JP4786538B2 - Lipophilic drug delivery vehicle and methods of use thereof - Google Patents

Description

(Cross-reference of related applications)
This application claims the benefit of US Provisional Patent Application No. 60 / 447,508 (filed Feb. 14, 2003) and US Provisional Patent Application No. 60 / 508,035 (filed Oct. 1, 2003). The disclosures of both provisional patent applications are hereby incorporated by reference in their entirety.

(Federal government commissioned research or commissioned development)
The present invention forms part of research supported by grant number HL65159 from the National Institutes of Health. The government may have certain rights to the invention.

(Field of Invention)
The present application relates to compositions and methods for delivery of bioactive agents. In particular, this application relates to bioactive agent delivery particles comprising a lipid binding polypeptide, a lipid bilayer, and a bioactive agent.

(Background of the Invention)
Bioactive substances (eg therapeutic agents, vaccine immunogens, and nutrients) often cannot be administered in pure form, but improve the solubility of the bioactive substance and make it suitable It must be packaged and incorporated into a biocompatible formulation that achieves optimal beneficial effects while minimizing undesirable side effects. Effective delivery of a bioactive factor often involves a short clearance time of the factor in the body, inefficient targeting to the site of action, or the nature of the bioactive factor itself (eg, poorly soluble or hydrophobic in aqueous media) Hindered by sex). Accordingly, many formulation methods have been developed to improve delivery, including sustained release formulations, emulsions, and liposome preparations.

  A liposome-based pharmaceutical delivery system has been described. Liposomes are spherical lipid bilayer membranes that are completely closed and contain an encapsulated aqueous volume. The lipid bilayer includes two lipid monolayers composed of lipids having a hydrophobic tail region and a hydrophilic head region. The structure of this membrane bilayer is the hydrophobic and non-polar tail of the lipid molecule oriented towards the center of the bilayer, while the hydrophilic head is both inside and outside of the liposome It's like pointing towards a certain water phase. The aqueous and hydrophilic core region of the liposome may contain a dissolved bioactive substance.

  Since pharmaceutically useful hydrophobic substances are insoluble or poorly soluble in an aqueous environment, delivery of pharmaceutically useful hydrophobic substances is often a particular problem. For hydrophobic compounds used as pharmaceuticals, direct injection can be impossible or very problematic, as can dangerous conditions (eg hemolysis, phlebitis, hypersensitivity, organ failure, and / or Death). There is a need for improved formulations for hydrophobic bioactive agents that enhance stability in aqueous environments and enable effective delivery of such agents to the desired site of action. Exists.

(Simple Summary of Invention)
The present invention provides compositions and methods for delivery of bioactive factors to an individual.

  In one aspect, the invention provides a bioactive agent delivery particle (including a lipid binding polypeptide, a lipid bilayer having an interior that includes a hydrophobic region, and a bioactive agent that binds to the hydrophobic region of the lipid bilayer). I will provide a. Bioactive agent delivery particles generally do not include a hydrophilic core or an aqueous core.

  The bioactive agent delivery particle comprises at least one hydrophobic region and has one or more bioactive agents incorporated into or associated with the hydrophobic interior of the lipid bilayer. Including. The hydrophobic region of a bioactive factor generally binds to a hydrophobic surface (eg, an acyl chain of a fatty acid) that is inside the lipid bilayer. In one embodiment, the bioactive factor is amphotericin B (AmB). In another embodiment, the bioactive factor is camptothecin.

  The particles are typically disc shaped and have a diameter in the range of about 7 to about 29 nm.

  Bioactive agent delivery particles include lipids (eg, phospholipids) that form a bilayer. In some embodiments, the bioactive agent delivery particle comprises both a lipid that forms a bilayer and a lipid that does not form a bilayer. In some embodiments, the lipid bilayer of the bioactive agent delivery particle comprises a phospholipid. In one embodiment, phospholipids incorporated into the delivery particle include dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG). In one embodiment, the lipid bilayer comprises DMPC and DMPG in a 7: 3 molar ratio.

  In a preferred embodiment, the lipid binding polypeptide is an apolipoprotein. The main interaction between a lipid binding polypeptide (eg an apolipoprotein molecule) and the lipid bilayer is generally a residue of a hydrophobic surface of an amphiphilic structure (eg a lipid binding polypeptide). Hydrophobic interaction around the particle between the fatty acid acyl chain of the lipid on the outer surface. The particles of the present invention may comprise exchangeable and / or non-exchangeable apolipoproteins. In one embodiment, the lipid binding polypeptide is apolipoprotein AI (ApoA-I).

  In some embodiments, particles comprising lipid-binding polypeptide molecules (eg, apolipoprotein molecules) that are modified to increase the stability of the particles are provided. In one embodiment, the modification includes the introduction of a cysteine residue that forms an intramolecular disulfide bond and / or an intermolecular disulfide bond.

  In another embodiment, having one or more linked functional moieties, such as one or more target moieties and / or one or more moieties that have a desired biological activity (eg, antimicrobial activity) Particles comprising a chimeric lipid binding polypeptide molecule (eg, a chimeric apolipoprotein molecule) are provided and the desired biological activity can be increased or the activity of a bioactive factor incorporated into the delivery particle. And can act synergistically.

  In another aspect, a pharmaceutical composition comprising bioactive agent delivery particles in a pharmaceutically acceptable carrier is provided. Also provided is a method for administering a bioactive agent to an individual, the method comprising administering to the individual a pharmaceutical composition comprising bioactive agent delivery particles in a pharmaceutically acceptable carrier. Is included. In some embodiments, the therapeutically effective amount of the bioactive agent is processed in a pharmaceutically acceptable carrier. In some embodiments, administration is parenteral (eg, intravenous, intramuscular, intraperitoneal, transmucosal, or intrathecal). In other embodiments, the particles are administered as an aerosol. In some embodiments, the bioactive factor is formulated for sustained release. In one embodiment, a method is provided for treating a fungal infection in an individual, wherein the method comprises the bioactive agent delivery particles of the invention (often in a pharmaceutically acceptable carrier in a therapeutically effective amount). The step of administering an antifungal agent, eg, AmB, incorporated in In another embodiment, a method is provided for treating a tumor in an individual, wherein the method is a bioactive agent delivery particle of the invention (often in a pharmaceutically acceptable carrier in a therapeutically effective amount). Administering an antineoplastic agent incorporated therein, eg, camptothecin. In one embodiment, the bioactive agent delivery particle comprises a lipid-binding polypeptide having a conjugated vasoactive intestinal peptide targeting moiety, and the tumor is a breast tumor.

  In yet a further aspect, a process is provided for formulating bioactive agent delivery particles as described above. In one embodiment, the formulation process comprises contacting a bioactive factor with a mixture comprising a lipid that forms a bilayer to form a mixture of lipid vesicles and bioactive factor, and the lipid vesicles And contacting the lipid-binding polypeptide with a mixture of a bioactive agent and a bioactive factor. In another embodiment, the formulation process involves the formation of a dispersion of lipid vesicles comprising a previously formed bilayer to which a bioactive factor (dissolved in a suitable solvent) is added. Suitable solvents for solubilizing the bioactive factor for this procedure include solvents with polar or hydrophilic characteristics that can solubilize the bioactive factor to be incorporated into the delivery particles of the present invention. Is mentioned. Examples of suitable solvents include, but are not limited to, dimethyl sulfoxide (DMSO) and dimethylformamide. To this vesicle / bioactive factor mixture, a lipid binding polypeptide is added and then incubated and / or sonicated. In one embodiment, the bioactive factor incorporated into the delivery particle by any of the processes described above is amphotericin B. In one embodiment, the amphotericin B is solubilized in DMSO. In another embodiment, the bioactive factor is camptothecin. In one embodiment, the camptothecin is solubilized in DMSO.

  The present invention includes bioactive agent delivery particles prepared by any of the processes described above, and pharmaceutical compositions containing particles prepared by any of the processes described above and a pharmaceutically acceptable carrier.

  In another aspect, the invention provides any of the above bioactive agent delivery particles or pharmaceutical compositions described above, or delivery particles prepared by any of the above methods and / or reagents for formulating the particles, And / or a kit with instructions for use in a method for administering a bioactive factor to an individual.

(Detailed description of the invention)
The present invention provides compositions and methods for delivery of bioactive agents to an individual. The delivery vehicle is provided in the form of a bioactive factor incorporated in a particle comprising a lipid binding polypeptide and a lipid bilayer. The inside of the particle contains the hydrophobic region of the lipid bilayer containing the hydrophobic portion of the lipid molecule (eg, the fatty acid acyl chain of the lipid), in contrast, the liposome is surrounded by the hydrophilic surface of the bilayer lipid Including a completely closed aqueous interior. The hydrophobic nature inside the particles of the invention can be determined, for example, by interaction between lipid molecules in the bilayer or by sequestration into the hydrophobic region between leaflets of the bilayer. Allow uptake. A bioactive factor comprising at least one hydrophobic region can be incorporated into the hydrophobic interior of the particle. As used herein, “incorporation” of a bioactive factor into the hydrophobic region of a lipid bilayer refers to the hydrophobic region or hydrophobic portion (eg, the bilayer of the bilayer lipid molecule). This refers to solubilization of the lipid to be formed in the fatty acid acyl chain), binding to the hydrophobic region or hydrophobic portion thereof, or interaction with the fatty acid acyl chain.

  The particles are generally disk-shaped with a diameter in the range of about 7 to about 29 nm, which is a known Stokes diameter standard (eg, Blanche et al. (1981) Biochim. Biophys. Acta. 665 ( 3): as described in 408-19) as determined by gradient gel electrophoresis with limited native pore. In some embodiments, the particles are stable in solution and can be lyophilized for long-term storage and then reconstituted in an aqueous solution. The lipid binding polypeptide component defines the boundaries of the discoid bilayer and provides structure and stability to the particles.

  Chimeric lipid binding polypeptide molecules (eg, apolipoprotein molecules) are also provided and can be used to incorporate a variety of additional functional properties in the delivery particles of the invention.

  The particles can be administered to an individual to deliver a bioactive factor to the individual.

(Bioactive factor delivery particles)
The present invention provides “particles” (also referred to herein as “delivery particles” or “bioactive agent delivery particles”), the particles comprising one or more types of lipid binding polypeptides. It comprises a lipid bilayer comprising lipids that form one or more types of bilayers, and one or more bioactive factors. In some embodiments, the delivery particles also include lipids that do not form one or more types of bilayers. Compositions comprising the particles are also provided. In one embodiment, a pharmaceutical composition containing delivery particles and a pharmaceutically acceptable carrier is provided.

  The interior of the particle includes a hydrophobic region (eg, composed of lipid fatty acid acyl chains). The particles of the present invention typically do not include a hydrophilic core or an aqueous core. This particle is a flat, disc-shaped, substantially circular lipid bilayer surrounded by an amphiphilic, α-helix and / or β-sheet of the lipid binding polypeptide. It is disc shaped and its lipid binding polypeptide binds to the hydrophobic surface of the bilayer around the periphery of the disc. An illustrative example of a disc-shaped bioactive agent delivery particle of the present invention is schematically illustrated in FIG.

  Typically, the diameter of the disc shaped delivery particle is from about 7 to about 29 nm, often from about 10 to about 25 nm, and often from about 15 to about 20 nm. “Diameter” refers to the diameter of one of the substantially circular surfaces of the disk.

(Lipid binding polypeptide)
As used herein, a “lipid-binding polypeptide” is an arbitrary that can form a stable interaction with a lipid surface and function to stabilize the lipid bilayer of a particle of the invention. Of synthetic peptides or proteins, or naturally occurring peptides or naturally occurring proteins. A particle can include one or more types of lipid binding polypeptides (ie, the lipid binding polypeptides in a single particle can be the same and are composed of two or more different polypeptide sequences. Also good). The lipid binding polypeptide surrounds the periphery of the particle.

  In some embodiments, lipid binding polypeptides useful for producing the delivery particles of the invention include naturally occurring proteins or fragments, natural variants, isoforms, analogs, or chimeras thereof. A protein having a form of amino acid sequence, a protein having a non-naturally occurring sequence, and any length of protein or peptide having the property of binding to lipids is consistent with known apolipoproteins, and it is May be purified from any source, recombinantly produced or synthetically produced. Naturally occurring protein analogs may be used. A lipid-binding polypeptide is one or more unnatural amino acids (wherein peptide bonds are replaced with structures that are more resistant to metabolic degradation, or individual amino acids are replaced with similar structures). For example, D-amino acids), amino acid analogs, or peptidomimetic structures.

