as biotin molecule 72.
  • the polypeptide is surrounded by, but not covalently linked to, the PEG chains which form the polymer layer on the liposome.
  • An important feature of this embodiment of the invention is high affinity of avidin for biotin which makes possible the attachment of polypeptides to a liposome surface with a polymer layer.
  • composition of the invention has been describe above with respect to a liposomal composition, i.e., one havin a liposomal particle support, it will be recognized that variety of other particles supports may be used.
  • the particle supports should be biodegradable, have surfac chemical groups through which polymer chains of polypeptid molecules can be attached to the surface, and preferably hav sizes in a selected size range between about 50-500 nm.
  • Exemplary particle supports of this type include size polylactic acid particles, or microcapsules formed of poly amin acids.
  • the liposome particle support is composed of three general types of vesicle-forming lipid components.
  • the first includes vesicle-forming lipids which will form the bulk of the vesicle structure in the liposome.
  • these vesicle-forming lipids include any amphipathic lipids having hydrophobic and polar head group moieties, and which (a) can form spontaneously into bilayer vesicles in water, as exemplified by phospholipids, or (b) are stably incorporated into lipid bilayers, with its hydrophobic moiety in contact with the interior, hydrophobic region of the bilayer membrane, and its polar head group moiety oriented toward the exterior, polar surface of the membrane.
  • the vesicle-forming lipids of this type are preferably ones having two hydrocarbon chains, typically acyl chains, and a polar head group.
  • phospholipids such as phosphatidylcholine (PC) , PE, phosphatidylglycerol (PG) , phosphatidic acid (PA) , phosphatidylinositol (PI) , and sphin- gomyelin (SM) , where the two hydrocarbon chains are typically between about 14-22 carbon atoms in length, and have varying degrees of unsaturation.
  • PC phosphatidylcholine
  • PE phosphatidylglycerol
  • PA phosphatidic acid
  • PI phosphatidylinositol
  • SM sphin- gomyelin
  • the above-described lipids and phospholipids whose acyl chains have a variety of degrees of saturation can be obtained commercially, or prepared according to published methods.
  • Other lipids that can be included in the invention are glycolipids and sterols, such as cholesterol.
  • the second general component includes a vesicle-forming lipid which is derivatized with a polymer chain which will form the polymer layer in the composition.
  • the vesicle-forming lipids which can be used as the second general vesicle-forming lipid component are any of those described for the first general vesicle-forming lipid component.
  • Vesicle forming lipids with diacyl chains, such as phospholipids, are preferred.
  • One exemplary phospholipid is phosphatidylethanolamine (PE) , which provides a reactive amino group which is convenient for coupling to the activated polymers.
  • An exemplary PE is distearyl PE (DSPE) .
  • the preferred hydrophilic polymer in the derivatized lipid is polyethyleneglycol (PEG) , preferably a PEG chain having a molecular weight between 1,000-10,000 daltons, more preferably between 2,000 and 5,000 daltons.
  • PEG polyethyleneglycol
  • Other hydrophilic polymers which may be suitable include polyvinylpyrrolidone, polyoxazoline, polymethacrylate, and polydimethylacrylamide, polylactic acid, polyglycolic acid, and derivatized celluloses, such as hydroxymethylcellulose or hydroxyethylcellulose. Additionally, block copolymers or random copolymers of these polymers, particularly including PEG segments, may be suitable.
  • Methods for preparing lipids derivatized with hydrophili polymers, such as PEG are well know e.g., as described in co owned U.S. Patent No. 5,013,556.
  • the third general vesicle-forming lipid component is a lipi anchor which serves to attach the polypeptide in the compositio to the liposome surface.
  • the lipid anchor has a hydrophobi moiety which serves to anchor the lipid in the outer layer o the liposome bilayer surface, and a head group which may be (i) a polar head group which can be activated for covalent couplin to the polypeptide, (ii) a hydrophilic polymer spacer arm whic carries an activatable chemical group at its free end, or (iii) a substrate by which the polypeptide can be attached to the lipid component via high-affinity binding, where the substrate may be attached directly to the lipid of through a spacer arm.
