EP2068832A2 - Complexes liposomiques contenant des agents pharmaceutiques et procédés - Google Patents

Complexes liposomiques contenant des agents pharmaceutiques et procédés

Info

Publication number
EP2068832A2
EP2068832A2 EP07811693A EP07811693A EP2068832A2 EP 2068832 A2 EP2068832 A2 EP 2068832A2 EP 07811693 A EP07811693 A EP 07811693A EP 07811693 A EP07811693 A EP 07811693A EP 2068832 A2 EP2068832 A2 EP 2068832A2
Authority
EP
European Patent Office
Prior art keywords
liposome
sialic acid
containing molecule
liposome complex
agent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP07811693A
Other languages
German (de)
English (en)
Inventor
Deepak Thakker
Michael Eric Benz
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Medtronic Inc
Original Assignee
Medtronic Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Medtronic Inc filed Critical Medtronic Inc
Publication of EP2068832A2 publication Critical patent/EP2068832A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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

Definitions

  • Liposomes also known as vesicles, have been designed to encapsulate pharmacological agents useful for in vivo purposes such as the diagnosis and treatment of various diseases and conditions. These cargo-carrying liposomes have experimentally shown potential for being site-specific carrier systems for a variety of such agents. Agents so delivered to designated sites in vivo demonstrate significantly enhanced therapeutic indices. Concurrently, a decrease in unwanted side effects and wasted portions of a pharmaceutical dosage are achieved. The advantages of encapsulation have been offset, however, by the deleterious effects of the body's reticuloendothelial system (RES), mainly the liver and spleen. The reticuloendothelial system acts to screen the body's circulation.
  • RES reticuloendothelial system
  • Liposomes have certain physical characteristics which render them susceptible to removal by the reticuloendothelial system. Once recognized, liposomes, whether given a site-specific molecule for so- called “targeted” delivery or not, are quickly phagocytosed by the reticuloendothelial system along with their cargo.
  • a liposome composition that delivers an encapsulated pharmaceutical agent into specific tissues (e.g., organs), particularly the brain, with reduced or no scavenging by the body's reticuloendothelial system.
  • specific tissues e.g., organs
  • o uivuvi/vix. i KJ ⁇ i ⁇ E A ⁇ v JJ, ⁇ i XKJIV Liposome complexes are provided for site-specific delivery of pharmaceutical agents (e.g., therapeutic or diagnostic agents) to a targeted site with improved efficiency.
  • Such liposome complexes include liposomes having associated therewith one or more types of sialic acid-containing molecules, preferably the sialic acid-containing molecules are attached to the external surface of the liposomes for surface modification.
  • the sialic acid-containing molecules form linkers for attachment of one or more targeting agents to the liposomes.
  • one or more pharmaceutical agents are located within the internal compartment of the liposome.
  • the liposome complexes of the present invention are particularly designed for delivering pharmaceutical agents across the blo ⁇ d-brain barrier, although they can also be used to target other organs such as the heart, liver, kidney, pancreas, etc., or other tissues (e.g., skin, spine).
  • the therapeutic management of almost all debilitating neurological and psychiatric disorders is largely hindered by the restricted access of pharmaceutical agents to the brain, imposed by the microvascular endothelial blood-brain barrier (BBB).
  • BBB microvascular endothelial blood-brain barrier
  • Preferred liposome complexes of the present invention enable a trans- vascular access and widespread distribution of a broad range of pharmaceutical chemical-based compounds, into the brain.
  • liposomal encapsulation protects the pharmaceutical agent from enzymatic degradation in vivo.
  • conjugation of anionic, non- immunogenic polysialic acids (PSAs) to the liposomal surface reduces and/or restricts the reticuloendothelial uptake of the liposomes and extends their viable time in the bloodstream.
  • PSAs anionic, non- immunogenic polysialic acids
  • binding the liposomal PSAs to one or more targeting agents facilitates entry into the brain.
  • targeting agents e.g., monoclonal antibodies, antibody fragments, or peptide analogues that specifically recognize receptors expressed in the brain micro vasculature
  • the liposome complex includes: a liposome having an exterior surface and an internal compartment; a pharmaceutical agent (e.g., a therapeutic agent or a diagnostic agent) associated with the liposome (in certain embodiments, located within the internal compartment of the liposome); a sialic acid-containing (e.g., terminated) molecule associated with the liposome (in certain embodiments, the sialic acid-containing molecule is attached at the external surface of the liposome); and a targeting agent; wherein the sialic acid-containing molecule forms a linker between the targeting agent and the external surface of the liposome.
  • a pharmaceutical agent e.g., a therapeutic agent or a diagnostic agent
  • the liposome complex includes: a liposome having an exterior surface and an internal compartment; a pharmaceutical agent (e.g., a therapeutic agent or a diagnostic agent) associated with the liposome (in certain embodiments, located within the internal compartment of the liposome); a sialic acid-containing (e.g., terminated) molecule associated with the liposome (in certain embodiments, the sialic acid-containing molecule is attached at the external surface of the liposome); and optionally a targeting agent; wherein the sialic acid-containing molecule comprises polysialic acid having a degree of polymerization of at least 8 (particularly when the complex does not include a targeting agent).
