WO2005074497A2 - Vesicules d'encapsulation renfermant des agents d'activation a auto-assemblage - Google Patents

Vesicules d'encapsulation renfermant des agents d'activation a auto-assemblage Download PDF

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Publication number
WO2005074497A2
WO2005074497A2 PCT/US2005/001927 US2005001927W WO2005074497A2 WO 2005074497 A2 WO2005074497 A2 WO 2005074497A2 US 2005001927 W US2005001927 W US 2005001927W WO 2005074497 A2 WO2005074497 A2 WO 2005074497A2
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recited
encapsulation
activation
agents
vesicle
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PCT/US2005/001927
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WO2005074497A3 (fr
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Timothy H. Joyce
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Hemolytics, Inc.
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Publication of WO2005074497A2 publication Critical patent/WO2005074497A2/fr
Publication of WO2005074497A3 publication Critical patent/WO2005074497A3/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
    • 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
    • A61K47/6921—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 particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6925—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 particulate, a powder, an adsorbate, a bead or a sphere the form being a microcapsule, nanocapsule, microbubble or nanobubble

Definitions

  • the present invention is in the field of encapsulation vesicles that contain self assembling activation agents such as pore forming agents that may be enclosed in encapsulation vesicles for treatment of disease.
  • Liposomes are small vesicular sacs that resemble tiny cells. These sacs have an aqueous or hydrophilic interior volume separated generally by a durable hydrophobic bilayer membrane. Both water-soluble drugs and insoluble drugs can, therefore, be incorporated into these vesicles. Depending upon the production process used, these vesicles may comprise a single membrane (unilamellar) or several membranes (multilamellar). This makes construction of such vesicles quite flexible. In addition, the typical size of these liposomes can be selected to range from 0.05 to several micrometers in diameter. The ability to design vesicles of varying size makes these vehicles an effective delivery agent for a variety of cellular targets.
  • liposomes Since their discovery, more than 35 years ago, liposomes have been used in a variety of ways to deliver a variety of different drugs. The prospect of targeting liposomes to cancer or tumor sites generated a considerable excitement in medical research in the 1960's and 1970's. Early liposome formulations, however, were no more effective than non-encapsulated formulations. For instance, studies of administering doxorubicin encapsulated liposomes showed little improved antitumor activity. The lack of improved antitumor activity was largely due to the fact that liposomes were unstable in blood and released a good portion of their drug contents as a consequence of rapid binding of plasma proteins (opsonization).
  • red blood cells erythrocytes
  • erythrocytes red blood cells
  • other similar cells that circulate for extended periods of time before degradation by macrophages. It was determined that red blood cells have a thick coat of carbohydrate on their surface and this allows them to circulate for extended periods of time (Allen, T.M., Chonn, A. "Large Unilamellar liposomes with low uptake by the reticuloendothelial system". FEBS Lett. 1987; 223:42-46).
  • Stepalth® liposomes One of the most effective liposomes called “Stealth® liposomes” were shown to stably encapsulate doxorubicin, recirculate for periods of several days after injection without releasing drug, penetrate into tumor cells, and release encapsulated drug within the tumor.
  • the long residence times of the Stealth® liposomes may be explained by the steric stabilization effect provided by the MPEG molecules on the surface of the vesicles.
  • the liposome surface comprises a protective hydrophilic layer that prevents interaction of the plasma components with the liposomes.
  • the Stealth® liposomes may circulate longer in the blood stream. Even today, the mechanism of why these liposomes work effectively remains unclear.
  • the liposome is quite small (the average is approximately 100 nm) and this allows for optimized drug carrying and circulation time.
  • most solid tumors exhibit unique pathoanatomic features, such as extensive angiogenesis, hyperpermeable and defective architecture, impaired lymphatic drainage, and greatly increased production of mediators that enhance vascular permeability.
  • pathoanatomic features such as extensive angiogenesis, hyperpermeable and defective architecture, impaired lymphatic drainage, and greatly increased production of mediators that enhance vascular permeability.
  • These conditions allow for Stealth® liposomes to extravasate in solid tumors through defects present in the endothelial barriers of newly forming blood vessels.
  • inflammatory tissue and tissues with local infections also contain vasculature with greatly enhanced permeability and, therefore, have been shown to be targets for efficient Stealth® liposome extravasation.
  • Extravasation of these and other type liposomes probably occur between gaps and other similar spaces that allow the liposomes to lodge themselves between tumor cells. Once there, it is believed that the enclosed drug material is released either by leakage or by liposome degradation caused by enzymes such as phospholipases (Working, P.K., Newman, M.S., Huang, S.K., et al.'Tharmacokinetics, biodistribution and therapeutic efficacy of doxorubicin encapsulated in Stealth® liposomes (DOXIL®). Liposomes Res. 1994; 4:667-687). Although there is no solid evidence, it is postulated that the release of drugs into tumor cells probably occurs over a period of days and possibly weeks.
  • Liposomes provide this function by encapsulating an active agent that typically does not change its active characteristics between the point of encapsulation and the point of delivery.
  • Other compositions rely on the ability to be administered without encapsulation, but to change activity upon exposure to a certain condition. These compounds, termed activation agents, change their biochemical characteristics in vivo.
  • activation agents change their biochemical characteristics in vivo.
  • non-targeted activation agents such as organic nanotubes are being used and developed to treat bacterial, viral and other diseases.
  • Certain activation agents such a pore forming agents are being developed to address drug resistance problems in bacteria and other microorganisms.
  • the present invention relates to a composition of matter for therapeutic treatment of humans and other mammals.
  • the therapeutic composition of the present invention comprises an encapsulation vesicle such as a liposome, and an active or inactive activation agent such as an organic nanotube enclosed in the encapsulation vesicle.
  • an encapsulation vesicle such as a liposome
  • an active or inactive activation agent such as an organic nanotube enclosed in the encapsulation vesicle.
  • the encapsulation vesicle must be large enough to allow an activation agent such as an organic nanotube within its interior.
  • the encapsulation vesicle may optionally allow the attachment of a targeting ligand and/or enclose a bioactive agent. In certain instances the encapsulation vesicle may be smaller than 600 nm in diameter.
  • the encapsulation vesicle may be from about 20 nm to about 100 nm, but no less than 10 nm. In most cases, the liposomes are most effectively constructed at about 100 nm. This will allow for maximum extravasation of the vesicles into cancer tumors and other sites of infection. Preferential localization of long-circulating liposomes at sites of infection and inflammation has been demonstrated in a variety of experimental models and human clinical trials. Anti- infective agents encapsulated in liposomes provide improved therapy, relative to that provided by unencapsulated drugs.
  • the size of these gaps has been measured in a variety of implanted tumors in mice and found to be no larger than about 0.6 microns. This size represents upper bounds for gaining access to tumor tissue.