  In a preferred embodiment, the lipid binding polypeptide is an apolipoprotein. Any apolipoprotein or fragment thereof or analog thereof may be used that can be combined with the lipid bilayer to form a discoidal particle. The particles may comprise exchangeable apolipoprotein molecules, non-exchangeable apolipoprotein molecules, or a mixture of exchangeable and non-exchangeable apolipoprotein molecules.

  Apolipoproteins generally have a class A amphipathic α-helical structural motif (Segrest et al. (1994) Adv. Protein Chem. 45: 303-369) and / or a β-sheet motif. Apolipoproteins generally contain a high content of α-helical secondary structures that have the ability to bind to hydrophobic surfaces. A unique feature of these proteins is their ability to interact with specific lipid bilayer vesicles and convert to disk-shaped complexes (for review see Narayanaswami and Ryan (2000) Biochimica et Biophysica. Acta 1483: 15-36). When in contact with lipids, this protein undergoes a conformational change that adapts its structure to accommodate liquid interactions.

  In general, the main interaction between this apolipoprotein and the lipid bilayer in the particle is the residues on the hydrophobic face of the amphiphilic α-helix of the apolipoprotein molecule and the hydrophobic surface of the lipid, For example, by hydrophobic interaction at the edge of the bilayer at the periphery of the bioactive agent delivery particle with the acyl chain of phospholipid fatty acids. The apolipoprotein molecule's amphiphilic α-helix faces the hydrophobic surface of the lipid bilayer at the periphery of the particle and the outside of the particle, and the particle is suspended in an aqueous medium. Including both hydrophilic surfaces that come into contact with the aqueous environment. In some embodiments, the apolipoprotein may comprise an amphiphilic β-sheet structure, wherein the hydrophobic residues of the β-sheet interact with the hydrophobic surface of the lipid at the periphery of the disc .

  Bioactive agent delivery particles often include from about 1 molecule to about 10 molecules of one or more types of apolipoprotein per particle. The amount of amphipathic α-helix due to apolipoprotein in the particle is generally determined by the other exposed hydrophobic surface of the lipid molecule located on the end face of the discoid lipid bilayer (ie, the periphery of the particle ) Is enough to cover. In one embodiment, where the apolipoprotein is human apolipoprotein AI (ApoA-I) and the lipid bilayer comprises palmitoyl oleoylphosphatidylcholine, the particles are directed against about 1 molecule of ApoA-I. It contains two ApoA-I molecules in a ratio of about 80 phospholipids.

  Examples of apolipoproteins that can be used to form the delivery particles of the present invention include ApoA-I, apolipoprotein E (ApoE), and apolipoprotein III (ApoIII), apolipoprotein A-IV (ApoA-IV), apolipoprotein A-V (ApoA-V), Apolipoprotein C-I (ApoC-I), Apolipoprotein C-II (ApoC-II), Apolipoprotein C-III (ApoC-III), Apolipoprotein D (ApoD), Apolipoprotein Protein A-II (ApoA-II), apolipoprotein B-100 (ApoB-100), apolipoprotein J (ApoJ), apolipoprotein H (ApoH), or fragments thereof, natural variants, isoforms, analogs or Texture Forms including but not limited to. In some embodiments, the apolipoprotein is human ApoA-I. In another embodiment, the apolipoprotein is a C-terminal domain or N-terminal domain of apolipoprotein E3, or an isoform thereof. In some embodiments, the apolipoprotein is a functional moiety that is synthetically or recombinantly linked (eg, a targeting moiety or a biologically active moiety that is not native to the apolipoprotein). (See, for example, FIG. 7).

  In some embodiments, exchangeable apolipoproteins are used. The “exchangeable apolipoprotein” can be replaced from the formed disk-shaped particle of the present invention by another protein or another peptide having a binding affinity for lipid without destroying the integrity of the particle. Exchangeable apolipoproteins include synthetic or natural peptides or proteins that can form stable binding interactions with lipids. More than 12 unique exchangeable apolipoproteins have been identified in both vertebrates and invertebrates (see, eg, Narayanaswami and Ryan above).

  In some embodiments, non-exchangeable apolipoproteins are used. As used herein, a “non-exchangeable apolipoprotein” forms a stable interaction with the lipid surface without destroying the original structure of the particle, and the particle of the invention Refers to a protein or peptide that can function to stabilize the phospholipid bilayer but cannot be removed from the surface of the particle.

(Bioactive factor)
The delivery particle includes one or more bioactive factors. As used herein, “bioactive agent” refers to any compound or any composition having biological (including therapeutic or diagnostic) activity. The bioactive factor can be a pharmaceutical agent, drug, compound or composition that is useful for medical treatment, diagnosis, or prevention.

  The bioactive agent incorporated into the delivery particle as described herein generally has at least one hydrophobicity that can bind to or be incorporated into the hydrophobic portion of the lipid bilayer. (Eg, lipophilic) regions. In some embodiments, at least one portion of the bioactive factor is inserted between lipid molecules in the interior of the delivery particle. Examples of bioactive agents that can be incorporated into the delivery particles of the present invention include antibiotic or antibacterial (eg, antibacterial, antifungal, and antiviral) agents, antimetabolites, antitumor agents, steroids, peptides, proteins (Eg, cellular receptor proteins, enzymes, hormones, neurotransmitters), radiolabels (eg, radioisotopes and compounds labeled with radioisotopes), fluorescent compounds, anesthetics, bioactive lipids, anticancer agents, anti-inflammatory Examples include, but are not limited to, agents, nutrients, antigens, pesticides, insecticides, herbicides, or photosensitizers used for photodynamic therapy. In one embodiment, the bioactive factor is the antifungal agent AmB. In other embodiments, the bioactive factor is camptothecin, all-trans retinoic acid, anamycin, nystatin, paclitaxel, docetaxel, or etiopurpurin. Bioactive factors comprising at least one hydrophobic region are known in the art and include ibuprofen, diazepam, griseofulvin, cyclosporine, cortisone, proleukin, etoposide, taxane, α- Tocopherol, vitamin E, vitamin A, and lipopolysaccharide are included, but are not limited to these. See, eg, Kagkadis et al. (1996) PDA J Pharm Sci Tech 50 (5): 317-323; Dardel (1976) Anaesth Scan 20: 221-24; Sweetana and Akers (1996) PDA J Pharm Sci 5: 330-342; see US Pat. No. 6,458,373.

  In some embodiments, the bioactive factor incorporated into the delivery particles of the invention is a non-polypeptide. In some embodiments, for administration to an individual, the bioactive factor and the delivery particle containing the bioactive factor are substantially non-immunogenic when administered to the individual.

  In some embodiments, the bioactive agent that can be incorporated into the delivery particles of the present invention exhibits improved solubility compared to the solubility of the bioactive agent in an aqueous medium. In many cases, formulation into delivery particles results in a decrease in the turbidity of the aqueous composition containing this bioactive agent. This is often reflected in the altered spectroscopic profile for the bioactive agent upon formulation into a delivery particle. The decrease in turbidity can be detected and / or quantified by measuring the optical density of the sample.

  The present invention provides a bioactive agent delivery particle comprising a lipid binding polypeptide, a lipid bilayer, and a bioactive factor, the inside of the lipid bilayer comprising a hydrophobic region, the bioactive factor comprising: Bound to the hydrophobic region of the lipid bilayer, and the bioactive agent delivery particle is larger for the aqueous medium than the bioactive agent in the aqueous medium alone (ie, not formulated in the bioactive agent delivery particle) It is soluble and contains bioactive factors. In one embodiment, AmB exhibits greater water solubility in the delivery particle than in an aqueous medium. In another embodiment, camptothecin exhibits greater water solubility in the delivery particle than in an aqueous medium.

  The present invention also provides a pharmaceutical composition containing a bioactive agent that has greater solubility by incorporation of the bioactive agent into the delivery particle than in an aqueous medium without incorporation into the delivery particle. To do. In some embodiments, increased solubility may be observed by the ability to filter precipitated material reduction, reduced light scattering, and / or reduced solid material upon centrifugation. In some embodiments, the improved solubility of the bioactive agent is not accompanied by a formulation in the delivery particle or is a different formulation (eg, an aqueous formulation, a liposomal formulation, a colloidal suspension, a cochlea, Administration with a cochleate, or complex with cyclodextrin, allows for the administration of bioactive factors at lower dosages than is possible and / or effective. In some embodiments, the improved solubility of the bioactive agent is such that when administered to an individual, such as a mammalian individual (eg, a human individual), the bioactive agent is incorporated into the delivery particle. Less toxicity and less than when administered in a different formulation (e.g., aqueous formulation, liposomal formulation, colloidal suspension, cochlear complex or complex with cyclodextrin). // produces an improved toxicity profile. In some embodiments, the improved solubility of the bioactive factor is such that when administered to an individual, such as a mammalian individual (eg, a human individual), the bioactive factor is present in the delivery particle. Greater efficacy than when administered in a formulation with or without a different formulation (eg, aqueous formulation, liposomal formulation, colloidal suspension, cochlea, or complex with cyclodextrin) Produces sex.

(Lipid bilayer)
The particles of the present invention comprise a lipid bilayer, which is generally a polar head group that faces away from the interior of the particle, as well as the lipid (s) and other that form the bilayer. It has a circular surface of the disc that includes the hydrophobic portion of the lipid component (if present) and the inside of the particle that includes the hydrophobic region of the lipid bilayer (ie, the gap between the circular surfaces). The hydrophobic surface of the lipid molecule at the end face of this bilayer (the circumferential surface of the bioactive agent delivery particle) is in contact with the lipid binding polypeptide of this particle, as discussed above. The particles can include lipids that form one or more types of bilayers, or a mixture of lipids that form one or more types of bilayers and lipids that do not form one or more types of bilayers. As used herein, a “lipid” is soluble or partially soluble in an organic solvent or, when present in an aqueous phase, separates into a hydrophobic environment. Refers to substances of biological or synthetic origin.

  Lipids that form any bilayer that can bind to a lipid-binding polypeptide to form a disc-shaped structure can be used by the present invention. As used herein, “bilayer-forming lipid” refers to a lipid capable of forming a lipid bilayer having a hydrophobic inner side and a hydrophilic outer side. Lipids forming the bilayer include, but are not limited to, phospholipids, sphingolipids, glycolipids, alkyl phospholipids, ether lipids, and plasmalogens. A lipid that forms one type of bilayer may be used, or a mixture of two or more types may be used. In some embodiments, the lipid bilayer comprises a phospholipid. Examples of suitable phospholipids include DMPC, DMPG, POPC, dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylserine (DPPS), cardiolipin, dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), egg yolk phosphatidylcholine (Egg PC), soybean phosphatidylcholine, phosphatidylinositol, phosphatidic acid, sphingomyelin, and cationic phospholipids, but are not limited to these. Examples of lipids that form other suitable bilayers include cationic lipids and glycolipids. In one embodiment, the particles comprise a phospholipid bilayer of DMPC and a phospholipid bilayer of DMPG, often in a molar ratio of about 7: 3. In another embodiment, the particle comprises a phospholipid bilayer of POPC. In some embodiments, the lipid mixture forming the bilayer is at least about 1: 100, about 1:50, about 1:20, about 1:10, about 1: 5, about 3: 7, about 1. : 2 or about 1: 1 molar ratio may be used.

  The particles can also include lipids that are lipids that do not form a bilayer. Such lipids include cholesterol, cardiolipin, phosphatidylethanolamine (this lipid can form a bilayer under certain circumstances), oxysterols, plant sterols, ergosterol, sitosterol, cationic lipids, cerebrosides , Sphingoserine, ceramide, diacylglycerol, monoacylglycerol, triacylglycerol, ganglioside, ether lipid, alkyl phospholipid, plasmalogen, prostaglandin, and lysophospholipid. In some embodiments, the lipid used to prepare the delivery particle can include one or more attached functional moieties (eg, targeting moieties, bioactive agents, or tags for purification or detection). .