  • a pharmaceutical agent e.g., a therapeutic agent or a diagnostic agent
  • the liposome is prepared from phospholipids selected from the group consisting of phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholines, phosphatidylglycerol, phosphatidic acid, phosphatidylmethanol, cardiolipin, ceramide, cholesterol, cerebroside, lysophosphatidylcholine, D-erythrosphingosine, sphingomyelin, dodecyl phosphocholine, N-biotinyl phosphatidylethanolamine, and combinations thereof.
  • a lipid head-group e.g., a phospholipid
  • sialic acid group e.g., sialic acid group.
  • the sialic acid-containing molecule is selected from the group consisting of capsular polysialic acid, sialic acid-containing gangliosides, colominic acid, and combinations thereof.
  • the sialic acid-containing molecule is a capsular polysialic acid.
  • the targeting agent is selected from the group consisting of peptides, peptide analogs, antibodies, antibody fragments, small organic or inorganic molecules, and combinations thereof.
  • the targeting agent is a compound that specifically recognizes receptors expressed in the brain microvasculature.
  • the liposome complex includes two or more different pharmaceutical agents.
  • the present invention also provides a pharmaceutical composition that includes a liposome complex of the present invention and an optional pharmaceutically acceptable carrier.
  • a pharmaceutical composition can also include a pharmaceutical agent that is not a part the liposome complex, which may be the same or different than the pharmaceutical agent associated with the liposome.
  • a pharmaceutical composition can crosslink, gel, or change in viscosity on or after administration to a subject.
  • a pharmaceutical composition can provide for delayed or extended release of the liposome complex after administration to a subject.
  • a pharmaceutical composition can include two or more different liposome complexes, each containing different pharmaceutical agents and/or targeting moieties.
  • the present invention also provides methods of treating and/or preventing an affliction by administering a liposome complex of the present invention or pharmaceutical composition containing such complex to a subject.
  • the affliction can be a psychiatric disorder, a neurological disorder, a disease affecting and/or originating in the brain, a disorder of the cardiac system, a disorder of the hepatic system, a disorder of the vascular system, a disorder of the orthopedic system, or combinations thereof.
  • agent to a subject by administering a liposome complex of the present invention or pharmaceutical composition containing such complex to the subject can be a psychiatric disorder, a neurological disorder, a disease affecting and/or originating in the brain, a disorder of the cardiac system, a disorder of the hepatic system, a disorder of the vascular system, a disorder of the orthopedic system, or combinations thereof.
  • the present invention also provides methods of making a liposome complex.
  • One method includes: preparing a liposome having an exterior surface and an internal compartment, a pharmaceutical agent associated therewith, and a sialic acid- containing molecule associated therewith; and attaching a targeting agent to the sialic acid-containing molecule; wherein sialic acid-containing molecule forms a linker between the targeting agent and the external surface of the liposome.
  • Another method includes: preparing a liposome having an exterior surface and an internal compartment, a pharmaceutical agent associated therewith, and a sialic acid-containing molecule associated therewith; and optionally attaching a targeting agent to the sialic acid-containing molecule; wherein the sialic acid- containing molecule comprises polysialic acid having a degree of polymerization of at least 8.
  • preparing a liposome having an exterior surface and an internal compartment and a pharmaceutical agent associated therewith involves encapsulating the pharmaceutical agent within the internal compartment of the liposome.
  • the step of preparing a liposome having an exterior surface and an internal compartment and a sialic acid-containing molecule associated therewith involves attaching a sialic acid-containing molecule to the external surface of the liposome during or after encapsulation of the pharmaceutical agent.
  • the method further includes a step of attaching a targeting agent to the sialic acid-containing molecule before, during, or after attaching the sialic acid- containing molecule to the external surface of the liposome.
  • sialic acid-containing molecule associated with the liposome means that such molecules are included within the structure of the liposome, through covalent bonds or through non-covalent interactions.
  • a polysialic acid unit could be incorporated into a liposome through non- bonded interactions by the presence of a lipid or lipid-like head group, as is found in the naturally occurring capsular polysialic acids. that such agent can be included within the structure of the liposome in a variety of manners.
  • association can be accomplished through encapsulation within the internal compartment of the liposome, or through attachment to the outer surface of the liposome through bonding or nonbonding interactions, or through intercalation between the double layer of lipid head groups, or through other methods known in the art of drug delivery using liposomes.
  • liposome refers to a vesicle (generally spherical in shape) in an aqueous medium, formed by a lipid bilayer enclosing an aqueous compartment.
  • sialic acid refers to the N-acyl derivative of neuraminic acid.
  • targeting agent refers to a compound that binds to a specific site in the body.
  • pharmaceutical agent refers to a therapeutic agent and/or a diagnostic agent.