  • the rate and extent of extravasation in solid tumors is critically related to liposome size (and plasma residence time). Particles with an average diameter greater than 600 nm do not extravasate at all. For particles with diameters below 600 nm, the rate of extravasation appears to increase with smaller diameters.
  • An important component of the invention is the activation agent.
  • the activation agent may be optionally activated by an activation condition.
  • the activation agent is an organic nanotube, it may be designed to activate by internal or external activation conditions that allow the nanotube to self-assemble in the membrane of a particular type of pathological cell such as a cancer cell.
  • the activation agent In its active form the activation agent is disposed in an internal matrix that prevents and/or delays its reaction with the encapsulation vesicle. If it is in inactive form the internal matrix is optional since it will not react or form supramolecular structures on the inside of the encapsulation vesicle.
  • the activation agent may also be associated with the encapsulation vesicle.
  • the invention also provides a method for therapeutic treatment using the composition of the invention.
  • the composition is formulated for parenteral administration (i.e.
  • the encapsulation vesicle is designed so that the activation agent remains in the encapsulation vesicle and is delivered to a desired tissue or cell.
  • the method for therapeutic treatment may also comprise contacting a cell membrane with a therapeutic composition that comprises an encapsulation vesicle and an active or inactive activation agent such as an organic nanotube enclosed in the encapsulation vesicle and allowing the cell membrane to incorporate the therapeutic composition so that the activation agent of the therapeutic composition may incorporate into the particular type of cell or be activated to incorporate in the particular type of cell membrane.
  • FIG. 1 shows a schematic representation of an encapsulation vesicle enclosing an activation agent of the present invention.
  • FIG. 2 shows a schematic representation of an encapsulation vesicle with and embodiment of the matrix layer for enclosing the activation agents of the present invention.
  • FIG. 3 shows how the encapsulation vesicle may be used to deliver an activation agent to a tumor. DESCRIPTION OF THE SPECIFIC EMBODIMENTS
  • compositions and methods for therapeutic treatment are provided.
  • the therapeutic composition may be used to contact a cell membrane.
  • the cell membranes may be in vitro or in vivo and include both pathogenic and nonpathogenic cells unless clearly stipulated otherwise.
  • activate or “activate by an activation condition” refers to the application of physical, chemical or biochemical conditions or processes that will cause an activation agent to open, close, assemble, self assemble, disassemble, assemble into a supramolecular structure, self assemble in a membrane, open and close, open or close, degrade, change from a lower to higher energy state, release a bioactive agent through or by the activation agent, release one or more molecules that may be photodynamically activated or activated by other activating conditions.
  • an activation agent may be activated by an external light source or laser to open and release a bioactive agent.
  • Active refers to the ability to self assemble into supramolecular structures without any additional physical, chemical or structural modification of the activation agent.
  • Inactive refers to the immediate inability to self assemble into a supramolecular structure, but capable of becoming active and activated by an activation condition.
  • ⁇ HL-K8A refers to a mutant hemolysin protein produced by replacing the lysine (K) at position 8 in the amino acid sequence with arginine (A).
  • C.HL-H5M refers to a mutant hemolysin protein produced by replacing the histidine (H) at position 5 in the amino acid sequence with methionine (M).
  • ⁇ HL(l-l 72*132-293) refers to a particular mutant ⁇ -hemolysin protein that has been produced using recombinant DNA techniques.
  • R104C refers to the replacement of arginine (R) 104 in the ⁇ -hemolysin protein with cysteine (C).
  • K168C refers to the replacement of lysine (K) 168 in the ⁇ -hemolysin protein with cysteine (C).
  • D 183C refers to the replacement of aspartate (D) 183 in the ⁇ -hemolysin protein with cysteine (C).
  • El 1C refers to the replacement of glutamate (E) 1 1 in the ⁇ -hemolysin protein with cysteine (C).
  • Bioactive agent refers to a substance that may be used in connection with an application that is therapeutic or diagnostic, such as, for example, in methods for diagnosing the presence or absence of a disease in a patient and/or methods for treatment of a disease in a patient.
  • the term also refers to a substance that is capable of exerting a biological effect in vitro or in vivo.
  • the bioactive agents may be neutral, positively or negatively charged.
  • Exemplary bioactive agents include for example prodrugs, targeting ligands, diagnostic agents, pharmaceutical agents, drugs, synthetic organic molecules, proteins, peptides, vitamins, steroids, steroid analogs and genetic material.
  • Biocompatible refers to materials that are generally not injurious to biological functions and which will not result in any degree of unacceptable toxicity, including allergenic responses and diseased states.
  • Biomolecule refers to molecules derived from a biological organism or source.
  • biomolecules include proteins, peptides, amino acids, nucleotides, nucleosides, polynucleotides, carbohydrates, lipids, sphingolipids, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), tRNA, mRNA, derivatives or these materials, collagen, fibrinogen, antibodies and other well known materials from biological organisms.
  • Carrier refers to a pharmaceutically acceptable vehicle, which is a nonpolar, hydrophobic solvent, and which may serve as a reconstituting medium.
  • the carrier may be aqueous based or organic based.
  • Carriers include, inter alia, lipids, proteins, polyscaccharides, sugars, polymers, copolymers, and acrylates.
  • Cell refers to any one of the minute protoplasmic masses that make up organized tissue, comprising a mass of protoplasm surrounded by a membrane, including nucleated and unnucleated cells and organelles.
  • Cell membrane refers the commonly described lipid based exterior boundary of a cell. The cell membrane may or may not comprise proteins or receptors.
  • Diseased cell refers to any cell that fails to operate in its naturally occurring condition or normal biochemical fashion. These cells should be capable of causing disease.
  • the word shall include cells that are subject to uncontrolled growth, cellular mutation, metastasis or infection.
  • the term shall also include cells that have been infected by a foreign virus or viral particle, bacteria, bacterial exotoxins or endotoxins, prions, or other similar type living or non-living materials.
  • the term may in particularly refer to cancer cells or cells infected by the polio virus, rhinovirus, piconavirus, influenza virus, or a retrovirus such as the human immunodeficiency virus (HIV).
  • HIV human immunodeficiency virus
  • Fusion refers to the joining together of components to form a single contiguous component. For instance, when two cell membranes contact each other the lipids, proteins or other cellular materials re-associate and/or reorganize to form a single contiguous membrane.
  • Genetic material or “therapeutic charge” refers to nucleotides and polynucleotides, including deoxyribonucleic acids (DNA) and ribonucleic acid (RNA).
  • the genetic material may be made by synthetic chemical methodology, may be naturally occurring, or may be made by commonly known recombinant DNA techniques.
  • the nucleotides, DNA, and RNA may contain one or more modified bases or base pairs, or unnatural nucleotides or biomolecules.