(Chimeric lipid binding polypeptide)
The present invention provides a chimeric lipid binding polypeptide, which can be used to prepare the delivery particles described above. A chimeric lipid binding polypeptide comprises one or more linked “functional moieties” (eg, one or more target moieties, moieties having a desired biological activity, affinity tags to assist in purification, and / or Or a reporter molecule for characterization studies or localization studies). A bound moiety having biological activity can have an activity that can be increased and / or synergized by the biological activity of the bioactive factor incorporated into the delivery particle. For example, a moiety having biological activity can have antibacterial (eg, antifungal, antibacterial, antigenic animal, bacteriostatic, fungistatic or antiviral) activity. In one embodiment, the bound functional portion of the chimeric lipid binding polypeptide does not contact the hydrophobic surface of the lipid bilayer when the lipid binding polypeptide is incorporated into a bioactive agent delivery particle. . In another embodiment, the bound functional moiety contacts the hydrophobic surface of the lipid bilayer when the lipid binding polypeptide is incorporated into a bioactive agent delivery particle. In some embodiments, the functional portion of the chimeric lipid binding polypeptide is native to a natural protein. In some embodiments, the chimeric lipid binding polypeptide interacts with a ligand or sequence recognized by a cell surface receptor or other cell surface portion, or those receptors or other cell surface portions. The resulting ligand or sequence.

  In some embodiments, the chimeric lipid binding polypeptide is a chimeric apolipoprotein. In one embodiment, the chimeric apolipoprotein is, for example, S. aureus. cerevisiae alpha-mating factor peptide, folic acid, transferrin, or a targeting moiety that is not native to native apolipoproteins such as lactoferrin. In another embodiment, the chimeric apolipoprotein is a bioactive factor (eg, histatin-5, magainin peptide, melittin, defensin, colicin, N-terminal lactoferrin peptide, echinocandin, hepcidin incorporated into the bioactive agent delivery particle. A moiety having a desired biological activity that is augmented and / or synergized by the activity of (such as bactenicin, or cyclosporine). In one embodiment, the chimeric lipid binding polypeptide may comprise a functional moiety native to an apolipoprotein. One example of an intrinsic functional part of an apolipoprotein is an intrinsic target moiety that is formed approximately by amino acids 130-150 of human ApoE, which is part of the low density lipoprotein receptor family. Contains the receptor binding region recognized by the member. Other examples of intrinsic functional parts of apolipoproteins include the region of ApoB-100 that interacts with the low density lipoprotein receptor and the region of ApoA-I that interacts with the scavenger receptor type B1. In other embodiments, functional moieties can be added synthetically or recombinantly to produce a chimeric lipid binding polypeptide.

  As used herein, a “chimeric” can be present in isolation and can be combined together to form a single molecule having all the desired functions of its constituent molecules. Refers to a molecule. The constituent molecules of the chimeric molecule are chemically conjugated, or the constituent molecules are all polypeptides or analogs thereof, and the polynucleotide encoding the polypeptide expresses a single continuous polypeptide. Can be combined synthetically by being able to be recombinantly fused together. Such chimeric molecules are referred to as fusion proteins. A “fusion protein” is a chimeric molecule whose constituent molecules are all polypeptides, and which are joined (fused) together so that the chimeric molecule forms a continuous single chain. The various components may be bonded directly to each other and may be bonded via one or more linkers.

  “Linker” or “spacer” as used herein with respect to a chimeric molecule refers to any molecule that links or joins the constituent molecules of the chimeric molecule. Many linker molecules are commercially available (eg, from Pierce Chemical Company, Rockford Illinois). Suitable linkers are well known to those skilled in the art and include, but are not limited to, linear or molecular chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. When the chimeric molecule is a fusion protein, the linker can be a peptide that binds proteins including the fusion protein. The spacer generally has no specific biological activity other than binding the protein or maintaining any minimal distance, or other spatial relationship between them, The constituent amino acids of the peptide spacer can be selected to affect several properties of the molecule such as folding, net charge, or hydrophobicity.

  In some embodiments, a chimeric lipid binding polypeptide (eg, a chimeric apolipoprotein) is prepared by chemically conjugating the lipid binding polypeptide molecule to a functional moiety to be bound. The Means for chemically conjugating molecules are well known to those skilled in the art. Such means will depend on the structure of the parts to be joined, but this can be easily ascertained by one skilled in the art.

Polypeptides typically contain a variety of functional groups (eg, carboxylic acid (—COOH), free amino (—NH ₂ ) groups, or sulfhydryl (—SH) groups), which contain the functional moiety or The moiety can be used to react with an appropriate functional group on the linker that binds to it, which can be N-terminal, C-terminal, or an internal residue of an apolipoprotein molecule (ie, N-terminal and C-terminal The apolipoprotein and / or the tagged moiety can be derivatized to expose additional reactive functional groups. Or additional reactive functional groups may be attached.

  In some embodiments, a lipid binding polypeptide fusion protein comprising a functional portion of a polypeptide is synthesized using a recombinant expression system. Typically, this involves generating a nucleic acid (eg, DNA) sequence encoding the lipid binding polypeptide and the functional portion such that, when expressed, the two polypeptides are in frame; The step includes placing the DNA under the control of a promoter, expressing the protein in a host cell, and isolating the expressed protein.

  Lipid binding polypeptide sequences and sequences encoding functional moieties as described herein can be, for example, polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification system (TAS) Alternatively, it may be cloned or amplified by in vitro methods such as a self-sustaining sequence replication system (SSR). A wide variety of cloning and in vitro amplification methodologies are well known to those of skill in the art. Examples of techniques sufficient to guide one of ordinary skill in the art by in vitro amplification methods include, for example, Mullis et al. (1987) US Pat. No. 4,683,202; PCR Protocols A Guide to Methods and Applications (Edited by Innis et al.) Academic. Press Inc. San Diego, CA (1990) (Innis); Arnheim & Levinson (October 1, 1990) C & EN 36-47; The Journal Of NIH Research (1991) 3: 81-94; (1989) Proc. Natl. Acad. Sci. USA 86: 1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomell et al. (1989) J. Clin. Chem., 35: 1826; (1988) Science, 241: 1077-1080; Van Brunt (1990) Biotechnology, 8: 291-294; Wu and Wallace, (1989) Gene, 4 : 560; and Barringer et al. (1990) Gene, 89: 117.

  In addition, DNA encoding the desired fusion protein sequence can be prepared synthetically using methods well known to those skilled in the art, including, for example, Narang et al. (1979) Meth. Enzymol. 68: 90-99, Brown et al. (1979) Meth. Enzymol. 68: 109-151, Beaucage et al. (1981) Tetra. Lett. 22: 1859-1862, direct chemical synthesis by methods such as the diethyl phosphoramidite method or the solid support method of US Pat. No. 4,458,066.

  Nucleic acid encoding a chimeric lipid binding polypeptide fusion protein can be incorporated into a recombinant expression vector in a form suitable for expression in a host cell. As used herein, an “expression vector” is a nucleic acid that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. The vector also includes regulatory sequences such as promoters, enhancers, or other expression control elements (eg, polyadenylation signals). Such regulatory sequences are known to those of skill in the art (see, eg, Goeddel (1990) Gene Expression Technology: Meth. Enzymol. 185, Academic Press, San Diego, CA; Berger and Kimmel, Guide to Molecule Moleto Mole Enzymology 152 Academic Press, Ink., San Diego, CA; Sambrook et al., (1989) Molecular Cloning-A Laboratory Manual (2nd edition), Volume 1 to Volume 3, Cold Spring Labor Harbor Label. (See ss, NY, etc.)

  In some embodiments, the recombinant expression vector for production of the chimeric lipid binding polypeptide is a plasmid or cosmid. In other embodiments, the expression vector is a virus, or part thereof, that can express a protein encoded by a nucleic acid introduced into the nucleic acid of the virus. For example, replication defective retroviruses, adenoviruses and adeno-associated viruses can be used. Expression vectors include bacteriophages (including all DNA phages and all RNA phages (eg, cosmids)), or all eukaryotic viruses (eg, baculoviruses and retroviruses, adenoviruses and adeno-associated viruses, herpes Viruses, vaccinia viruses and all single-stranded DNA viruses, double-stranded DNA viruses, and partially double-stranded DNA viruses, all + (negative) and-(negative) strand RNA viruses, and replication defective retroviruses It can be derived from a viral vector derived from a virus. Another example of an expression vector is the yeast artificial chromosome (YAC), which contains both a centromere and two telomeres that can replicate YAC as a small linear chromosome. Another example is the bacterial artificial chromosome (BAC).

  The chimeric lipid binding polypeptide fusion protein of the invention can be expressed in a host cell. As used herein, the term “host cell” refers to a recombinant expression vector for production of a chimeric apolipoprotein fusion protein, as described above, which can be transfected for expression. Refers to any cell or any cell line. Host cells include the progeny of a single host cell, which progeny is not necessarily completely identical (morphologically or morphologically) to natural, accidental, or intentional mutations. In the complementarity of whole genomic DNA). Host cells include cells transfected or transformed in vivo with an expression vector as described above. Suitable host cells include bacterial cells (eg, E. coli), fungal cells (eg, S. cerevisiae), invertebrate cells (eg, insect cells such as SF9 cells), and vertebrae including mammalian cells. Examples include, but are not limited to, animal cells.

  Expression vectors encoding chimeric lipid binding polypeptide fusion proteins can be transfected into host cells using standard techniques. “Transfection” or “transformation” refers to the insertion of an exogenous polynucleotide into a host cell. This exogenous polynucleotide can be maintained as a non-integrated vector such as, for example, a plasmid, or alternatively can be integrated into the genome of the host cell. Examples of transfection techniques include, but are not limited to, calcium phosphate coprecipitation, DEAE-dextran mediated transfection, lipofection, electroporation and microinjection. Suitable methods for transfecting host cells can be found in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory press and other laboratory textbooks. Nucleic acids are also described in retroviral vectors (see, eg, Ferry et al. (1991) Proc. Natl. Acad. Sci., USA, 88: 8377-881; and Kay et al. (1992) Human Gene Therapy 3: 641-647. Adenoviral vectors (see, eg, Rosenfeld (1992) Cell 68: 143-155; and Herz and Gerard (1993) Proc. Natl. Acad. Sci., USA, 90: 2812-2816), Receptor-mediated DNA uptake (eg, Wu and Wu, (1988) J. Biol. Chem. 263: 14621; Wilson et al., (1992) J. Biol. Chem. 267: 963-967; US Pat. No. 5,166,320), direct injection of DNA (eg, Acsadi et al. (1991) Nature 332: 815-818; and Wolff et al. (1990) Science 247: 1465-1468) or Particle bombardment (genelists) (eg, Cheng et al., (1993) Proc. Natl. Acad. Sci., USA, 90: 4455-4259; and Zelenin et al., (1993) FEBS Letts. 315 : See 29-32) and can be transported into the cell via a delivery mechanism suitable for introduction of the nucleic acid into the cell in vivo.

  Once expressed, the chimeric lipid binding polypeptide can be obtained by standard procedures in the art, including but not limited to affinity purification, ammonium sulfate precipitation, ion exchange chromatography, or gel electrophoresis. Can be purified.

  In some embodiments, chimeric lipid binding polypeptides can be generated using cell-free expression systems or via solid peptide synthesis.

(Modified lipid binding polypeptide)
In some embodiments of the invention, if the polypeptide is incorporated into a bioactive agent delivery particle as described above, the modification will increase the stability of the particle or provide targeting capabilities. A lipid binding polypeptide is provided which is modified. In some embodiments, this modification allows the lipid-binding polypeptide of the particle to stabilize the disc-shaped structure or conformation of the particle. In one embodiment, the modification includes the introduction of a cysteine residue into the apolipoprotein molecule that allows for the formation of intramolecular disulfide bonds or intermolecular disulfides (eg, by site-directed mutagenesis). In another embodiment, a chemical cross-linking agent is used to form intermolecular linkages between apolipoprotein molecules, improving the stability of the particles. Intermolecular crosslinking prevents or reduces the dissociation of apolipoprotein molecules from the particles and / or prevents the particles from being replaced by apolipoprotein molecules within the individual to whom they are administered.

  In other embodiments, the lipid binding polypeptide is a chemical derivatization of one or more amino acid residues or site-directed mutations that confer targeting ability to or recognition by the cell surface receptor. Modified by any of the triggers.

(Delivery system for delivery of bioactive factor to an individual)
The present invention provides a delivery system for delivery of a bioactive agent to an individual, the system comprising bioactive agent delivery particles and a carrier as described above, optionally containing a pharmaceutically acceptable carrier. Including. In some embodiments, the delivery system includes an effective amount of the bioactive agent.

