Classifications

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Definitions

• the disclosure relates generally to compositions comprising coated particles and to methods of making and using such compositions for targeted drug delivery.
• Nanoparticles are solid or semi-solid particles having a diameter from about 1 nm to about 10,000 nm (10 microns), preferably from about 1 nm to about 2,000 nm (2 microns).
• Such particles can be formed from a variety of materials, including proteins, synthetic polymers, polysaccharides, nucleic acids, small molecules, and combinations thereof, and have been used in many different applications, primarily separations, diagnostics, and drug delivery.
• compositions comprising such particles have been found to be useful for drug delivery.
• U.S. Patent Publication No. 2006/0073199 discloses that particles comprising an active agent can be formulated as aqueous suspensions, and stabilized against aggregation and particle growth by providing surfactant coatings on or about the particles.
• compositions comprising particles and methods for making and using same, particularly in delivering drugs of interest.
• One aspect of the invention is directed to a surface-modified particle comprising a particle core and a coating associated with the particle core.
• the particle core comprises an active agent, such as a therapeutic agent or a diagnostic agent (e.g., a small organic molecule or a biomacromolecule).
• the surface-modified particles according to the present invention generally have an average size from about 1 nm to about 2,000 nm.
• Another aspect of the invention is directed to a method of enhancing cellular uptake of an active agent.
• the method comprises contacting cells with surface-modified particles under conditions sufficient to enhance cellular uptake of the surface-modified particles.
• the particles comprise a particle core and a coating associated with the particle core, wherein the particle core comprises an active agent, the coating comprises a surfactant of formula I or a salt thereof, and the surface-modified particle has an average size from about 1 nm to about 2,000 nm.
• These methods may be carried out ex vivo or in vivo.
• Each of the methods of enhancing cellular uptake of an active agent may be carried out ex vivo, i.e., without any treatment of the human body or animal body by surgery or therapy.
• the methods of enhancing cellular uptake of an active agent of the invention can also be carried out in vivo.
• the cells may be phagocytic cells such as macrophages, monocytes, granulocytes, agranulocytes, neutrophils and combinations thereof.
• the cells may be tumor cells.
• Another aspect of the invention is directed to use of a plurality of surface-modified particles in the preparation of a medicament for enhancing cellular uptake of an active agent, wherein said surface-modified particle comprises a particle core and a coating associated with the particle core, the particle core comprises an active agent selected from the group consisting of small molecules, peptides, and proteins, the coating comprises a surfactant of formula I or a salt thereof, and the surface-modified particle has an average size from about 1 nm to about 2,000 nm.
• Another aspect of the invention is directed to a plurality of surface-modified particles for use in enhancing cellular uptake of an active agent, wherein said surface- modified particle comprises a particle core and a coating associated with the particle core, the particle core comprises an active agent selected from the group consisting of small molecules, peptides, and proteins, the coating comprises a surfactant of formula I or a salt thereof, and the surface-modified particle has an average size from about 1 nm to about 2,000 nm.
• Another aspect of the invention is directed to a method for treating a subject having an inflammatory disease or disorder comprising administering to said subject a plurality of surface-modified particles, said surface-modified particles comprising a particle core and a coating associated with the particle core, wherein the particle core comprises an active agent (e.g., an anti-inflammatory agent), the coating comprises a surfactant of formula I or a salt thereof, the surface-modified particle has an average size from about 1 nm to about 2,000 nm, and said administration is effective in alleviating, treating, and/or preventing symptoms or pathologies associated with said inflammatory disease or disorder.
• an active agent e.g., an anti-inflammatory agent
• the coating comprises a surfactant of formula I or a salt thereof
• the surface-modified particle has an average size from about 1 nm to about 2,000 nm
• said administration is effective in alleviating, treating, and/or preventing symptoms or pathologies associated with said inflammatory disease or disorder.
• Another aspect of the invention is directed to use of a plurality of surface-modified particles in the preparation of medicament used for treating an inflammatory disease or disorder
• the medicament comprises an amount of the surface-modified particles effective in alleviating, treating, and/or preventing symptoms or pathologies associated with an inflammatory disease or disorder
• said surface-modified particle comprises a particle core and a coating associated with the particle core
• the particle core comprises an active agent (e.g., an anti-inflammatory agent)
• the coating comprises a surfactant of formula I or a salt thereof
• the surface-modified particle has an average size from about 1 nm to about 2,000 nm.
• Another aspect of the invention is directed to a plurality of surface-modified particles or a pharmaceutical composition thereof for use in treating a subject having an inflammatory disease or disorder, wherein said surface-modified particle comprises a particle core and a coating associated with the particle core, the particle core comprises an active agent (e.g., an anti-inflammatory agent), the coating comprises a surfactant of formula I or a salt thereof, and the surface-modified particle has an average size from about 1 nm to about 2,000 nm.
• an active agent e.g., an anti-inflammatory agent
• the coating comprises a surfactant of formula I or a salt thereof
• the surface-modified particle has an average size from about 1 nm to about 2,000 nm.
• Another aspect of the invention is directed to a method for treating a subject having a proliferative disease or disorder comprising administering to said subject a plurality of surface-modified particles, said surface-modified particles comprising a particle core and a coating associated with the particle core, wherein the particle core comprises an active agent (e.g., an anti-proliferative such as an antineoplastic agent), the coating comprises a surfactant of formula I or a salt thereof, the surface-modified particle has an average size from about 1 nm to about 2,000 nm, and said administration is effective in alleviating, treating, and/or preventing symptoms or pathologies associated with said proliferative disease or disorder.
• an active agent e.g., an anti-proliferative such as an antineoplastic agent
• the coating comprises a surfactant of formula I or a salt thereof
• the surface-modified particle has an average size from about 1 nm to about 2,000 nm
• said administration is effective in alleviating, treating, and/or
• Another aspect of the invention is directed to use of a plurality of surface-modified particles in the preparation of medicament used for treating a proliferative disease or disorder in wherein the medicament comprises an amount of surface-modified particles effective in alleviating, treating, and/or preventing symptoms or pathologies associated with a proliferative disease or disorder, wherein said surface-modified particle comprises a particle core and a coating associated with the particle core, the particle core comprises an active agent (e.g., an anti-proliferative such as an antineoplastic agent), the coating comprises a surfactant of formula I or a salt thereof, and the surface-modified particle has an average size from about 1 nm to about 2,000 nm.
• an active agent e.g., an anti-proliferative such as an antineoplastic agent
• the coating comprises a surfactant of formula I or a salt thereof
• the surface-modified particle has an average size from about 1 nm to about 2,000 nm.
• Another aspect of the invention is directed to a plurality of surface-modified particles or a pharmaceutical composition thereof for use in treating a subject having a proliferative disease or disorder, wherein said surface-modified particle comprises a particle core and a coating associated with the particle core, the particle core comprises an active agent (e.g., an anti-proliferative such as an antineoplastic agent), the coating comprises a surfactant of formula I or a salt thereof, and the surface-modified particle has an average size from about 1 nm to about 2,000 nm.
• an active agent e.g., an anti-proliferative such as an antineoplastic agent
• the coating comprises a surfactant of formula I or a salt thereof
• the surface-modified particle has an average size from about 1 nm to about 2,000 nm.
• Another aspect of the invention is directed to a method for treating a subject having an infectious disease or disorder comprising administering to said subject a plurality of surface-modified particles, said surface-modified particles comprising a particle core and a coating associated with the particle core, wherein the particle core comprises an active agent (e.g., an anti-infective agent), the coating comprises a surfactant of formula I or a salt thereof, the surface-modified particle has an average size from about 1 nm to about 2,000 nm, and said administration is effective in alleviating, treating, and/or preventing symptoms or pathologies associated with said infectious disease or disorder.
• an active agent e.g., an anti-infective agent
• the coating comprises a surfactant of formula I or a salt thereof
• the surface-modified particle has an average size from about 1 nm to about 2,000 nm
• said administration is effective in alleviating, treating, and/or preventing symptoms or pathologies associated with said infectious disease or disorder.
• Another aspect of the invention is directed to use of a plurality of surface-modified particles in the preparation of medicament used for treating an infectious disease or disorder
• the medicament comprises an amount of the surface-modified particles effective in alleviating, treating, and/or preventing symptoms or pathologies associated with an infectious disease or disorder
• said surface-modified particle comprises a particle core and a coating associated with the particle core, wherein the particle core comprises an active agent (e.g., an anti-infective agent), the coating comprises a surfactant of formula I or a salt thereof, and the surface-modified particle has an average size from about 1 nm to about 2,000 nm.
• an active agent e.g., an anti-infective agent
• the coating comprises a surfactant of formula I or a salt thereof
• the surface-modified particle has an average size from about 1 nm to about 2,000 nm.
• Another aspect of the invention is directed to a plurality of surface-modified particles or a pharmaceutical composition thereof for use in treating a subject having an infectious disease or disorder
• said surface-modified particle comprises a particle core and a coating associated with the particle core, wherein the particle core comprises an active agent (e.g., an anti-infective agent), the coating comprises a surfactant of formula I or a salt thereof, and the surface-modified particle has an average size from about 1 nm to about 2,000 nm.
• an active agent e.g., an anti-infective agent
• the coating comprises a surfactant of formula I or a salt thereof
• the surface-modified particle has an average size from about 1 nm to about 2,000 nm.
• the invention is directed to a method for treating a subject having a neurodegenerative disease or disorder comprising administering to said subject a plurality of surface-modified particles, said surface-modified particles comprising a particle core and a coating associated with the particle core, wherein the particle core comprises an active agent (e.g., an anti-neurodegenerative agent), the coating comprises a surfactant of formula I or a salt thereof, the surface-modified particle has an average size from about 1 nm to about 2,000 nm, and said administration is effective in alleviating, treating, and/or preventing symptoms or pathologies associated with said neurodegenerative disease or disorder.
• an active agent e.g., an anti-neurodegenerative agent
• the coating comprises a surfactant of formula I or a salt thereof
• the surface-modified particle has an average size from about 1 nm to about 2,000 nm
• said administration is effective in alleviating, treating, and/or preventing symptoms or pathologies associated with said neurodegenerative disease or disorder.
• Another aspect of the invention is directed to use of a plurality of surface-modified particles in the preparation of medicament used for treating of a neurodegenerative disease or disorder, wherein the medicament comprises an amount of the surface-modified particles effective in alleviating, treating, and/or preventing symptoms or pathologies associated with a neurodegenerative disease or disorder, wherein said surface-modified particle comprises a particle core and a coating associated with the particle core, wherein the particle core comprises an active agent (e.g., an anti-neurodegenerative agent), the coating comprises a surfactant of formula I or a salt thereof, and the surface-modified particle has an average size from about 1 nm to about 2,000 nm.
• an active agent e.g., an anti-neurodegenerative agent
• the coating comprises a surfactant of formula I or a salt thereof
• the surface-modified particle has an average size from about 1 nm to about 2,000 nm.
• Another aspect of the invention is directed to a plurality of surface-modified particles or a pharmaceutical composition thereof for use in treating a subject having a neurodegenerative disease or disorder, wherein said surface-modified particle comprises a particle core and a coating associated with the particle core, wherein the particle core comprises an active agent (e.g., an anti-neurodegenerative agent), the coating comprises a surfactant of formula I or a salt thereof, and the surface-modified particle has an average size from about 1 nm to about 2,000 nm.
• an active agent e.g., an anti-neurodegenerative agent
• the coating comprises a surfactant of formula I or a salt thereof
• the surface-modified particle has an average size from about 1 nm to about 2,000 nm.
• Another aspect of the invention is directed to a method for treating a subject having an infectious disease or disorder, an inflammatory disease or disorder, a neurodegenerative disease or disorder, or a proliferative disease or disorder comprising administering to said subject a plurality of surface-modified particles into a body cavity having a site of disease or inflammation, said surface-modified particles comprising a particle core and a coating associated with the particle core, wherein the particle core comprises an active agent, the coating comprises a surfactant of formula I or a salt thereof, the surface-modified particle has an average size from about 1 nm to about 2,000 nm, and said administration is effective in alleviating, treating, and/or preventing symptoms or pathologies associated with said disease or disorder.
• Another aspect of the invention is directed to use of a plurality of surface-modified particles in the preparation of medicament used for the treating of an infectious disease or disorder, an inflammatory disease or disorder, a neurodegenerative disease or disorder, or a proliferative disease or disorder
• the medicament comprises an amount of the surface- modified particles effective in alleviating, treating, and/or preventing symptoms or pathologies associated with said disease or disorder
• said surface-modified particle comprises a particle core and a coating associated with the particle core, wherein the particle core comprises an active agent, the coating comprises a surfactant of formula I or a salt thereof, and the surface-modified particle has an average size from about 1 nm to about 2,000 nm.