  • Incorporate refers to one or more processes for taking up a component, agent, material, cell membrane or biomolecule. Incorporation processes may include invagination, phagocytosis, endocytosis, exocytosis or fusion processes. These processes may or may not further include one or more clathrate coated pits or receptors.
  • “Intracellular” or “intracellularly” refers to the area within the plasma membrane of a cell, including the protoplasm, cytoplasm and/or nucleoplasm.
  • “Intracellular delivery” refers to delivery of a bioactive agent, such as a targeting ligand and/or prodrug or drug, into the area within the plasma membrane of the cell.
  • Lipid refers to a naturally occurring, synthetic or semi-synthetic (i.e. modified natural) compound that is generally amphipathic.
  • the lipids typically comprise a hydrophilic component and a hydrophobic component.
  • Exemplary lipids include, for example, fatty acids, neutral fats, phosphatides, oils, glycolipids, surface active agents (surfactants), aliphatic alcohols, waxes, terpenes and steroids.
  • semi-synthetic denotes a natural compound that has been chemically modified in some fashion.
  • Liposome refers to a generally spherical or spheroidal cluster or aggregate of amphipathic compounds, including lipid compounds, typically in the form of one or more concentric layers, for example bilayers. They may also be referred to as lipid vesicles.
  • the liposome may be formulated, for example, from ionic lipids and/or non-ionic lipids. Liposomes formulated from non-ionic lipids may be referred to as niosomes.
  • “Nanoerythrosome” refers to a vesicle structure that is derived from erythrocytes and substantially free of hemoglobin.
  • vesicles have a size of less than about 1 micrometer to about 0.1 micrometer and are substantially spherical or spheroidal.
  • the term refers to any bioactive agent carrier described in United States Patent No. 5,653,999 and associated patents or patent applications (herein incorporated by reference in their entirety).
  • Nanocomposites refers to composite structures whose characteristic dimensions are found on the nanoscale. An example is the suspension of carbon nanotubes in a soft plastic host.
  • Nanodot refers to nanoparticles that consist of homogenous material, especially those that are almost spherical or cubical in shape.
  • Nanoparticle refers to any material that can be made, ground or produced on the nanoscale.
  • Nanopore refers to a pore or passage through the structure that has a nanoscale inner diameter, where the inner diameter ranges, in many embodiments from about 0.1 to about 400 nanometers, such as from 10 to 30 nanometers, or from 5 to 10 nanometers.
  • Nanorod refers to nanostructures that are shaped like long sticks or dowels, with a diameter in the nanoscale and a length not very much longer.
  • Nanoscale refers to phenomena that occur on the length scale between 1 and 100 nanometers.
  • Nanostructure refers to structures whose characteristic variation in design length is on the nanoscale.
  • Nanowire refers to nanorods that can conduct electricity.
  • Patient refers to animals, including mammals, preferably humans.
  • Polymer refers to molecules formed from chemical union of two or more repeating units. Accordingly, included within the term “polymer” may be, for example, dimers, trimers and oligomers. The polymer may be synthetic, naturally occurring or semi-synthetic. The term may refer to molecules that comprise 10 or more repeating units.
  • Protein refers to molecules comprising essentially alpha-amino acids in peptide linkages. Included within the term “protein” are globular proteins such as albumins, globulins and histones, fibrous proteins such as collagens, elastins and keratins.
  • Receptor refers to a molecular structure within a cell or on the surface of a cell that is generally characterized by the selective binding of a specific substance.
  • exemplary receptors include cell surface receptors for peptide hormones, neurotransmitters, antigens, complement fragments, immunoglobulins and cytoplasmic receptors for steroid hormones.
  • Receptors may also comprise intracellular receptors such as those on the surface of the nuclear membrane (i.e. PPARs).
  • Regular of a patient refers to a particular area or portion of the patient and in some instances to regions throughout the entire patient. Examples of such regions include the eye, gastrointestinal regions, cardiovascular regions (including myocardial tissue), circulatory system, bladder, mucosa, renal region, vascular tissues, as well as disease tissue such as cancerous tissue including prostate, breast, gallbladder, and liver.
  • the term includes, for example, areas to be targeted by a drug delivery device or a bioactive agent.
  • the term refers to both topical and internal organs and tissues.
  • vascular or “vasculature” denotes blood vessels (including arteries, veins, and the like).
  • gastrointestinal region includes the region defined by the esophagus, stomach, small intestine, large intestine, and rectum.
  • renal region denotes the region defined by the kidney and the vasculature that leads directly to and from the kidney and includes the abdominal aorta.
  • Regular to be targeted or “targeted region” refers to a region where delivery of a therapeutic is desired.
  • Reverse pegylated or reverse m-pegylated refers to an encapsulation vesicle that comprises one or more polymers attached to the exterior of a liposome.
  • the polymers may comprise polyethylene glycol, methylpolyethylene glycol or one of their derivatives.
  • the polymers may comprise one or more targeting ligands such as a monoclonal antibody.
  • This type of encapsulation vesicle may be designed so that the polymers are attached to the exterior of the liposome and degrade or break off of the liposome over time, exposing the liposome membrane it is attached to.
  • Solid-state or solid state material refers to materials that are not biological, biologically based or biological in origin.
  • Such materials may include carbon based materials, synthetic fibers, polymers, plastics, semiconductor materials, silica or silicon based substrates or materials, carbon based nanotubes, quantum dots, artificial bone cylinders, magnetic nanoparticles, nanocrystals, suicide inhibitors, nanodots, nanotubes, nanostructures, or nanowires. These structures may be enclosed within, inserted into, comprise a portion of or be attached to the encapsulation vesicles or activation agents. In certain instances they may also comprise the activation agent. These materials should be capable of activation by an activation condition.
  • Supramolecular structures are multi-subunit structures, e.g. nanotubes, barrels and carpets of nanotubules, which are believed to be formed through "noncovalent” assembly. These structures are thermodynamically controlled assemblies that can undergo reversible structural assembly and disassembly. This process will depend upon the environment, subunit structure, side group selection, side group interaction, and the nature and combination of noncovalent forces acting on the system. Hence, an important and attractive feature of these structures is their ability to select amongst various cell membrane types.
  • “Surface” or “on the surface of the encapsulation vesicle” refers to being covalently or noncovalently attached to the exterior, associated with the exterior, embedded or partially embedded or forming a pore or channel through.
  • a activation agent on the surface of an encapsulation vesicle may be covalently or noncovalently attached to the exterior of the encapsulation vesicle, it may be embedded or partially embedded in the encapsulation vesicle, or it may create a channel or pore through the encapsulation vesicle. Channels or pores may allow for release of bioactive agents.
  • Activation agents on the surface of an encapsulation vesicle should be capable of activation by an activation condition.
  • Targeting ligand refers to any material or substance that may promote targeting of tissues and/or receptors in vivo or in vitro with the therapeutic compositions of the present invention.