  • Lipid polar head groups which can be activated for coupling of a polypeptide include amine, acid, and hydroxyl groups.
  • One preferred lipid is phosphatidylinositol (PI) which has hydroxyl groups which can be oxidized to reactive aldehyde groups.
  • PI phosphatidylinositol
  • Lipid anchors having a spacer arm with a reactive free end are preferably lipids which are derivatized with PEG, as above, but where the PEG is substantially shorter than the PEG forming the polymer layer.
  • the spacer arm is generally of 100-1,500 daltons, preferably 600-1,000 daltons.
  • Lipid anchors in which the head group is a substrate can be prepared by a variety of well-known methods for attaching a substrate, such as biotin, short peptides, or non-peptide enzyme substrates to the polar head groups of lipids.
  • a substrate such as biotin, short peptides, or non-peptide enzyme substrates
  • the coupling methods discussed below with respect to coupling polypeptides to activated lipid groups, after liposome formation are suitable for attaching peptide substrates to free lipids.
  • Other lipid anchors, such as biotinylated PE are commercially available.
  • the substrates are relatively small, e.g., less than about 5,000 daltons, to allow their incorporation into multilamellar liposomes with a minimum of disruption of the lipid bilayer structures.
  • the substrate is preferably one capable of binding irreversibly to the polypeptide, to ensure that the polypeptide remains bound to the liposomes over its lifetime in the bloodstream.
  • the liposomes may be prepared by a variety of technigues, such as those detailed in Szoka et al, 1980.
  • Multilamellar vesicles can be formed by simple lipid-film hydration technigues. In this procedure, a mixture of liposome-forming lipids of the type detailed above dissolved in a suitable organic solvent is evaporated in a vessel to form a thin film, which is then covered by an agueous medium. The lipid film hydrates to form MLVs, typically with sizes between about 0.1 to 10 microns.
  • the lipids components used in forming the liposomes are preferably present in a molar ratio of about 70-90 percent vesicle forming lipids, 1-25 percent polymer derivatized lipid, and 0.1-5 percent lipid anchor.
  • One exemplary formulation includes 50-70 mole percent underivatized PE, 20-40 mole percent cholesterol, .0.1-1 mole percent of a PE-PEG (150) spacer polymer with a chemically reactive group at its free end for polypeptide or substrate coupling, 5-10 mole percent PE derivatized by PEG 3500 polymer chains, and 1 mole percent ⁇ -tocopherol.
  • the liposomes are preferably prepared to have substantially homogeneous sizes in a selected size range, typically between about 0.03 to 0.5 microns.
  • One effective sizing method for REVs and MLVs involves extruding an agueous suspension of the lipo ⁇ somes through a series of polycarbonate membranes having a selected uniform pore size in the range of 0.03 to 0.2 micron, typically 0.05, 0.08, 0.1, or 0.2 microns.
  • the pore size o the membrane corresponds roughly to the largest sizes of lipo somes produced by extrusion through that membrane, particularl where the preparation is extruded two or more times through th same membrane.
  • Homogenization methods are also useful for down sizing liposomes to sizes of lOOnm or less (Martin) .
  • the polypeptide in the composition is an immunogeni polypeptide.
  • This polypeptide can be an antibody, or F antibody fragment. These antibodies may be obtained fro nonhuman sources, or may be chimeric polypeptides containin peptide regions derived from both human and nonhuman sources The antibodies may be conjugated with a toxin, a radionuclide or a chemotherapeutic drug.
  • Exemplary antibodies include murine monoclonal antibodies t (i) tumor-specific antigens used for therapy of cancer of th colon, ovary, or breast, (ii) CD3 for treatment of rena allograft rejection, or (iii) IL-2 for treatment of autoimmun diseases (Sehon, 1992) .
  • the polypeptide is therapeutic enzyme, such as streptokinase, tissue plasminoge activator (TPA) , or arginase.
  • TPA tissue plasminoge activator
  • arginase arginase
  • the polypeptide is a allergen, such as an isolated protein or protein fragment fro a known allergenic source.
  • allergen such as an isolated protein or protein fragment fro a known allergenic source.