• Another aspect of the invention is directed to a plurality of surface-modified particles or a pharmaceutical composition thereof for use in treating a subject having an infectious disease or disorder, an inflammatory disease or disorder, a neurodegenerative disease or disorder, or a proliferative disease or disorder, wherein said surface-modified particle comprises a particle core and a coating associated with the particle core, wherein the particle core comprises an active agent, the coating comprises a surfactant of formula I or a salt thereof, and the surface-modified particle has an average size from about 1 nm to about 2,000 nm.
• Each of the aforementioned methods for treating can be effected by using cellular transport to deliver the surface-modified particles to a target tissue of the subject, or by localized administration of the surface-modified particles into a body cavity having a site of disease (e.g., cancer, infection) and/or inflammation in the subject such that the surface- modified particles can be taken up by diseased or inflammatory cells located within the body cavity.
• a site of disease e.g., cancer, infection
• FIG. 1 provides graphs showing uptake of DSPE-mPEG2000/poloxamer 188- coated paclitaxel particles labeled with Oregon Green (No DOTAP) and DOTAP-coated paclitaxel particles labeled with Oregon Green (DOTAP).
• FIG. 2 provides graphs showing uptake of DSPE-mPEG2000/poloxamer 188- coated paclitaxel particles (DSPE-mPEG2000/poloxamer 188), DOTAP-coated paclitaxel particles labeled with Oregon Green and stored for 3 months (DOTAP Sample 1), freshly prepared DOTAP-coated paclitaxel particles labeled with Oregon Green (DOTAP Sample 2), and protamine-coated paclitaxel particles labeled with Oregon Green (Protamine).
• FIG. 3 provides graphs showing uptake of DSPE-mPEG2000/poloxamer 188- coated paclitaxel particles.
• the Oregon Green coated particles either contained DOTAP or did not contain DOTAP (No DOTAP). Cells were cultured for 1, 2, or 6 days prior to exposing the cells to the paclitaxel particles.
• FIG. 4 provides graphs showing uptake of DSPE-mPEG2000/poloxamer 188- coated paclitaxel particles labeled with Oregon Green (DSPE-mPEG2000/poloxamer 188), DOTAP-coated paclitaxel particles labeled with Oregon Green (DOTAP/DSPE- mPEG2000/poloxamer 188), polylactic-co-glycolic acid-coated paclitaxel particles labeled with Oregon Green (PLGA/poloxamer 188), and phosphatidylserine-coated paclitaxel particles labeled with Oregon Green (PS/DSPE-mPEG2000/poloxamer 188).
• FIG. 5 provides graphs showing uptake of DSPE-mPEG2000/poloxamer 188- coated paclitaxel particles labeled with Oregon Green (DSPE-mPEG2000/poloxamer 188), DOTAP-coated paclitaxel particles labeled with Oregon Green (DOTAP/DSPE- mPEG2000/poloxamer 188), and cetyl trimethylammonium bromide-coated paclitaxel particles labeled with Oregon Green (CTAB/DSPE-mPEG2000/poloxamer 188).
• DETAILED DESCRIPTION DETAILED DESCRIPTION
• One aspect of the invention provides a surface-modified particle comprising a particle core and a coating associated with the particle core.
• the particle core comprises an active agent which is typically selected from the group consisting of small molecules, peptides, and proteins
• alkenyl refers to straight chained and branched hydrocarbon groups containing at least one carbon-carbon double bond, nonlimiting examples of which include straight chain and branched hexadecenyl and octadecenyl groups.
• alkynyl refers to straight chained and branched hydrocarbon groups containing at least one carbon-carbon triple bond, nonlimiting examples of which include straight chain and branched hexadecynyl and octadecynyl groups.
• R 1 , R 2 , and R 3 alkyl groups of formula I can have, for example, from 1 to 8 carbon atoms, from 1 to 6 carbon atoms, and/or from 1 to 4 carbon atoms.
• R 1 , R 2 , and R 3 are independently selected from the group consisting of methyl and ethyl.
• R 4 and R 5 alkyl groups of formula I can have, for example, from 6 to 40 carbon atoms, from 10 to 24 carbon atoms, from 14 to 18 carbon atoms, from 5 to 39 carbon atoms, from 9 to 23 carbon atoms, and/or from 13 to 17 carbon atoms.
• R 4 and R 5 alkenyl groups of formula I can have, for example, 1, 2, 3, 4, 5, 6, or more double bonds.
• the R 4 and R 5 alkenyl groups can have, for example, from 6 to 40 carbon atoms, from 10 to 24 carbon atoms, from 14 to 18 carbon atoms, from 5 to 39 carbon atoms, from 9 to 23 carbon atoms, and/or from 13 to 17 carbon atoms.
• R 4 and R 5 alkynyl groups of formula I can have, for example, 1, 2, 3, 4, 5, 6, or more triple bonds.
• the R 4 and R 5 alkynyl groups can have, for example, from 6 to 40 carbon atoms, from 10 to 24 carbon atoms, from 14 to 18 carbon atoms, from 5 to 39 carbon atoms, from 9 to 23 carbon atoms, and/or from 13 to 17 carbon atoms.
• R 4 and R 5 are independently selected from the group consisting of octyl, 2-ethylhexyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, cis-9- hexadecenyl, octadecyl, 16-methylheptadecyl, trans-9-octadeceny ⁇ , ds-9-octadecenyl, d ⁇ ds ⁇ , 12-octadecadienyl, trans, tr ⁇ ns-9,12-octadecadienyl, cis,cis,cis-9, 12,15- octadecatrienyl, trans, trans, tr ⁇ ns-9,12,15-octadccatxicnyl, 12-hydroxy-9-octadecenyl, eicosanyl, doco
• m and n are 1; R 1 , R 2 , and R 3 are methyl; and R 4 and R 5 are ds-Q-octadecenoyl, i.e., the surfactant of formula I is N-[l-(2,3-dioleoyloxy)propyl]-N,N,N- trimethylammonium (DOTAP) or a salt thereof (e.g., N-[l-(2,3-dioleoyloxy)propyl]-N,N,N- trimethylammonium methylsulfate or N-[l-(2,3-dioleoyloxy)propyl]-N,N,N- trimethylammonium chloride).
• DOTAP N-[l-(2,3-dioleoyloxy)propyl]-N,N,N- trimethylammonium
• DOTAP N-[l-(2,3-dioleoyloxy)propyl]
• m and n are 1; R 1 , R 2 , and R 3 are methyl; and R 4 and R 5 are ds-Q-octadecenyl, i.e., the surfactant of formula I is N- [1 -(2,3- dioleyloxy)propyl]-N,N,N-trimethylammonium (DOTMA), or a salt thereof (e.g., N-[l-(2,3- dioleyloxy)propyl]-N,N,N-trimethylammonium methylsulfate or N- [1 -(2,3- dioleyloxy)propyl]-N,N,N-trimethylammonium chloride).
• DOTMA N-[l-(2,3- dioleyloxy)propyl]-N,N,N-trimethylammonium methylsulfate or N- [1 -(2,3- dioleyloxy)propyl]-N,N,N-trimethylammoni
• the active agent is paclitaxel; m and n are 1; R 1 , R 2 , and R 3 are methyl; and R 4 and R 5 are cis-9- octadecenoyl, i.e., the surfactant of formula I is N-[l-(2,3-dioleoyloxy)propyl]-N,N,N- trimethylammonium (DOTAP) or a salt thereof (e.g., N-[l-(2,3-dioleoyloxy)propyl]-N,N,N- trimethylammonium methylsulfate or N-[l-(2,3-dioleoyloxy)propyl]-N,N,N- trimethylammonium chloride).
• DOTAP N-[l-(2,3-dioleoyloxy)propyl]-N,N,N- trimethylammonium
• DOTAP N-[l-(2,3-dio
• the active agent is paclitaxel; m and n are 1; R 1 , R 2 , and R 3 are methyl; and R 4 and R 5 are ds-Q-octadecenyl, i.e., the surfactant of formula I is N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium (DOTMA), or a salt thereof (e.g., N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium methylsulfate or N- [l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride).
• DOTMA N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium methylsulfate or N- [l-(2,3-dioleyloxy)propyl
• Another aspect of the present invention provides methods for enhancing uptake of an active agent by phagocytic or non-phagocytic cells by exposing the cells to a surface- modified particle comprising a particle core and a coating associated with the particle core, thereby forming cells loaded with the surface modified particles.
• the particle core comprises an active agent which is typically selected from the group consisting of small molecules, peptides, and proteins
• the coating comprises a surfactant of formula I or a salt thereof
• the surface-modified particle has an average size from about 1 nm to about 2,000 nm.
• Enhanced uptake by the cells of the active agent is observed at least as compared to cells contacted with particles not having a coating comprising a surfactant of formula I or a salt thereof.
• the invention provides for use of a plurality of surface- modified particles in the preparation of a medicament for enhancing cellular uptake of an active agent by phagocytic or non-phagocytic cells.
• the invention also provides methods for delivery of an active agent or a pharmaceutical composition comprising a plurality of surface-modified particles to a target tissue of a mammalian subject through cellular transport using the aforementioned cells loaded with the surface modified particles.
• Efficient methods of targeted delivery of surface-modified particles allow for administration of increased concentrations of the therapeutic agent to a site of disease, tumor, or infection with less adverse systemic effects and less toxicity.
• various methods of administration such as intravenous administration, intramuscular administration, subcutaneous administration, intraperitoneal, oral and the like will facilitate enhanced uptake of particles by cells that traffic to the lymphatic system, the liver, and other tissue targets.
• Subcutaneous administration for example, is contemplated for various diseases, including head and neck cancers which invade locoregionally along the lymphatics.
• target tissue refers to the particular tissue of the subject to be treated.
• target tissues include, but are not limited to, the brain and other portions of the central nervous system, the lymphatic system (e.g., lymph nodes, bone marrow, spleen, thymus, etc.), the liver, and any site of infection, inflammation, or tumor.
• delivery to a target tissue can be effected by localized administration of the surface-modified particles into a body cavity having a site of disease (e.g., cancer, infection) and/or inflammation in the subject such that the surface-modified particles can be taken up by diseased or inflammatory cells located within the body cavity so as to deliver the active agent in close proximity to the diseased tissue target.
• a site of disease e.g., cancer, infection
• cancers of the peritoneal cavity such as ovarian cancer, peritoneal mesothelioma, peritoneal carcinomatosis, and the like can be treated by intraperitoneally administering the particles or pharmaceutical compositions comprising the particles into the peritoneal cavity.
• bladder cancers, infections, and/or inflammation can be treated by administering the particles or pharmaceutical compositions comprising the particles into the bladder cavity;
• pulmonary cancers, infections, and/or inflammation can be treated by administering the particles into the pulmonary cavity (e.g., via inhalation);
• cancers, infections, and/or inflammation of the pleural cavity can be treated by administering particles into the pleural cavity;
• cancers, infections, and/or inflammation of the cardiac cavity can be treated by administering particles or pharmaceutical compositions comprising the particles into the cardiac cavity;
• ophthalmic cancers, infections, and/or inflammation can be treated by administering the particles or pharmaceutical compositions comprising the particles into the aqueous humor or vitreous humor of the eye.
• gastrointestinal cancers, infections, and/or inflammation can be treated by administering the particles or pharmaceutical compositions comprising the particles into the stomach and/or intestines (e.g., via oral administration).
• the surface-modified particles or pharmaceutical compositions comprising the surface-modified particles are administered proximate to and/or adjacent to a site of disease or inflammation via administration to a body cavity containing the site of disease or inflammation, the surface-modified particles can be taken up by the diseased (e.g., cancerous, infected) or inflammatory cells located at the site of disease or inflammation such that enhanced uptake by the diseased or inflammatory cells of the surface modified particles according to the invention is observed at least as compared to cells contacted with particles not having a coating comprising a surfactant of formula I or a salt thereof.
• a body cavity refers to a relatively empty space surrounded by a supporting tissue or a fluid-filled space surrounded by a supporting tissue.
• a body cavity encompasses both the tissue surrounding (and defining) the cavity and the complete interior of the cavity.
• Exemplary body cavities include the peritoneal cavity, the bladder cavity, the pulmonary cavity, the pleural cavity, the cardiac cavity, the gastrointestinal cavity, the aqueous humor of the eye, and the vitreous humor of the eye.
• the invention contemplates methods, compositions and medicaments for treating a subject having an inflammatory disease or disorder comprising administering to said subject a plurality of surface-modified particles, said surface-modified particles comprising a particle core and a coating associated with the particle core, wherein the particle core comprises an active agent which is typically selected from the group consisting of small molecules, peptides, and proteins, the coating comprises a surfactant of formula I, as defined herein, or a salt thereof, the surface-modified particle has an average size from about 1 nm to about 2,000 nm, and said administration is effective in alleviating, treating, and/or preventing symptoms or pathologies associated with said inflammatory disease or disorder.