  • the targeting ligand may be synthetic, semi-synthetic, or naturally occurring.
  • Materials or substances which may serve as targeting ligands include, for example, proteins, including antibodies, antibody fragments, hormones, hormone analogues, glycoproteins and lectins, peptides, polypeptides, amino acids, sugars, saccharides, including monosaccharides and polysaccharides, carbohydrates, vitamins, steriods, steriod analogs, hormones, cofactors, bioactive agents, genetic material, including nucleotides, nucleosides, nucleotide acid constructs and polynucleotides.
  • “Therapeutic” refers to any pharmaceutical, drug or prophylactic agent which may be used in the treatment (including the prevention, diagnosis, alleviation, or cure) of a malady, affliction, disease or injury to a patient.
  • Therapeutic includes contrast agents and dyes for visualization, Therapeutically useful peptides, polypeptides and polynucleotides may be included within the meaning of the term pharmaceutical or drug.
  • tissue refers generally to specialized cells that may perform a particular function. The term refers to an individual cell or plurality or aggregate of cells, for example, membranes, blood or organs. The term also includes reference to an abnormal cell or plurality of abnormal cells.
  • Exemplary tissues include myocardial tissue, including myocardial cells, membranous tissues, including endothelium and epithelium, laminae, connective tissue, including interstitial tissue, and tumors.
  • "Vesicle” or “encapsulation vesicle” refers to an entity that is generally characterized by the presence of one or more walls or membranes that form one or more internal voids. Vesicles may be formulated, for example, from a stabilizing material such as a lipid, including the various lipids described herein, a proteinaceous material, including the various proteins described herein, and a polymeric material, including the various polymeric materials described herein.
  • vesicles may also be formulated from carbohydrates, surfactants, and other stabilizing materials, as desired.
  • the lipids, proteins, polymers and/or other vesicle forming stabilizing materials may be natural, synthetic or semi-synthetic.
  • Preferred vesicles are those which comprise walls or membranes formulated from lipids.
  • the walls or membranes may be concentric or otherwise.
  • the stabilizing compounds may be in the form of one or more monolayers or bilayers. In the case of more than one monlayer or bilayer, the monolayers or bilayers may be concentric.
  • Stabilizing compounds may be used to form a unilamellar vesicle (comprised of one monolayer or bilayer), an oligolamellar vesicle (comprised of more than about three monolayers or bilayers).
  • the walls or membranes of vesicles may be substantially solid (uniform), or referred to as, for example, liposomes, lipospheres, nanoliposomes, particles, micelles, bubbles, microbubbles, microspheres, nanospheres, nanostructures, microballoons, microcapsules, aerogels, clathrate bound vesicles, hexagonal/cubic/hexagonal II phase structures, and the like.
  • the internal void of the vesicle may be filled with a wide variety of materials including, for example, water, oil, gases, gaseous precursors, liquids, fluorinated compounds or liquids, liquid perfluorocarbons, liquid perfluoroethers, therapeutics, bioactive agents, if desired, and/or other materials.
  • the vesicles may also comprise a targeting ligand if desired.
  • Vehicle stability refers to the ability of vesicles to retain the gas, gaseous precursor and/or other bioactive agents entrapped therein after being exposed, for about one minute, to a pressure of about 100 millimeters (mm) of mercury (Hg).
  • Vesicle stability is measure in percent (%), this being the fraction of the amount of gas which is originally trapped in the vesicle and which is retained after release of the pressure. Vesicle stability also includes "vesicle resilience" which is the ability of a vesicle to return to its original size after release of the pressure.
  • Activation agents can be in active or inactive form. In active form they must be associated with an internal matrix or material in the encapsulation vesicle to prevent them from self assembling and forming supramolecular structures in the encapsulation vesicles. In the inactive or inert form no internal matrix or material is required, but the activation agent must be capable of being activated by an activation condition to form supramolecular structures, change conformation, change energy state, or be altered by a chemical, physical or electrical property.
  • the activation agent in its active form must also be capable of destroying or disrupting the cellular biochemistry of the cell or cell membrane it is in or becomes incorporated into.
  • Activation agents may be capable of being transferred or incorporated into the cell membranes or cellular interior of other cells.
  • the activation agent may also be capable of being used as a transmembrane channel to regulate or deliver a drug.
  • the activation agent may be designed to open and close by an activation condition. They may also be activated to assemble, disassemble, organize, form supramolecular structures or incorporate into membranes after being activated by an activation condition.
  • the activation agents may for instance comprise a pore forming agent, a solid state material, a zeolite, a nanotube, a nanorod, a nanocomposite, a nanowire, an ionophore, a nanodot, a quantum dot, a nanostructure, a plastic, a polymer, a synthetic material, silica or silicon materials, artificial bone or bone material, suicide inhibitors and other similar materials known and previously described in the art.
  • the activation agent may comprise a nanowire positioned in the encapsulation vesicle that may be activated by an exogenous or endogenous source.
  • the nanowire may become incorporated into a cancer cell (by endocytosis, fusion, or phagocytosis) and then may be irradiated by an external light source to "burn out" the tumor.
  • the activation agents may also comprise a pore forming agent.
  • the pore forming agents may be in inactive form and then are activated by an activation condition.
  • the pore forming agents then self assemble on nearby membranes to form supramolecular structures that disrupt the cellular biochemistry or polarity of the cell that they incorporate into.
  • the invention should not be interpreted to be limited to the above described embodiments and materials and includes other embodiments and materials that maintain the above described properties that are know in the art or that may be developed.
  • Active pore forming agents must be capable of self-assembling in cell membranes or walls. They generally comprise monomeric units that aggregate together to form more highly ordered or organized supramolecular structures (i.e heptamer, hexamer, nantubes, sheets, carpets, etc.). The structures and assembly process may comprise various stages of assembly.
  • the pore forming agents generally work synergistically to form channels that span the cell membrane or wall. The monomeric units may disrupt but in many conditions do not form the pores individually. These agents may be organic or synthetic in nature. In inactive form pore forming agents will not readily associate or aggregate to form highly ordered or organized supramolecular structures.
  • the active pore forming agent may have lytic activity and the structures it forms may be capable of being activated to open, close or both. It also may be capable of releasing chemicals or molecules that may prove toxic to a pathogenic cell. Pore forming agents must be capable of self assembling. Active or inactive pore forming agents may be designed to hold bioactive agents or degrade to release bioactive agents or other materials that may be potentially toxic to a pathogenic cell upon activation by an activation condition. Pore forming agents may be produced synthetically (i.e. a peptide, peptide fragment, organic nanotube etc.). The pore forming agent can be a molecule or fragment, derivative or analog of such molecules.