  • a liposome complex that comprises “a” pharmaceutical agent can be interpreted to mean that the liposome complex includes “one or more” pharmaceutical agents.
  • the te ⁇ n "and/or” means one or all of the listed elements (e.g., preventing and/or treating an affliction means preventing, treating, or both treating and preventing further afflictions). disclosed embodiment or every implementation of the present invention.
  • the description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.
  • FIG. 1 is a representation of a polysialylated immuno-liposome encapsulating a therapeutic, expression plasmid DNA, and having the distal ends of liposome-conjugating polysialic acid (PSA) strands conjugated to a monoclonal antibody, which specifically recognizes a BBB endothelial receptor.
  • PSA polysialic acid
  • FIG. 2 depicts the size distribution of liposome complexes with a mean size of 150 nm (standard deviation of 30 nm). These complexes encapsulate a plasmid DNA that enables the expression of green fluorescent protein (GFP), and incorporate sialic acid-containing molecules (gangliosides), which are further conjugated to a monoclonal antibody that specifically recognizes the transferrin receptor known to express at high levels in the BBB endothelium.
  • FIG. 3 is a representative of an agarose gel electrophoresis performed in order to confirm the encapsulation of plasmid DNA by liposome complexes.
  • FIG. 4 is a representative transmission electron microscopy image of exemplary liposome complexes of the present invention.
  • transferrin receptor- targeting monoclonal antibodies tethered to sialic acid-containing molecules are bound by an anti-mouse secondary antibody, which is in turn conjugated 10 nm gold.
  • the position of gold particles indicates the association of transferrin receptor- targeting monoclonal antibody with the liposome complexes.
  • FIG. 5 demonstrates the expression of GFP in mouse Neuro2a neuroblastoma cells upon treatment with liposome complexes that encapsulate the GFP-expressing plasmid DNA.
  • Image A depicts the GFP-expressing cells brightly lit upon excitation required for visualizing GFP.
  • Image B is a corresponding bright- field image. Scale bar: 10 microns or micrometers ( ⁇ m).
  • the invention provides a liposome complex for delivering a pharmaceutical agent (e.g., a diagnostic and/or therapeutic agent), and preferably a therapeutic agent, to a targeted area in a subject.
  • a pharmaceutical agent e.g., a diagnostic and/or therapeutic agent
  • the liposome complex includes a liposome having an exterior surface and an internal compartment, at least one pharmaceutical agent associated with the liposome (in certain embodiments, one or more pharmaceutical agents are located within the internal compartment of the liposome), and at least one sialic acid-containing molecule that is associated with the liposome.
  • the liposome complex also includes at least one targeting agent, wherein the at least one targeting agent binds to a specific site in the body of the subject and is attached to (e.g., covalently bonded to) the at least one sialic acid-containing molecule (i.e., the sialic acid-containing molecule forms a linker between the targeting agent and the external surface of the liposome).
  • Liposome complexes of the present invention can be used to target various organs (e.g., brain, heart, liver, kidney, pancreas) or other tissues of the body (e.g., skin, spine).
  • organs e.g., brain, heart, liver, kidney, pancreas
  • other tissues of the body e.g., skin, spine
  • a liposome complex can be administered to a subject for preferential uptake by an organ of interest.
  • the liposome complexes of the present invention are particularly designed for delivering pharmaceutical agents across the blood-brain barrier.
  • the blood-brain barrier (BBB) poses a central problem in the unresponsiveness of almost all debilitating neurological disorders to conventional drug therapy.
  • the liposomes can form nanocontainers, such as nanoparticles, and are commonly used for encapsulation of pharmaceutical agents. They are typically spherical in shape, and preferably have an average particle size (i.e., the average of the longest dimension, which is the diameter for spherical particles) of no greater than 1000 nanometers (nm). Liposomes having an average particle size of 50 nm are desired, although other sizes may also be useful for crossing the blood brain barrier.
  • Suitable types of liposomes may be prepared from, for example, phospholipids selected from the group consisting of phosphatidyl serine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholines, phosphatidylglycerol, phosphatidic acid, phosphatidylmethanol, cardiolipin, ceramide, cholesterol, cerebroside, lysophosphatidylcholine, D-erythrosphingosine, synthetic analogs of these molecules, derivatives of these molecules, and combinations thereof.
  • phospholipids selected from the group consisting of phosphatidyl serine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholines, phosphatidylglycerol, phosphatidic acid, phosphatidylmethanol, cardiolipin, ceramide, cholesterol, cerebroside, lysophosphatidylcholine, D-erythros
  • Liposomes may be prepared according to any of the well known conventional processes. For example, liposomes may be made by depositing a thin film of lipid on the inner wall of a flask, adding an aqueous phase, and shaking vigorously (e.g., by hand). Another method may include, for example, sonication of a lipid film in an aqueous solution, followed by extrusion through a series of filters (e.g., of decreasing pore size). Yet another method of making liposomes is to dialyze an aqueous solution of lipids in the presence of a detergent such as sodium cholate. As the detergent is depleted, the lipids form liposomes.
  • a detergent such as sodium cholate