  As used herein, “individual” refers to any prokaryotic or any eukaryotic organism in which it is desired to deliver a bioactive factor. In some embodiments, the individual is a prokaryote such as a bacterium. In other embodiments, the individual is a eukaryote such as a fungus, plant, invertebrate (eg, insect), or vertebrate. In some embodiments, the individual is a human, non-human primate, laboratory animal (eg, mouse or rat), companion animal (eg, cat or dog), or livestock (eg, horse, sheep, cow, or Vertebrates such as pigs), birds (ie birds individuals), or reptiles (ie reptile individuals).

  In some embodiments, the delivery particles are formulated in a carrier suitable for administration to an individual. As used herein, “carrier” refers to a relatively inert substance that facilitates administration of a bioactive agent. For example, the carrier can impart form or consistency to the composition, or act as a diluent. “Pharmaceutically acceptable carrier” refers to a carrier that is biocompatible (ie, not toxic to the host) and suitable for a particular route of administration for a pharmacologically effective substance. Suitable pharmaceutically acceptable carriers include, but are not limited to, stabilizers, wetting and emulsifying agents, salts for varying osmolarity, encapsulating agents, buffers, and skin penetration enhancers. Not. Examples of pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (Alfonso R. Gennaro, Ed., 18th edition, 1990).

  As used herein, “effective amount” refers to the amount of bioactive agent sufficient to achieve the desired result. A “therapeutically effective amount” or “therapeutic dose” is an organism that is sufficient to achieve beneficial clinical results (such as reduction or alleviation of disease symptoms, reduction or alleviation of fungal or bacterial infection, etc.). Refers to the amount of active factor.

  In some embodiments, the delivery system is a pharmaceutical composition containing bioactive agent delivery particles and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition contains bioactive agent delivery particles comprising a bioactive agent that is not a polypeptide and a pharmaceutically acceptable carrier. In some embodiments, the bioactive agent delivery particle and the bioactive agent are non-immunogenic when administered to an individual. Immunogenicity can be measured by methods well known in the art. For example, immunogenicity can be determined by ELISA methods (eg, sera from individuals administered for antibodies that bind to an equivalent amount of bioactive agent delivery particles to which bioactive agent delivery particles are bound to an immunosorbent plate). Can be evaluated).

  In some embodiments, the present invention provides a pharmaceutical composition containing a bioactive agent delivery particle that is incorporated into the delivery particle by incorporation of the bioactive agent into the delivery particle. It exhibits greater solubility in aqueous solvents than the bioactive factor in aqueous media without uptake into the aqueous medium. In some embodiments, the present invention provides a pharmaceutical composition containing bioactive agent delivery particles, wherein the bioactive agent is directed against an individual such as a mammalian individual (eg, a human individual). Administered with bioactive agent delivery particles, or with different formulations (eg, aqueous formulations, liposomal formulations), colloidal suspensions, cochlea, or cyclodextrins It exhibits a lower toxicity and / or improved toxicity profile than is administered in the complex). In some embodiments, the present invention provides a pharmaceutical composition containing a bioactive agent delivery particle that does not involve a formulation in the bioactive agent delivery particle or a different formulation. Rather than being administered in a product (eg, aqueous formulation, liposomal formulation), aqueous formulation, liposomal formulation, colloidal suspension, cochlear, or complex with cyclodextrin) Show improved efficacy in the treatment of infections such as infections or fungal infections, disease states, tumors, etc. In some embodiments, the bioactive agent with improved solubility, toxicity profile, and / or efficacy is AmB. In another embodiment, the bioactive factor with improved solubility, toxicity profile, and / or efficacy is camptothecin.

(how to use)
The present invention provides a method for administering a bioactive factor to an individual. The method of the invention includes the step of administering a delivery particle as described above comprising a lipid binding polypeptide, a lipid bilayer, and a bioactive agent, wherein the inside of the particle is the hydrophobicity of the lipid bilayer. Including the surface. Optionally, a therapeutically effective amount of the particles is administered, and optionally a therapeutically effective amount of the particles is administered in a pharmaceutically acceptable carrier. In general, the particles are disk-shaped with a diameter of about 7 to about 29 nm as measured by gradient gel electrophoresis with native pore restriction. Typically, the bioactive factor includes at least one hydrophobic region that can be integrated into the hydrophobic region of the lipid bilayer.

  The route of administration can vary depending on the nature of the bioactive factor to be administered, the individual, and the condition to be treated. When the individual is a mammal, administration is generally parenteral. Routes of administration include, but are not limited to, intravenous, intramuscular, intraperitoneal, subcutaneous, transmucosal, nasal, intrathecal, topical, and transdermal. In one embodiment, the particles are administered as an aerosol. The delivery particles can be formulated in a pharmaceutically acceptable form (optionally in a pharmaceutically acceptable carrier or excipient) for administration to an individual. The present invention provides pharmaceutical compositions that are in the form of delivery particles in solution for parenteral administration. For the preparation of such compositions, methods well known in the art can be used and any pharmaceutically acceptable carrier, diluent, excipient, or other additive commonly used in the art. Agents can be used.

  The delivery particles of the invention can be made into a pharmaceutical composition by combination with a suitable medical carrier or medical diluent. For example, the delivery particles can be solubilized in a solvent commonly used in the preparation of injectable solutions, such as saline, water, or aqueous dextrose. Other suitable pharmaceutical carriers and their formulations are described in Remington's Pharmaceutical Sciences, supra. Such formulations can be constructed in sterile vials containing the delivery particles and optionally containing excipients in dry powder form or lyophilized powder form. Prior to use, the physiologically acceptable diluent is added and the solution is collected by syringe for administration to an individual.

  Delivery particles can also be formulated for sustained release. As used herein, “sustained release” means that the blood concentration of the factor in an individual is for an extended duration (over a period of hours, days, weeks, or longer) Refers to the release of the bioactive factor from the formulation at a rate maintained within the therapeutic window. The delivery particles can be formulated in a biodegradable control matrix or a non-biodegradable control matrix, many of which are well known in the art. The sustained release matrix can include synthetic polymers or synthetic copolymers (eg, in the form of a hydrogel). Examples of such polymers are polyesters, polyorthoesters, polyanhydrides, polysaccharides, poly (phosphoesters), polyamides, polyurethanes, poly (imide carbonates) and poly (phosphazenes), and poly-lactide-co- Examples include glycolide (PLGA) and a copolymer of poly (lactic acid) and poly (glycolic acid). Collagen, albumin, and fibrinogen containing substances can also be used.

  The delivery particles can be administered by the methods described herein to treat a number of conditions, including but not limited to bacterial infections, fungal infections, disease states, metabolic disorders, Or, for example, it can be administered as a prophylactic medication (eg, before or after surgery) to prevent bacterial or fungal infection. Delivery particles can be used, for example, to deliver an anti-tumor agent (eg, chemotherapeutic agent, radionuclide) to a tumor. In one embodiment, the lipid binding polypeptide includes a moiety that targets the particle to a particular tumor. The delivery particles can also be used for administration of dietary supplements (ie, food supplements that provide food or health benefits). In some embodiments, the delivery particles are co-administered with other conventional therapies (eg, as part of multiple drug “cocktails”), or one or more orally administered agents (eg, In combination with factors for the treatment of fungal infections). Delivery particles can also be administered as insecticides or herbicides.

  In one aspect, the present invention also provides a method for treating a fungal infection in an individual. The method comprises administering to an individual a therapeutically effective amount of an antifungal agent in a pharmaceutically acceptable carrier, the antifungal agent comprising a particle comprising a lipid binding polypeptide and a lipid bilayer. The lipid bilayer inside is entrapped and is hydrophobic. In one embodiment, the antifungal agent is AmB, which is incorporated into the hydrophobic interior of the lipid bilayer. In some embodiments, the lipid binding polypeptide is a chimeric protein comprising a targeting moiety and / or a moiety having biological activity. In one embodiment, the lipid binding polypeptide comprises a targeting moiety, such as a yeast α-mating factor peptide. In another embodiment, the lipid binding polypeptide comprises histatin 5, which is an antimicrobial peptide.

  In another aspect, the present invention provides a method for treating a tumor in an individual. The method includes administering a therapeutically effective amount of a chemotherapeutic agent in a bioactive agent delivery particle as described above in a pharmaceutically acceptable carrier. In one embodiment, the chemotherapeutic agent is camptothecin. The lipid binding polypeptide component of the delivery particle can include a targeting moiety that targets the particle to tumor cells. In one embodiment, vasoactive intestinal peptide (VIP) is bound to the lipid binding polypeptide. Because breast cancer cells often overexpress the VIP receptor, in one embodiment, a bioactive agent delivery particle comprising a camptothecin and lipid binding polypeptide-VIP chimera is a method for treating breast cancer. used.

(Targeting)
The delivery particles of the invention can include a targeting functional group (eg, a targeting functional group for targeting the particle to a particular cell type or tissue type, or to the infectious agent itself). . In some embodiments, the particle includes a targeting moiety that is bound to a lipid binding polypeptide or lipid component. In some embodiments, the bioactive factor incorporated into the particle has targeting ability.

  In some embodiments, the particles can be targeted to specific cell surface receptors by manipulating the property of recognizing the receptors in lipid binding polypeptides (eg, apolipoprotein molecules). For example, a bioactive agent delivery particle modifies the lipid-binding polypeptide component of the particle so that it can interact with a receptor on the surface of the cell type to be targeted by It can be targeted to specific cell types known to have sex factors inside.

  In one aspect, a receptor-mediated method of targeting can be used to deliver anti-Leishmania factors to macrophages, which macrophages are responsible for infection of protozoan parasites by Leishmania. It is a part. Examples of such species include Leishmania major, Leishmania donovani, and Leishmania braziliansis. Bioactive agent delivery particles, including anti-Leishmania factors, alter macrophage lipid-binding polypeptide components of the particles to provide recognition by class A scavenger receptors (SR-A) in macrophage endocytosis. Can be targeted to. For example, an apolipoprotein that has been chemically modified or genetically modified to interact with SR-A is one or more bioactive factors that are effective against Leishmania species (eg, AmB, pentavalent antimony, and / or Can be incorporated into delivery particles containing (such as hexadecylphosphocholine). Targeting delivery particles containing anti-Leishmania factors specific for macrophages can be used as a means of inhibiting the growth and proliferation of Leishmania species.

  In one embodiment, SR-A targeted bioactive agent delivery particles comprising AmB are administered to an individual in need of treatment for Leishmania infection. In another embodiment, another anti-Leishmania factor such as hexadecylphosphocholine is administered before, during, or after treatment with the AmB-containing particles.

  In some embodiments, targeting modifies a lipid binding polypeptide (eg, apolipoprotein) to be incorporated into the bioactive agent delivery particle, thereby conferring SR-A binding capability to the particle. Is achieved. In some embodiments, targeting is achieved by chemically modifying one or more lysine residues with, for example, malondialdehyde, maleic anhydride, or acetic anhydride at an alkaline pH. This is accomplished by varying the charge density of the polypeptide (see, eg, Goldstein et al. (1979) Proc. Natl. Acad. Sci. 98: 241-260). In one embodiment, Apo B-100 or a truncated form thereof (eg, 17% of the N-terminus of ApoB-100 (residues 1 to 782 of apoB-17)) is modified by reaction with malondialdehyde. . In other embodiments, the apolipoprotein molecule (eg, any of the apolipoproteins described herein) is also chemically modified, eg, by acetylation or maleylation, and includes a biological activity comprising an anti-Leishmania factor Can be incorporated into agent delivery particles.

  In other embodiments, the SR-A binding ability allows the lipid-binding polypeptide to be altered by site-directed mutagenesis that replaces one or more positively charged amino acids with neutral or negatively charged amino acids. It is given to the delivery particle by modification.

  In other embodiments, recognition by SR-A is a chimera comprising an N-terminal extension or a C-terminal extension with a ligand recognized by SR-A or an amino acid sequence having a high concentration of negatively charged residues. Is provided by preparing a lipid binding polypeptide. The extension of the negatively charged polypeptide is not bound to the lipid surface of the bioactive agent delivery particle, thus making it more accessible to the ligand binding site of the receptor.

(Method for preparing bioactive agent delivery particles)
The present invention provides a method for formulating bioactive agent delivery particles. In one embodiment, a process is provided that includes adding a lipid binding polypeptide molecule to a mixture comprising a lipid and a bioactive agent molecule that forms a bilayer.