  • the pore forming agents may be capable of making one or more lesions or pores in the encapsulation vesicle(s). These pore forming agents may be derived from a variety of bacteria including ⁇ -hemolysin, E.coli hemolysin, E.coli colicin, B. thuringensis toxin, aerolysin, perfringolysin, pneumolysin, streptolysin O, and listeriolysin. Eucaryotic pore forming agents capable of lysing cells include defensin, magainin, complement, gramicidin, mellitin, perforin, yeast killer toxin and histolysin.
  • Synthetic organic molecules that are capable of forming a lytic pore in encapsulation vesicles can also be used.
  • the composition of the invention can also include fragments of naturally occurring or synthetic pore forming agents that exhibit lytic activity.
  • the invention provides for biologically active and inactive fragments of polypeptides. Biologically active fragments are active if they are capable of forming one or more lesions or pores in synthetic or naturally occurring membrane systems. Inactive fragments are pore forming agents that are capable of being activated or cleaved into activity by some internal or external event, physical activity, or chemical modification.
  • the biologically active fragments of lytic pore forming agents can be generated by methods know to those skilled in the art such as proteolytic cleavage or recombinant plasmids.
  • the invention also includes analogs of naturally occurring pore forming agents that may be capable of lysing cells. These analogs may differ from the naturally occurring pore forming agents by amino acid sequence differences or by modifications that do not affect sequence, or both. Modifications include in vivo or in vitro chemical derivatization of polypeptides, e.g., acetylation, or carboxylation. Also included in the spirit of the invention are modifications of glycosylation and those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing steps.
  • the invention also includes analogs in which one or more peptide bonds have been removed and replaced with an alternative type of bond or an alternative type of covalent bond such as a "peptide mimetic". These mimetics are well known in the art. Similarly, the replacement of the L-amino acid residues is a standard way of rendering the polypeptide less sensitive to proteolysis.
  • blocking groups that are used at the amino terminal end including: t-butyloxycarbonyl, acetyl, theyl, succinyl, methoxysuccinyl, suberyl, adipyl, azelayl, dansyl, benzyloxcarbonyl, fluorenylmethoxycarbonyl, methoxyazelayl, methoxyadipyl, methoxysuberyl, and 2,4 dinitrophenyl.
  • the present invention also includes modifications that may be used to enhance such modifications or degradations.
  • naturally or synthetically occurring organic and inorganic molecules that may be combined with proteins or constructs of the present invention to make them less susceptible to immunological attack.
  • the compound of the present invention may be coupled to molecules such as polyethylene glycol
  • the invention also includes modifications that result in an inactive pore forming agent that can be activated by cell associated substances or conditions. Such modifications can include peptides containing enzymatic cleavage sites (lysine and arginine bonds that can be cleaved) or chemically reactive groups that can be photo-activated. Modifications also include peptides that may be modified to optimize solubility properties or to mediate activation by cell-associated substances.
  • the invention includes peptides and genetic variants both natural and induced. Induced mutants can be made in a variety of methods known in the art including random mutangenesis or polymerase chain reaction.
  • the invention also includes the use of organic and non-organic nanotubes.
  • these molecules may comprise hollow coiled molecules, linear D,L peptides from cylindrical ⁇ or TI helices, helices folding of linear oligophenylacetylenes, ring stacking motifs, tubular ensembles form cyclic D,L- ⁇ -peptides, microcrystalline peptide nanotubes, self assembling transmembrane ion channels, pore structures from D,L peptides, cystine macrocycles, serinophanes, carbohydrate nanotubes, tubular mesophases from macrocyclic precursors, sector assembly motifs, nanotubes from block copolymers, folded sheet motifs, and others.
  • the activation agents may also comprise solid-state materials that are both capable of being on the surface of an encapsulation vesicle or enclosed by the encapsulation vesicles. These material generally are in inactive form.
  • the activation agent may be or may comprise a zeolite, a nanorod, a nanocomposite, a nanowire, a nanodot, a quantum dot, a nanostructure, a plastic, a light pipe or tube, a synthetic material, silica or silicon materials, artificial bone or bone material, suicide inhibitors and other similar materials known and previously described in the art. Each of these materials must be capable of activation by an activation condition.
  • Activation may include lytic activity, change or energy state by irradiation and/or degradation or release of materials that may prove toxic to a pathogenic cell.
  • the activation agent may also comprise a combination or mixture of one or more of these agents.
  • Activation may be by light or irradiation by UV, IR, heat or other physical or chemical agent to focus energy or change the material from a lower to higher electronic state.
  • a targeting ligand may be optionally employed with the present invention.
  • Targeting ligand refers to any material or substance that may promote targeting of tissues and/or receptors in vivo or in vitro with the compositions of the present invention.
  • the targeting ligand may be optional employed with the present invention.
  • a key property of the targeting ligand is the ability for the ligand to bind, attach or associate with the surface of a pathogenic cell.
  • the targeting ligand may be synthetic, semi-synthetic, or naturally occurring.
  • Materials or substances that may serve as targeting ligands include, for example, proteins, antibodies, antibody fragments, hormones, hormone analogues, glycoproteins and lectins, peptides, polypeptides, amino acids, sugars, saccharides, including monosaccharides and polysaccharides, carbohydrates, vitamins, steriods, steriod analogs, hormones, cofactors, bioactive agents, genetic material, including nucleotides, nucleosides, nucleotide acid constructs and polynucleotides.
  • the targeting ligands may include fusion proteins, monoclonal or polyclonal antibodies, Fv fragments, Fab' or (Fab') 2 or any similar reactive immunolgically derived component that may be used for targeting the constructs.
  • Targeting ligands can also include other ligands, hormones, growth hormones, opiod peptides, insulin, epidermal growth factor, insulin like growth factor, tumor necrosis factors, cytokines, fibroblasts or fibroblast growth factors, interleukins, melanocyte stimulating hormone, receptors, viruses, cancer cells, immune cells, B cells, T-cells, CD4 or CD4 soluble fragments, lectins, concavalins, glycoproteins, molecules of hemopoetic origin, integrins and adhesion molecules.
  • ligands may be used in conjunction with the photodynamic pore forming agents.
  • the seringe portion of the diphtheria toxin may be attached to a ligand and the constructs inserted into the encapsulation vesicles. These constructs could then be used to target or deliver the vesicles with the photodynamic pore forming agents.
  • ligands may be employed to form immunoliposomes.
  • ligands may comprise the Anti-HER2 antibody that provides specific binding to the HER2-overexpressing cancer cells.
  • immunoliposomes may be designed and coated with non-antibody ligands such as the folic acid/folate receptor, CKD 602 antibody/receptor, antintegrins and sialyl lewis (x)-oligosaccharide ligands attached to mPEG or PEG ligands extending from the liposome surface.
