WO1999059643A2 - Complexe d'apport par liposomes - Google Patents

Complexe d'apport par liposomes Download PDF

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Publication number
WO1999059643A2
WO1999059643A2 PCT/US1999/011177 US9911177W WO9959643A2 WO 1999059643 A2 WO1999059643 A2 WO 1999059643A2 US 9911177 W US9911177 W US 9911177W WO 9959643 A2 WO9959643 A2 WO 9959643A2
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Prior art keywords
antibody
liposome
liposomal delivery
liposomal
delivery vehicle
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PCT/US1999/011177
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English (en)
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WO1999059643A3 (fr
Inventor
John R. Lau
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Sdg, Inc.
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Publication date
Application filed by Sdg, Inc. filed Critical Sdg, Inc.
Priority to AU40902/99A priority Critical patent/AU4090299A/en
Publication of WO1999059643A2 publication Critical patent/WO1999059643A2/fr
Publication of WO1999059643A3 publication Critical patent/WO1999059643A3/fr

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    • 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
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
    • 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

  • This invention relates generally to liposomal delivery complexes providing enhanced delivery of biologically active diagnostic and therapeutic agents.
  • a liposomal delivery complex comprising a liposome, an antibody, and a connecting moiety which specifically binds the Fc region of the antibody to connect the antibody to the liposome.
  • Liposomes are lipid molecules formed into a typically spherically shaped arrangement defining aqueous and membranal inner compartments. Liposomes can be used to encapsulate pharmacological agents within the inner compartments, and deliver such agents to desired in vivo sites. However, liposomes are susceptible to removal by the body's reticuloendothelial system (RES), mainly the liver and the spleen. As part of the body's immune system, the RES will quickly phagocytize the liposome along with its cargo, thus hampering the treatment or diagnostic regime.
  • RES reticuloendothelial system
  • the therapeutic index of the agent is decreased by non site-specific delivery.
  • One approach to improve the site specificity of liposomal carrier systems has been to use antibodies to target the liposomal system to specific cell-surface antigens in a mammal. For example, targeting antibodies on a liposomal complex enhance the delivery of a cytotoxic agent to a malignant cell while avoiding the non-malignant or healthy cells.
  • the antibody is derivatized, using procedures well-known in the art, in an effort to anchor it to the liposome surface.
  • the liposomal delivery complex generally comprises a liposome, an antibody, and a connecting moiety which specifically binds the Fc region of the antibody.
  • the connecting moiety binds the liposome to the antibody, such that the antibodies are not derivatized in any way that minimizes their effectiveness.
  • the antibody is derivatized in order to covalently bond to the liposomal surface.
  • the derivatization procedures necessarily disrupt the structural integrity of the antibody. Derivatization of the antibody produces debris and antibody fragments that not only compete with the liposomal delivery complex for attachment to the target cell-surface antigen, but also compete for attachment to the liposomal carrier construct.
  • the connecting moiety comprises a protein, known as protein G' (i.e., "protein G prime"), which possesses binding specificity for the Fc region of antibodies.
  • protein G' i.e., "protein G prime”
  • the gene for protein G from Streptococcus strain G 148 has been cloned and expressed in Escherichia coli.
  • the regions on the gene corresponding to the albumin-binding domains and the Fab-binding region have been deleted by site-directed mutagenesis.
  • the translation of regions corresponding to the cell-wall and membrane-binding domains has been prevented by introducing stop codons upstream of these domains.
  • this recombinant DNA sequence encodes protein G', which binds only the Fc portion of an antibody, Eliasson, M. et al. (1988) J. Bio. Chem. 263, pp. 4323-4327), and see, Goward, C.R., et al (1990) Biochem. J., 267, pp. 171-177, for the nucleotide sequence and deduced amino acid sequence of Protein G', incorporated herein in their entireties by reference.
  • protein G' shields the Fc regions of the attached antibodies from non-specific binding to cell-surfaces, other proteins, and anatomical structures.
  • the molecular structure of protein G' enables the molecule to accept the Fc region of an antibody without introducing chemical procedures that might denature either protein.
  • Each protein G' molecule can accept one or two Fc regions from monoclonal antibodies.
  • the liposomal delivery complex further includes a nonsense antibody which provides RES avoidance without competing with the targeting antibody for the intended antigen.