In some embodiments, the lipid-bioactive factor mixture also includes a surfactant (such as sodium cholate, cholic acid, or octyl glucoside), and the process comprises The method further includes removing the surfactant after the peptide has been added. Typically, this surfactant is removed by dialysis or gel filtration. In one embodiment, the process comprises combining a lipid that forms a bilayer and a bioactive agent molecule in a solvent to form a bioactive agent mixture, drying the mixture (eg, under a stream of N ₂ and Removing the solvent (by lyophilization), contacting the dried mixture with a solution comprising a surfactant to form a lipid-bioactive factor-surfactant mixture, lipid binding to the mixture Adding a functional polypeptide molecule, and then removing the surfactant.

  In some embodiments, the particles are prepared using a microfluidizer processor. This procedure utilizes high pressure that pressurizes the components together in a reaction chamber.

  In some embodiments, the particles are prepared by incubation of a suspension of lipid vesicles comprising a bioactive factor in the presence of a lipid binding polypeptide (eg, apolipoprotein). In one embodiment, the suspension is sonicated.

  In other embodiments, the delivery particles are prepared from a preformed vesicle dispersion. Lipids (eg, phospholipids) are hydrated with a buffer and dispersed by stirring or sonication. To the lipid bilayer vesicle dispersion, solubilized bioactive factor is added in a suitable solvent to form a lipid-bioactive factor complex. In some embodiments, the solvent is volatile or dialyzable for convenient removal after addition of the bioactive factor to the lipid bilayer vesicle dispersion. After further agitation, lipid binding polypeptide is added and the sample is incubated, mixed by agitation, and / or sonicated. Typically, the vesicles and apolipoproteins are incubated at the liquid-crystal phase transition temperature of a lipid that forms a particular bilayer or a mixture of lipids that form a used bilayer, or Incubate near the gel to temperature. This phase transition temperature can be determined by calorimetry.

  Preferably, the lipid composition that forms a suitable bilayer is such that, when dispersed in an aqueous medium, the lipid vesicles do not biodegrade from the carrier solvent into the aqueous environment without precipitation or phase separation of the bioactive factor. Used to provide a suitable environment for active agent transfer. This preformed lipid bilayer vesicle is also preferably capable of undergoing lipid-binding polypeptide-induced transformation to form the delivery particles of the invention. Furthermore, the lipid-bioactive factor complex preferably has the properties of a lipid vesicle that allows conversion to a bioactive agent delivery particle upon incubation with a lipid binding polypeptide under appropriate conditions. To maintain. The unique combination of lipid substrate-bioactive factor complex organization and properties of lipid binding polypeptides is tailored to create a system, whereby pH, ionic strength, temperature and lipid-bioactive factor-lipid Under appropriate conditions of the binding polypeptide concentration, the reorganization of the tertiary structure of these substances occurs, and the lipid bilayer surrounded by the stable lipid binding polypeptide is incorporated into the lipid environment of this bilayer. Produced with bioactive factors. For a discussion of the effects of pH, ionic strength, and lipid-binding polypeptide concentration on the ability of lipid-binding polypeptides to induce the conversion of different types of phospholipid vesicles into discoid particles, see Weers et al., ( 2001) Eur. J. et al. Biochem. 268: 3728-35.

  Particles prepared by any of the above processes can be further purified (eg, by dialysis, density gradient centrifugation, and / or gel permeation chromatography).

  In a method of preparation for the formulation of bioactive agent delivery particles, preferably at least about 70%, more preferably at least about 80%, and even more preferably at least about 90% of the bioactive agent used in this procedure. Yet more preferably at least about 95% is incorporated into the particles.

  The present invention provides bioactive agent delivery particles prepared by any of the methods described above. In one embodiment, the present invention provides a pharmaceutical composition comprising delivery particles prepared by any of the methods described above and a pharmaceutically acceptable carrier.

(Storage and stability)
The particles of the present invention are stable for a long time under a variety of conditions (see, eg, FIG. 5). The particles, or compositions comprising the particles of the invention, can be stored at room temperature, refrigerated (eg, about 4 ° C.), or frozen (eg, about −20 ° C. to about −80 ° C.). They can be stored in solution or in a dried state (eg, lyophilized). Bioactive agent delivery particles can be lyophilized under an inert atmosphere, frozen, or stored in solution at 4 ° C. The particles may be in a liquid medium such as a buffer (eg, phosphate buffer or other suitable buffer) or a pharmaceutically acceptable carrier, eg, for use in a method of administration of a bioactive agent to an individual. Can be stored in a carrier such as Alternatively, the particles can be stored in a dried or lyophilized form and then reconstituted in a liquid medium prior to use.

(kit)
The reagents and particles described herein can be packaged in kit form. In one aspect, the present invention provides kits comprising delivery particles and / or reagents useful for preparing delivery particles in suitable packaging. The kit of the invention comprises either separately or in combination: a lipid binding polypeptide (eg, apolipoprotein), phospholipid, bioactive factor, vector, reagent, enzyme, host cell and / or Growth medium for cloning and / or expression of recombinant lipid binding polypeptides (eg, recombinant apolipoprotein) and / or chimeras of lipid binding polypeptide (eg, apolipoprotein chimeras) and for administration to individuals Reagents and / or pharmaceutically acceptable carriers for formulating the delivery particles.

  Each of the reagents or formulations is in a solid form, liquid buffer, or pharmaceutically acceptable carrier suitable for storage in stock, or as required for reaction, culture, or replacement or addition to an injectable medium. Supplied in. Appropriate packaging is provided. As used herein, “packaging” is customarily used in systems and prepares or formulates bioactive agents, or delivery particles (eg, apolipoprotein molecules, phospholipids, bioactive agents). A solid matrix or substance that can hold one or more reagents or components (eg, delivery particles) to be used within defined limits for use in the method of delivery of one or more reagents to do so. Such materials include, but are not limited to, glass bottles and plastic (eg, polyethylene, polypropylene, and polycarbonate) bottles, vials, paper, plastic, and packaging materials laminated with plastic foil.

  The kit optionally provides additional components (eg, means for purifying buffers, reaction surfaces, or delivery particles) that are useful in the methods and formulation procedures of the invention.

  Further, the kit may optionally include instructions or instructional materials (ie, providing labels and / or instructions for performing the methods of the invention (eg, preparation, formulation and / or use of delivery particles)). Protocol). This teaching material includes representatively described media or printed materials, but they are not limited to these formats. Media that can record such instructions and communicate them to the end user are contemplated by the present invention. Such media include, but are not limited to, electrical recording media (eg, magnetic disks, tapes, cartridges, chips), optical media (eg, CD ROM), and the like. Such media may include addresses for internet sites that provide such instructions.

  The following examples are illustrative and are not intended to limit the invention.

Example 1. Preparation and characterization of ApoA-I-phospholipid-amphotericin B particles
(Preparation of recombinant ApoA-I)
Recombinant ApoA-I is prepared as described in Ryan et al. (2003) Protein Expression and Purification 27: 98-103 and described below to prepare ApoA-I-phospholipid-AmB particles. Used to do.

(Preparation of ApoA-I-phospholipid-AmB particles)
ApoA-I-phospholipid-AmB particles were prepared as follows:
Dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG) in a molar ratio of 7: 3 were dissolved in chloroform: methanol (3: 1, v / v). To 10 mg of the above DMPC / DMPG mixture was added 0.25 ml of AmB (2 mg / ml; solubilized in acidified chloroform: methanol (3: 1, v / v)). The mixture was dried under a stream of N ₂ gas to form a thin film on the container wall. The dried sample was then lyophilized for 16 hours to remove a trace amount of solvent.

  The dried lipid mixture was resuspended in 0.5 ml Tris-Saline buffer (10 mM Tris base, 150 mM NaCl, pH 8) and the mixture was vortexed for 30 seconds.

  To this resuspended lipid mixture, 0.5 ml of 22 mM sodium cholate was added and vortexed for 3 minutes. The mixture was incubated at 37 ° C. with vortexing every 10 minutes for 1.25 hours or until the mixture was clear. To this clear solution 2 ml of isolated recombinant ApoA-I (prepared as described in Example 1) was added at a concentration of 1.5 mg / ml and the mixture was added for an additional hour 37 Incubated at 0 ° C. To remove sodium cholate, the sample was dialyzed against 4 liters of Tris-Saline for 72 hours at 4 ° C., changing the dialysis buffer every 24 hours.

  This sample was further purified by density gradient ultracentrifugation. This solution was adjusted to a density of 1.30 g / ml by the addition of solid KBr in 1.5 ml. The sample was transferred to a 3 ml centrifuge tube, overlayered with saline and centrifuged in a Beckman L7-55 centrifuge at 275,000 × g for 3 hours.

(Particle stability)
The particles prepared by this procedure were stable for over 3 months in lyophilized form.

(Particle characterization)
UV / visible light scans were performed on ApoA-I-phospholipid particles (prepared as described above, but no AmB added) and compared to scans for AmB-containing particles. FIG. 1 shows a scan for particles that do not contain AmB. The only peak observed was a protein peak near 280 nm. FIG. 2 shows a scan for AmB-containing particles prepared as described above. In addition to the peak near 280 nm, many additional peaks were observed in the spectrum in the 300-400 nm region, confirming the presence of AmB. Free AmB is insoluble in aqueous media and it has spectral characteristics that are different from the spectral characteristics observed in FIG. Madden et al. (1990) Chemistry and Physics of Lipids, 52: 189-98.

  Characterization studies showed that ApoA-I, phospholipids, and AmB migrate as separated particle populations when subjected to density gradient ultracentrifugation (FIG. 3). This complex floats to a characteristic density in the gradient, depending on the protein / lipid ratio in the particle.

  Furthermore, gradient gel electrophoresis under non-denaturing conditions showed that the major complex produced was of uniform size and showed a Stokes diameter of 8.5 nm (FIG. 4). Analysis of the isolated particles showed that no major deviation from the initial molar ratio of AmB, phospholipid, and apolipoprotein occurred.

Example 2. Antifungal activity of AmB-containing bioactive agent delivery particles against Saccharomyces cerevisiae
ApoA-I-DMPC / DMPG-AmB particles were prepared as described in Example 1 and used to determine the antifungal activity of this complex. S. C. cerevisiae cultures were grown in YPD medium in the presence of varying amounts of ApoA-I-DMPC / DMPG-AmB particles (0-25 μg AmB / ml). The culture was grown at 30 ° C. for 16 hours and the extent of culture growth was monitored spectrophotometrically. As shown in FIG. 8, the AmB-containing particles were very effective at inhibiting fungal growth in a dose-dependent manner.

Example 3. Long-term stability of bioactive agent delivery particles
Recombinant ApoE3NT terminal domain (ApoE3NT) was prepared as Fisher et al. (1997) Biochem Cell Biol 75: 45-53. ApoE3NT-AmB containing particles were prepared by the cholic acid dialysis method as described in Example 1 and used to evaluate long-term stability.

FIG. 5 shows particles stored at 4 ° C. in phosphate buffer (lane 1), particles stored at −20 ° C. in phosphate buffer (lane 2), or −80 in phosphate buffer. ℃ frozen at, lyophilized and prior to analysis of the particles was redissolved in _{H 2} O, it shows a native PAGE 4 to 20% gradient slab gels. The size and mobility of the AmB-containing particles are not affected by freezing and thawing, nor by lyophilization and resolubilization, which means that the particles are It was shown to maintain integrity. These are important parameters regarding scale-up and long-term storage of AmB delivery particles.

Example 4. Preparation of AmB-containing bioactive agent delivery particles using POPC
ApoA-I-POPC particles were prepared using the cholate dialysis method described in Example 1. A native PAGE gradient gel analysis of ApoA-I-POPC particles is shown in FIG. Particles not containing AmB are shown in lane 1 and particles containing AmB are shown in lane 2. This gel shows that AmB incorporation into the particles does not change its size. However, this gel suggests that the POPC-containing particles are of a different size than the DMPC / DMPG particles (shown in lane 3).

(Example 5. Preparation of AmB-containing particles using a microfluidizer processor)
ApoA-I, AmB, egg PC, DPPG, and cholesterol were combined in a microfluidizer sample holder and passed through the reaction chamber of the microfluidizer processor at 18,000 psi. The resulting solution was collected and characterized with respect to particle formulation, hydrophobic material uptake, size, and stability. AmB-containing particles with a diameter of about 16 nm were obtained and were stable to lyophilization and reconstitution in aqueous solvents.