  • Still another method is based on high pressure homogenization of a lipid solution using commercially available equipment. Additional methods may include, for example, re-hydration of freeze-dried vesicles and reverse-phase evaporation. Descriptions and protocols for these methods are well known to those of skill in the art. See, for example,
  • Liposomes A Practical Approach (2 nd edition, 2003), edited by Vladimir Torchilin and Volkmar Weissig, Oxford University Press, Oxford, UK. Materials for making liposomes are commercially available, for example, from Avestin Inc., Ottawa, Canada, Microfluidics, a division of MFIC Corp., Newton, MA, and Harvard Apparatus, a Harvard Bioscience, Inc. company, Holliston, MA.
  • One or more pharmaceutical agents may be associated with a liposome, such as encapsulated within a liposome, using a wide variety of mechanisms, including encapsulation within the internal compartment of the liposome, or attachment to the outer surface of the liposome through bonding or nonbonding interactions, intercalation between the double layer of lipid head groups, and the like.
  • methods of associating one or more pharmaceutical agents with liposomes include, but are not limited to: encapsulating an agent within the aqueous core of the liposome, which can occur by preparing the liposome in the presence of the agent; causing a non-bonded interaction (e.g., van der Waals) between an agent and the hydrophilic tail of a lipid used to form the liposome, either within the core or at the outer surface of the liposome; causing an interaction between an agent and the lipid head group of a lipid used to form the liposome; intercalating an agent between the double layer of lipid head groups in a liposome; bonding (e.g., covalent, ionic, or hydrogen bonding) an agent to a molecule that makes up the structure of the and/or causing complex formation between an agent and a cationic salt that may be a part of the structure of the liposome.
  • a non-bonded interaction e.g., van der Waals
  • agents employed do not impose any significant limitation upon the scope of the invention.
  • a wide variety of agents can be used. Such agents that are particularly susceptible to liposomal entrapment or association are useful. Such agents can be for therapeutic and/or diagnostic purposes.
  • agents include oligonucleotide-based compounds (e.g., DNA and RNA), amino acid- based compounds (e.g., proteins and peptides), polysaccharides, small molecule organic and inorganic compounds, organometallic species, and the like.
  • Various combinations of pharmaceutical agents may be incorporated into liposomes according to the present invention.
  • Therapeutic agents include, for example, antibiotics, antidepressants, antitumorigenics, antivirals, cytokines, hormones, imaging agents, neurotransmitters, nucleic acids, stimulants, regulating agents that turn genes and/or protein production on or off, and the like.
  • Suitable therapeutic agents may include, for example, poly-functional alkylating agents, such as mechlorethamine, chlorambucil, melphalan, thiotepa, busulfan, cyclophosphamide, ifosfamide; antimetabolites, such as methotrexate, 6-mercaptopurme, 6-thioguanme, 5-fluorouracil, 5-fluorodeoxyuridine, cytarabine, fludarabine, 2-chlorodeoxyadenosine, 2-deoxycoformycin, genicitabine: 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,
  • Diagnostic agents include, for example, metals (e.g., gadolinium) that can be detected by MRI, agents for enhancing radiopacity (e.g., barium), radioisotopes, and the like. Techniques of using liposomes with diagnostic agents are known in the art and described, for example, in Arti, J.Clin. Oncol., 24, 3299-3308 (2006).
  • the number of pharmaceutical agents associated with (e.g., encapsulated within) the liposomes may vary from one to many, depending on the desired result (e.g., the affliction being treated).
  • liposome complexes may include at least one pharmaceutical agent at a suitable level to produce the desired result.
  • sialic acid-containing molecules are associated with liposomes according to the present invention.
  • the sialic acid- containing molecule(s) are attached to the liposome external surface after the liposomes are prepared.
  • sialic acid-containing molecules such as polysialic acids, can be used to make the liposomes and thereby be incorporated into the structure of the liposome during preparation of the liposome.
  • Various combinations of such mechanisms of associating sialic acid-containing molecules can be used in any one liposome complex.
  • the liposome complexes of the present invention experience less scavenging by the body's reticulo-endothelial system than occurs with liposomes that do not have sialic acid-containing molecules associated therewith. While not being limited to theory, it is believed that while the liposomal encapsulation reduces and even prevents enzymatic degradation of the pharmaceutical agent in vivo, the hydrophilic nature of the sialic acid-containing molecule forms a protective "watery" coating around the liposome, thereby providing protection from reticulo-endothelial uptake.
  • this combination of liposome and sialic acid surface modification increases the blood residence time of the pharmaceutical agent.
  • sialic acid-containing molecules present a negligible risk of generating antigenic or immunogenic responses and are generally biodegradable, in contrast to currently employed polymers such as poly(ethylene glycol). link one or more targeting agents to the external surface of the liposome.
  • one or more targeting agents can be directly attached to the liposomes or attached to linkers other than sialic acid-containing molecules.
  • Various combinations of attaching targeting agents to the liposomes can be used in any one liposome complex.