• the subject has an inflammatory disease or disorder, and m and n are 1; R 1 , R 2 , and R 3 are methyl; and R 4 and R 5 are ds-Q-octadecenoyl.
• the active agent is an antiinflammatory agent. Delivery of the active agent can be affected via cellular transport, as described herein, or by local administration to the site of inflammation, as described herein.
• the invention contemplates methods, compositions and medicaments for treating a subject having a neurodegenerative disease or disorder comprising administering to said subject a plurality of surface-modified particles, said surface-modified particles comprising a particle core and a coating associated with the particle core, wherein the particle core comprises an active agent which is typically selected from the group consisting of small molecules, peptides, and proteins, the coating comprises a surfactant of formula I, as defined herein, or a salt thereof, the surface-modified particle has an average size from about 1 nm to about 2,000 nm, and said administration is effective in alleviating, treating, and/or preventing symptoms or pathologies associated with said neurodegenerative disease or disorder.
• the subject has a neurodegenerative disease or disorder, and m and n are 1; R 1 , R 2 , and R 3 are methyl; and R 4 and R 5 are ds-Q-octadecenoyl.
• the active agent is an anti-neurodegenerative agent. Delivery of the active agent can be affected via cellular transport, as described herein.
• the invention contemplates methods, compositions and medicaments for treating a subject having a proliferative disease or disorder comprising administering to said subject a plurality of surface-modified particles, said surface-modified particles comprising a particle core and a coating associated with the particle core, wherein the particle core comprises an active agent which is typically selected from the group consisting of small molecules, peptides, and proteins, the coating comprises a surfactant of formula I, as defined herein, or a salt thereof, the surface-modified particle has an average size from about 1 nm to about 2,000 nm, and said administration is effective in alleviating, treating, and/or preventing symptoms or pathologies associated with said proliferative disease or disorder.
• the particle core comprises an active agent which is typically selected from the group consisting of small molecules, peptides, and proteins
• the coating comprises a surfactant of formula I, as defined herein, or a salt thereof
• the surface-modified particle has an average size from about 1 nm to about 2,000 nm
• the subject has a proliferative disease or disorder, and m and n are Ij R 1 , R 2 , and R 3 are methyl; and R 4 and R 5 are ds-Q-octadecenoyl.
• the active agent is an anti-proliferative agent such as an antineoplastic agent. Delivery of the active agent can be affected via cellular transport, as described herein, or by local administration to the site of disease, as described herein.
• the invention contemplates methods, compositions and medicaments for treating a subject having an infectious disease or disorder comprising administering to said subject a plurality of surface-modified particles, said surface-modified particles comprising a particle core and a coating associated with the particle core, wherein the particle core comprises an active agent which is typically selected from the group consisting of small molecules, peptides, and proteins, the coating comprises a surfactant of formula I, as defined herein, or a salt thereof, the surface-modified particle has an average size from about 1 nm to about 2,000 nm, and said administration is effective in alleviating, treating, and/or preventing symptoms or pathologies associated with said infectious disease or disorder.
• the subject has an infectious disease or disorder, and m and n are 1; R 1 , R 2 , and R 3 are methyl; and R 4 and R 5 are ds-Q-octadecenoyl.
• the active agent is an anti-infective agent such as an anti-fungal agent, an anti- viral agent, an antibacterial agent, or an anti-parasitic agent. Delivery of the active agent can be affected via cellular transport, as described herein, or by local administration to the site of disease, as described herein.
• the methods of administration disclosed herein contemplate administration of a therapeutically effective amount of said surface modified particles.
• therapeutically effective amount refers to an amount of surface-coated particles that is sufficient to alleviate, ameliorate, clear, treat, and/or prevent symptoms or pathologies associated with a disease or disorder contemplated for treatment in accordance with the treatment methods disclosed herein. Determination of therapeutically effective amounts is well within the capability of those skilled in the art, especially in light of the disclosure provided herein.
• the active agent of the surface-modified particle can be poorly water soluble or water soluble.
• the active agent can be a therapeutic agent or a diagnostic agent.
• the active agent can be a small molecule or a biologic, such as a protein, a peptide, a carbohydrate, or a complex, conjugate, or combination thereof.
• DNA, RNA, oligonucleotides, and polynucleotides are not suitable active agents for use with the surface modified particles of the invention.
• Active agents used in accordance with the compositions and methods disclosed herein exhibit the pharmaceutical activities normally associated with such active agents notwithstanding that the active agents can be taken up and subsequently delivered to target tissues by phagocytic or non-phagocytic cells.
• active agents also can be administered locally at a site of disease(e.g., cancer, infection) and/or inflammation in a mammalian subject and taken up by diseased cells (such as infected or cancerous cells), or inflammatory cells, located at the site of disease and/or inflammation.
• the active agent can be selected from a variety of known pharmaceutical compounds such as, but not limited to: analgesics, anesthetics, analeptics, adrenergic agents, adrenergic blocking agents, adrenolytics, adrenocorticoids, adrenomimetics, anticholinergic agents, anticholinesterases, anticonvulsants, alkylating agents, alkaloids, allosteric inhibitors, anabolic steroids, anorexiants, antacids, antidiarrheals, antidotes, antifolics, antipyretics, antirheumatic agents, psychotherapeutic agents, neural blocking agents, anti-inflammatory agents, antihelmintics, anticoagulants, antidepressants, antiepileptics, antifibrotic agents, anti- infective agents (e.g., antifungals, antiviral agents such as antiretroviral agents such as nucleoside reserve transcriptase inhibitors, non-nucle
• antineoplastic agents include, but are not limited to, paclitaxel, paclitaxel derivative compounds, alkaloids, antimetabolites, enzyme inhibitors, alkylating agents, and combinations thereof.
• the active agent also can be a protease inhibitor, such as an HIV protease inhibitor.
• protease inhibitors include, but are not limited to, indinavir, ritonavir, saquinavir, nelfinavir, atazanavir and combinations thereof.
• the active agent can be a nucleoside reverse transcriptase inhibitor. Examples of nucleoside reverse transcriptase inhibitors include, but are not limited to, zidovudine, didanosine, stavudine, zalcitabine, lamivudine, and combinations thereof.
• the active agent can be a non-nucleoside reverse transcriptase inhibitor.
• non-nucleoside reverse transcriptase inhibitors include, but are not limited to, efavirenz, nevirapine, delaviradine, and combinations thereof.
• anti-inflammatory agents include, but are not limited to, non-steroidal anti-inflammatory drugs, non- selective cycloxygenase (COX) inhibitors, COX-I inhibitors, COX-2 inhibitors, lipoxygenase inhibitors, corticosteroids, anti-oxidants, tumor necrosis factor (TNF) inhibitors, and combinations thereof.
• COX-2 inhibitors include, but are not limited to, celecoxib, rofecoxib, valdecoxib, parecoxib, lumiracoxib, etoricoxib, and combinations thereof.
• Diagnostic agents include x-ray imaging agents and contrast media.
• x-ray imaging agents include WIN-8883 (ethyl 3,5-diacetamido-2,4,6-triiodobenzoate) also known as the ethyl ester of diatrazoic acid (EEDA), WIN 67722, i.e., (6-ethoxy-6-oxohexyl- 3,5-bis(acetamido)-2,4,6-triiodobenzoate; ethyl-2-(3,5-bis(acetamido)-2,4,6-triiodo- benzoyloxy) butyrate (WIN 16318); ethyl diatrizoxyacetate (WIN 12901); ethyl 2-(3,5- bis(acetamido)-2,4,6-triiodobenzoyloxy)propionate (WIN 16923); N-ethyl 2-(3,5- bis(acetamidamido)
• Contrast agents include those that are expected to disintegrate relatively rapidly under physiological conditions, thus minimizing any particle associated inflammatory response. Disintegration can result from enzymatic hydrolysis, solubilization of carboxylic acids at physiological pH, or other mechanisms. Thus, poorly soluble iodinated carboxylic acids such as iodipamide, diatrizoic acid, and metrizoic acid, along with hydrolytically labile iodinated species such as WIN 67721, WIN 12901, WIN 68165, and WIN 68209 are included.
• Other contrast media include, but are not limited to, particulate preparations of magnetic resonance imaging aids such as gadolinium chelates, or other paramagnetic contrast agents. Examples of such compounds are gadopentetate dimeglumine (MA GNEVIS T®) and gadoteridol (PROHANCE®).
• the active agent is a poorly water-soluble compound.
• “poorly water soluble” is a solubility of the compound in water of less than about 10 mg/mL, and preferably less than about 1 mg/mL.
• These poorly water-soluble compounds are particularly suitable for aqueous suspension preparations since there are limited alternatives of formulating these compounds in an aqueous medium.
• surfactants of formula I, or salts thereof, which provide the coatings in accordance with the invention can adsorb to the surface of particles comprising such poorly water soluble active agents to form a substantially uniform coating thereon.
• the hydrophobic tail moieties of surfactants of formula I or salts thereof can associate with hydrophobic regions on the particle surface.
• surfactants of formula I or salts thereof are positively charged, and thus electrostatic interactions between the surfactant and negatively charged regions on the particle surface can stabilize the coating comprising the surfactant of formula I or salt thereof.
• the poorly water soluble active agent compound is an organic compound having a molecular weight less than 2500 grams/mol, less than 2000 grams/mol, and most typically less than 1000 grams/mol, for example, between 200 grams/mol and 900 grams/mol. Such organic compounds are referred to herein as "small molecules.”
• the invention can be practiced with water-soluble compounds.
• the water soluble active compounds can be entrapped in a solid carrier matrix (for example, polylactate-polyglycolate copolymer, albumin, starch), or encapsulated in a surrounding vesicle that is substantially impermeable to the active agent.
• This encapsulating vesicle can be a polymeric coating such as polyacrylate.
• the small particles prepared from these water soluble compounds can be modified to improve chemical stability and control the pharmacokinetic properties of the compounds by controlling the release of the compounds from the particles.
• water-soluble compounds include, but are not limited to, simple organic compounds, proteins, peptides, nucleotides, and carbohydrates.
• the following description of particles also applies to all embodiments disclosed herein.
• the particles can be amorphous, semicrystalline, crystalline, or a combination thereof as determined by suitable analytical methods such as differential scanning calorimetry (DSC) or X-ray diffraction.
• DSC differential scanning calorimetry
• X-ray diffraction X-ray diffraction.
• the particles Prior to administration, can be homogenized through a homogenization process.
• the particles can also be homogenized through a microprecipitation/homogenization proces s .
• the coated particles generally have an average effective particle size of generally from about 1 nm to about 2 ⁇ m (or 2000 nanometers) as measured by dynamic light scattering methods (e.g., photocorrelation spectroscopy, laser diffraction, low-angle laser light scattering (LALLS), medium-angle laser light scattering (MALLS)), light obscuration methods (Coulter method, for example), rheology, or microscopy (light or electron).
• the preferred average effective particle size depends on factors such as the intended route of administration, formulation, solubility, toxicity and bioavailability of the compound.
• Other suitable particle sizes include, but are not limited to, about 10 nm to about 1 ⁇ m, about 50 nm to about 500 nm, and/or about 100 nm to about 250 nm.
• the coated particles are solid or semi-solid particles comprising active agents.
• the coated particles generally consist of at least 5% (w/w) active agent, for example, at least 10% (w/w), at least 25% (w/w), at least 50% (w/w), and/or at least 75% (w/w) or more active agent.
• the method of preparing small particle dispersions using energy addition techniques includes the step of adding the active agent or pharmaceutically active compound, which sometimes shall be referred to as a drug, in bulk form to a suitable vehicle such as water or aqueous solution generally containing one or more of the surfactants set forth below, or other liquid in which the pharmaceutical compound is not appreciably soluble, to form a first suspension, which shall be referred to as a presuspension.
• a suitable vehicle such as water or aqueous solution generally containing one or more of the surfactants set forth below, or other liquid in which the pharmaceutical compound is not appreciably soluble
• a first suspension which shall be referred to as a presuspension.
• Energy is added to the presuspension to form a particle dispersion which is physically more stable than the presuspension.
• Energy is added by mechanical grinding (e.g., pearl milling, ball milling, hammer milling, fluid energy milling, jet milling, or wet grinding).
• Energy addition techniques further include subjecting the presuspension to high shear conditions including cavitation, shearing or impact forces utilizing a microfluidizer.
• the present invention further contemplates adding energy to the presuspension using a piston gap homogenizer or counter current flow homogenizer such as those disclosed in U.S. Pat. No. 5,091,188 which is incorporated herein by reference and made a part hereof.
• Suitable piston gap homogenizers are commercially available under the product names EMULSIFLEXTM (Avestin) and FRENCH® Pressure Cell (Thermo Spectronic).