  • Example include ovalbumin, or mixture of allergenic peptides, such as nondialysabl constituents of an agueous extract of ragweed pollen.
  • Polypeptide compositions of this invention can be attached to liposome surfaces by covalent attachment or by polypeptide binding to a substrate carried on the liposome surfaces.
  • lipid anchor components in the liposomes are reacted with the polypeptide under conditions favoring covalent attachment to the lipid anchor.
  • the polar head group of the lipid anchor is activated by a suitable activating agent, and the activated liposomes are then reacted with the polypeptide.
  • Fig. 2 illustrates the activation of DSPE (compound I) by malei ido propionate N-hydroxysuccinimide ester (compound II) (MPS, Pierce) , to form an activated maleimido-DSPE (compound III) .
  • the maleimido group at the polar head group is reactive towards thiol-containing polypeptides, to attach the polypeptide to the liposome surface via a thioether linkage.
  • Fig. 3 illustrates the activation of DSPE (compound I) by bromoacetyl N-hydroxysuccinimide ester (compound IV) to form the bromo acetamide of DSPE (compound V) .
  • the bromoacetamide group at the polar head group is also reactive towards thiol- containing polypeptides, to attach the polypeptide to the liposome surface via a thioether linkage.
  • Fig. 4 shows the synthesis of a DSPE derivatized with a PEG chain and having an activated chemical group at the chain's free end.
  • PEG bis (amine) compound VI
  • 2-nitrobenzene sulfonyl chloride compound VII
  • TEA triethylamine
  • the compound is reacted with DSPE in TEA to form the derivatized PE lipid protected at one end with 2 nitrobenzyl sulfonyl chloride.
  • the protective group is remove by treatment with acid to give the D ⁇ PE-PEG (compound IX) havin a terminal a ine on the PEG chain.
  • Reaction with maleic aci anhydride gives the corresponding maleamic (compound X)
  • whic on reaction with acetic anhydride gives the PE-PEG-maleimid (compound XI) .
  • the compound is reactive with sulhydryl groups, for couplin polypeptides through a thioether linkage to generate compoun XXI, as illustrated in Fig. 7.
  • FIG. 5 Another reaction method for coupling a protected poly alkylether to a lipid amine is shown in Fig. 5.
  • PEG compound XII
  • thi reaction scheme PEG (compound XII) is initially protected a one of its terminal OH ends by a trimethylsilane group, as show at the top in Fig. 5.
  • the protected PEG (compound XIII) i reacted with the anhydride of trifluoromethyl sulfonate t activate the free PEG end with trifluoromethyl sulfonat (compound XIV) .
  • Reaction of the activated compound with a lipi amine, such as PE in the presence of triethylamine, gives th desired derivatized PE product.
  • the trimethylsilyl protective group can be released by aci treatment, yielding the desired PE-PEG (compound XV) with a fre terminal OH.
  • Reaction of compound XV with acetic anhydride i DMSO converts the terminal OH to an aldehyde group (compoun XVI) which can be coupled to a peptide via reductive amination, as illustrated in Fig. 8, to generate compound XXIII. Reactio details are given in Example 2.
  • the aldehyde-based coupling reaction just described may also be employed in a liposome composition containing PI, PG, or other lipid anchor component with an oxidizable head group.
  • the preformed liposomes are oxidized, e.g., b reaction with periodate.
  • the aldehyde groups formed by the oxidation may then be used for protein coupling, as illustrate in Fig. 8.
  • the fre hydroxyl group is activated by reaction with disuccinimidy carbonate to activate the terminal hydroxyl group (compound XIX) prior to reaction with DSPE to generate product (compound XX) .
  • Hydrazide groups obtained by deprotection of compound XX (Bo group removal) are reactive towards aldehydes, which can b generated on numerous biologically relevant compounds. Hydrazides can also be acylated by active esters o carbodiimide-activated carboxyl groups. Acyl azide group reactive as acylating species can be easily obtained fro hydrazides thus allowing conjugation of amino containin ligands.
  • FIG. 9A-9C One exemplary method for attaching polypeptides noncovalently to liposomes is illustrated in Figs. 9A-9C. Fig.