  • non-antibody ligands such as the folic acid/folate receptor, CKD 602 antibody/receptor, antintegrins and sialyl lewis (x)-oligosaccharide ligands attached to mPEG or PEG ligands extending from the liposome surface.
  • ligands once bound to the target molecule on the surface of tumor cells, result in uptake and internalization of the entire immunoliposome (including the encapsulate drug or activation agent).
  • Once internalized immunoliposomes comprising the self assembling activation agents can be activated to self assemble and destroy the cell or
  • ligand technique called reverse pegylation. It may be desirable in certain instances to remove the surface grafted polymers or ligands after the ligands have contacted target tissue or the liposomes have extravasated into target tissues. Reversible pegylation is intended to protect liposomes while they are circulating in the blood compartment just long enough for them to reach the target tissue.
  • the PEG coating which covers a reactive layer of molecules on the immediate surface of the liposomes (e.g. cationic molecules as in the case of gene delivery), then falls off after the liposomes have reached the intended target tissue, exposing the reactive layer.
  • the reactive layer is then free to interact with the target cells to exert its effects (e.g., causing liposome cell or liposome-endosomal membrane fusion in gene delivery applications).
  • dithiobenzyl (DTB)-linked pegylated lipids can be cleaved with cysteine under mild conditions attainable in vivo.
  • DTB dithiobenzyl
  • liposomes coated with DTB-linked lipopolymers may be effective either through passive targeting or via ligand mediated targeting.
  • nanoshells may be employed with encapsulation vesicles with or without the self assembling activation agents.
  • Nanoshells comprising layered gold atoms wrapped around a tiny globe of silica may comprise or be attached to the encapsulation vesicles.
  • the nanoshell can be attached to the encapsulation vesicles using a variety of techniques know in the art. In other embodiments they may be enclosed by the encapsulation vesicles. This provides the advantage of not requiring the use of targeting ligand for the nanoshell since extravasation can be employed. However, it is within the scope of the invention the separate targeting ligands may also be attached to the encapsulation vesicles.
  • compositions then can be activated in place to destroy cancer tissues (burning out the tumor) or by allowing the compositions to perform endosomal fusion and then irradiating the compositions to destroy the tumors or diseased cells. Irradiation may be performed by IR, UV or any other light source.
  • ligands may be one member of a binding pair where the other member is and endogenous structure that is on the external cell membrane of the target tissue.
  • the encapsulation vesicle is important to the present invention and has a few important properties.
  • the encapsulation vesicle must be capable of accomodating an activation agent inside its membrane. This may include the option of being able to attach a targeting ligand to the surface of the encapsulation vesicle.
  • the encapsulation vesicle may also have the ability to encapsulate a bioactive compound.
  • the encapsulation vesicle need not be a synthesized material. For instance, it may be naturally occurring or comprise parts of naturally occurring cells.
  • the encapsulation vesicle may comprise a red blood cell, a white blood cell, a red blood cell ghost, a white blood cell ghost, a pathogenic cell, a diseased cell, or any other cell that has been infected or not infected.
  • the encapsulation vesicle must be capable of associating or retaining one or more activation agents within its membrane.
  • the encapsulation vesicles may be synthetic
  • vesicle or "encapsulation vesicle” refers to an entity that is generally characterized by the presence of one or more walls or membranes that form one or more internal voids.
  • Vesicles may be formulated, for example, from a stabilizing material such as a lipid, including the various lipids described herein, a proteinaceous material, including the various proteins described herein, and a polymeric material, including the various polymeric materials described herein. As discussed herein, vesicles may also be formulated from carbohydrates, surfactants, and other stabilizing materials, as desired.
  • the lipids, proteins, polymers and/or other vesicle forming stabilizing materials may be natural, synthetic or semi-synthetic Preferred vesicles are those which comprise walls or membranes formulated from lipids. The walls or membranes may be concentric or otherwise.
  • the stabilizing compounds may be in the form of one or more monolayers or bilayers.
  • the monolayers or bilayers may be concentric.
  • Stabilizing compounds may be used to form a unilamellar vesicle (comprised of one monolayer or bilayer), an oligolamellar vesicle (comprised of more than about three monolayers or bilayers).
  • the walls or membranes of vesicles may be substantially solid (uniform), or referred to as, for example, liposomes, lipospheres, stealth® liposomes, nanoliposomes, particles, nanoparticles, micelles, bubbles, microbubbles, microspheres, nanospheres, nanostructures, microballoons, microcapsules, aerogels, clathrate bound vesicles, hexagonal/cubic/hexagonal II phase structures, and the like.
  • the internal void of the vesicle may be filled with a wide variety of materials including, for example, water, oil, gases, gaseous precursors, liquids, fluorinated compounds or liquids, liquid perfluorocarbons, liquid perfluoroethers, therapeutics, bioactive agents, if desired, and/or other materials.
  • the vesicles may also comprise a targeting ligand if desired.
  • the encapsulation vesicles may also include nanoerythrosomes and other lipid based or cellular derived materials.
  • the vesicles may comprise parts of cell, other diseased or pathogenic cells capable of fusion or having receptors or fusion proteins on their surfaces.
  • a potential encapsulation vesicle may comprise a virus such as a T4 phage, an adenovirus, a polio virus, an influenza virus, an HIV virus or other viruses, bacteria, fungi, or pathogenic cells capable of membrane fusion.
  • viruses such as a T4 phage, an adenovirus, a polio virus, an influenza virus, an HIV virus or other viruses, bacteria, fungi, or pathogenic cells capable of membrane fusion.
  • These vesicles may be naturally occurring or may have been altered physically or chemically through recombinant DNA technology.
  • Naturally occurring encapsulation vesicles may include erythrocytes, leukocyte, melanocytes, fibroblasts or components of these cells.
  • encapsulation vesicles may include synthetically designed organic molecules and biodegradable polymers are also within the scope of the present invention.
  • the vesicle may comprise a solid, substantially solid, gel, sol-gel, composite, nanocomposite, nanostructure, nanoporous material, porous nanostructure, nanoshell, nanocrystal, degradable polymer, biodegradable polymer, or device as taught in United States Patent No. 3,948,254 (herein incorporated by reference).
  • Other structures that are well known in the art include nanostructures that self-assemble. For instance such structures are described by Whitesides et al., Science (1991) 254: 1312-1319.
  • the encapsulation vesicle may comprise an internal matrix that helps to improve the stability of the vesicle (See FIG. 2). If the activation agent is in an active form the internal matrix must be used. An important function of the internal matrix is that it prevents the activation agents from self assembling in the encapsulation vesicle and destroying it. If the activation agent is inactive it is optional. For instance, the pH of the interior of the encapsulation vesicle may be changed so that a pore forming agent does not assemble in the membrane of the encapsulation vesicle. The pore forming agent is in inactive form and does not react or associate with the wall of the encapsulation vesicle.