• Suitable microfluidizer s are available from Microfluidics Corp.
• the step of adding energy can also be accomplished using sonication techniques.
• the step of sonicating can be carried out with any suitable sonication device. Suitable devices include Branson Model S-450A and Cole-Parmer 500/750 Watt Model. Such devices are well known in the industry.
• the sonication device has a sonication horn or probe that is inserted into the presuspension to emit sonic energy into the solution.
• the sonicating device in a preferred form of the invention, is operated at a frequency of from about 1 kHz to about 90 kHz and more preferably from about 20 kHz to about 40 kHz or any range or combination of ranges therein.
• the probe sizes can vary and preferably are in distinct sizes such as 1/2 inch or 1/4 inch or the like.
• the dispersion of small particles can be sterilized prior to administering. Sterilization can be accomplished by heat sterilization, gamma irradiation, filtration (either directly as a dispersion having particle sizes under 200 nm, or by sterile filtration of the solutions used in the precipitation process, prior to forming the solid dispersion), and by application of very high pressure (greater than 2000 atmospheres), or by a combination of high pressure and elevated temperature.
• Small particle dispersions can also be prepared by precipitation techniques. The following is a description of examples of precipitation techniques.
• microprecipitation Methods One example of a microprecipitation method is disclosed in U.S. Pat. No. 5,780,062, which is incorporated herein by reference and made a part hereof.
• the '062 patent discloses an organic compound precipitation process including: (i) dissolving the organic compound in a water- miscible first solvent; (ii) preparing a solution of polymer and an amphiphile in an aqueous second solvent and in which second solvent the organic compound is substantially insoluble whereby a polymer/amphiphile complex is formed; and (iii) mixing the solutions from steps (i) and (ii) so as to cause precipitation of an aggregate of the organic compound and the polymer/amphiphile complex.
• the continuous phase of the dispersion can be optionally replaced by another continuous phase by removing the first continuous phase using methods such as centrifugation and filtration, and adding a second continuous phase and subsequently redispersing the solid material in the second continuous phase.
• One or more optional surfactants set forth below can be added to the first organic solvent, to the second aqueous solution, or to both the first organic solvent and the second aqueous solution.
• the process includes the steps of: (1) providing a multiphase system having an organic phase and an aqueous phase, the organic phase having a pharmaceutically active compound therein; and (2) sonicating the system to evaporate a portion of the organic phase to cause precipitation of the compound in the aqueous phase to form a dispersion of small particles.
• the step of providing a multiphase system includes the steps of: (1) mixing a water immiscible solvent with the pharmaceutically active compound to define an organic solution, (2) preparing an aqueous based solution with one or more surface active compounds, and (3) mixing the organic solution with the aqueous solution to form the multiphase system.
• the step of mixing the organic phase and the aqueous phase can include the use of piston gap homogenizers, colloidal mills, high speed stirring equipment, extrusion equipment, manual agitation or shaking equipment, microfluidizer, or other equipment or techniques for providing high shear conditions.
• the crude emulsion will have oil droplets in the water of a size of approximately less than 1 ⁇ m in diameter.
• the crude emulsion is sonicated to define a microemulsion and eventually to provide a dispersion of small particles.
• the step of providing a multiphase system includes the steps of: (1) mixing a water immiscible solvent with the pharmaceutically effective compound to define an organic solution; (2) preparing an aqueous based solution with one or more surface active compounds; and (3) mixing the organic solution with the aqueous solution to form the multiphase system.
• the step of mixing the organic phase and the aqueous phase includes the use of piston gap homogenizers, colloidal mills, high speed stirring equipment, extrusion equipment, manual agitation or shaking equipment, microfluidizer, or other equipment or techniques for providing high shear conditions.
• Small particle dispersions can also be prepared using a solvent-antisolvent precipitation technique disclosed by Fessi et al. in U.S. Pat. No. 5,118,528 and by Leclef et al. in U.S. Pat. No. 5,100,591 which are incorporated herein by reference and made a part hereof.
• Both processes include the steps of: (1) preparing a liquid phase of a biologically active substance in a solvent or a mixture of solvents to which may be added one or more surfactants; (2) preparing a second liquid phase of a non- solvent or a mixture of non-solvents, the non-solvent is miscible with the solvent or mixture of solvents for the substance; (3) adding together the solutions of (1) and (2) with stirring; and (4) removing of unwanted solvents to produce a dispersion of small particles.
• These methods are distinguished from those described under the above section, "Microprecipitation Methods", in that they do not provide for a last step of adding energy to the suspension in the form of high- shear mixing or heat.
• Phase Inversion Precipitation Small particle dispersions can be formed using phase inversion precipitation as disclosed in U.S. Pat. Nos. 6,235,224, 6,143,211 and 6,616,869, each of which is incorporated herein by reference and made a part hereof.
• Phase inversion is a term used to describe the physical phenomena by which a polymer dissolved in a continuous phase solvent system inverts into a solid macromolecular network in which the polymer is the continuous phase.
• One method to induce phase inversion is by the addition of a nonsolvent to the continuous phase. The polymer undergoes a transition from a single phase to an unstable two phase mixture: polymer rich and polymer poor fractions.
• Micellar droplets of nonsolvent in the polymer rich phase serve as nucleation sites and become coated with polymer.
• the '224 patent discloses that phase inversion of polymer solutions under certain conditions can bring about spontaneous formation of discrete microparticles, including nanoparticles.
• the '224 patent discloses dissolving or dispersing a polymer in a solvent.
• a pharmaceutical agent is also dissolved or dispersed in the solvent.
• the agent is dissolved in the solvent.
• the polymer, the agent and the solvent together form a mixture having a continuous phase, wherein the solvent is the continuous phase.
• the mixture is then introduced into at least tenfold excess of a miscible nonsolvent to cause the spontaneous formation of the microencapsulated microparticles of the agent having an average particle size of between 10 nm and 10 ⁇ m.
• the particle size is influenced by the solvent:nonsolvent volume ratio, polymer concentration, the viscosity of the polymer- solvent solution, the molecular weight of the polymer, and the characteristics of the solvent-nonsolvent pair.
• Small particle dispersions can be formed by pH shift precipitation techniques. Such techniques typically include a step of dissolving a drug in a solution having a pH where the drug is soluble, followed by the step of changing the pH to a point where the drug is no longer soluble.
• the pH can be acidic or basic, depending on the particular pharmaceutical compound.
• the solution is then neutralized to form a dispersion of small particles.
• pH shifting precipitation process is disclosed in U.S. Pat. No. 5,665,331, which is incorporated herein by reference and made a part hereof.
• the process includes the step of dissolving of the pharmaceutical agent together with a crystal growth modifier (CGM) in an alkaline solution and then neutralizing the solution with an acid in the presence of suitable surface-modifying surface- active agent or agents to form a small particle dispersion of the pharmaceutical agent.
• CGM crystal growth modifier
• the precipitation step can be followed by steps of diafiltration clean-up of the dispersion and then adjusting the concentration of the dispersion to a desired level.
• a precipitating nonsolvent miscible with the organic solvent is infused into the solvent mixture at a temperature between about -10° C and about 100° C and at an infusion rate of from about 0.01 ml per minute to about 1000 ml per minute per volume of 50 ml to produce a suspension of precipitated non-aggregated solid particles of the compound with a substantially uniform mean diameter of less than 10 ⁇ m. Agitation (e.g., by stirring) of the solution being infused with the precipitating nonsolvent is preferred.
• the nonsolvent may contain a surfactant to stabilize the particles against aggregation. The particles are then separated from the solvent.
• the parameters of temperature, ratio of nonsolvent to solvent, infusion rate, stir rate, and volume can be varied according to the invention.
• the particle size is proportional to the ratio of nonsolvent: solvent volumes and the temperature of infusion and is inversely proportional to the infusion rate and the stirring rate.
• the precipitating nonsolvent may be aqueous or non-aqueous, depending upon the relative solubility of the compound and the desired suspending vehicle.
• Temperature Shift Precipitation may also be used to form small particle dispersions. This technique is disclosed in U.S. Pat. No. 5,188,837, which is incorporated herein by reference and made a part hereof.
• lipospheres are prepared by the steps of: (1) melting or dissolving a substance such as a drug to be delivered in a molten vehicle to form a liquid of the substance to be delivered; (2) adding a phospholipid along with an aqueous medium to the melted substance or vehicle at a temperature higher than the melting temperature of the substance or vehicle; (3) mixing the suspension at a temperature above the melting temperature of the vehicle until a homogenous fine preparation is obtained; and then (4) rapidly cooling the preparation to room temperature or below.
• Solvent Evaporation Precipitation Solvent evaporation precipitation techniques are disclosed in U.S. Pat. No. 4,973,465 which is incorporated herein by reference and made a part hereof.
• the '465 Patent discloses methods for preparing microcrystals including the steps of: (1) providing a solution of a pharmaceutical composition and a phospholipid dissolved in a common organic solvent or combination of solvents, (2) evaporating the solvent or solvents and (3) suspending the film obtained by evaporation of the solvent or solvents in an aqueous solution by vigorous stirring to form a dispersion of small particles.
• the solvent can be removed by evaporating a sufficient quantity of the solvent to cause precipitation of the compound.
• the solvent can also be removed by other well known techniques such as applying a vacuum to the solution or blowing nitrogen over the solution.
• Reaction Precipitation includes the steps of dissolving the pharmaceutical compound, and optionally other excipients, into a suitable solvent to form a solution.
• the compound may be added in an amount at or below the saturation point of the compound in the solvent.
• the compound or any of the excipients is precipitated from solution by reacting with a chemical agent or by modification in response to adding energy such as heat or UV light or the like such that the modified compound has a lower solubility in the solvent and precipitates from the solution to form a small particle dispersion.
• Precipitation of excipient provides a solid matrix into which the drug is sorbed.
• Compressed Fluid Precipitation A suitable technique for precipitating by compressed fluid is disclosed in WO 97/14407 to Johnston, which is incorporated herein by reference and made a part hereof.
• the method includes the steps of dissolving a water- insoluble drug in a solvent to form a solution.
• the solution is then sprayed into a compressed fluid, which can be a gas, liquid or supercritical fluid.
• the addition of the compressed fluid to a solution of a solute in a solvent causes the solute to attain or approach supersaturated state and to precipitate out as fine particles.
• the compressed fluid acts as an antisolvent which lowers the cohesive energy density of the solvent in which the drug is dissolved.
• the drug can be dissolved in the compressed fluid which is then sprayed into an aqueous phase.
• the rapid expansion of the compressed fluid reduces the solvent power of the fluid, which in turn causes the solute to precipitate out as small particles in the aqueous phase.
• the compressed fluid acts as a solvent.
• a surface modifier such as a surfactant, is included in this technique.
• Particles utilized in this invention can also be produced from a process involving mixing or dissolving macromolecules such as proteins with a water soluble polymer. This process is disclosed in U.S. Pat. Nos. 5,981,719, 6,090,925, 6,268,053, 6,458,387, and U.S. Patent Pub. No. 2004/0043077, which are incorporated herein by reference and made a part hereof.
• particles are prepared by mixing a macromolecule in solution with a polymer or a mixture of polymers in solution at a pH near the isoelectric point of the macromolecule. The mixture is incubated in the presence of an energy source, such as heat, radiation, or ionization, for a predetermined amount of time. The resulting particles can be removed from any unincorporated components present in the solution by physical separation methods.
• the categories of processes are distinguished based upon the physical properties of the organic compound as determined through x-ray diffraction studies, differential scanning calorimetry (DSC) studies, or other suitable study conducted prior to the energy- addition step and after the energy-addition step.
• DSC differential scanning calorimetry
• the organic compound in the presuspension takes an amorphous form, a semi-crystalline form or a supercooled liquid form and has an average effective particle size.
• the organic compound is in a crystalline form having an average effective particle size essentially the same or less than that of the presuspension.
• the organic compound prior to the energy-addition step is in a crystalline form and has an average effective particle size.
• the organic compound is in a crystalline form having essentially the same average effective particle size as prior to the energy- addition step but the crystals after the energy- addition step are less likely to aggregate or form large crystals.
• the lower tendency of the organic compound to aggregate or form large crystals is observed by laser dynamic light scattering and light microscopy.
• the organic compound prior to the energy- addition step the organic compound is in a crystalline form that is friable and has an average effective particle size. What is meant by the term “friable” is that the particles are fragile and are more easily broken down into smaller particles.
• the organic compound is in a crystalline form having an average effective particle size smaller than the crystals of the pre- suspension.
• the first solution and second solvent are simultaneously subjected to the energy-addition step.
• the physical properties of the organic compound before and after the energy addition step were not measured.
• the energy-addition step can be carried out in any fashion wherein the presuspension or the first solution and second solvent are exposed to cavitation, shearing or impact forces.