  • FIG. 9B shows a similar reaction for biotinylating a polypeptide, in this case, an IgG to generate compound XXVI Coupling is achieved by first binding avidin to the liposomes then incubating the avidin-coated liposomes with th polypeptide.
  • an IgG to generate compound XXVI Coupling is achieved by first binding avidin to the liposomes then incubating the avidin-coated liposomes with th polypeptide.
  • the liposomes are incubated with the polypeptid under conditions effective to allow polypeptide binding to th substrate.
  • End-functionalized PEG-DSPE contains a chemicall active group which can be used for attaching a variety o compounds to liposomes. From these studies it has bee determined that end-functionalization does not affect th extended lifetime in the bloodstream of liposomes containin PEG-DSPE, monomethoxy PEG-DSPE, or other similarly modifie vesicle-forming lipids.
  • liposomes containing PEG-DSPE end-functionalized b hydrazide were prepared.
  • the hydrazide group at the end of PEG chain can be used for the introduction of other functiona groups, or can be used in numerous types of conjugation scheme (Inman) .
  • Particularly useful is hydrazide's reactivity towar various glycoproteins, such as immunoglobulins ( ilchek) , fo attaching these molecules to liposomes.
  • Gallium 67-labelled, hydrazide end-functionalized PE liposomes were injected in rats by tail vein injection at abou 10-20 micromolar phospholipid/kg body weight. Blood sample was obtained by retroobital bleeding at defined times. The percen of gallium labelled liposomes remaining in the bloodstream wa determined at 0, 15 minutes, l, 3, 5, and 24 hours and i presented in Table 1. The percent injected gallium 67-labelle liposome dose remaining in the blood stream at different time is illustrated in a half log plot versus time in Fig. 10.
  • a fluid liposom composition was prepared from partially hydrogenated eg phosphatidylcholine (HPEPC) .
  • HPEPC partially hydrogenated phosphatidylcholine
  • a typical liposome compositio contains the hydrazide PEG-DSPE lipid, partially hydrogenate egg PC (PHEPC), and cholesterol in a lipid:lipid:lipid mol ratio of about 0.15:1.85:1.
  • a rigid liposome composition was prepared by substituting hydrogenated serum phosphatidylcholine (HSPC) for PHEPC at the same mole ratio.
  • HSPC hydrogenated serum phosphatidylcholine
  • the fluidity of the liposome composition does not affect the blood retention time of the liposomes. However, the fluidity of the liposome composition does appear to affect the tissue distribution of the end- functionalized liposome. For example, rigid liposomes are preferentially retained by live, spleen and bone tissue. Fluid liposomes are preferentially retained by the kidneys, heart, skin and muscle tissue. Table 1
  • the invention includes an improved method fo treating a subject by parenteral administration of a immunogenic polypeptide.
  • the improvement includes pretreatin the subject by parenteral administration of a polymer polypeptide composition of the type described above, i.e., a composition containing the polypeptide and polyethylene glycol (PEG) chains which surround, but are not covelently attached to, said polypeptide.
  • a polymer polypeptide composition of the type described above, i.e., a composition containing the polypeptide and polyethylene glycol (PEG) chains which surround, but are not covelently attached to, said polypeptide.
  • the polymer-polypeptide composition is administered intravenously several days before treatment with the peptide in free form. This pretreatment is designed to desensitize the subject to the peptide, i.e., suppress the subject's immune response to the free peptide.
  • the composition is preferably injected in an amount corresponding to between about 0.1 to 2 mg polypeptide/kg body weight.
  • the composition may be administered at periodic intervals during the pretreatment period. After the pretreatment period, the peptide in free form can be administered for periods of up to 20 and 40 days, with reduced immune response to the free polypeptide.
  • the polymer-polypeptide composition can be administered at intervals throughout the treatment.
  • the desired product eluted at 20% (1% cone. NH 4 OH in
  • the wax was dissolved in 2.5 ml chloroform and placed on a silicate column which had been wetted with chloroform.