  • the sialic acid-containing molecule may include, for example, a wide variety of materials that include sialic acid, polysialic acid, sialic acid analogues and polysialic acid analogues (such analogues are materials containing synthetically modified sialic acid), phospholipids and polysaccharides containing sialic acid units
  • sialic acid-containing molecules can be used if desired.
  • the sialic acid-containing molecule may be oligomeric or polymeric and include at least 2 sialic acid units, more preferably at least 4 sialic acid units, and even more preferably at least 8 sialic acid units.
  • Such materials are also referred to as "polysialic acids," or polymers of sialic acid whose degree of polymerization (DP) is preferably at least 2, more preferably at least 4, and even more preferably at least 8.
  • DP degree of polymerization
  • the number of sialic acid units is no greater than 1000, and in some embodiments no greater than 200.
  • the sialic acid-containing molecule includes, for example, a lipid head-group and a sialic acid group.
  • the lipid head-group may be a terminal phospholipid group for insertion into the lipid layer.
  • Suitable terminal phospholipids groups may include, for example, those listed above, and combinations thereof.
  • sialic acid-containing molecules may include, for example, capsular polysialic acid, sialic acid-containing gangliosides, colominic acid (i.e., a polysialic acid in which all sialic acid residues are linked in 2->8 fashion), and combinations thereof.
  • the sialic acid-containing molecules are anionic polymers of N-acetylneuraminic acid.
  • Capsular polysialic acids are generally referred to by reference to the microorganisms that produce them. Suitable examples include Serogroup B polysialic acid from N. meningitidis C, and polysialic acid from E. coli K92, which are abbreviated as PSB, PSC, and PSK92, respectively. See, for example, Gregoriadis et al., FEBS Letter, 315(3), January 1993, pp 271-76. Synthetic analogs of capsular polysialic acid are also suitable. One such analog is described as a twin- tailed, lipid-linked polysialic acid in Matthews et al., Biopolymers, 33, pp 453-7 (1993).
  • Sialic acid-containing gangliosides are commercially available from sources such as Avanti Polar Lipids, Alabaster, AL.
  • sources such as Avanti Polar Lipids, Alabaster, AL.
  • One such product has the structure
  • Attachment of the sialic acid-containing molecule(s) to the liposome external surface can occur during or after encapsulation of the pharmaceutical agent(s).
  • Suitable methods for attaching the sialic acid-containing molecule to the liposome may include, for example, the method described in the articles described above.
  • the liposome complexes of the present invention may include at least one sialic acid-containing molecule at a suitable level to produce the desired result. More than one type of sialic acid-containing molecule can be used if desired.
  • targeting agents in the liposome complexes. For example, to provide transport of the liposomes that contain pharmaceutical agent(s) across the blood-brain barrier containing molecule(s).
  • the targeting agent is selected from the group consisting of peptides, peptide analogs, antibodies (whether monoclonal or polyclonal) or fragments thereof, small organic or inorganic or organometallic molecules (i.e., compounds or portions thereof), and combinations thereof. More preferably, the targeting agent is a compound or a portion thereof that specifically recognizes receptors expressed in the brain microvasculature (e.g., receptors for bradykinin, insulin, insulin-like growth factors, leptin, low-density lipoproteins, and transferrin).
  • receptors expressed in the brain microvasculature e.g., receptors for bradykinin, insulin, insulin-like growth factors, leptin, low-density lipoproteins, and transferrin.
  • Targeting agents may be endogenous peptide ligands of the receptors, analogs of the endogenous ligands, or peptidomimetic monoclonal antibodies (MAbs such as 8D3, Rl 7, and OX26 antibodies) that bind the same receptor of the endogenous ligand.
  • MAbs such as 8D3, Rl 7, and OX26 antibodies
  • FIG. 4 One such example is depicted by Figures 4 and 5, wherein the targeting agent for liposomal complexes is a monoclonal antibody that recognizes the transferrin receptor.
  • Neuro2a neuronal cells express transferrin receptors on their surface that enable the delivery of liposome-encapsulated plasmid DNA in these cells. Such a delivery is abolished upon downregulation of the cell surface transferrin receptors.
  • Suitable targeting agents may include, for example, insulin, transferrin, insulin-like growth factors, and leptin. Such compounds or portions thereof facilitate the receptor-mediated transcytosis of the liposome complexes of the present invention across the BBB, and subsequent entry into the brain.
  • a molecule that includes sialic acid could be conjugated with two different targeting agents, one peptide targeting an endogenous blood-brain barrier receptor (such as those discussed above) and the other targeting an endogenous brain cell membrane peptide.
  • the latter could be specific for particular cells within the brain, such as neurons, glial cells, pericytes, smooth muscle cells, or microglia.
  • the latter could also be added to enhance the cellular uptake, e.g., cell-penetrating peptides that include those described in Dietz et al., MoI. & Cell. Neurosci., 27, 85- 131 (2004). Accordingly, therapeutic delivery can further be exacted to specific cell organ, for example).
  • Attachment of the targeting agent(s) to the liposome external surface can occur before, during, or after the sialic acid-containing molecule(s) are associated with to the liposomes.
  • Suitable methods for attaching the targeting agent(s) to the sialic acid-containing molecule(s) may include, for example, generation of an aldehyde on the terminal sialic acid unit (e.g., by reaction with sodium periodate), which may react with an amine of the targeting agent to yield an imine. This imine may be subsequently reduced by a reducing agent such as sodium borohydride to yield a hydrolytically stable secondary or tertiary amine.