• the energy-addition step is an annealing step.
• Annealing is defined in this invention as the process of converting matter that is thermodynamically unstable into a more stable form by single or repeated application of energy (direct heat or mechanical stress), followed by thermal relaxation. This lowering of energy may be achieved by conversion of the solid form from a less ordered to a more ordered lattice structure. Alternatively, this stabilization may occur by a reordering of the surfactant molecules at the solid-liquid interface.
• the first process category as well as the second, third, and fourth process categories, can be further divided into two subcategories, Method A and B.
• the first solvent according to the following processes is a solvent or mixture of solvents in which the organic compound of interest is relatively soluble and which is miscible with the second solvent.
• solvents include, but are not limited to water-miscible protic compounds, in which a hydrogen atom in the molecule is bound to an electronegative atom such as oxygen, nitrogen, or other Group VA, VIA and VII A in the Periodic Table of elements.
• solvents include, but are not limited to, alcohols, amines (primary or secondary), oximes, hydroxamic acids, carboxylic acids, sulfonic acids, phosphonic acids, phosphoric acids, amides and ureas.
• first solvent also include aprotic organic solvents. Some of these aprotic solvents can form hydrogen bonds with water, but can only act as proton acceptors because they lack effective proton donating groups.
• aprotic solvents is a dipolar aprotic solvent, as defined by the International Union of Pure and Applied Chemistry (IUPAC Compendium of Chemical Terminology, 2nd Ed., 1997): a solvent with a comparatively high relative permittivity (or dielectric constant), greater than ca. 15, and a sizable permanent dipole moment, that cannot donate suitably labile hydrogen atoms to form strong hydrogen bonds, e.g. dimethyl sulfoxide.
• Dipolar aprotic solvents can be selected from the group consisting of: amides (fully substituted, with nitrogen lacking attached hydrogen atoms), ureas (fully substituted, with no hydrogen atoms attached to nitrogen), ethers, cyclic ethers, nitriles, ketones, sulfones, sulfoxides, fully substituted phosphates, phosphonate esters, phosphoramides, nitro compounds, and the like.
• DMSO Dimethylsulfoxide
• NMP N-methyl-2-pyrrolidinone
• 2- pyrrolidinone 1,3-dimethylimidazolidinone
• DMA dimethylacetamide
• DMF dimethylformamide
• HMPA hexamethylphosphoramide
• Solvents may also be chosen that are generally water-immiscible, but have sufficient water solubility at low volumes (less than 10%) to act as a water-miscible first solvent at these reduced volumes.
• Examples include aromatic hydrocarbons, alkenes, alkanes, and halogenated aromatics, halogenated alkenes and halogenated alkanes.
• Aromatics include, but are not limited to, benzene (substituted or unsubstituted), and monocyclic or polycyclic arenes. Examples of substituted benzenes include, but are not limited to, xylenes (ortho, meta, or para), and toluene.
• alkanes include but are not limited to hexane, neopentane, heptane, isooctane, and cyclohexane.
• halogenated aromatics include, but are not restricted to, chlorobenzene, bromobenzene, and chloro toluene.
• halogenated alkanes and alkenes include, but are not restricted to, trichloroethane, methylene chloride, ethylenedichloride (EDC), and the like.
• solvents suitable for use as the first solvent include, but are not limited to: N-methyl-2-pyrrolidinone (also called N-methyl-2-pyrrolidone), 2- pyrrolidinone (also called 2-pyrrolidone), l,3-dimethyl-2-imidazolidinone (DMI), dimethylsulfoxide, dimethylacetamide, acetic acid, lactic acid, methanol, ethanol, isopropanol, 3-pentanol, n-propanol, benzyl alcohol, glycerol, butylene glycol (butanediol), ethylene glycol, propylene glycol, monoacylated and diacylated monoglycerides (such as glyceryl caprylate), dimethyl isosorbide, acetone, dimethylsulfone, dimethylformamide, 1,4- dioxane, tetramethylenesulfone (sulfolane), acetonitrile
• the second solvent is an aqueous solvent.
• This aqueous solvent may be water by itself.
• This solvent may also contain buffers, salts, surfactant(s), water-soluble polymers, and combinations of these excipients.
• Method A the organic compound ("active agent" or "drug") is first dissolved in the first solvent to create a first solution.
• the organic compound can be added from about 0.1% (w/v) to about 50% (w/v) depending on the solubility of the organic compound in the first solvent. Heating of the concentrate from about 30° C to about 100° C may be necessary to ensure total dissolution of the compound in the first solvent.
• a second aqueous solvent is provided with one or more optional surface modifiers such as an anionic surfactant, a cationic surfactant, a zwitterionic surfactant, a nonionic surfactant or a biologically surface active molecule added thereto.
• surface modifiers such as an anionic surfactant, a cationic surfactant, a zwitterionic surfactant, a nonionic surfactant or a biologically surface active molecule added thereto.
• Suitable anionic surfactants include but are not limited to alkyl sulfonates, alkyl phosphates, alkyl phosphonates, potassium laurate, triethanolamine stearate, sodium lauryl sulfate, sodium dodecylsulfate, alkyl polyoxyethylene sulfates, sodium alginate, dioctyl sodium sulfosuccinate, phosphatidyl glycerol, phosphatidyl inosine, phosphatidylinositol, diphosphatidylglycerol, phosphatidylserine, phosphatidic acid and their salts, sodium carboxymethylcellulose, cholic acid and other bile acids (e.g., cholic acid, deoxycholic acid, glycocholic acid, taurocholic acid, glycodeoxycholic acid) and salts thereof (e.g., sodium deoxycholate, etc.).
• cholic acid e.g.,
• Zwitterionic surfactants are electrically neutral but possess local positive and negative charges within the same molecule.
• Suitable zwitterionic surfactants include but are not limited to zwitterionic phospholipids.
• Suitable phospholipids include phosphatidylcholine, phosphatidylethanolamine, diacyl-glycero-phosphoethanolamine (such as dimyristoyl-glycero-phosphoethanolamine (DMPE), dipalmitoyl-glycero- phosphoethanolamine (DPPE), distearoyl-glycero-phosphoethanolamine (DSPE), and dioleolyl-glycero-phosphoethanolamine (DOPE)).
• DMPE dimyristoyl-glycero-phosphoethanolamine
• DPPE dipalmitoyl-glycero- phosphoethanolamine
• DSPE distearoyl-glycero-phosphoethanolamine
• DOPE dioleolyl-glycero-phosphoethanolamine
• phospholipids that include anionic and zwitterionic phospholipids may be employed in this invention. Such mixtures include but are not limited to lysophospholipids, egg or soybean phospholipid or any combination thereof.
• the phospholipid, whether anionic, zwitterionic or a mixture of phospholipids, may be salted or desalted, hydrogenated or partially hydrogenated, or natural, semisynthetic, or synthetic.
• the phospholipid may also be conjugated with a water-soluble or hydrophilic polymer to specifically target the delivery to macrophages in the present invention. However, conjugated phospholipids may be used to target other cells or tissue in other applications.
• a preferred polymer is polyethylene glycol (PEG), which is also known as the monomethoxy polyethyleneglycol (mPEG).
• PEG polyethylene glycol
• mPEG monomethoxy polyethyleneglycol
• the molecular weights of the PEG can vary, for example, from 200 to 50,000.
• Some commonly used PEG's that are commercially available include PEG 350, PEG 550, PEG 750, PEG 1000, PEG 2000, PEG 3000, and PEG 5000.
• the phospholipid or the PEG-phospholipid conjugate may also incorporate a functional group which can covalently attach to a ligand including but not limited to proteins, peptides, carbohydrates, glycoproteins, antibodies, or pharmaceutically active agents.
• These functional groups may conjugate with the ligands through, for example, amide bond formation, disulfide or thioether formation, or biotin/streptavidin binding.
• the ligand-binding functional groups include but are not limited to hexanoylamine, dodecanylamine, 1,12- dodecanedicarboxylate, thioethanol, 4-(p-maleimidophenyl)butyramide (MPB), 4-(p- maleimidomethyl)cyclohexane-carboxamide (MCC), 3-(2-pyridyldithio propionate (PDP), succinate, glutarate, dodecanoate, and biotin.
• Suitable cationic surfactants include but are not limited to quaternary ammonium compounds, such as benzalkonium chloride, cetyltrimethylammonium bromide, chitosans, lauryldimethylbenzylammonium chloride, acyl carnitine hydrochlorides, dimethyldioctadecylammomium bromide (DDAB), dioleoyltrimethylammonium propane (DOTAP, also known as N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium), N-[I- (2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium (DOTMA), dimyristoyltrimethylammonium propane (DMTAP), dimethylaminoethanecarbamoyl cholesterol (DC-Choi), l,2-diacylglycero-3-(O-alkyl)phosphoric acid,
• Suitable nonionic surfactants include: glyceryl esters, polyoxyethylene fatty alcohol ethers (MACROGOLTM and BRIJTM), polyoxyethylene sorbitan fatty acid esters (polysorbates), polyoxyethylene fatty acid esters (MYRJTM), sorbitan esters (SPANTM), glycerol monostearate, polyethylene glycols, polypropylene glycols, cetyl alcohol, cetostearyl alcohol, stearyl alcohol, aryl alkyl polyether alcohols, polyoxyethylene -polyoxypropylene copolymers (poloxamers), poloxamines, methylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, noncrystalline cellulose, polysaccharides including starch and starch derivatives such as hydroxyethyl starch (HES), polyvinyl alcohol, and polyvinylpyrrolidone.
• MACROGOLTM and BRIJTM polyoxyethylene sorb
• the nonionic surfactant is a polyoxyethylene and polyoxypropylene copolymer and preferably a block copolymer of propylene glycol and ethylene glycol.
• Such polymers are sold under the trade name POLOXAMER also sometimes referred to as PLURONIC®, and sold by several suppliers including Spectrum Chemical and Ruger.
• polyoxyethylene fatty acid esters is included those having short alkyl chains.
• SOLUTOL® HS 15 polyethylene-660-hydroxystearate, manufactured by BASF Aktiengesellschaft.
• Surface-active biological molecules include such molecules as albumin, casein, hirudin or other appropriate proteins.
• proteins having hydrophilic and hydrophobic domains also can be used.
• Polysaccharide surface active biologies are also included, and consist of but are not limited to, starches, heparins, and chitosans.
• Other suitable surfactants include any amino acids such as leucine, alanine, valine, isoleucine, lysine, aspartic acid, glutamic acid, methionine, phenylalanine, or any derivatives of these amino acids such as, for example, amide or ester derivatives and polypeptides formed from these amino acids.
• pH adjusting agents include, but are not limited to, hydrochloric acid, sulfuric acid, phosphoric acid, monocarboxylic acids (such as, for example, acetic acid and lactic acid), dicarboxylic acids (such as, for example, succinic acid), tricarboxylic acids (such as, for example, citric acid), THAM (tris(hydroxymethyl)aminomethane), meglumine (N- methylglucosamine), sodium hydroxide, and amino acids such as glycine, arginine, lysine, alanine, histidine and leucine.
• the second solvent should have a pH within the range of from about 3 to about 11.
• the aqueous medium may additionally include an osmotic pressure adjusting agent, such as but not limited to glycerin, a monosaccharide such as dextrose, a disaccharide such as sucrose, a trisaccharide such as raffinose, and sugar alcohols such as mannitol, xylitol and sorbitol.
• an osmotic pressure adjusting agent such as but not limited to glycerin, a monosaccharide such as dextrose, a disaccharide such as sucrose, a trisaccharide such as raffinose, and sugar alcohols such as mannitol, xylitol and sorbitol.
• one or more of the following excipients may be utilized: gelatin, casein, lecithin (phosphatides), gum acacia, cholesterol, tragacanth, stearic acid, benzalkonium chloride, calcium stearate, glyceryl monostearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, polyoxyethylene alkyl ethers, e.g., macrogol ethers such as cetomacrogol 1000, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, e.g., the commercially available TWEENSTM, polyethylene glycols, polyoxyethylene stearates, colloidal silicon dioxide, phosphates, sodium dodecylsulfate, carboxymethylcellulose calcium, carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, non
• the method for preparing small particles of an organic compound includes the steps of adding the first solution to the second solvent.
• the addition rate is dependent on the batch size, and precipitation kinetics for the organic compound. Typically, for a small-scale laboratory process (preparation of 1 liter), the addition rate is from about 0.05 cc per minute to about 10 cc per minute.
• the solutions should be under constant agitation. It has been observed using light microscopy that amorphous particles, semi-crystalline solids, or a supercooled liquid are formed to create a pre-suspension.
• the method further includes the step of subjecting the pre- suspension to an energy- addition step to convert the amorphous particles, supercooled liquid or semicrystalline solid to a more stable, crystalline solid state.