(Example 6. Preparation of AmB-containing particles from phospholipid vesicles)
A suspension of AmB-containing phospholipid vesicles is aliquoted with a 20-40 mg / ml solution of AmB in DMSO (corresponding to 2.5 mg AmB) and containing a 7: 3 molar ratio of DMPC: DMPG. Prepared by adding to an aqueous dispersion of the phospholipid formed. The vesicles were incubated on the gel to the liquid phase transition temperature (about 24 ° C.) of the phospholipid. The addition of 4 mg of apolipoprotein resulted in a time-dependent decrease in sample turbidity, consistent with the formation of AmB-containing bioactive agent delivery particles. Complete clarity of the sample was achieved by sonication in a gentle bath for 1-20 minutes at 21-25 ° C or a gentle bath for 4-16 hours without sonication at 24 ° C. The resulting particles showed> 90% AmB uptake efficiency (ie,% of AmB starting material recovered in the delivery particles) and no material was lost upon filtration, centrifugation, or dialysis. . Other studies have shown that similar results can be achieved with AmB concentrations adjusted to the same height as 5 mg / 10 mg phospholipid. This procedure consists of five apolipoproteins tested (ApoA-I, ApoE3NT, Bombyx mori ApoIII, and a modified form of human ApoA-I containing a C-terminal extension containing an antifungal peptide, histatin 5, and S. cerevisiae α -A modified form of human ApoA-I containing a C-terminal extension containing the conjugating factor peptide) worked equally well.

  Density gradient ultracentrifugation of ApoA-I or ApoE3NT containing particles is a single particle suspension suspended at a characteristic density in the range of 1.21 g / ml consistent with the formulation of lipid-protein complexes. Showed the population. Characterization of the fractions obtained according to density gradient ultracentrifugation showed that phospholipid, AmB, and apolipoprotein migrated to the same position in this gradient, consistent with the AmB-containing particle formulation.

  A comparison of the relative mobility of ApoA-I-AmB-containing particles on the native PAGE with known standards indicated that over 90% of the particles had a Stokes diameter of about 8.5 nm. This value is similar to particles produced in the absence of AmB, indicating that the addition of this bioactive factor does not significantly change the particle size distribution.

As a measure of the overall stability of the ApoA-I-AmB containing bioactive agent delivery particles, the particles were either frozen at -20 ° C or lyophilized. Freezing / thawing had no effect on the particle size distribution. Similarly, subjecting the particles to lyophilization and re-dissolving in H ₂ O did not affect size distribution or sample appearance.

  These data indicate that AmB, phospholipid, and apolipoprotein combine to form a homogeneous population of bioactive agent delivery particles in which AmB is fully integrated into the bilayer portion of the particle. Strongly suggest. Spectrophotometric analysis of AmB-containing particles showed a set of characteristic peaks in the visible light range consistent with AmB solubilization in bioactive agent delivery particles.

(Example 7. Comparison of AmB-containing particles prepared as in Example 6 with particles prepared by an alternative procedure)
Incorporation of bioactive agents into bioactive agent delivery particles using the method described in Example 6 was prepared according to “neo-HDL prepared by Schouten et al. (1993) Molecular Pharmacology 44: 486-492, as follows. Compared to incorporation into the particles: 3 mg egg yolk phosphatidylcholine, 0.9 mg cholesterol, and 1.5 mg AmB (dissolved in chloroform) were mixed in a 20 ml glass vial and the solvent was Evaporated under a stream of nitrogen. Degassed and nitrogen-saturated 10 ml of sonication buffer (10 mM Tris HCI pH 8.0, 100 mM KCI, 1 mM EDTA, and 0.025% NaN ₃ ) and added The contents of the vial were sonicated by Macrotip (14 μm average power) under a stream of nitrogen. The temperature was maintained above 41 ° C. and below 50 ° C. The sonication was stopped after 60 minutes and the temperature was adjusted to 42 ° C. Sonication was continued and 20 mg of ApoA-I (dissolved in 2 ml of 4M urea) was added in 10 equivalents over 10 minutes. Sonication was continued for 30 minutes at 42 ° C. after all protein was added.

  The sonication mixture is then centrifuged for 3 minutes to remove large particles and insoluble material, and the supernatant is analyzed by UV / visible spectroscopy to solubilize amphotericin B solubilized in the product particles. The amount was evaluated. It was noted that this solution was slightly opaque. The sample was scanned from 250 nm to 500 nm. For comparison, AmB-containing particles prepared by the procedure described in Example 6 were tested. The result is shown in FIG. The spectral range (300-500 nm) arising from AmB was quite distant between the two samples. AmB-containing bioactive agent delivery particles produced using the protocol described in Example 6 had a characteristic strong extinction maximum indicative of AmB solubilization and incorporation into the particles (Madden et al., Supra). Samples prepared according to Schouten et al. (FIG. 12B) did not produce these characteristic spectral maxima (FIG. 12A). In fact, this obtained spectrum is very similar to the spectrum reported by Madden et al. (Supra) for an aqueous dispersion of AmB in the absence of lipid. Thus, AmB was not incorporated into lipid particles using this procedure, but the procedure described in Example 6 resulted in sufficient AmB incorporation.

Example 8. Comparison of antifungal activity between AmB-containing bioactive agent delivery particles and liposomal AmB formulations
The antifungal activity of ApoA-I-AmB particles (prepared as in Example 6) and the commercial liposome formulation of AmB (AmBismome®), their ability to inhibit the growth of yeast (S. cerevisiae) Compared with ability. The data in FIG. 10 shows that ApoA-I-AmB bioactive agent delivery particles FIG. 5 shows that cerevisiae growth was more effectively inhibited than the same amount of AmB formulation (such as AmBisome®). The ApoA-I-AmB bioactive agent delivery particles achieved 90% growth inhibition at 1 μg / ml, but this level of inhibition required 25 μg / ml AmBisome®.

  The antifungal activity of ApoA-I-AmB particles and AmBisome® was also demonstrated against all two pathogenic fungi, Candida albicans (C. albicans) and Aspergillus fumigatus (A. fumigatus). Comparison in cell assays. As a control, particles without AmB were also tested. The results are shown in Table 1.

  (Table 1. Inhibition of growth of pathogenic fungi by amphotericin B)

得られた結果は、ＡｍＢ含有生物活性因子送達粒子が、ＡｍＢｉｓｏｍｅ（登録商標）より、より低濃度にて両方の病原性真菌の種に対して有効であることを示した。ＡｍＢを欠くコントロール粒子は、有効ではなかった。ＡｍＢ含有生物活性因子送達粒子は、Ｃ．ａｌｂｉｃａｎｓに対して０．１μｇ／ｍｌの濃度にてＥＤ_９０（９０％の増殖阻害が観察される濃度）を示したが、同じレベルの増殖阻害を達成するのに、０．８μｇ／ｍｌのＡｍＢｉｓｏｍｅ（登録商標）を必要とした。Ａ．ｆｕｍｉｇａｔｕｓに関して、ＡｍＢ含有生物活性因子送達粒子は、０．２μｇ／ｍｌの濃度にて９０％の真菌の増殖を阻害したが、同じ効果を達成するのに、１．６μｇ／ｍｌのＡｍＢｉｓｏｍｅ（登録商標）を必要とした。

The results obtained showed that AmB-containing bioactive agent delivery particles are more effective against both pathogenic fungal species at lower concentrations than AmBisome®. Control particles lacking AmB were not effective. AmB-containing bioactive agent delivery particles include C.I. Although ED ₉₀ (concentration at which 90% growth inhibition was observed) was shown at a concentration of 0.1 μg / ml against albicans, 0.8 μg / ml AmBisome to achieve the same level of growth inhibition (Registered trademark) was required. A. For Fumigatus, AmB-containing bioactive agent delivery particles inhibited 90% fungal growth at a concentration of 0.2 μg / ml, but to achieve the same effect, 1.6 μg / ml AmBisome® ) Was required.

  In another experiment, AmB-containing bioactive agent delivery particles comprising apolipoprotein III as the lipid-binding polypeptide were obtained from three pathogenic fungi, C.I. albicans, A.M. Fumigatus, and Cryptococcus neoformans (C. neoformans) were compared to AmBisome® for their ability to inhibit the growth. This data is shown in Table 2.

  (Table 2. Inhibition of growth of pathogenic fungi by amphotericin B)

ＡｍＢ含有生物活性因子送達粒子は、０．０３μｇ／ｍｌにてＣ．ａｌｂｉｃａｎｓの増殖を９０％阻害した。相当するＥＤ_９０を、０．４μｇ／ｍｌのＡｍＢｉｓｏｍｅ（登録商標）で得た。Ａ．ｆｕｍｉｇａｔｕｓの場合において、ＡｍＢ含有生物活性因子送達粒子は、０．１μｇ／ｍｌにて真菌の増殖を９０％阻害したが、同じ効果を達成するのに２．５μｇ／ｍｌのＡｍＢｉｓｏｍｅ（登録商標）の濃度を必要とした。同様の様式において、ＡｍＢ含有粒子は、ＡｍＢｉｓｏｍｅ（登録商標）より、５倍低いＡｍＢ濃度にてＣ．ｎｅｏｆｏｒｍａｎｓ増殖の阻害において有効であった。

AmB-containing bioactive agent delivery particles are produced at 0.03 μg / ml C.I. 90% of the growth of albicans was inhibited. The corresponding ED ₉₀ was obtained with 0.4 μg / ml AmBisome®. A. In the case of Fumigatus, AmB-containing bioactive agent delivery particles inhibited fungal growth by 90% at 0.1 μg / ml, but 2.5 μg / ml of AmBisome® to achieve the same effect. Concentration required. In a similar manner, AmB-containing particles are produced at a C.I. It was effective in inhibiting neoformans growth.

  All samples tested were soluble in the RPMI medium used for this experiment, and no precipitation or interference was observed in any of the samples tested against any of the fungal species. These data suggest that the formulation of AmB in the bioactive agent delivery particles of the present invention has a stronger antifungal activity than the liposomal formulation.

Example 9. Incorporation of camptothecin into bioactive agent delivery particles
Camptothecin-containing bioactive agent delivery particles were prepared as follows: 7: 3 molar ratio of DMPC: DMPG (total 5 mg) in buffer (20 mM sodium phosphate, pH 7.0) for 1 minute. Dispersion by vortexing produced a dispersion of phospholipid bilayer vesicles. 10 μl of a 10 mg / ml camptothecin solution in DMSO was added to the phospholipid bilayer dispersion. Thereafter, 2 mg of recombinant human apolipoprotein A-I (0.5 ml of 4 mg / ml solution in 20 mM sodium phosphate, pH 7.0) was added and the sample was then subjected to sonication. . The clarified sample was then centrifuged at 13,000 xg for 3 minutes and the supernatant was removed and stored at 4 ° C.

  The fluorescence spectrum of camptothecin-containing particles is shown in FIG. 11 (compared to camptothecin solubilized in sodium dodecyl sulfate (SDS)). Fluorescence measurements were obtained on a Perkin Elmer LS 50B emission spectrometer at an excitation wavelength of 360 nm with emission monitored at 400-600 nm. The blue shift in fluorescence maximum induced by camptothecin in SDS micelles (FIG. 11A) compared to camptothecin incorporated in bioactive agent delivery particles (FIG. 11B) indicates that the drug is micelles relative to the delivery particles. Some suggest localization in a more hydrophobic environment.

Example 10 Electron Microscopy of Freezing Cleavage of AmB-Containing Bioactive Agent Delivery Particles
A preparation of AmB-containing bioactive agent delivery particles was prepared for freezing electron microscopy as follows: DMPC: DMPG (7: 3 molar ratio) AmB bioactive agent delivery particles (3 mg / mg). A sample of ml (prepared as in Example 6) was quenched using sandwich technology and liquid nitrogen cooled propane. Cryofixed samples were stored in liquid nitrogen for less than 2 hours before processing. This cleaving process was performed in a JOEL JED-900 freeze etcher and the exposed cleaved plane was covered with Pt for 30 seconds at an angle of 25-35 degrees and covered with carbon (2 kV / 60-80 mA for 35 seconds). 1 × 10 ⁻⁵ Torr). Replicas made in this way were cleaned with concentrated fuming HNO ₃ for 24 hours, after which stirring was repeated at least 5 times with fresh chloroform / methanol (1: 1 by weight). Replicas cleaned in this way were tested on a JOEL 100 CX or Philips CM 10 electron microscope.

  An electron micrograph obtained from freezing cleaving of the AmB-containing particles as described above is shown in FIG. Electron micrographs taken from several frozen cleaved preparations show the presence of high concentrations of small protein-lipid complexes. The apparent diameter is in the range of about 20-60 nm, which is frequently around 40 nm. When observed by electron microscopy of freeze-fracturing, the apparent diameter of the particles is greater than that obtained by gradient gel electrophoresis with native pore restriction. This difference may be due to the influence of the staining procedure used for sample handling or particle visualization by electron microscopy.