  • the native, targeting antibody molecule and nonsense antibody may be RES avoidance enhanced together or separately by the selective addition of derivatized sialic acid molecules or macrophage avoidance molecules in general.
  • the addition of naturally occurring sialic acid coated antibodies as well as antibodies that have been enhanced with derivatized sialic acid lessens any apparent antigenicity that might be attributed to the protein G' moiety.
  • Another aspect of this invention is a pharmaceutical composition of the liposomal delivery complex with pharmaceutically acceptable excipients, and a method of treating mammalian conditions with the composition.
  • One embodiment of the invention is the site-specific delivery of a liposomal complex containing a cytotoxic agent to a malignant cell in vivo in order to achieve an increase in the therapeutic index.
  • Another aspect of this invention is a method of forming a liposomal carrier comprising sonicating a mixture of a liposomal component, a linking moiety, and a connecting moiety, in order to facilitate bonding between the liposomal surface and me linking moiety.
  • the liposomal component includes a thiocholesterol group for bonding to the linking moiety, and the sonication energy breaks up the liposomal components into smaller liposomes so that the thiocholesterol binding groups are exposed on the surface of the liposome. Additionally, the sonication energy provides an energized system which increases bonding between the linking moiety and the liposome.
  • an antibody is associated with the surface of a liposome in order that the antibody can direct the liposome to the intended cell-surface antigen for site-specific delivery thereto. Due to the use of a connecting moiety which specifically binds the Fc region of antibodies, the presence of undesirable antibodies, antibody fragments and other debris are eliminated that may otherwise interfere with the binding of a targeted antibody liposomal complex to a cell-surface antigen, or the binding of the targeting antibody to the liposomal carrier construct. Additionally, in one embodiment, the liposomal delivery complex of the invention also provides for improved RES avoidance.
  • Figure 1 illustrates the nucleotide sequence and deduced amino acid sequence of protein G', as reported in Goward, C.R., et al (1990), supra.
  • the invention generally comprises a construct for connecting an antibody or antibody fragment to a liposome, comprising a connecting moiety which specifically binds to the Fc region of the antibody and a linking moiety for connecting the connecting moiety to the liposome.
  • a liposomal delivery vehicle comprising a liposome bound to a connecting moiety, and optionally including a linking moiety for connecting the connecting moiety to the liposome.
  • the invention further comprises a liposomal delivery complex comprising a liposome, an antibody, and a connecting moiety which specifically binds to the Fc region of the antibody, used, for example, for delivering a diagnostic or therapeutic agent to a mammal.
  • protein G' is the connecting molecule.
  • Protein G' available from Pierce Chemicals, comprises a recombinant protein having a molecular weight of approximately 22,000. Due to the small molecular weight of protein G', the molecule is less antigenic and more amenable to derivatization than other protein connectors.
  • the nucleotide sequence of protein G' is listed in SEQ ID NO: 1 ( Figure 1). The deduced amino acid sequence of protein G ', coded for by the recombinant DNA sequence of SEQ ID NO: 1 ( Figure 1).
  • Protein G ' has an N-terminal primary amino functional group, whereby attachment to a liposome can be obtained.
  • the liposome and protein G' are connected by a linking agent.
  • linking moieties may be used, including sulfo- succinimidyl p-maleimidophenylbutyrate (sulfo-SMPB), p-maleimidophenylbutyrate phosphatidylethanolamine (MPB-PE), 2- iminothiolane and succinimidylacetylthio acetic acid (SAT A).
  • sulfo-SMPB succinimidyl p-maleimidophenylbutyrate
  • MPB-PE p-maleimidophenylbutyrate phosphatidylethanolamine
  • 2- iminothiolane and succinimidylacetylthio acetic acid (SAT A).
  • the linking moiety forms a bond, such as a thioether bond, which is nonhydrolyzable in cellular media, although the linking moieties may involve other bonds, such as disulfide bonds, which may be reduced in blood serum or plasma.
  • the linking moiety serves to attach the protein G ' molecule to the liposomal surface via a membrane sequestered thiocholesterol anchoring molecule.
  • the 2- iminothiolane and SATA linking moieties are capable of expressing a free sulfhydryl functional group and can be coupled to thiocholesterol in the liposomal membrane by a disulfide bond.