  • the liposome complexes of the present invention may include at least one targeting agent at a suitable level to produce the desired result. More than one type of targeting agent can be used if desired.
  • Figure 1 shows a particular example of one embodiment of the present invention in which a liposome complex encapsulates a therapeutic, expression plasmid DNA.
  • Distal ends of the liposome-conjugating polysialic acid (PSA) strands are further conjugated to a monoclonal antibody, which specifically recognizes a BBB endothelial receptor.
  • PSA polysialic acid
  • the invention also provides methods of making liposome complexes for delivering a pharmaceutical agent.
  • such methods typically involve preparing a liposome having an exterior surface and an internal compartment and associating a pharmaceutical agent with the liposome (preferably, incorporating a pharmaceutical agent within the internal compartment of the liposome).
  • a sialic acid-containing molecule can be associated with (e.g., attached at the external surface of) the liposome during or after association of the pharmaceutical agent.
  • a targeting agent can be attached to the sialic acid- containing molecule before, during, or after association of the sialic acid-containing molecule with the liposome.
  • the invention also provides methods of in vivo administration of a liposome complex of the present invention for delivering a pharmaceutical agent to a subject.
  • administration could be used for treating and/or preventing one or more afflictions such as a psychiatric or neurological disorder or any disease affecting and/or originating in the brain such as Parkinson's Disease and orthopedic systems.
  • liposome complexes could be used, each containing a different pharmaceutical agent, for desired effect.
  • various combinations of pharmaceutical agents (not associated with a liposome) and liposome complexes of the present invention could be administered to a subject.
  • a pharmaceutical agent could be added to the delivery solution that contains a liposome complex of the present invention.
  • the liposome complex is administered intra-vascularly and crosses the blood brain barrier, which is particularly desirable for preventing and/or treating neurological disorders.
  • the liposome complexes of this invention provide useful mechanisms for pharmaceutical applications for administering a therapeutic or diagnostic agent to a subject. Accordingly, the liposome complexes of this invention are useful as pharmaceutical compositions, optionally in combination with pharmaceutically acceptable carriers. Such pharmaceutical compositions can include two or more types of liposome complexes, each containing different pharmaceutical agents and/or targeting moieties. Such pharmaceutical compositions can also include one or more pharmaceutical agents that are not a part of the liposome complex. Liposome complexes of the present invention can be included in pharmaceutical compositions that crosslink, gel, or change in viscosity on or after administration to a subject. This can occur, for example, through covalent bond formation, hydrogen bond formation, pH change, temperature change, complex formation, and the like.
  • Liposome complexes of the present invention can be included in pharmaceutical compositions that provides for delayed or extended release of the liposome complex, and, hence, the pharmaceutical agent(s) associated therewith after administration to a subject. This can occur, for example, using pH sensitivity as a trigger for release.
  • Administration of the liposome 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, although IV administration is preferred.
  • compositions 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 administration of precise dosages.
  • the pharmaceutical compositions will include the liposome complexes as described herein and optionally a pharmaceutical acceptable excipient, other medicinal agents, pharmaceutical agents, carriers, adjuvants, etc.
  • Topical formulations composed of the liposome complexes described herein, 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 and 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.
  • 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%.
  • 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 may 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 stearate, and the like; and a binder such as starch, gum acacia, gelatin, polyvinylpyrrolidone, cellulose and derivatives thereof, and the like.
  • Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, etc., the liposomes 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, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, etc.
  • nontoxic auxiliary substances such as wetting agents, emulsifying agents, or solubilizing agents, pH buffering agents, and the like, for example, acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, etc.
  • composition or formulation to be administered will, in any event, contain a quantity of the active pharmaceutical agent in an amount sufficient to effectively treat the disorder or disease of the subject being treated.
  • the suspension in, for example, propylene carbonate, vegetable oils or triglycerides, is preferably encapsulated in a gelatin capsule.
  • a gelatin capsule Such suspensions and the preparation and encapsulation thereof, can be prepared by methods that are well known to those of skill in the art.
  • 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 liposome complexes 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.
  • Nasal solutions of the liposome complexes alone or in combination with pharmaceutically acceptable excipients can also be administered.