  • the following solvents were passed through the column in sequence: 100 ml of 100% chloroform 0% (1% CONC. NH 4 OH in methanol)
  • the mixture was filtered by suction to separate crystals of triethylammonium chloride and the crystals were washed with 5 ml benzene. Filtrate and benzene wash liquids were combined. This solution was evaporated to dryness under vacuum to provide 15.83 grams of colorless oil which solidified on standing.
  • the TLC assay system used silica gel coated glass plates, with solvent combination butanone/acetic acid/water; 40/25/5; v/v/v. Iodine vapor absorption served for visualization.
  • the desired N-1-trimethylsilyloxy PEG 1500 PE was a chief constituent of the 170-300 ml portions of column effluent.
  • PE compound was dissolved in 2 ml of tetrahydrofuran. To this, 6 ml acetic acid and 2 ml water was added. The resulting solution was let to stand for 3 days at
  • the mixture was applied to the top of a 10 mm X 250 mm chroma- tographic absorption column packed with octadecyl bonded phase silica gel and column was developed with ethanol water 80:20% by volume, collecting sequential 20 ml portions of effluent.
  • Polyethylene glycol (Fluka, PEG-2000, 42 g, 42 meguiv OH) is dissolved in toluene (200 ml) and azeotropically dried (Zalipsky, 1987) , and treated with ethyl isocyanotoacetate (2.3 ml, 21 mmol) and triethylamine (1.5 ml, 10 mmol). After overnight reaction at 25°C the solution is evaporated to dryness. The residue is dissolved in 0.2 M NaOH (100 ml) and any trace of toluene is evaporated. The solution is maintained at pH 12 with periodical dropwise additions of 4 M NaOH.
  • Succinimidyl carbonate groups content 4.15 • 10 "4 meguiv/g (98% of theoretical value) was determined by titration (Zalipsky, 1991) .
  • PE Phosphatidylethanolamine
  • DMSO dimethylsulfoxide
  • Biotin-PE is utilized in forming liposomes by the reverse phase evaporation method.
  • Liposomes containing 1 mole percent biotin-PE are incubated in the presence of avidin. Liposome-bound avidin is separated from free avidin on a Sepharose CL-B column (25 x 1 cm) in TES-buffered saline (pH 7.4).
  • Antibody 5 mg IgG in 1 ml sodium borate buffer (0.1 M, ph8.8), is incubated with 100 microliters N-hydroxysuccinimide biotin (10 mg/ml in dimethyl sulfoxide) at room temperature for four hours. Biotinylated antibodies are purified by passage through a Sephadex G-25 column (25 x 1 cm) in TES buffered saline. C. Preparation of Liposome-bound IgGs.
  • the hydrazide PEG-DSPE lipid was combined with partially hydrogenated egg PC (PHEPC) and cholesterol in a lipid:lipid:lipid mole ratio of about 0.15:1.85:1 and the lipid mixture was hydrated.
  • PHEPC partially hydrogenated egg PC
  • lipid hydration occured in the presence of desferal mesylate, followed by sizing to 0.1 micron, and removal of non-entrapped desferal by gel filtration with subsequent loading of Ga-oxide into the liposomes.
  • the unencapsulated Ga was removed during passage through a .Sephadex G-50 gel exclusion column. Both compositions contained 10 micromoles/ml in 0.15 M NaCl, 5 mM desferal.
  • a second lipid mixture was prepared in a similar manner but with HSPC instead of PHEPC.
  • the liposome composition (0.4 ml) was injected IV in animals. At times 0, 0.25, 1, 3, or 5 and 24 hours after injection, blood samples were removed and assayed for the amount of Ga-desferal remaining in the blood, expressed as a percentage of the amount measured immediately after injection. Hydrazide-PEG liposome have a blood halflife of about 15 hours, and nearly 30% of the injected material is present in the blood after 24 hours.

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Abstract

Amélioration apportée à une méthode de traitement thérapeutique. Ledit traitement consiste à administrer à un sujet une composition polymère-polypeptide, avant d'administrer audit sujet le polypeptide sous forme libre. La composition polymère-polypeptide comporte le polypeptide et des chaînes de polyéthylène glycol qui entourent le polypeptide, mais ne sont pas liées à ce dernier de manière covalente.