  • the liposomal delivery complex of the invention includes at least one protein G' molecule, although typically, a plurality of protein G' molecules are used. Determination of the number of protein G' molecules required would be obvious to one of ordinary skill in the art.
  • the liposomal delivery complex of the invention is prepared by mixing an excess number of antibodies with a predetermined number of protein G' molecules, thereby assuring saturation of all available Fc binding sites on protein G'. It is then possible, using known separation techniques, to remove the excess unbound antibodies which results in a high level of purified liposomal delivery complex. Thus, a high degree of targeting efficiency is achieved by separating the unbound antibodies from the fully functional liposomal complex. This step prevents free antibody competition for cell-surface antigens. Sepharose C1-2B-300 gel filtration chromatography may be used to fractionate the various components of the mixture.
  • a primary amino group on protein G' reacts with the linking moiety sulfo- succinimidyl p-maleimidophenylbutyrate (sulfo-SMPB) at physiological pH to form a protein G ' p-maleimidophenylbutyrate construct.
  • This construct expresses a maleimido functional group which is useful for creating new covalent bonds to sulfhydryl functionalities.
  • the maleimido functional group of the construct When energy is imparted into the liposomal suspension by sonication or microfluidization, the maleimido functional group of the construct is capable of reacting with energized thiocholesterol, an anchoring component located in the liposomal membrane.
  • This newly synthesized conjugate consisting of the liposome, p-maleimidophenylbutyrate and protein G' can couple with the Fc region of a monoclonal antibody to produce the liposomal p-maleimidophenylbutyrate protein G ' monoclonal antibody complex.
  • antibodies or antibody fragments which are preferably monoclonal antibodies, having an Fc region may be attached to the protein G' connecting moiety of the invention.
  • the term antibody refers to a protein or protein fragment. While primarily discussed in terms of monoclonal antibodies, the liposomal delivery complex of the invention should be understood to include polyclonal antibodies as well. In a presently preferred embodiment of the invention, the antibody is IgG, although other suitable immunoglobulins may be used.
  • the Fc region of the antibody binds non-covalently to the protein G' Fc receptors.
  • the antibodies are selected for their specificity to cell surface antigens based on the results of a biopsy evaluation of the patient's tissue or organ.
  • the protein G' connecting moiety provides a liposomal delivery complex having improved targeting efficiency.
  • the antibody is oriented on the liposomal delivery complex such that binding of the antibody to cell-surface antigens is optimized.
  • the antibody targeting determinants on the variable regions of the A and B chains are expressed toward the bulk phase media.
  • the immobilized Fc region of the antibody establishes the correct molecular and spacial orientation for the determinant A and B chains on the antibody to make the correct and specific interaction with the appropriate cell surface antigen.
  • the protein G ' connecting moiety of the invention allows the liposomal carrier to be connected to a non-derivatized antibody. By not invoking harsh chemical reactions to derivatize the antibodies, the native structure of the antibody is preserved.
  • Antibodies that have been derivatized with functional linking groups for the purpose of attaching an antibody to a liposome are encumbered by potential functional group interactions. It is possible for covalent binding to occur if antibodies with free sulfhydryl groups interact with neighboring antibodies and form disulfide bonds. This interaction produces multiple antibody aggregates and negates the effectiveness of the targeting moiety.
  • an antibody that has been derivatized with a deacylated succinimidylacetylthio acetic acid (SAT A) molecule expresses a free sulfhydryl group and is capable of interacting with other native sulfhydryl groups in addition to neighboring antibodies which also contain deacylated SATA moieties.
  • SAT A succinimidylacetylthio acetic acid
  • Other types of cross-linking pose similar problems and make the exact or specific derivatization of an antibody at the molecular level difficult to control. For example, it is relatively simple to place a few SATA molecules on the exposed e- amino groups of lysine residues residing in the native structure of a protein.
  • the association of the Fc portion of the antibody with the Fc binding portion of the protein G' molecule obviates the need to derivatize the antibody for connection to the liposome and the prerequisite to reduce the antibody disulfide bonds. Therefore, the effectiveness of the delivery system is improved. Consequently, lower concentrations of the therapeutic or diagnostic agents can be administered to the patient, with a resulting decrease in the negative effects associated with the therapeutic regime. Additionally, the step of removing reducing agents, such as dithiothreitol or mercaptoethanol, by chromatography, centrifugation or dialysis is eliminated, thus greatly simplifying the preparation procedure.