• the resulting particles will have an average effective particles size as measured by dynamic light scattering methods (e.g., photocorrelation spectroscopy, laser diffraction, low-angle laser light scattering (LALLS), medium-angle laser light scattering (MALLS)), light obscuration methods (Coulter method, for example), rheology, or microscopy (light or electron) within the ranges set forth above.
• dynamic light scattering methods e.g., photocorrelation spectroscopy, laser diffraction, low-angle laser light scattering (LALLS), medium-angle laser light scattering (MALLS)
• light obscuration methods Coulter method, for example
• rheology rheology
• microscopy light or electron
• the energy-addition step involves adding energy through sonication, homogenization, countercurrent flow homogenization, microfluidization, or other methods of providing impact, shear or cavitation forces.
• the sample may be cooled or heated during this stage.
• the energy-addition step is effected by a piston gap homogenizer such as the one sold by Avestin Inc. under the product designation EmulsiFlex-C160.
• the energy-addition step is accomplished by ultrasonication using an ultrasonic processor such as the Vibra-Cell Ultrasonic Processor (600W), manufactured by Sonics and Materials, Inc.
• the energy- addition step is accomplished by use of an emulsification apparatus as described in U.S. Pat. No. 5,720,551, which is incorporated herein by reference and made a part hereof.
• the temperature of the processed sample may be desirable to within the range of from approximately -30° C to 30° C.
• Method B differs from Method A in the following respects.
• the first difference is a surfactant or combination of surfactants is added to the first solution.
• the surfactants may be selected from the groups of anionic, nonionic, cationic surfactants, and surface-active biological modifiers set forth above.
• amorphous particles recrystallize as large crystals. Adding energy to the pre-suspension in the manner disclosed above typically affords particles that show decreased rates of particle aggregation and growth, as well as the absence of recrystallization upon product storage.
• Methods A and B are further distinguished from the process of the '062 patent by the absence of a step of forming a polymer-amphiphile complex prior to precipitation.
• a complex cannot be formed as no polymer is added to the diluent (aqueous) phase.
• the surfactant which may also act as an amphiphile, or polymer, is dissolved with the organic compound in the first solvent. This precludes the formation of any amphiphile-polymer complexes prior to precipitation.
• successful precipitation of small particles relies upon the formation of an amphiphile-polymer complex prior to precipitation.
• the '062 Patent discloses the amphiphile-polymer complex forms aggregates in the aqueous second solution.
• the '062 Patent explains the hydrophobic organic compound interacts with the amphiphile-polymer complex, thereby reducing solubility of these aggregates and causing precipitation.
• the inclusion of the surfactant or polymer in the first solvent (Method B) leads, upon subsequent addition to second solvent, to formation of a more uniform, finer particulate than is afforded by the process outlined by the '062 Patent.
• the processes for coating the particles prepared by the present invention can be accomplished through various techniques known to those skilled in the art.
• the coating can be associated with the particle through various associations, including covalent and/or non- covalent associations (e.g., covalent bonding, ionic interactions, electrostatic interactions, dipole-dipole interactions, hydrogen bonding, van der Waal's forces, hydrophobic/hydrophobic domain interactions, cross-linking, and/or any other interactions).
• Non-covalently bound coatings can be prepared, for example, by the methods for preparing particle cores disclosed herein provided that a surfactant according to formula I or salt thereof is used to manufacture the particles, or by mixing a plurality of pre-fabricated particles with a solution comprising a surfactant of formula I, as defined herein, or salt thereof, to form surface-modified particles according to the invention.
• the solution can be mixed under high-shear conditions using, for example, a microfluidizer, a piston gap homogenizer, a counter-current flow homogenizer, or an ultrasonic processor.
• the surface electrical potential of the particles can be determined by measuring the zeta potential before and after the coating process.
• the surfactant of formula I or salt thereof can be modified with a fluorescent label and absorption of the fluorescently-labeled surfactant of formula I or salt thereof can be detected by fluorescence microscopy.
• the coatings comprising a surfactant of formula I or a salt thereof can associate with the particle core, for example by adsorbing to the surface of particles, which is an efficient method for associating coatings comprising surfactants according to formula I or a salt thereof to particle cores, particularly particles comprising poorly water soluble active agents, as explained above.
• the coating can further include one or more additional surfactants, including additional surfactants of formula I or salts thereof, by adding the additional surfactants to the solution comprising the surfactant of formula I or salt thereof, and then mixing the prefabricated particles with said solution.
• additional surfactant(s) can be selected from a variety of known anionic surfactants, cationic surfactants, zwitterionic surfactants, nonionic surfactants and surface active biological modifiers .
• Suitable additional surfactants include the surfactants previously set forth herein.
• Exemplary additional surfactants include, but are not limited to, poloxamers, phospholipids, polyethylene glycol-conjugated phospholipids, and polysorbates.
• Exemplary combinations of additional surfactants include, but are not limited to, poloxamers and phospholipids, poloxamers and polyethylene glycol-conjugated phospholipids, and poloxamers and polysorbates.
• One embodiment of the present invention is directed to a method of enhancing cellular uptake of an active agent, comprising contacting cells with a plurality of surface- modified particles, said particles comprising a particle core and a coating associated with the particle core.
• the cells can be phagocytic cells, weakly phagocytic cells, or non-phagocytic cells.
• the particle core comprises an active agent which is typically selected from the group consisting of small molecules, peptides, and proteins
• the coating comprises a surfactant of formula I, as defined herein, or a salt thereof and the surface-modified particle has an average size from about 1 nm to about 2,000 nm. Uptake of the active agent by the cells is thereby enhanced, at least relative to the uptake of active agent when particles that do not comprise the aforementioned coating are used.
• Uptake by cells allows the active agent to be delivered to target tissues in need of treatment because the various cell types capable of enhanced uptake of the coated particles in accordance with the disclosure also traffic to diseased (e.g., cancerous, infected) or inflamed tissues.
• diseased e.g., cancerous, infected
• inflamed tissues e.g., neutrophils predominate early in infection or inflammation, followed by monocyte-derived phagocytes that leave the blood vasculature and enter infected tissues, and such cells demonstrate enhanced uptake of the surface-modified particles according to the invention at least relative to particles not having a coating comprising a surfactant according to formula I or a salt thereof.
• Tissues that are most affected by bacterial, viral or fungal pathogens and which are inflamed can be targeted by delivery of drug-loaded cells (granulocytes, for example) having a propensity to be directed to these inflammation sites by chemotaxis.
• drug-loaded cells granulocytes, for example
• Such diseases and disorders include, but are not limited to, infectious diseases or disorders, inflammatory diseases or disorders, neurodegenerative diseases or disorders, and proliferative diseases or disorders.
• phagocytic cell types that are capable of enhanced uptake of coated particles. These cells include, but are not limited to, macrophages, monocytes, granulocytes, agranulocytes and neutrophils.
• the present invention also encompasses weakly phagocytic cells and non-phagocytic cells.
• suitable cell types include, but are not limited to, T-lymphocytes, B-lymphocytes, null cells, natural killer cells, lymphocytes, red blood cells, muscle cells, bone marrow cells, stem cells, bone cells, vascular cells, organ tissue cells, neuronal cells, basophils, eosinophils, dendritic cells, and endothelial cells.
• Still other cells can be used to deliver the pharmaceutically active compounds to a subject.
• Any cell type may be used in the present invention so long as it is capable of uptake of the particle.
• Uptake by the cells of the particles may include phagocytosis, or other means of endocytosis, or attachment/adsorption of the particle onto the surface of the cells.
• Particles associated with the cell surface can also be taken into the cells by pinocytosis, which is an invagination of the cell membrane to form an intracellular capsule around the particle.
• pinocytosis In pinocytosis ("cell drinking"), the engulfed particle is relatively small (e.g., 20 nm) (Watts et al., Endocytosis: what goes in and how?, Journal of Cell Science, 1992, volume 103(1), pages 1-8). Pinocytosis occurs continuously in almost all eukaryotic cells.
• Diseased cells can also demonstrate enhanced uptake of the surface modified particles according to the invention at least as compared to cells contacted with particles not having a coating comprising a surfactant of formula I or a salt thereof.
• Tumor cells are not conventionally considered to be phagocytic.
• surface-modified particles were taken up by ovarian tumor cells within the peritoneal cavity of mice. The exact mechanism of the enhanced uptake is unknown, but may involve phagocytosis, endocytosis, pinocytosis, or other cellular uptake mechanisms.
• the particles advantageously include a coating which facilitates cellular uptake.
• the coating can facilitate uptake by cells such as monocytes, macrophages, and T-lymphocytes, which are capable of trafficking by known mechanisms such as chemotaxis to a site of inflammation, infection, and/or tumor and thereby deliver the particles to a particular target tissue.
• the contacting of the cells to the surface-modified particle is carried out ex vivo (i.e., outside of a mammalian subject).
• the contacting of the cells to the surface- modified particle can be carried out in vivo (i.e., inside a mammalian subject).
• An amount of the surface modified particle that is effective to treat a disease or disorder is used during the contacting step.
• a certain amount of the particles may be taken up by a cell type that does not traffic to a target tissue of interest, or is not released by the cell at the target tissue of interest. Therefore, one of ordinary skill understands that the amounts of particles administered may be optimized by routine protocols, provided that such amounts are within established administration protocols.
• the cells can be isolated from a mammalian subject using a cell separator or apheresis device.
• a cell separator or apheresis device For instance, the CS-3000TM cell separator (Fenwal Inc., Lake Zurich, 111.) or the ISOLEXTM cell separator (Baxter Healthcare Corp., Deerfield, 111.) can be used to isolate various cells.
• Other methods known to those skilled in the art of ex-vivo cell isolation can be employed to obtain cells useful in the present invention.
• Such methods include, but are not limited to, apheresis of peripheral blood, mobilization of bone marrow cells through, e.g., G-CSF, M-CSF, or GM-CSF, or direct removal of marrow cells by spinal, sternal, lumbar, or iliac crest puncture.
• the ex-vivo cells can be maintained in a cell culture medium or other isolating system known to those skilled in the art.
• Examples of such media are Alserver's Solution, Ames' Medium, Eagle's Basal Medium, CHO (Chinese Hamster Ovary) cell culture media, Click's Medium, Dulbecco's Modified Eagle's Medium, phosphate-buffered saline, phosphate-buffered dextrose or sucrose, Earle's Balanced Salt Solution, Gene Therapy Medium-3, Gey's Balanced Salt Solution, Glasgow Minimum Essential Medium, Hanks' Balanced Salt Solutions, Hybridoma Media, Iscove's Modified Dulbecco's Medium, Krebs-Henseleit Buffer with sugars, Leibovitz Media (L-15), M16 Medium, McCoy's Medium, MCDB, MDBK (Madin-Darby Bovine Kidney), MDCK (Madin-Darby Canine Kidney), Medium 199, NCTC, Ham's Media (e.g., Nutrient Mixture F- 10), Coon's Modified Ham's Medium, RPMI,
• the purpose of the culture so described may be for the purpose of simple storage without loss of cells, or for cell proliferation or expansion, by appropriate addition of growth factors, cytokines, and nutrients, to encourage cell expansion. Such expansion would minimize the number of times that a patient would have to be prepared for removal of cellular samples.
• the cells can be contacted with the coated particles and incubated for a short period of time to allow for cell uptake of the particles.
• concentrations of particles used in the ex-vivo procedure will vary due to several factors, including, but not limited to, type of cells used, concentration of cells, active agent employed, size of the small particle dispersions, disease to be treated, and so on.
• the cellular isolates are contacted with about 1 to about 300 mg/ml of particles of the present invention.
• the particles are at a concentration higher than the thermodynamic saturation solubility, thereby allowing the particles to remain in particulate form during uptake and delivery to the mammalian subject.
• the cells can be incubated with the particles for up to 24 hours or longer to permit sufficient cell uptake of the drug particles.
• any method to effect uptake of particles of active agent by ex vivo cells can be used with the requirement that the method does not destroy or otherwise make the cells non- useful for administration to a subject.
• site-specific delivery of the particle via a biorecognition molecule may be used. See, e.g., U.S. Patent Publication No. 2003/0092069, incorporated herein by reference, which discloses the transferring of genes into specific cells or tissues via a hollow nanoparticle.
• Other methods of loading the ex-vivo cells include electroporation, sonoporation, and other mechanical means that disrupt the cell membrane (sonication, for example) and enable insertion of solid particulates into the cells. Ultrasound was successfully used by Zarnitsyn et al.
• Transfection reagents contain surface active agents and include 293FECTINTM Transfection Reagent and LIPOFECTAMINETM, both products of Invitrogen Corporation (Carlsbad, Calif.).