PCT/US1994/003102 1993-03-23 1994-03-22 Composition polymere-polypeptide et procede d'administration de ladite composition WO1994021281A1 (fr)

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WO1998016201A1 (fr) * 1996-10-11 1998-04-23 Sequus Pharmaceuticals, Inc. Composition de liposomes therapeutiques et procede
US5820873A (en) * 1994-09-30 1998-10-13 The University Of British Columbia Polyethylene glycol modified ceramide lipids and liposome uses thereof
US5885613A (en) * 1994-09-30 1999-03-23 The University Of British Columbia Bilayer stabilizing components and their use in forming programmable fusogenic liposomes
US6673364B1 (en) 1995-06-07 2004-01-06 The University Of British Columbia Liposome having an exchangeable component
US6734171B1 (en) 1997-10-10 2004-05-11 Inex Pharmaceuticals Corp. Methods for encapsulating nucleic acids in lipid bilayers
US6906024B1 (en) * 1998-05-01 2005-06-14 Procter & Gamble Company Fabric care compositions comprising cellulose binding domains
US6936272B2 (en) 1996-10-11 2005-08-30 Alza Corporation 10139483Therapeutic liposome composition and method of preparation
US7432331B2 (en) 2002-12-31 2008-10-07 Nektar Therapeutics Al, Corporation Hydrolytically stable maleimide-terminated polymers
US7432330B2 (en) 2002-12-31 2008-10-07 Nektar Therapeutics Al, Corporation Hydrolytically stable maleimide-terminated polymers
CN113264997A (zh) * 2021-04-13 2021-08-17 康汉医药(广州)有限公司 一种蛋白药物在常温、高温条件下保存的方法

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Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5820873A (en) * 1994-09-30 1998-10-13 The University Of British Columbia Polyethylene glycol modified ceramide lipids and liposome uses thereof
US5885613A (en) * 1994-09-30 1999-03-23 The University Of British Columbia Bilayer stabilizing components and their use in forming programmable fusogenic liposomes
US6673364B1 (en) 1995-06-07 2004-01-06 The University Of British Columbia Liposome having an exchangeable component
US6958241B2 (en) 1996-10-11 2005-10-25 Martin Francis J Therapeutic liposome composition and method
US6043094A (en) * 1996-10-11 2000-03-28 Sequus Pharmaceuticals, Inc. Therapeutic liposome composition and method
US6936272B2 (en) 1996-10-11 2005-08-30 Alza Corporation 10139483Therapeutic liposome composition and method of preparation
WO1998016201A1 (fr) * 1996-10-11 1998-04-23 Sequus Pharmaceuticals, Inc. Composition de liposomes therapeutiques et procede
US7122202B2 (en) 1996-10-11 2006-10-17 Alza Corporation Therapeutic liposome composition and method of preparation
US6734171B1 (en) 1997-10-10 2004-05-11 Inex Pharmaceuticals Corp. Methods for encapsulating nucleic acids in lipid bilayers
US6906024B1 (en) * 1998-05-01 2005-06-14 Procter & Gamble Company Fabric care compositions comprising cellulose binding domains
US7432331B2 (en) 2002-12-31 2008-10-07 Nektar Therapeutics Al, Corporation Hydrolytically stable maleimide-terminated polymers
US7432330B2 (en) 2002-12-31 2008-10-07 Nektar Therapeutics Al, Corporation Hydrolytically stable maleimide-terminated polymers
US8106131B2 (en) 2002-12-31 2012-01-31 Nektar Therapeutics Hydrolytically stable maleimide-terminated polymers
US8227555B2 (en) 2002-12-31 2012-07-24 Nektar Therapeutics Hydrolytically stable maleimide-terminated polymers
US8835556B2 (en) 2002-12-31 2014-09-16 Nektar Therapeutics Hydrolytically stable maleimide-terminated polymers
CN113264997A (zh) * 2021-04-13 2021-08-17 康汉医药(广州)有限公司 一种蛋白药物在常温、高温条件下保存的方法

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