  • reducing agents such as dithiothreitol or mercaptoethanol
  • the Fc portion of the antibody molecule has a natural affinity for the non-specific binding sites on other proteins as well as for cellular membranes that may be encountered in vivo.
  • the Fc region of the monoclonal antibody is sequestered within the Fc binding portion of protein G', the adverse binding characteristics of the Fc portion are eliminated.
  • the result is accomplished by not chemically altering the monoclonal antibody in any fashion, and this ensures maintenance of the native structure of the monoclonal antibody. Therefore, the antibody attachment and orientation in relation to the liposome carrier is achieved in a simple and efficacious manner.
  • Fc portion of the antibody is masked and nonspecific antibody binding is prevented.
  • enzymes such as pepsin or papain are no longer needed to cleave the Fc fragment from the respective F(ab prime)2 or Fab antibody binding regions in order to prevent nonspecific antibody binding.
  • the liposomal delivery complex further comprises a masking agent to provide RES avoidance and thereby protect the complex from immunoreactive attack.
  • the masking agent comprises a RES avoidance moiety such as sialic acid on the native targeting antibody.
  • Another embodiment of the invention involves using nonsense, preferably monoclonal, antibodies that have no targeting capability but rather have an attachment of naturally occurring sialic acid that provides RES avoidance capability. Thus, by employing the nonspecific Fc binding domain of these nonsense sialic acid-coated antibodies, direct attachment can be made to protein G'.
  • macrophage avoidance antibodies containing sialic acid and targeting antibodies containing binding sites which attach to cell-surface antigens can be equally or proportionately mixed as they are added to a liposomal conjugate.
  • This use of a native antibody molecule that has no targeting capability yet possesses macrophage avoidance characteristics has been found in this invention to prolong the circulation time of targeted liposomes and thus increase the likelihood of the liposomal complex locating the designated cell-surface antigens.
  • the interplay on the liposome surface between alternating target and macrophage avoidance antibodies can be made to function optimally and synergistically by using concentrations of antibodies that reflect the appropriate mole ratios.
  • This new procedure provides an ease of synthesis and utility, since targeting and macrophage avoidance capability can be achieved in a single-step by the simultaneous addition of different antibodies to the surface of a liposome.
  • individual protein G ' molecules are linked to the liposome, and to targeting antibodies as described above and to macrophage avoidance moieties.
  • the sialic acid of the native antibody molecule functions to inhibit uptake of the entire liposomal complex by the various macrophage systems.
  • native proteinaceous structures of the monoclonal antibody and protein G' contain lysine and cysteine residues that provide reactive sites where additional derivatized sialic acid moieties can be covalently attached to create an enhanced macrophage avoidance system for the entire complex as well as for individual components.
  • the new macrophage avoidance molecules are optimally oriented toward the bulk phase media and are not sterically restricted by other molecules of the liposomal complex.
  • native sialic acid of the liposomal complex functions as an integral structural component of the native antibody, protein G' and the liposomal carrier.
  • the native antibody molecule or nonsense antibodies can be RES enhanced together or separately by the selective addition of derivatized sialic acid molecules.
  • These molecules contain specific maleimido or sulfhydryl functional groups which facilitate sialic acid attachment to proteinaceous molecules as well as to membrane anchoring moieties.
  • the derivatized molecules react with proteinaceous surfaces by utilizing amino acid functional groups on either protein G', monoclonal antibodies, or the liposomal surface. In either circumstance, because of the attachment to protein G', sialic acid is oriented in a sterically correct and optimal manner in order to perform the macrophage masking function.
  • the sialic acid is not intercalated in the liposomal membrane, but rather is bound to an antibody, protein G', or the liposomal surface.
  • the antibody does not coat the liposomal membrane per se, but is oriented about 35 A to about 50 A from the liposomal surface by the appropriate linking moieties. In this manner, macrophage masking is achieved because the sialic acid moiety is optimally spaced and oriented a specific distance from the liposomal surface.