• SAINTTM reagent from Synvolux Therapeutics B. V. L. J. (Groningen, The Netherlands), which is based on a pyridinium surfactant.
• the cell loading procedure can be utilized provided that the cells are able to take up the coated active agent particles at a faster rate than the competing dissolution process.
• the particles should be of an appropriate size to allow for the cells to take up the coated particles and deliver them to the target tissue before complete dissolution of the particle. Because cells which are known to traffic to the target tissue of interest are capable of taking up the particles, the active agent is ultimately released from the cells or otherwise delivered in the vicinity of the target tissue. Furthermore, the concentration of the active agent composition should be kept higher than the saturation solubility of the composition so that the particle is able to remain in the crystalline state during uptake.
• Administering of the surface-modified particle can be performed by various techniques known in the art for administering particles.
• Administering includes administering the surface-modified particle to a mammalian subject.
• Suitable methods for administering of the surface-modified particle or pharmaceutical compositions thereof include, but are not limited to, administering the particle intravenously, intraarterially, intramuscularly, subcutaneously, intradermally, intraarticularly, intrathecally, epidurally, intracerebrally, buccally, rectally, topically, transdermally, orally, intranasally, via the pulmonary route, intraperitoneally, and/or intraophthalmically.
• the step of administering can be by bolus injection, by intermittent infusion, or by continuous infusion.
• the amount of surface- modified particle and method of delivery can be determined by skilled clinicians. Various factors will affect the amount and method of delivery including, but not limited to, the type of cells used (for ex vivo methods of administration), the sex, weight and age of the subject to be treated, the type and maturity of the disease or disorder to be treated, the active agent to be administered, and so on.
• the active agent can be provided in doses ranging from 1 pg compound/kg body weight to 1000 mg/kg, 0.1 mg/kg to 100 mg/kg, 0.1 mg/kg to 50 mg/kg, and 1 to 20 mg/kg, given in daily doses or in equivalent doses at longer or shorter intervals, e.g., every other day, twice weekly, weekly, or twice or three times daily.
• Various diseases or disorders can be treated by the present methods including, but not limited to, infectious diseases or disorders, inflammatory diseases or disorders, neurodegenerative diseases or disorders, and proliferative diseases or disorders.
• symptoms of such diseases or disorders can be alleviated by the present methods.
• infectious diseases or disorder refers to a condition caused by pathogenic microorganisms, such as bacteria, viruses, parasites or fungi.
• Infectious diseases or disorders that can benefit from the disclosed methods include, but are not limited to, viral infections (including retroviral infections) such as dengue, enterovirus infections, HIV, hepatitis B, hepatitis C, and influenza; fungal infections; parasitic infections such as African trypanosomiasis and malaria; and bacterial infections such as cholera, meningitis, and tuberculosis.
• Inflammatory disease or disorder refers to a condition characterized by redness, heat, swelling, and pain (i.e., inflammation) that typically involves tissue injury or destruction. Inflammatory diseases or disorders are notably associated with the influx of leukocytes and/or leukocyte chemotaxis. Inflammatory conditions may result from infection with pathogenic organisms or viruses and from noninfectious events including but not limited to trauma or reperfusion following myocardial infarction or stroke, immune responses to foreign antigens, and autoimmune responses. Accordingly, inflammatory conditions amenable to treatment with the methods and compounds of the invention encompass conditions associated with reactions of the specific defense system, conditions associated with reactions of the non-specific defense system, and conditions associated with inflammatory cell activation.
• the term "specific defense system” refers to the component of the immune system that reacts to the presence of specific antigens.
• inflammatory conditions resulting from a response of the specific defense system include but are not limited to the classical response to foreign antigens, autoimmune diseases, and delayed type hypersensitivity response mediated by B-cells and/or T-cells (i.e., B-lymphocytes and/or T- lymphocytes).
• Chronic inflammatory diseases, the rejection of solid transplanted tissue and organs including but not limited to kidney and bone marrow transplants, and graft versus host disease (GVHD) are further examples of inflammatory conditions resulting from a response of the specific defense system.
• non-specific defense system refers to inflammatory conditions that are mediated by leukocytes that are incapable of immunological memory (e.g., granulocytes including but not limited to neutrophils, eosinophils, and basophils, mast cells, monocytes, macrophages).
• granulocytes including but not limited to neutrophils, eosinophils, and basophils, mast cells, monocytes, macrophages.
• inflammatory conditions that result, at least in part, from a reaction of the non-specific defense system include but are not limited to adult (acute) respiratory distress syndrome (ARDS), multiple organ injury syndromes, reperfusion injury, acute glomerulonephritis, reactive arthritis, dermatitis with acute inflammatory components, acute purulent meningitis, other central nervous system inflammatory conditions including but not limited to stroke, thermal injury, inflammatory bowel disease, granulocyte transfusion associated syndromes, and cytokine-induced toxicity.
• ARDS adult (acute) respiratory distress syndrome
• multiple organ injury syndromes reperfusion injury
• acute glomerulonephritis reactive arthritis
• dermatitis with acute inflammatory components acute purulent meningitis
• other central nervous system inflammatory conditions including but not limited to stroke, thermal injury, inflammatory bowel disease, granulocyte transfusion associated syndromes, and cytokine-induced toxicity.
• the therapeutic methods of the invention include methods for the amelioration of conditions associated with inflammatory cell activation.
• “Inflammatory cell activation” refers to the induction by a stimulus (including but not limited to cytokines, antigens, and auto-antibodies) of a proliferative cellular response, the production of soluble mediators (including but not limited to cytokines, oxygen radicals, enzymes, prostanoids, and vasoactive amines), or cell surface expression of new or increased numbers of mediators (including but not limited to major histocompatability antigens and cell adhesion molecules) in inflammatory cells (including but not limited to monocytes, macrophages, T lymphocytes, B lymphocytes, granulocytes (polymorphonuclear leukocytes including neutrophils, basophils, and eosinophils), mast cells, dendritic cells, Langerhans cells, and endothelial cells).
• a stimulus including but not limited to cytokines, antigens, and
• diseases or disorders which can be successfully treated include diseases or disorders characterized by inflammation or infection, including but not limited to, rheumatoid arthritis, Graves' disease, myasthenia gravis, thyroiditis, diabetes, inflammatory bowel disease, autoimmune oophoritis, systemic lupus erythematosus, and Sjogren's syndrome.
• neurodegenerative diseases or disorders which can be successfully treated include, but are not limited to, Parkinson's disease, Alzheimer's disease, multiple sclerosis, encephalomyelitis, encephalitis (including HIV encephalitis), Huntington's disease, amyotrophic lateral sclerosis (also known as Lou Gehrig's disease), frontotemporal dementia, prion diseases, Creutzfeldt-Jakob disease, and adrenoleukodystrophy.
• Other neurodegenerative diseases or disorders which can be successfully treated include Pick's disease, frontotemporal lobar degeneration, progressive aphasia, and semantic dementia.
• TSEs transmissible spongiform encephalopathies
• Creutzfeldt-Jakob disease new variant Creutzfeldt-Jakob disease
• Gerstmann-Straussler- Scheinker syndrome fatal familial insomnia
• kuru kuru
• the neurodegenerative diseases or disorders also can be Alexander disease, Alper's disease, ataxia telangiectasia, Batten disease (also known as Spielmeyer-Vogt-Sjogren-Batten disease), Canavan disease, Cockayne syndrome, corticobasal degeneration, HIV-associated dementia, Kennedy's disease, Krabbe disease, Lewy body dementia, Machado-Joseph disease (spinocerebellar ataxia type 3), multiple system atrophy, neuroborreliosis, Pelizaeus-Merzbacher disease, primary lateral sclerosis, Refsum's disease, Sandhoff disease, Schilder's disease, schizophrenia, spinocerebellar ataxia, spinal muscular atrophy, Steele-Richardson-Olszewski disease, and tabes dorsalis.
• Batten disease also known as Spielmeyer-Vogt-Sjogren-Batten disease
• Canavan disease Cockayne syndrome
• Proliferative diseases or disorders that can benefit from the disclosed methods include, but are not limited to, colon cancer, kidney cancer, non small cell lung cancer, small cell lung cancer, lung adenocarcinoma, head and neck cancer, cancers of the peritoneal cavity (such as ovarian cancer, gastrointestinal cancer, abdominal cancer and mesothelioma), cervical cancer, breast cancer, prostate cancer, brain cancer (such as glioma, meningioma, astrocytoma), stomach cancer, intestinal cancer, sarcoma, melanoma, leukemia, acute lymphocytic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, myeloma, and glioblastoma.
• cancers of the peritoneal cavity such as ovarian cancer, gastrointestinal cancer, abdominal cancer and mesothelioma
• Thyroiditis includes Hashimoto's thyroiditis, subacute thyroiditis (also known as de Quervain's thyroiditis), silent thyroiditis (also known as painless thyroiditis), post partum thyroiditis, drug-induced thyroiditis, radiation-induced thyroiditis, and acute suppurative thyroiditis.
• Paclitaxel particles were prepared using a microprecipitation/homogenization procedure. Specifically, paclitaxel (0.5 g) was dissolved in N-methyl pyrrolidone (NMP) (3 g) and then added, with rotor- stator mixing, to aqueous surfactant solution A (25 rnL).
• Solution A pH -7.8 to 8.0
• Solution A contained sodium phosphate, dibasic, anhydrous (0.13 g), sodium phosphate, monobasic, monohydrate (0.01 g), glycerin (2.2 g), DSPE-mPEG 2000 (0.2 g), and poloxamer 188 (0.5 g) in 100 mL water (Table 1).
• the resulting suspension was transferred to a homogenizer (Avestin C5) and circulated at static pressure until the suspension temperature reached at least 5O 0 C.
• the suspension was then homogenized at a target pressure of 20,000 + 2,000 psi and a target temperature of 6O 0 C for 60 minutes.
• the suspension was collected and centrifuged for 30 minutes at 10,000 rpm. Upon completion of the centrifuge cycle, the supernatant was decanted and replaced with an equal volume of aqueous surfactant solution B.
• Solution B (pH -7.8 to 8.0) contained the same components as solution A and additionally contained N- [l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP, 0.1 g) (Table 1).
• the pellet was resuspended, and the centrifugation was repeated twice more, using solution B as the replacement surfactant each time.
• the nanosuspension was homogenized for 30 minutes at a target pressure of 20,000 + 2,000 psi and a target temperature of 6O 0 C.
• the final suspension contained particles having a size of -160-170 nm.
• Fluorescently-labeled paclitaxel particles were prepared according to the procedure above by adding fluorescently-labeled paclitaxel to the drug concentrate. Specifically, 400 ⁇ g Oregon Green-labeled paclitaxel (available from Invitrogen, Carlsbad, CA) was added to the drug concentrate described above to yield fluorescently-labeled paclitaxel particles with adequate fluorescence intensity to be detected in flow cytometry and fluorescent microscopy.
• the uptake of DOTAP-coated paclitaxel particles by human mononuclear cells was compared to the uptake of protamine-coated paclitaxel particles and DSPE-mPEG 2000/poloxamer 188-coated paclitaxel particles.
• the protamine-coated paclitaxel particles were prepared by adding 0.08 mL of a 25 mg/mL protamine solution to 0.01 mL of an Oregon Green-labeled paclitaxel suspension at 10 mg/mL.
• Human mononuclear cells for use in the uptake experiments were purified from the whole blood of human donors. These cells were cultured in tissue culture treated 6-well plates (BD Biosciences) for 5-7 days in Media A, with media exchanged every 2-3 days. Media A contained DMEM (Gibco BRL cat. no. 11960-051) supplemented with the following to make IL: 1000U/ml recombinant human macrophage-colony stimulating factor- 1 (rhM-CSF-1) (Chemicon), 100 mL heat-inactivated human serum, 10 mL 200 mM L- glutamine (Gibco BRL cat. no. 25030-081), 2 niL 50 mg/ml Gentamicin (Sigma cat. no. G1397), and 400 ⁇ L 25 mg/niL Ciprofloxacin (Bayer code no. 89-001-1). The cells also were cultured on glass coverslips for microscopy applications.
• DMEM Gibco BRL cat. no.
• paclitaxel formulations paclitaxel particles having a DOTAP coating, paclitaxel particles having a protamine coating, or paclitaxel particles having a DSPE-mPEG 2000/poloxamer 188 coating
• the suspension formulations contained paclitaxel (doped with Oregon Green-labeled paclitaxel) at a final concentration of -10 ⁇ M.
• paclitaxel doped with Oregon Green-labeled paclitaxel
• the cells were washed at least three times with 2 mL/well phosphate- buffered saline (PBS). The cells were then scraped in PBS and transferred to microfuge tubes (or fixed and mounted if the cells were adherent to coverslips).