  • Formulations of the liposome complexes 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.
  • DNA-containing liposomes composed of POPC (l-palmitoyl-2-oleoyl-sn- glycerol-3-phosphocholine), DDAB (didodecyldimethylammonium bromide), and ganglioside were prepared at a ratio of 82: 15:3. Lipid films were first created and then rehydrated.
  • POPC l-palmitoyl-2-oleoyl-sn- glycerol-3-phosphocholine
  • DDAB didodecyldimethylammonium bromide
  • ganglioside were prepared at a ratio of 82: 15:3. Lipid films were first created and then rehydrated.
  • Each lipid was separately dissolved in chloroform/methanol in a 10 milliliters (mL) round-bottomed flask with a 20/24 outer joint as follows: To 16.4 micromoles ( ⁇ mol) (or 12.42 milligrams (mg)) of POPC in chloroform (purchased from Avanti Polar Lipids, Alabaster, AL), additional chloroform (583.8 ⁇ L) and methanol (583.8 microliters ( ⁇ L)) were added to adjust the total chloroform :mcthanol ratio to 2:1.
  • Green fluorescent protein (GFP)-encoding supercoiled plasmid DNA 100 micrograms ( ⁇ g)
  • pEGFP-Cl cationic lipid solution
  • pEGFP-Cl cationic lipid solution
  • dialysis membranes of 5000 Dalton MWCO (all purchased from Harvard Apparatus, Hollister, MA) per manufacturer's instructions, for at least 48 hours, changing the dialysis buffer (containing 20 mM HEPES and 140 mM NaCl, pH 7.5) at least twice a day.
  • the liposome/DNA complexes were then sequentially extruded using a mini- extruder (Product No. 610000, purchased from Avanti Polar Lipids) and 400 nm, 200 nm, and 100 nm polycarbonate membranes (all purchased from Avanti Polar Lipids) per the manufacturer's instructions.
  • the resultant liposomes produced were in the size range of 100-200 nm, as measured by the Partica LA-950 laser diffraction particle size analyzer (purchased from Horiba Jobin Yvon, Edison, NJ).
  • Figure 2 depicts the size distribution of liposome complexes with a mean size of 150 nm (standard deviation of 30 nm).
  • Unencapsulated DNA was removed by treating the liposome/DNA complexes with nuclease digestion, based on the protocol described in the example of U.S. Patent No. 6,372,250.
  • Lane 5 (Positive control for lane 1) Complexes made using a commercially available reagent L1POFECTAMINE 2000 (Invitrogcn, Carlsbad, CA) to encapsulate the GFP-expressing plasmid DNA per the manufacturer's instructions.
  • Lane 6 (Positive control for lane 2) L1POFECTAMINE 2000 complexes lyzed, using the same detergent as described earlier, to release the encapsulated GFP- expressing plasmid DNA.
  • Sialic acid residues on the surface of liposomes were conjugated to a monoclonal antibody (ACL8971, purchased from Accurate Chemical & Scientific, Westbury, NY) targeting the transferrin receptor that is highly expressed in the blood brain barrier.
  • ACL8971 monoclonal antibody
  • the liposome suspension (approximately 1.0 mL) was incubated with sodium periodate solution (0.2 mL, 0.2 M in water) in a brown vial at the ambient temperature for 30 minutes. The material was then transferred to a 10,000 molecular weight cut off Slide-A-Lyzer (purchased from Pierce, Rockford, IL) and dialyzed overnight (approximately 24 hours) against borate buffer (1 L, 20 mM sodium borate Na 2 B 4 O 7 .10H 2 O with 120 mM NaCl, pH 8.4).
  • borate buffer (1 L, 20 mM sodium borate Na 2 B 4 O 7 .10H 2 O with 120 mM NaCl, pH 8.4
  • the material (approximately 1.4 mL) was transferred to a vial and mixed with 1.0 mL transferrin receptor antibody (ACL8971 ).
  • ACL8971 transferrin receptor antibody
  • sodium cyanoborohydride solution (0.4 mL, 2 M in water) was added. The materials were shook well and stored at 4 0 C overnight.
  • the DNA-encapsulating liposomes (50 ⁇ L) were added to approximately 70% confluent mouse Neuro2a neuroblastoma cells (American Type Culture Collection, Manassas, VA) plated in a 6- well plate with 1 mL of culture media. After approximately 48 hours of culturing at 37°C, cells were imaged under a fluorescent microscope to visualize the green fluorescence emitted by cells expressing GFP encoded by the DNA from liposomes (Figure 5). Image A depicts Image B is a corresponding bright-field image. Scale bar: 10 ⁇ m.
  • the same transfection procedure was followed except that the cells were initially cultured for 24 hours with 1 ⁇ g/mL of transferrin receptor antibody that was not conjugated to the liposomes.
  • This pre-treatment leads to a substantial downregulation of transferrin receptors on the surface of Neuro2a cells.
  • incubation of such Neuro2a cells with the DNA-containing liposomes conjugated to the transferrin receptor antibody did not result in any GFP-expressing cells, suggesting the requirement of transferrin receptors on the cell surface for an uptake of DNA-encapsulating liposomes conjugated to the transferrin receptor antibody.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Dispersion Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Preparation (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne des complexes liposomiques destinés à la délivrance d'agents pharmaceutiques dans des sites spécifiques avec une efficacité de ciblage améliorée. Les complexes liposomiques incluent un agent pharmaceutique associé au liposome ; une molécule contenant de l'acide sialique associée au liposome ; et facultativement un agent de ciblage fixé à la molécule contenant de l'acide sialique. L'invention concerne également des procédés de préparation des complexes liposomiques et leur administration in vivo.
EP07811693A 2006-09-11 2007-09-06 Complexes liposomiques contenant des agents pharmaceutiques et procédés Withdrawn EP2068832A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US84380806P 2006-09-11 2006-09-11
PCT/US2007/019449 WO2008033253A2 (fr) 2006-09-11 2007-09-06 Complexes liposomiques contenant des agents pharmaceutiques et procédés