  • the antibody concentrations for cell targeting is limited, this limitation may result in filling the protein G' molecules to less than full capacity.
  • the balance of the binding sites can then be filled by different antibodies directed to other designated antigens on the same cell or by nonsense-antibodies which will serve as RES masking agents.
  • the two available binding sites on protein G' are filled which shield the antibody Fc region from nonspecific binding.
  • One embodiment of the invention comprises the integrated liposomal complex that contains the targeting antibodies, nonsense sialic acid laden antibodies, protein G' molecules, linking moieties, and the liposome carrying the appropriate therapeutic agent.
  • the protein G ' molecules provide a coupling means which creates multiple binding sites for attaching the portion of a variety of antibodies. These antibodies exhibit the necessary cell surface antigen specificity.
  • the flexibility offered by this integrated drug delivery system provides for the attachment of single or multiple numbers of antibody molecules and augments the potential for correct and multiple antibody cell-surface interactions.
  • Another aspect of this invention is the method of forming a liposomal carrier in which sonication energy is applied to a mixture of the liposomal, linking moiety, and connecting moiety components which form the liposomal carrier.
  • the connecting moiety comprises protein G'
  • the liposome has a thiocholesterol moiety.
  • a protein G' construct comprising protein G' bonded to a linking moiety is combined with the liposome, and sonication energy, is applied to the mixture.
  • the sonication energy breaks up the lipid structures into smaller liposome structures, typically about 200 A to about 1500 A in diameter. Consequently, the thiocholesterol groups, which otherwise would not be presented to the external phase media due to the hydrophobicity of the cholesterol, are exposed on the liposome surface.
  • the availability of the thiocholesterol groups is enhanced, to thereby enhance the binding between the linking moiety and the liposome.
  • the liposomal delivery complexes of this invention provide useful agents for pharmaceutical applications for administering an active agent to a host. Accordingly, the complexes of this invention are useful as pharmaceutical compositions in combination with pharmaceutically acceptable carriers. Administration of the complexes described herein can be via any of the accepted modes of administration for the biologically active substances that are desired to be administered. These methods include oral, topical, parenteral, ocular, transdermal, nasal and other systemic or aerosol forms.
  • compositions used may be in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, suspensions, or the like, preferably in unit dosage forms suitable for single admimstration of precise dosages.
  • the pharmaceutical compositions will include the protein G' liposomal complex as described and a pharmaceutical acceptable excipient, and, optionally, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, etc.
  • Topical formulations composed of the protein G' liposomal complexes hereof, penetration enhancers, and other biologically active drugs or medicaments can be applied in many ways.
  • the solution can be applied dropwise, from a suitable delivery device, to the appropriate area of skin or diseased skin or mucous membranes and rubbed in by hand or simply allowed to air dry.
  • a suitable gelling agent can be added to the solution and the preparation can be applied to the appropriate area and rubbed in.
  • the solution formulation can be placed into a spray device and be delivered as a spray. This type of drug delivery device is particularly well suited for application to large areas of skin, to highly sensitive skin or to the nasal or oral cavities.
  • Parenteral adrninistration is generally characterized by injection, either subcutaneously, intramuscularly or intravenously.
  • Injectables can be prepared in conventional forms, either as liquid solution or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like.
  • the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, etc.
  • the amount of active compound administered will, of course, be dependent on the subject being treated, the type and severity of the affliction, the manner of administration the judgment of the prescribing physician.
  • the dosage form is intended to give a sustained-release effect, the total dose given will be integrated over a total time period of the sustained-release device in order to compute the appropriate dose required.
  • effective dosage ranges for specific biologically active substances of interest are dependent upon a variety of factors, and are generally known to one of ordinary skill in the art, some dosage guidelines can be generally defined.
  • the protein prime liposomal component will be suspended in an aqueous solution and generally not exceed 30% (w/v) of the total formulation.
  • the drug component of the formulation will most likely be less than 20% (w/v) of the formulation and generally greater than 0.01% (w/v).
  • topical formulations are prepared in gels, creams or solutions having an active ingredient in the range of from 0.001% to 10% (w/v), preferably 0.01 to 5%, and most preferably about 1% to about 5%. (Of course, these ranges are subject to variation depending upon the potency of the therapeutic agent, and could in appropriate circumstance fall within a range as broad as from 0.001% to 20%.)