  • the pore forming agents becomes active and can then self assemble in the encapsulation vesicle and/or certain targeted membranes (i.e. pH changes conformation or charge of the agent to now incorporate in cell membranes or walls).
  • certain targeted membranes i.e. pH changes conformation or charge of the agent to now incorporate in cell membranes or walls.
  • the activation agent is in active form, the internal matrix is necessary to prevent, slow or time assembly of the activation agents in the encapsulation vesicles themselves.
  • the internal matrix may comprise a series of layers, gel materials, sol gel materials, drugs, mixture of drugs, pH alterations or changes, plastics, carbohydrates, proteins, polymers, biopolymers, metals, nanoparticles, nanocomposites, chemical moieties, lipids, glycoproteins and/or glycolipids. These materials may be synthetic or naturally derived. The only limited exception to the above properties is when an active activation agent is disposed in an encapsulation vesicle which it does not readily associate (i.e. self assemble or form supramolecular structures).
  • Active activation agents may be mixed with an internal matrix comprising a variety of liquid carriers, solid matrices, semi-solid matrices, semi-solid carriers, finely divided solid carriers or combinations, diluents, excipients, salts, suspensions, powders, buffers, fillers, extenders such as starch, cellulose, sugars, mannitol, silic derivatives, binding agents (carboxymethyl cellulose, hydroxymethylcellulose, hydroxypropyl, methylcellulose, alginates, gelatin, polyvinyl-pyrolidone, and glycerol), calcium calcium carbonate, resorption accelerators such as quartenary ammonium compounds, surface active agents, cetyly alcohol, glycerol monstearate, oils, emuls
  • an internal matrix comprising a variety of liquid carriers, solid matrices, semi-solid matrices, semi-solid carriers, finely divided solid carriers or combinations, diluents, excipients, salts, suspensions, powders
  • an activation agent such as a pore forming agent may be activated before or after release from the encapsulation vesicle. This may be accomplished by conditions or substances that are endogenously provided by the system or target cell or exogenously provided by a source other than the target cell. Physical, chemical or biochemical conditions may be used to activate the lytic activity. Physical conditions include heat, light or temperature changes. Chemical activators include changes in pH or reduction potential, metal ions or protecting groups that may be activated or de-activated. Light sources may include lasers, red lasers, infrared sources, ultraviolet lights, and other optical materials or substances well known in the art.
  • activation may be accomplished by the insertion of a stent into a patient.
  • the stent is inserted and then can be switched on or off to emit a light, UV or IR beam to activate the activation agent.
  • a removable photoactivatable protecting group may be employed.
  • the pore forming agent or protein becomes inactive by addition of the protecting group.
  • the protecting group Upon irradiation by an external light or UV source the protecting group is removed and the pore forming agent becomes activated.
  • Lytic pore forming activity can also be activated biochemically by any substance secreted by a pathogenic cell.
  • biochemical activators include: proteases, esterases, glycosidase, ectokinases, phosphatases and similar type substance or parts of these substances.
  • activation agents can be loaded into encapsulation vesicles at different stages, depending on their physical and chemical properties.
  • the activation agent can be included in the aqueous solution during hydration.
  • passive loading encapsulation vesicle formation and drug encapsulation occur at the same time.
  • the drug can be loaded after the encapsulation vesicles are formed, a process called active loading. This strategy is used in the encapsulation of a number of modern drugs.
  • the hydration step is performed to encapsulate an ammonium sulfate solution.
  • extraliposomal ammonium sulfate is removed by diafiltration.
  • the activation agent is then added to the liposome preparation.
  • the absence of the ammonium sulfate in the extra- encapsulation phase establishes a chemical gradient that induces the drug to diffuse into the encapsulation vesicle and become trapped inside.
  • Active loading is usually more effective than passive loading. More than 90% of the added activation agent becomes encapsulated during the loading, while the typical efficiency of passive loading ranges from 20% to 40%.
  • the unencapsulated drug can be removed by diafiltration or ionic exchange methods if needed.
  • the preparation may then be sterilized by passage through a 0.2 micron sterilization membrane and filled into final product vials.
  • the product can be lyophilized for added stability.
  • Components (i.e. monomers, cyclic peptides etc.) of the composition of the present invention are self-assembling. Under certain conditions they may be designed so that they do not self assemble in cell membranes or walls.
  • the composition may be assembled in any order. Self-assembly may be molecular based where there is a spontaneous association of molecules under equilibrium conditions that form stable, structurally well defined aggregates joined by covalent or non-covalent bonds.
  • the nanotube may be delivered site specifically to a tumor or cancer cell and then allowed to self assemble.
  • the nanotubes can be designed to self assemble in particular kinds of cell membranes.
  • the nanotubes can also be designed so that they could be activated by an activation condition and then, after chemical medication, would be allowed to self assemble.
  • the therapeutic composition comprises an encapsulation vesicle for encapsulating a bioactive agent and an activation agent such as a self assembling nanotube enclosed in the encapsulation vesicle.
  • FIG. 1 shows a schematic view of a first embodiment of the present invention. The figure shows an encapsulation vesicle such as a liposome and an activation agent such as a nanotube enclosed inside the membrane of the encapsulation vesicle.
  • the activation agent may comprise a D,L- ⁇ - peptide or similar type molecule.
  • the nanotube may be designed to be activatable to self assemble by an activation condition.
  • FIG.2 shows the therapeutic composition and how it may be employed.
  • the therapeutic composition circulates in the blood stream and is extravasated by tumor tissues. Once in place they are believed to be generally degraded by phospholipases (Stealth® liposome).
  • the activation agents can then be released to the tumor cells to self assemble in their membranes.
  • Activation agents can directly self assemble or can be derivatized or modified so that an activation condition can be employed to activate them to self assemble.
  • composition and methods can be administered to an animal or human suffering from a medical disorder or disease.
  • the composition may be used alone or in combination with other chemotherapeutic or cytotoxic agents.
  • the encapsulation vesicles can contain a bioactive agent used to treat a disease.
  • an oligomeric antisense DNA or nucleic acid could be used in the carrier for delivery to a diseased or pathogenic cell.
  • Other bioactive agents used for treating cancer and HIV could also be used.
  • the composition and methods may also be administered by intravenous infusion, subcutaneous injection, or direct injection to the site of infection by a stint, laparoscope or other similar type device.
  • the present invention could also be applied topically or aspirated to a tumor site via bronchial passages to treat cancers of the lung.
  • the administration may be by oral, nasal, parenteral (including subcutaneous, intravenous, intramuscular, and intraperitoneal), rectal, dermal, transdermal, intrathoracic, intrapulmonary, intrananasal (respiratory routes).
  • the therapeutic may also be formulated for sustained release (microencapsulation, See WO 94/07529).