Publications (1)

Publication Number Publication Date
EP2068832A2 true EP2068832A2 (fr) 2009-06-17

Family

ID=39184261

Family Applications (1)

Application Number Title Priority Date Filing Date
EP07811693A Withdrawn EP2068832A2 (fr) 2006-09-11 2007-09-06 Complexes liposomiques contenant des agents pharmaceutiques et procédés

Country Status (2)

Country Link
EP (1) EP2068832A2 (fr)
WO (1) WO2008033253A2 (fr)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2100597A3 (fr) * 2008-03-11 2009-12-30 Medtronic, Inc. Complexes de liposomes
WO2010049562A1 (fr) 2008-10-28 2010-05-06 Universidade De Santiago De Compostela Systèmes nanoparticulaires élaborés à base de polymères anioniques
EP2488210A4 (fr) 2009-10-12 2014-04-30 Smith Holdings Llc Procédés et compositions de modulation de l'expression génique en utilisant des médicaments à base d'oligonucléotides administrés in vivo ou in vitro
WO2011149461A1 (fr) * 2010-05-27 2011-12-01 Medtronic, Inc. Anticorps anti-amyloïde bêta conjugués à des molécules contenant de l'acide sialique
ES2403544B2 (es) 2011-11-11 2013-12-13 Universidade De Santiago De Compostela Sistemas nanoparticulares elaborados a base de ésteres de sorbitán.
EP3501495A1 (fr) 2017-12-21 2019-06-26 InnoMedica Holding AG Liposomes contenant de la sphingomyéline
CN112741821B (zh) * 2021-01-15 2023-03-24 厦门诺康得生物科技有限公司 一种唾液酸纳米颗粒及其制备方法和应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5567432A (en) * 1991-08-02 1996-10-22 Lau; John R. Masking of liposomes from RES recognition
AU4090299A (en) * 1998-05-20 1999-12-06 Sdg, Inc. Liposomal delivery complex
AU1776801A (en) * 1999-11-24 2001-06-04 Liposome Company, Inc., The Modular targeted liposomal delivery system
EP1341497A4 (fr) * 2000-11-02 2005-10-19 Smithkline Beecham Corp Conjugues antagoniste du recepteur-lipide et vehicules d'apport contenant lesdits conjugues

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2008033253A3 *

Also Published As

Publication number Publication date
WO2008033253A3 (fr) 2008-08-07
WO2008033253A2 (fr) 2008-03-20

Similar Documents

Publication Publication Date Title
Chen et al. Nanobowl-supported liposomes improve drug loading and delivery
US11406597B2 (en) Fusogenic liposome-coated porous silicon nanoparticles
US9655848B2 (en) Liposomes for in-vivo delivery
US5785987A (en) Method for loading lipid vesicles
Sheikholeslami et al. Exploring the impact of physicochemical properties of liposomal formulations on their in vivo fate
Lai et al. Molecular design of layer-by-layer functionalized liposomes for oral drug delivery
EP2068832A2 (fr) Complexes liposomiques contenant des agents pharmaceutiques et procédés
US20080213349A1 (en) Liposome Complexes Containing Pharmaceutical Agents and Methods
US20180161274A1 (en) Liposome Compositions Encapsulating Modified Cyclodextrin Complexes and Uses Thereof
WO2005094265A2 (fr) Agents d'activation a auto-assemblage cibles au moyen d'une liberation de medicaments actifs
Zhao et al. Tuning the membrane fluidity of liposomes for desirable in vivo fate with enhanced drug delivery
JP2010518012A (ja) カンプトテシン誘導体を含む医薬組成物
González-Rioja et al. The development of highly dense highly protected surfactant ionizable lipid RNA loaded nanoparticles
US20100209494A1 (en) Liposomes and Uses Thereof
Binaymotlagh et al. Liposome–Hydrogel Composites for Controlled Drug Delivery Applications
JP5253716B2 (ja) ｐＨ応答性分子集合体
US20160271072A1 (en) Polymer nanoparticles containing multiple agents and methods thereof
JP4827277B2 (ja) リポソーム
JP2020521004A (ja) ｃ（ＲＧＤ−ＡＣＰ−Ｋ）修飾血中滞留型リポソーム
Singh et al. Cubosomes: An Emerging and Promising Drug Delivery System for Enhancing Cancer Therapy
Gan et al. Liposomal Nanomaterials: A Rising Star in Glioma Treatment
EP2100597A2 (fr) Complexes de liposomes
CN109568598A (zh) 用于药物流产的胎盘靶向纳米颗粒及其制备方法和应用
Abriata et al. Liposomal Drug Carriers: Principles, Formulation Perspectives, and Potentials for Brain Drug Delivery
Afonin et al. OPEN ACCESS EDITED BY

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20080331

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC MT NL PL PT RO SE SI SK TR

DAX Request for extension of the european patent (deleted)
17Q First examination report despatched

Effective date: 20121106

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20130319