  • the total dose given will depend upon the size of the affected area of the skin and the number of doses per day. The formulations be applied as often as necessary, but preferably not more than about three times per day.
  • a pharmaceutically acceptable, non-toxic composition is formed by the incorporation of any of the normally employed excipients, such as, for example, mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, sodium crosscarmellose, glucose, gelatin, sucrose, magnesium carbonate, and the like.
  • excipients such as, for example, mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, sodium crosscarmellose, glucose, gelatin, sucrose, magnesium carbonate, and the like.
  • Such compositions include solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained-release formulations and the like.
  • compositions will take the form of a pill or tablet.
  • the composition will contain along with the active ingredient: a diluent such as lactose, sucrose dicalcium phosphate, or the like; a lubricant such as magnesium stearate or the like; and a binder such as starch, gum acacia, gelatin, polyvinylpyrrolidone, cellulose and derivatives thereof, and the like.
  • Liquid pharmaceutically adrninistrable compositions can, for example, be prepared by dissolving, dispersing, etc. the protein G' liposomal complex as described above and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like to thereby form a suspension.
  • a carrier such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like to thereby form a suspension.
  • the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, or solubilizing agents, pH buffering agents and the like, for example, acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, tiiethanola ine sodium acetate, ttiethanolamine oleate, etc.
  • auxiliary substances such as wetting agents, emulsifying agents, or solubilizing agents, pH buffering agents and the like, for example, acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, tiiethanola ine sodium acetate, ttiethanolamine oleate, etc.
  • auxiliary substances such as wetting agents, emulsifying agents, or solubilizing agents, pH buffering agents and the like, for example, acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, tii
  • Dosage forms or compositions containing active ingredient in the range of 0.005%o 50% with the balance made up from no-toxic carrier may be prepared.
  • the exact composition of these formulations may vary widely depending on the particular properties o the therapeutic agent in question. However, they will generally comprise from 0.01% to 50%, and preferably from 0.05% to 10% active ingredient for highly potent drugs, and fro 25-50% for moderately active drugs.
  • the suspension in for example propylene carbonate, vegetable oils or triglycerides, is preferably encapsulated in a gelatin capsule.
  • Such suspensions and the preparation and encapsulation thereof can be prepared by methods that are disclosed in U.S. Patents Nos. 4,328,245; 4,409,239; and 4,410,545.
  • the suspension may be diluted with a sufficient quantity of a pharmaceutically acceptable liquid carrier, e.g. water, to be easily measured for administration.
  • liquid or semi-solid oral formulations may be prepared by dissolving or dispersing the protein G' liposomal complex in vegetable oils, glycols, triglycerides, propylene glycol esters (e.g., propylene carbonate) and the like, and encapsulating these solutions or suspensions in hard or soft gelatin capsule shells.
  • the percentage of active agent contained in such parenteral compositions is highly dependent on the specific nature thereof, as well as the activity of the compound and the needs of the subject. However, percentages of active ingredient of 0.01% to 10% in solution are employable, and will be higher if the composition is a solid which will be subsequently diluted to the above percentages. Preferably the composition will comprise 0.2 - 2% of the active agent in solution. Nasal solutions of the liposomal complex along or in combination with pharmaceutically acceptable excipients can also be administered.
  • Formulations of the protein G' liposomal complex may also be administered to the respiratory tract as an aerosol for a nebulizer.
  • the particles of the formulation have diameters of less than 50 microns, preferably less than 10 microns.
  • the particular diagnostic or therapeutic agent employed does not impose any significant limitation upon the scope of the invention. Any agent which is susceptible to liposomal entrapment or association, and the delivery of which may benefit from such association, is expected to be useful, e.g. antibiotics, antidepressants, antitumorigenics, antivirals, cytokines, hormones, imaging agents, neurotransmitters, stimulants, and the like.