  • activation agents have been discussed in WO 2003092632 A2 entitled “Cyclic Peptide Anticancer Agents and Methods", Filed June 5, 2003; WO 2003092631 A2 entitled “Cyclic Peptide Anti- Viral Agents and Methods", Filed June 5, 2003; and WO 2003093300 A2, Entitled “Anti-Microbial Peptides and Compositions", Filed June 5, 2003 (these references are herein incorporated by reference in their entirety).
  • activation agent may be employed, the present invention may find cyclic D,L- ⁇ -peptides or organic nanotube particularly useful.
  • Cell specific activation agents may be selected by testing in vitro activity. This is not a requirement of the invention.
  • Lopez et al. "Antibacterial agents based on the cyclic D,L- ⁇ - peptide architecture, Letters to Nature, Volume 412, July 26, 2001, describes a methodology for selection of particular cyclic peptides that self assemble in bacterial membranes as opposed to mammalian cell membranes. Selectivity is based on substitution of various amino acids in the cyclic peptides. Similar screening activities can be conducted for other types of bacteria and diseased cells. For instance, screening activity has been done for cancer cells, viral cells, and bacterial cells.
  • Encapsulation vesicles The use of an encapsulation vesicle with an activation agent such as a cyclic D,L- ⁇ - peptides have a number of problems because cyclic D,L- ⁇ - peptides are capable of self assembly in their membranes. However, as discussed, it would be possible to produce certain encapsulation vesicles that do not allow self assemble or rapid self assembly in their membranes. This may be accomplished by membrane composition (i.e altering membranes to comprise various lipids, cholesterol, sterols, steroids or components that do not allow self assembly or a particular cyclic D,L- ⁇ - peptides) or use of materials such as various polymeric materials that do not allow these agents to assemble at all in the material.
  • membrane composition i.e altering membranes to comprise various lipids, cholesterol, sterols, steroids or components that do not allow self assembly or a particular cyclic D,L- ⁇ - peptides
  • materials such as various polymeric materials that do
  • the activation agents made in inactive form to trigger or activate under light, pH, protease degradation, etc..
  • various polymer, gel, or chemical moieties within the encapsulation vesicle to prevent, hinder or slow the self assembly of the activation agents in the membrane of the encapsulation vesicle (in certain cases this would be from the inside out and form pores or holes in the encapsulation vesicle).
  • the interior of the encapsulation vesicle may be altered (i.e pH is changed or chemical composition) so that the pore forming agent will not self assemble in the encapsulation vesicle wall.
  • cyclic peptides and nanotubes have been design to be triggered from inactive form to active form by proteases, chemical reactions or changes in pH.
  • the nanotubes then self assemble into larger supramolecular structures when pH is changed. For instance when placed under physiological pH conditions; See WO 2003092632 A2 entitled “Cyclic Peptide Anticancer Agents and Methods", Filed June 5, 2003; WO 2003092631 A2 entitled “Cyclic Peptide Anti-Viral Agents and Methods", Filed June 5, 2003; and WO 2003093300 A2, Entitled “Anti- Microbial Peptides and Compositions", Filed June 5, 2003. In other embodiments this technique may be useful in helping to deliver the drug.
  • the activation agent could be buried within a polymer, various chemical or polymeric membranes or layers within the center of the encapsulation vesicle. In combination with these materials may or may not be a drug that is to be delivered. Over time and after the encapsulation vesicle has extravasated into a tumor, the activation agents can then be released to self assemble and bore holes in the encapsulation vesicle to deliver a drug. Stable drug encapsulation is essential for successful targeting of liposomes. Drug released from liposomes en route to the target site may not contribute effectively to a therapeutic effect at the intended disease sites.
  • the drug carrier may circulate in blood for up to a week following intravenous administration, ensuring stability upon exposure to such dynamic conditions for an extended duration can be challenging.
  • Many drugs that can be encapsulated into liposomes in vitro are released rapidly when exposed to plasma components.
  • the lipid composition may be chosen to impart a solid-phase morphology of the bilayer's hydrocarbon core at body temperature. Phospholipid compositions with these characteristics are more resistant to leakage than those composed of unsaturated lipids, whose bilayer core tends to be more fluid.
  • the internal buffer system used to retain a drug inside of a liposome is also critical to the stability of the system.
  • the internal buffer system is capable of inducing drug complexation and/or precipitation within the internal aqueous compartment.
  • Complexation and/or precipitation may be effective ways to prevent active forms of activation agents from reacting with encapsulation vesicles they readily associate with or form supramolecular structures on or in.
  • An important purpose of encapsulating these activation agents is that the agents need not be targeted using expensive ligands such as antibodies. For instance, by extravasation various liposomes have been shown to infiltrate and degrade in cancer cells or tumors over time. To date such use of liposomes for delivery of cyclic D,L- ⁇ - peptides, pore forming agents and activation agents is unprecedented.

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Abstract

L'invention concerne une composition et une méthode destinées au traitement thérapeutique d'êtres humains et d'autres mammifères. La composition thérapeutique selon l'invention comprend une vésicule d'encapsulation et un agent d'activation, tel qu'un peptide ?-DL renfermé dans ladite vésicule. L'agent d'activation peut se présenter sous la forme active ou inactive. L'invention concerne également une méthode de traitement thérapeutique.
PCT/US2005/001927 2004-01-30 2005-01-21 Vesicules d'encapsulation renfermant des agents d'activation a auto-assemblage WO2005074497A2 (fr)

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Cited By (3)

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Publication number Priority date Publication date Assignee Title
US20140010879A1 (en) * 2012-06-12 2014-01-09 The Methodist Hospital Research Institute Compositions and methods of treating therapy resistant cancer and uses thereof
US20220308001A1 (en) * 2021-03-25 2022-09-29 The Regents Of The University Of California Apparatus and method for single cell discrimination
US11994484B2 (en) * 2022-03-24 2024-05-28 The Regents Of The University Of California Apparatus and method for single cell discrimination

Citations (1)

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Publication number Priority date Publication date Assignee Title
US5777078A (en) * 1993-04-28 1998-07-07 Worcester Foundation For Experimental Biology Triggered pore-forming agents

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5777078A (en) * 1993-04-28 1998-07-07 Worcester Foundation For Experimental Biology Triggered pore-forming agents

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140010879A1 (en) * 2012-06-12 2014-01-09 The Methodist Hospital Research Institute Compositions and methods of treating therapy resistant cancer and uses thereof
US10842749B2 (en) * 2012-06-12 2020-11-24 The Methodist Hospital Research Institute Compositions and methods of treating therapy resistant cancer and uses thereof
US20220308001A1 (en) * 2021-03-25 2022-09-29 The Regents Of The University Of California Apparatus and method for single cell discrimination
US11994484B2 (en) * 2022-03-24 2024-05-28 The Regents Of The University Of California Apparatus and method for single cell discrimination

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