  The substantially spherical complex did not show a concave or convex surface (shadows in front of and behind the structure, respectively) as characteristic in liposomes. Furthermore, no evidence for micelle structure was observed.

Example 11. In vivo evaluation of antifungal activity of AmB-containing bioactive agent delivery particles in immunoresponsive mice
The in vivo antifungal activity of AmB-containing bioactive agent delivery particles is evaluated as follows.

(animal)
6-8 week old female BALB / c mice (20-25 g) are bred and maintained under standard laboratory conditions.

(Study on toxicity)
Each group of 3 mice is dosed in AmB-containing bioactive agent delivery particles in saline buffered to pH 7.4 with 10 mM sodium phosphate (eg, 1 mg / kg, 2 mg / kg, Receive control particles containing 5 mg / kg, 10 mg / kg, or 15 mg / kg AmB), or no AmB. A single dose is administered intraperitoneally in an amount of 0.1 ml. Preliminary studies have shown that this bioactive agent delivery particle is completely soluble under these conditions.

  Following injection, the mice are observed for any general response (eg, abnormal behavior or posture, dyspnea, fur, or ability to obtain food or beverage). Observations for abnormalities or death begin immediately after administration and continue twice a day for 7 days. Body weight is recorded at the same time every day.

  Blood is collected from the mice before euthanasia. The blood is assayed for liver enzymes such as lactate dehydrogenase to assess the extent of liver specific damage.

(Effects of AmB-containing bioactive factor delivery particles in the treatment of systemic cryptococcus)
The therapeutic range of AmB-containing particles is determined as follows and compared to AmBisome®.

C. sensitive to AmB Neoformans clinical isolates are cultured and prepared as inoculum for infection at a concentration of 2 × 10 ⁶ conidia / ml. Each mouse receives 1 × 10 ⁵ conidial inoculum in 0.05 ml normal saline intracranially under general anesthesia.

  The antifungal agent is administered from 2 hours after infection, and is administered intraperitoneally in an amount of 0.1 ml per day for 5 days. The dosage of AmB used is determined based on the toxicity studies described above. One treatment group of mice receives AmBisome®, one treatment group receives AmB-containing bioactive agent delivery particles, and the control group receives no treatment.

  Infected mice are monitored twice a day and any signs of illness or death are recorded for up to 28 days. Body weight is recorded every day at the same time. A moribund animal that cannot move normally or cannot eat food or beverage is euthanized. Based on the results of these studies, a second set of studies is conducted to verify and reproduce the study results. The AmB dose utilized and the number of mice in the control and treatment groups can be adjusted to reflect the knowledge gained from previous experiments.

(Determination of fungal burden on tissues)
Mice are sacrificed on day 1 after the last day of treatment. The kidney and brain are aseptically removed and weighed. The tissue is homogenized and serially diluted in normal saline. This homogenate is incubated for 48 hours on PDA (potato dextrose agar) plates to determine colony forming units (CFU). The fungal burden of tissue CFU / g is determined.

(Statistical analysis)
Differences in viable bacteria and mean CFU in the kidney or brain are compared using an appropriate statistical test.

(Pharmacokinetic study)
Blood samples, liver samples, kidney samples, lung samples, and cerebrospinal fluid samples were obtained from infected mice at doses of 0.8 mg / kg and 2.0 mg / kg, respectively. Collected at 10 minutes, 2 hours, 8 hours, and 24 hours after intravenous infusion of delivery particles or AmBisome®. Mice are placed under general anesthesia and whole blood is collected from the blood vessels under the arm. A thoracotomy is performed and the tissue sample is perfused with normal saline and then surgically removed. Tissue is homogenized using methanol containing 1-amino-4-nitronaphthalene. Serum and tissue homogenate supernatants are stored until analysis. The concentration of AmB in each sample was determined by Granich et al. (1986) Antimicrob. Agents Chemother. 29: 584-88 as determined by high performance liquid chromatography (HPLC). In summary, serum samples (0.1 ml) are combined with 1.0 ml methanol containing 1.0 mg internal standard (1-amino-4-nitronaphthalene) per ml and mixed by vortexing. After centrifugation, the supernatant is dried under reduced pressure and then redissolved with 0.2 ml of methanol for injection onto an HPLC column ( _C18 reverse phase). Tissue samples containing weighed water are homogenized with a glass homogenizer in 10 volumes of methanol containing 5.0 mg of internal standard per ml and centrifuged. The mobile phase is a mixture of acetonitrile and 10 mM sodium acetate buffer (pH 4.0; 11:17 (vol / vol)) (1.0 ml / min flow rate). The AmB concentration is determined by the ratio of the AmB peak height to the internal standard peak height.

Example 12. Targeting tumor cells with camptothecin-containing bioactive agent delivery particles
Bioactive agent delivery particles are prepared using a VIP targeting moiety that binds to the lipid binding polypeptide component.

  The lipid binding polypeptide component of the camptothecin-containing particles can be produced in recombinant form in Escherichia coli (E. coli) transformed by a plasmid vector having the coding sequence of the lipid binding polypeptide therein. For example, recombinant human ApoA-I can be utilized. An E. coli containing an ApoA-I expression plasmid inside. E. coli cells are cultured at 37 ° C. in medium. When the optical density of the culture at 600 nm reaches 0.6, ApoA-I synthesis is induced by the addition of isopropylthiogalactoside (0.5 mM final concentration). After an additional 3 hours of culture, the bacteria are pelleted by centrifugation and disrupted by sonication. The cell lysate is centrifuged at 20,000 × g for 30 minutes at 4 ° C. and apoA-I is isolated from the supernatant fraction.

  Recombinant lipid-binding polypeptide chimera includes N-terminal peptide extension and / or C-terminal peptide extension that manipulates ApoA-I and corresponds to a 28 amino acid neuropeptide, vasoactive intestinal peptide (VIP) Generated by. ApoA-I-VIP chimeras can be utilized to generate bioactive agent delivery particles including phospholipids, camptothecins and ApoA-I-VIP chimeras.

  For example, an ApoA-I-VIP chimera can be constructed by synthesizing complementary oligonucleotide primers corresponding to the coding sequence of a VIP sequence having a Hind III site and an Xba I site at the ends. This oligonucleotide (approximately 100 base pairs) is annealed to produce double stranded DNA with the desired “sticky ends” and has appropriately placed Hind III and Xba I restriction enzyme sites. Subcloned into a plasmid vector containing the ApoA-I coding sequence. After ligation, transformation and screening for positive chimeric constructs, plasmid DNA is isolated and subjected to automated dideoxy method sequence analysis. After confirming that this sequence corresponds to the sequence predicted for the desired chimera, production of recombinant ApoA-I-VIP chimeras was performed as described above for wild-type ApoA-I. performed in E. coli. The purified recombinant chimera is then evaluated by gel electrophoresis, mass spectrometry, and as described in Example 8, in a manner similar to wild type ApoA-I, bioactive agent delivery particles of the invention. Is evaluated for the ability to produce

ApoA-I-VIP chimera-camptothecin-containing bioactive agent delivery particles can be used in breast cancer cell growth inhibition studies to measure the degree of lipid particle targeting. For example, the human breast cancer cell line MCF-7 was obtained from the American Type Culture Collection and was humidified as a monolayer culture in modified Eagle's medium supplemented with 10% fetal calf serum and antibiotics (penicillin and streptomycin). Maintained at 37 ° C. in% CO ₂ incubator. Isolated wild type ApoA-I or ApoA-I-VIP chimeras are radioiodinated and incorporated into camptothecin-containing bioactive agent delivery particles of the invention and incubated with the cells. Radioactivity bound to the cells is determined after incubation of labeled camptothecin-containing bioactive agent delivery particles with MCF-7 cells cultured at 4 ° C. The ability of VIP to compete with the binding of ApoA-I-VIP chimera or ApoA-I-VIP chimera bound to bioactive agent delivery particles to MCF cells is determined in a competitive binding assay. Cell binding data is assessed by Scatchard analysis. The extent of MCF-7 cell internalization of ApoA-I-VIP chimeric bioactive agent delivery particles is assessed in incubation with radioiodinated ApoA-I-VIP chimer containing bioactive agent delivery particles at 37 ° C. After incubation and washing, the radioactivity soluble in trichloroacetic acid is determined, providing a measure of lipid binding polypeptide degradation.

Growth inhibition studies and cytotoxicity studies with different bioactive agent delivery particles are evaluated by clonogenic assays. Exponentially growing cells are resuspended in medium and the number of cells is determined using an electronic instrument. Alternatively, inhibition of clonal growth of MCF-7 by camptothecin-ApoA-I-VIP chimeric bioactive agent delivery particles can be assessed based on a decrease in ³⁵ S-methionine uptake. Aliquots of cells are seeded in triplicate in culture dishes. After incubation, specific lipid particles are added to this dish from stock solutions to achieve final concentrations of camptothecin of 0 nM, 0.1 nM, 1 nM, 5 nM, 10 nM, 50 nM, 100 nM, 250 nM. After a specific time interval ranging from 0 to 72 hours, the medium is removed by aspiration and fresh medium is added. The percent survival at each drug concentration with different exposure times is determined from the ratio of the number of trypan blue excluded cells and compared to the results obtained with control particles lacking camptothecin.

Example 13. In vivo evaluation of toxicity of AmB-containing bioactive agent delivery particles
Studies were conducted to determine the safety and toxicity of AmB-containing bioactive agent delivery particles. The particles were prepared as in Example 6.

  Female BALB / c mice (6-8 weeks old, weighing 20-25 grams) were divided into groups of 3 mice and each group was divided into 1 mg / kg, 2 mg / kg, 5 mg / kg, 10 mg / Treated with AmB formulations in kg, or 15 mg / kg bioactive agent delivery particles, and intraperitoneally (IP) once in a volume of 0.1 ml in phosphate buffered saline (PBS) Delivered as a dose. The control group received PBS only.

  Mice were observed for weight loss or appearance and behavioral abnormalities immediately after dosing, 2 hours after dosing and 6 hours after dosing, and then at least twice a day for 7 days. Blood was collected 24 hours after administration. Markers for liver damage (alanine aminotransferase (ALT) and aspartate aminotransferase (AST)) and markers for kidney damage (urea and creatinine) were quantified.

  Table 3 shows the in vivo safety / toxicity profile of bioactive agent delivery particles containing AmB below.

  (Table 3. Toxicity of AmB-containing bioactive agent delivery particles in mice)

１５ｍｇ／ｋｇの投薬量が、マウスにおいて有毒であることを見出した。この投薬量レベルおいて、即死または異常性は存在しなかったが、３匹のマウスの内２匹が投与した次の日に死亡した。１０ｍｇ／ｋｇ以下の用量にて、ＡｍＢ含有粒子は、安全であることを見出した。図１４に示されるように、名目上の体重減少を５ｍｇ／ｋｇおよびそれ以下の投薬量にて観察した。１０ｍｇ／ｋｇで処置したマウスにおいて、顕著な体重減少を２日目に観察し、その後、その週の終わりには体重の９０％までの回復を観察した。薬物のこの濃度にて、腎毒性のわずかな兆候（１０ｍｇ／ｋｇにて０．１６ｍｇ／ｄｌのクレアチニン対０ｍｇ／ｋｇにて０．１ｍｇ／ｄｌ；尿素には変化無し）が観察され、そして肝毒性は、観察されなかった。この結果は、毒性において劇的な減少を記録した。なぜならこれまでに報告された実験は、ＡｍＢが界面活性剤のミセル中に処方された同様の実験条件下で、４．４ｍｇ／ｋｇのＡｍＢにて、マウスにおいて４％の生存率しか生じなかったからである（Ｂａｒｗｉｃｚら、（１９９２）Ａｎｔｉｍｉｃｒｏｂ Ａｇｅｎｔｓ Ｃｈｅｍｏｔｈｅｒ ３６：２３１０−２３１５）。

A dosage of 15 mg / kg was found to be toxic in mice. There was no immediate death or anomaly at this dosage level, but 2 of 3 mice died the next day after administration. At doses of 10 mg / kg or less, AmB-containing particles were found to be safe. As shown in FIG. 14, nominal weight loss was observed at dosages of 5 mg / kg and lower. In mice treated with 10 mg / kg, significant body weight loss was observed on day 2, after which a recovery to 90% of body weight was observed at the end of the week. At this concentration of drug, slight signs of nephrotoxicity were observed (0.16 mg / dl creatinine at 10 mg / kg vs. 0.1 mg / dl at 0 mg / kg; no change in urea) and liver Toxicity was not observed. This result recorded a dramatic decrease in toxicity. Because the experiments reported so far produced only 4% survival in mice at 4.4 mg / kg AmB under similar experimental conditions where AmB was formulated in surfactant micelles. (Barwickz et al. (1992) Antimicrob Agents Chemother 36: 2310-2315).