  • Therapeutic agents include, for example, polyfunctional alkylating agents, such as mecWorethamine, chlorambucil, melphalan, thiotepa, busulfan, cyclophosphamide, ifosfamide; antimetabolites, such as methotrexate, 6-mercaptopurine, 6-thioguanine, 5- fluorouracil, 5-fluorodeoxyuridine, cytarabine, fludarabine, 2- chlorodeoxyadenosine, 2-deoxycoformycin, gemcitabine: antibiotics, such as doxorubicin, bleomycin, dactinomycin, daunorubicin, plicamycin, mitomycin C, mitoxantrone; steroid and hormonally active compounds such as the androgen, fluoxymesterone; the antiandrogen, flutamide; the estrogens, ethinyl estradiol and diethylstilbestrol; the antiestrogen, tamoxifen
  • sulfo-succinimidyl-p-maleimidophenylbutyrate sulfo- SMPB was solubilized in 0.4 ml of PBS buffer with accompanying sonication.
  • Liposomal Components Dipalmitoyl phosphatidyl choline (DPL)
  • Ultrasonic/Cell Disruptor equipped with a transducer and cuphorn which was temperature regulated by a Lauda K-2R heated water bath.
  • Norleu-10 antibody stock solution was prepared at a concentration of 0.5 mg/23 ⁇ l. This solution was then diluted to a total volume of 250 ⁇ l with PBS buffer pH 7.5.
  • the liposomal suspension was then chromatographed over a (1.5 x 25 cm) Sepharose C1-2B-300 column equilibrated with PBS buffer pH 7.5 to remove any free or unbound antibody. 12. Following chromatography fraction #11, which contained 1.5 ml of the liposomal protein G' antibody complex, was determined to have the highest lipid concentration and was used as the incubation media in the LS-18 colon cancer cell culture binding study.
  • the LS-180 colon cancer cell culture study protocol provided for 1 x 10 +5 LS-180 cells to be plated per sample well. After a suitable cell growth phase, the study was started and it was determined that there were 3.3 x 10 +n cells per sample well. Into each experimental well 19.24 x 10 +1 ° liposomes were introduced that averaged 1980 A in diameter. The number of liposomes was determined from a previous calculation that yielded 5.97 x 10 "19 moles of lipid per liposome. The experimental sample well also contained 1.09 ⁇ g of monoclonal antibody which was attached to the liposomal complex. This was equivalent to 7.27 x 10 "12 moles of antibody per sample well or 43.8 x 10 + ⁇ antibodies per well.
  • the reaction media in the sample well contained 22.8 molecules of antibody per liposome.
  • the results of the LS-180 colon cancer cell binding study focused on the comparison of the two methods employed to attach antibodies to the liposomes.
  • the first method used dithiothreitol (DTT), a disulfide bond reducing agent, to cleave some of the disulfide bonds in the Norleu-10 monoclonal antibody in order to expose free sulfhydryl functionalities that could be subsequently attached via a thioether bond to the liposomal membrane anchoring molecule represented by p-maleimidophenylbutyrate phosphatidylethanol amine (MPB-PE).
  • DTT dithiothreitol
  • MPB-PE p-maleimidophenylbutyrate phosphatidylethanol amine
  • the second method used the protein G' construct to attach the monoclonal antibodies to the liposomal membrane.
  • the protein G' liposomes At 4 °C there was 2.8-fold increase in the binding of the protein G' liposomes to the LS-180 colon cancer cells as compared to liposomes expressing DTT-treated antibodies, i.e., 774 ng 14 C CHOL for protein G ' as compared to 276 ng 14 C CHOL for DTT.
  • the tests conducted at 37 °C to determine the extent of internalization of the liposomal carrier show that when protein G' liposomes with attached monoclonal antibodies were compared to liposomes with DTT-treated antibodies, the protein G' complexes expressed a three-fold increase in the number of liposomes internalized into the LS-180 colon cancer cells, i.e., 1076 ng 14 C CHOL for protein G' as compared to 356 ng 14 C CHOL for DTT.

Abstract

On décrit un complexe d'apport par liposome qui permet de transporter à un site spécifique sur la surface d'une cellule un agent pharmacologique avec une meilleure efficacité de ciblage. Le complexe d'apport par liposome comprend de manière générale un liposome, un anticorps, et une fraction de connexion qui se lie à la région Fc de l'anticorps pour lier le liposome à l'anticorps. Dans une forme de réalisation préférée, la fraction de connexion est une protéine G'.
PCT/US1999/011177 1998-05-20 1999-05-19 Complexe d'apport par liposomes WO1999059643A2 (fr)

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