• paclitaxel uptake was assessed by both the ratio of CD14+ cells (monocyte-derived macrophages) positive for Oregon Green fluorescence (% paclitaxel+/CD14+), i.e., the percentage of cells that have internalized or adsorbed paclitaxel particles, and the Mean Fluorescence Intensity (MFI).
• MFI Mean Fluorescence Intensity
• FIGS. 1 and 2 The uptake kinetics of the paclitaxel suspensions are shown in FIGS. 1 and 2 (results are shown as both percentages of paclitaxel positive cells after nanosuspension uptake and MFI of cell-associated/internalized particles).
• FIG. 1 cells were exposed to the paclitaxel particles for 0, 3, 5 hours, while in FIG. 2, cells were exposed to the paclitaxel particles for 0, 0.25, 0.5, and 1 hour.
• the DOTAP coating substantially improved the uptake of paclitaxel particles as compared to DSPE-mPEG 2000/poloxamer 188-coated particles (FIGS. 1 and 2) and protamine-coated particles (FIG. T).
• FIG. 2 demonstrates the stability of the paclitaxel formulations upon storage.
• DOTAP Sample 1 was stored for approximately 3 months prior to the uptake experiments.
• DOTAP Sample 2 was used in the uptake experiments shortly after preparation. These results indicate that storage of DOTAP-coated paclitaxel particles for several months does not significantly affect the particle uptake kinetics.
• Uptake of paclitaxel particles was quantified by reverse phase HPLC.
• Paclitaxel uptake was measured after incubating the cells with the paclitaxel suspensions for 15, 30, and 60 minutes. Samples were prepared by adding acetonitrile (500 ⁇ L) to a 500 ⁇ L aliquot of each cell suspension and vortexing to mix. The samples were then centrifuged at 10,000 rpm for 30 minutes at 25 0 C and the supernatants were analyzed by reverse phase HPLC to determine the amount of paclitaxel in the sample (Table T).
• FIG. 3 shows uptake by monocyte-derived macrophages of DOTAP-coated paclitaxel nanosuspensions after 1, 2, or 6 days of culture.
• the cells were exposed to the paclitaxel particles for various periods of time from 0 and 3.5 hours.
• the results indicate that the longer the cells are cultured, the less responsive they are to DOTAP-coated particles. It is theorized that all phagocytosis is expected to increase as the cells mature from monocytes to macrophages, therefore the DOTAP coating may not provide as great of an advantage for enhancing cellular uptake in more mature cells when compared to younger cells (cells which have been cultured in vitro for relatively short periods of time).
• DOTAP-coated paclitaxel particles were compared to the uptake of polylactic-co-glycolic acid (PLGA)-coated paclitaxel particles and to the uptake of phosphatidylserine (PS)-coated paclitaxel particles.
• PLGA polylactic-co-glycolic acid
• PS phosphatidylserine
• PLGA-coated paclitaxel particles and PS-coated paclitaxel particles were prepared in accordance with the procedure described in Example 1, except that the PLGA particles were sonicated, rather than homogenized, and were formulated using a solution containing phosphate buffer, glycerin, PLGA, and Poloxamer 188, and the PS particles were formulated using a solution containing phosphate buffer, glycerin, DSPE-mPEG 2000, Poloxamer 188, and phosphatidylserine.
• the uptake kinetics of the paclitaxel suspensions are shown in FIG. 4 (results are shown as both percentages of paclitaxel positive cells after nanosuspension uptake and MFI of cell associated/internalized particles).
• the DOTAP coating substantially improved the uptake of particles compared to PLGA-coated or phosphatidylserine-coated particles.
• CTAB-coated paclitaxel particles were prepared in accordance with the procedure described in Example 1, except that the CTAB particles were formulated using a solution containing phosphate buffer, glycerin, DSPE-mPEG 2000, Poloxamer 188, and CTAB.
• Peritoneal macrophages were isolated from mice and exposed to paclitaxel particles having a DOTAP coating and to paclitaxel particles without such a coating. Fluorescence images showed that peritoneal macrophages exposed to the DOTAP-coated particles took up greater amounts of paclitaxel than those exposed to DSPE-mPEG 2000/poloxamer 188-coated particles (data not shown). This example supports that DOTAP enhances uptake of particles by peritoneal macrophages.
• a mouse model was established wherein test mice were implanted with RFP- OVCAR-3 cells and tumors were allowed to grow. The tumors expressed RFP and had red fluorescence. Oregon Green-labeled paclitaxel particles having a DOTAP coating were administered by intraperitoneal injection to mice having RFP-expressing tumors. The presence and location of the DOTAP-coated paclitaxel particles were detected relative to the tumors using fluorescence.
• both tumors and paclitaxel particles were observed by fluorescence microscopy (red fluorescence for tumors, green fluorescence for particles).
• the DOTAP-coated paclitaxel particles were present in the tumor-bearing mice up to 30 days post-injection, indicating that the tumors present in the peritoneal cavity of the mouse were partially responsible for the increased residence time.
• the DOTAP-coated paclitaxel particles frequently co- localized with tumors.
• this example supports that the DOTAP-coated paclitaxel particles target tumor sites as opposed to healthy tissues, and are able to persist within the targeted tumor sites for significant periods of time such that they can effectively deliver a sustained release of the therapeutic drug.
• a dose ranging study using various doses of the DOTAP-coated paclitaxel particles was also carried out to identify effective doses for inducing ovarian tumor cell death in the mouse model.
• the size of the tumors was measured to determine efficacy of the given dose.
• a dose of 25 mg/kg administered on day 1 reduced the size of the tumor 25 days after administration.
• This study further supports that administration of DOTAP-coated paclitaxel particles effectively treated cancer in vivo.
• Particles of protease inhibitors indinavir (IDV) and ritonavir (RTV) were prepared using a microprecipitation/ homogenization procedure (previously described herein). The particles were then coated with various surfactants including DOTAP. The uptake and effectiveness of the different protease inhibitor particle formulations were compared.
• the protease inhibitor formulations used in this study are set out in Table 3.
• the particles were suspended in a solution comprising Lipoid E80 (1.4%), P- 188 (0.5%), DSPE-mPEG 2 ooo (0.2%), and/or DOTAP (0.1%) in order to coat the particles.
• the particles were added to the solution in a weight- to -volume of approximately 2%.
• the suspensions of the surface-modified particles were prepared by adding particles to the coating solution and mixing for 4-7 minutes using an Ultra-Turrax T- 18 (IKA Works Inc., Wilmington, NC, USA) rotor-stator mixer to reduce initial particle size. The suspensions were then homogenized at 20,000 pounds per square inch for approximately 30 passes or until desired particle size was reached.
• DOTAP-containing suspensions For DOTAP-containing suspensions, the homogenized suspension was centrifuged (12,100 x g for 30 minutes at 5 0 C) to pellet the drug particles. The drug was resuspended in surfactant containing DOTAP by mixing with an Ultra-Turrax T-18. The nanosuspensions were formulated at a slightly alkaline pH of 7.8 using either 10 mM sodium phosphate or 10 mM HEPES as a buffer. Tonicity was adjusted with glycerin (2.25%) or sucrose (9.25%).
• rDHPE triethylammonium salt
• Human monocytes were obtained by leukapheresis from HIV-I and hepatitis seronegative donors and were purified by counter-current centrifugal elutriation as described in Dou et al. (Blood, 108(8):2827-2835, 2006). Wright-stained cytospins were prepared and cell purity assayed by immunolabeling with anti-CD68 (clone KP-I).
• Monocytes were cultivated at a concentration of 1 x 10 6 cells/ml at 37 0 C in a humidified atmosphere (5% CO 2 ) in Dulbecco's modified Eagle medium supplemented with 10% heat-inactivated pooled human serum, 1% glutamine, 50 ⁇ g/ml gentamicin 10 ⁇ g/ml ciprofloxacin and 1000 U/ml recombinant human macrophage colony stimulating factor.
• monocytes were cultured for 7 days in the presence of macrophage colony stimulating factor as described in Gendelman et al. (J. Exp. Med. 167:1428-1441, 1988).
• MDM (2 x 10 6 per well) were cultured with the surface-modified protease inhibitor particles at concentrations of 1, 10 and 100 ⁇ M. Uptake of the particles was assessed without medium change for 24 hours with cell collection occurring hourly.
• Adherent MDM were collected by washing three times with 1 ml of phosphate-buffered saline, followed by scraping cells into 1 ml phosphate-buffered saline. Samples were centrifuged at 950 x g for 10 minutes at 4 0 C, and the supernatant removed. Cell pellets were sonicated in 200 ⁇ l of methanol and centrifuged at 10,000 x g for 10 minutes at 4 0 C. The methanol extract was stored at -8O 0 C until RP-HPLC analysis was performed.
• RTV-I The RTV formulation with the slowest rate and lowest absolute amount of uptake was RTV-I, which was coated with DSPE-mPEG2ooo alone, had a size of 200 nm and a zeta-potential of -25.6 mV (Table 3).
• the RTV formulation with the fastest absorption rate and greatest accumulation of RTV was RTV-4, which was coated with a combination of P-188, DSPE, HiPEG 2 OOo, and DOTAP, had a size of 620 nm and a zeta-potential of +15.5 mV. Maximum uptake occurred at 8 hours for RTV-4 and at 12 hours for RTV-I.
• Absolute amount of uptake was approximately 1.5-times greater for RTV-4 (40.98 ⁇ g/1 x 10 6 cells) than for RTV-I (24.68 ⁇ g/1 x 10 6 cells).
• the physical properties of RTV-4 were similar to IDV-4, which also contained DOTAP. This suggests that the physical properties that optimize the uptake of IDV particles also optimize the uptake of RTV particles.
• ALAMARBLUETM assay (AbD Serotec, Raleigh, NC, USA) according to the manufacturer's instructions.
• the ALAMARBLUE reagents incorporate an oxidation-reduction indicator that both fluoresces and changes color in response to chemical reduction of growth medium resulting from cell growth. Thus, the intensity of the oxidation-reduction indicator is proportional to cell growth and cell viability.
• Results show that each of the IDV particle formulations did not significantly change the viability of monocytes or MDM at the highest concentration used (100 ⁇ M). Additionally, all RTV particle preparations also tested at 100 ⁇ M did not alter macrophage viability but decreased monocyte viability by 15%.
• HBMECs human brain microvessel endothelial cells
• HBMEC monolayers are not visible on these inserts, manual readings of trans-epithelial electric resistance were assessed with an EVOM voltmeter (World Precision Instruments, Sarasota, FL, USA) to confirm monolayer formation and confluence.
• Monocytes were labeled with calcein-AM (Invitrogen) at 5 ⁇ M/1 x 10 6 cells for 45 minutes and washed with phosphate- buffered saline.
• media samples (10 ⁇ l) were mixed with 10 ⁇ l of a solution containing 100 mM Tris-HCl (pH 7.9), 300 mM KCl, 10 mM dithiothreitol (DTT), 0.1% nonyl phenoxylpolyethoxylethanol-40 and water.
• the reaction mixture was incubated at 37 0 C for 15 minutes and 25 ⁇ l of a solution containing 50 mM Tris-HCl (pH 7.9), 150 mM KCl, 5 mM DTT, 15 mM MgCl 2 , 0.05% nonyl phenoxylpolyethoxylethanol-40, 10 ⁇ g/ml poly(A), 0.250 U/ml oligo d(T)i 2 -i 8 , and 10 ⁇ Ci/ml 3 H- thymidine 5 '-triphosphate (TTP) was added to each well; plates were incubated at 37 0 C for 18 hours.
• TTP triphosphate
• HIV-I p24 antigen was also used to substantiate antiretroviral activity in MDM that were treated with surface-modified particles and subsequently infected with HIV-I.
• Cells were fixed with 4% phosphate-buffered paraformaldehyde 10 days after HIV-I infection.
• Mouse monoclonal antibodies to HIV-I p24 (1:10, Dako, Carpinteria, CA, USA) were used to determine the density of HIV-I- infected cells. Quantification of immuno staining was performed by densitometry using Image-Pro Plus, v. 4.0 (Media Cybernetics Inc., Bethesda, MD, USA). Expression of p24 was quantified by determining the positive area (index) as a percentage of the total image area per microscopy field.
• IDV-4 and RTV-4 inhibited HIV-I progeny virion production in infected cells for 15 days, while IDV-I and RTV-I did not. This was demonstrated by the gradual loss of p24 inhibition as well as the breakthrough of viral spread over time as demonstrated by increased density of p24 labeling. This data set mirrors that of the RT analysis and demonstrates decreased rates of viral replication for IDV-4 and RTV-4 by showing reductions in both RT activity and HIV-I p24 over time.
• the DOTAP-coated protease inhibitor particles retain retroviral activity and MDM loaded with DOTAP-coated protease inhibitor particles are effective in treating HIV infection.

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