Example 14. In vivo effect of AmB-containing bioactive agent delivery particles
Experiments were performed to determine whether the formulation of AmB into bioactive agent delivery particles would impair its antifungal effect.

Female BALB / c mice (6-8 weeks old) were divided into 4 groups of 10 mice each. Each mouse was inoculated with 5 × 10 ⁵ Candida albicans ATCC strain 90028 spore. Two hours after inoculation, mice were treated with fluconazole (orally administrable antifungal treatment (30 mg / kg via oral gavage)), AmBisome (5 mg / kg IP), as described in Example 13. AmB-containing bioactive agent delivery particles (5 mg / kg IP) formulation, or control “empty” bioactive agent delivery particles without AmB but with a protein load equivalent to particles containing AmB. Treatment was continued once a day for 5 days. Through this study, mice were monitored for mortality and abnormalities in appearance and behavior. At 24 hours after the last treatment, mice were sacrificed and kidney and brain tissues were excised for assessment of fungal burden. For assessment of survival, mice were observed for 29 days and tested twice daily for mortality, weight loss, and food or beverage ingestion.

  As shown in FIG. 16, all mice treated with fluconazole survived for the duration of this study. One of the mice treated with AmB-containing bioactive agent delivery particles died on the second day of the study. Death was associated with the effect of this particle due to the time of death of this mouse and the lack of toxicity associated with AmB-containing particles at 5 mg / kg (see Example 13 above). I can't think of it. Conversely, all mice treated with “empty” disc particles died and only one AmBisome treated mouse survived.

  As shown in FIG. 17, mice treated with AmB-containing bioactive agent delivery particles showed only a slight weight loss (<2%) over the course of this study. Conversely, fluconazole-treated mice and AmBisome-treated mice showed a maximum weight loss of 14% and 23%, respectively, during the course of this experiment.

  As shown in FIG. 18, evaluation of fungal load showed low fungal levels in the brain and kidney of mice treated with AmB-containing bioactive agent delivery particles. Aside from the brain for fluconazole-treated mice, treatment with AmB-containing particles produced the lowest levels of brain and kidney fungal load among different therapeutic regimens.

  The results of this study demonstrated that AmB-containing bioactive agent delivery particles are effective for antifungal treatment. The mortality observed with AmBisme treatment was higher than previously reported studies using a similar regimen for treatment of Cryptococcosis infection (observed> 90% survival) (Clemons and Stevens (1998) Antimicrob Agents Chemother 42: 899-902). However, treatment of Candida albicans can be difficult due to the fact that the effect of AmBisome depends on the level of infection at the time of treatment (van Etten et al. (1998) Antimicrob Agents Chemother 42: 2433-1433). The inoculum used in this study may indicate that the level of fungal load exceeds the ability to effectively treat AmBisome, but the efficacy of AmB-containing disc particles was not affected by the level of this infection.

  Although the present invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, those skilled in the art will recognize that certain changes and modifications may be practiced without departing from the spirit and scope of the invention. Is obvious. Accordingly, this description should not be construed as limiting the scope of the invention which is indicated by the appended claims.

  All publications, patents, and patent applications cited herein are hereby incorporated by reference for all purposes and each publication, patent, or patent application specifically and individually. To the same extent as indicated, are hereby incorporated by reference in their entirety.

FIG. 1 shows the UV / visible absorption spectrum at 250-450 nm of ApoA-I-phospholipid particles containing no bioactive factor, prepared as in Example 1. FIG. 2 shows the UV / visible absorption spectrum at 250-450 nm of ApoA-I-phospholipid-AmB particles prepared as in Example 1. FIG. 3 shows a plot of the number of fractions versus protein concentration for ApoA-I-phospholipid-AmB particles after density gradient ultracentrifugation. The particles were prepared as described in Example 2 and adjusted to a density of 1.3 g / ml by the addition of KBr. This solution was centrifuged in a discontinuous gradient at 275,000 xg for 5 hours at 10 ° C. After centrifugation, the tube contents were fractionated from the top and fractionated into the protein content of each determined fraction. FIG. 4 shows native polyacrylamide gel electrophoresis (PAGE) analysis of ApoA-I-phospholipid particles (on a slab gel with a 4-20% acrylamide gradient). The particles were prepared with ApoA-I and two different lipid preparations (DMPC / DMPG or palmitoyl oleylphosphatidylcholine (POPC)). The gel was stained with Coomassie Blue. Lane 1: ApoA-I POPC particles; Lane 2: ApoA-I-POPC-AmB particles; Lane 3: ApoA-I-DMPC / DMPG-AmB particles. The relative movement of the size reference is shown on the left. FIG. 5 shows a comparison of the effect of different storage conditions on the size and structural integrity of apolipoprotein E N-terminal domain (ApoE3NT) -DMPC / DMPG-AmB particles. The particles were isolated by density ultracentrifugation and then subjected to electrophoresis on native PAGE (with a 4-20% gradient slab gel). The gel was stained with Amido Black. Lane 1: particles stored in phosphate buffer at 4 ° C. for 24 hours; Lane 2: particles stored in phosphate buffer at −20 ° C. for 24 hours; Lane 3: lyophilized and −80 Particles frozen for 24 hours at 0 ° C. and then redissolved in H ₂ O. The relative movement of the size reference is shown on the left. FIG. 6 schematically shows the shape and molecular mechanism of the bioactive agent delivery particle. FIG. 7 schematically illustrates chimeric lipid binding polypeptides and their incorporation into bioactive agent delivery particles. The chimeric protein can include a target portion (FIG. 7A) or a portion having the desired biological activity (FIG. 7B). FIG. 7C schematically illustrates incorporation of the chimeric polypeptide shown in FIGS. 7A and 7B into bioactive agent delivery particles. FIG. 8 graphically illustrates the antifungal activity of bioactive delivery particles comprising AmB against Saccharomyces cerevisiae (S. cerevisiae) in culture as described in Example 2. FIG. 9 is an electron micrograph of a cryogenic stage of bioactive delivery particles containing AmB prepared as described in Example 10. FIG. 10 shows the S. cerevisiae between ApoA-I-DMPC / DMPG-AmB particles and AmBisome® as described in Example 8. A comparison of the ability to inhibit cerevisiae growth is shown. FIG. 11 shows a comparison of fluorescence spectra between camptothecin solubilized in SDS (FIG. 11A) and bioactive agent delivery particles comprising camptothecin (FIG. 11B) as described in Example 9. . FIG. 12 shows AmB incorporation into lipid particles prepared as described in Example 7 (FIG. 12A) and AmB into bioactive agent delivery particles prepared as described in Example 6. A comparison of the UV / visible spectrum of the uptake of (FIG. 12B). FIG. 13 is an illustration of an embodiment of a procedure for preparing bioactive agent delivery particles. FIG. 14 shows the change in body weight of mice administered bioactive agent delivery particles containing the indicated dosages of AmB as described in Example 13. FIG. 15 shows urea serum levels (FIG. 15A), creatinine serum levels (FIG. 15A) in mice administered bioactive agent delivery particles containing the indicated dosages of AmB as described in Example 13. FIG. 15B), serum levels of aspartate aminotransferase (AST) (FIG. 15C), and serum levels of alanine aminotransferase (FIG. 15D). FIG. 16 shows the survival rate of mice that received the indicated treatment as described in Example 14. AMB-ND: bioactive agent delivery particles containing AmB; AmB: AmBisome; FLCZ: fluconazole; ND: disc particles without AmB. FIG. 17 shows the change in body weight of mice that received the indicated treatment as described in FIG. AMB-ND: bioactive agent delivery particles containing AmB; AmB: AmBisome; FLCZ: fluconazole; ND: disc particles without AmB. FIG. 18 shows the fungal burden on the tissue in mice that received the indicated treatment as described in FIG. AMB-ND: bioactive agent delivery particles containing AmB; AmB: AmBisome; FLCZ: fluconazole; ND: disc particles without AmB. FIG. 19 shows the effect of apolipoprotein AI on the light scattering intensity of AmB phospholipid vesicles. 200 micrograms of phospholipids (DMPC and DMPG (7: 3 molar ratio)) and 50 micrograms of AmB were dispersed by vortexing in 20 mM sodium phosphate (pH 7.4) and the apolipoprotein Incubated in the presence and absence at 24 ° C. The normal light scattering intensity of the samples was measured as a function of time in a Perkin-Elmer Model LS50b luminescence spectrometer. Was set to 600 nm with a slit width of 4 nm Curve A) AmB phospholipid vesicle preparation only. Curve B) AmB phospholipid vesicle preparation with 80 milligrams of apolipoprotein AI.

Claims (22)

A composition for treating a fungal infection in an individual comprising a lipid binding polypeptide, a lipid bilayer, and a bioactive factor delivery particle comprising a bioactive factor, wherein the bioactive factor is at least one hydrophobic An antifungal agent comprising a sex region, wherein the antifungal agent is a non-polypeptide, the delivery particle does not comprise a hydrophilic core, and the inside of the lipid bilayer comprises a hydrophobic region. The bioactive factor binds to the hydrophobic region of the lipid bilayer, the lipid binding polypeptide comprises a class A amphiphilic α-helix structural motif and / or β-sheet motif, and said particles are surrounded by the α- helix and / or β- sheet lipid binding polypeptide, a flat, a disk-shaped having a disk-shaped lipid bilayers, lipid binding Poripepu De bind around the lipid bilayer of the hydrophobic surface of the particles of the periphery, the composition is administered to said individual at a dosage of less 10 mg / kg, the bioactive agent is amphotericin B der A composition characterized by the above.   The composition of claim 1, wherein the discoidal particles have a diameter of 7 nm to 29 nm.   The composition according to claim 1, wherein the hydrophobic region of the bioactive factor binds to a hydrophobic surface inside the lipid bilayer. The composition according to any one of claims 1 to 3 , wherein the lipid-binding polypeptide is an apolipoprotein. 5. The composition of claim 4 , wherein the apolipoprotein is a replaceable apolipoprotein. 6. The composition according to claim 5 , wherein the apolipoprotein is human apolipoprotein AI. The composition of claim 4 , wherein the apolipoprotein is a chimeric apolipoprotein comprising a functional moiety. 8. The composition of claim 7 , wherein the functional moiety is a target moiety. 8. The composition of claim 7 , wherein the functional moiety comprises biological activity. The apolipoprotein has been modified to improve the stability of the particle , the modification introducing an cysteine residue into the apolipoprotein molecule that forms an intermolecular or intramolecular disulfide bond; and apolipoprotein It is selected from the group consisting of the use of chemical cross-linking agent which forms a link between molecules during molecular Ru composition of claim 4. 11. The composition of claim 10 , wherein the modification comprises introduction of a cysteine residue that forms an intermolecular disulfide bond or an intramolecular disulfide bond. The composition according to any one of claims 1 to 11 , wherein the lipid bilayer comprises a phospholipid. 13. The composition of claim 12 , wherein the phospholipid comprises dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG). The composition according to any one of claims 1 to 13 , further comprising a pharmaceutically acceptable carrier. 15. The composition of claim 14 , wherein the composition is formulated for sustained release. 15. A composition according to claim 14 for administering a bioactive factor to an individual. 17. The composition of claim 16 , wherein the composition contains a therapeutically effective amount of the bioactive factor. 18. A composition according to any one of claims 16 to 17 , wherein the composition is suitable for parenteral administration. 19. The composition of claim 18 , wherein the parenteral administration is selected from the group consisting of intravenous administration, intramuscular administration, intraperitoneal administration, transmucosal administration, and intrathecal administration. 18. A composition according to claim 16 or 17 , wherein the composition is suitable for administration as an aerosol. 18. A composition according to claim 16 or 17 , wherein the composition is formulated for sustained release. A kit for treating a fungal infection in an individual comprising the composition of claim 14 or 15 and instructions for use in a method for administering the bioactive factor to the individual.

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