WO2003105765A2 - Micelles phospholipidiques dans des liposomes, servant d'agents de solubilisation pour des composes insolubles dans l'eau - Google Patents

Micelles phospholipidiques dans des liposomes, servant d'agents de solubilisation pour des composes insolubles dans l'eau Download PDF

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Publication number
WO2003105765A2
WO2003105765A2 PCT/US2003/018686 US0318686W WO03105765A2 WO 2003105765 A2 WO2003105765 A2 WO 2003105765A2 US 0318686 W US0318686 W US 0318686W WO 03105765 A2 WO03105765 A2 WO 03105765A2
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Prior art keywords
lipid
water
composition
polynucleotide
protein
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PCT/US2003/018686
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English (en)
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WO2003105765A3 (fr
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Hayat Onyuksel
Israel Rubinstein
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The Board Of Trustees Of The University Of Illinois
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Priority to AU2003253645A priority Critical patent/AU2003253645A1/en
Publication of WO2003105765A2 publication Critical patent/WO2003105765A2/fr
Publication of WO2003105765A3 publication Critical patent/WO2003105765A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers

Definitions

  • paclitaxel On top of poor solubility, paclitaxel is not absorbed effectively from the GI tract due to the effects of multidrug resistant (MDR) transporters, and as a result, it is mainly administered by the intravenous route.
  • MDR multidrug resistant
  • the commercially available formulation of paclitaxel, Taxol®, is a concentrated solution of paclitaxel in cremophor EL (polyoxyethylated castor oil) and dehydrated alcohol (1:1 v/v) which has to be further diluted 5 to 20 fold with 0.9% sodium chloride before intravenous administration.
  • cremophor EL elicits severe hypersensitivity reactions and hypotension
  • cremophor EL has also been shown to affect the pharmacokinetics and biodistribution of paclitaxel causing a non-linear disposition of Taxol® in patients.
  • aqueous formulations of paclitaxel devoid of cremophor EL include bile salt and phospholipid mixed-micellar formulations, complexes with cyclodextrins, emulsions, liposomes and the formation of water-soluble prodrugs of paclitaxel.
  • Most of the carriers such as cyclodextrins and mixed micelles can accommodate only a limited amount of the drug or amphiphilic compound due to the limited hydrophobic regions in the carrier resulting in lower concentrations of the drug or compound in solution.
  • the amount of carrier would have to be increased, which could lead to vehicle side effects similar to cremophor.
  • Liposomal formulations are relatively non-toxic, but could incorporate only a low amount of paclitaxel, possibly due to the difficulty in packing of the bulky structure of the drug into the lipid bilayer. Emulsion formulations of paclitaxel may also not prove very useful due to the toxicity of the emulsifiers that are used.
  • the formation of hydrolysable prodrugs of paclitaxel, although feasible, is a costly and difficult procedure. Also, this approach may cause undesirable alterations in the pharmacokinetic and pharmacodynamic properties of the drug.
  • Phospholipid micelles composed of PEGylated phospholipids such as poly (ethylene glycol)-grafted distearoyl-phosphatidylethanolamine (PEG-DSPE) are safe, biocompatible and non-toxic and are relatively new drug delivery systems that are being explored to solubilize water-insoluble drugs and amphiphilic peptides and proteins.
  • PEG-DSPE poly (ethylene glycol)-grafted distearoyl-phosphatidylethanolamine
  • SSM sterically stabilized micelles
  • micellar systems are shown to be relatively stable on dilution due to their low critical micellar concentration (CMC) values in contrast to conventional detergent micelles.
  • compositions comprising water-insoluble drugs, amphiphilic peptides, amphiphilic proteins, or polynucleotides incorporated in a sterically- stabilized simple micelle (SSSM), said SSSM encapsulated in a sterically-stabilized liposome (SSL).
  • SSSM sterically- stabilized simple micelle
  • SSL sterically-stabilized liposome
  • the SSSM is composed of distearoylphosphatidylethanolamine (DSPE).
  • DSPE distearoylphosphatidylethanolamine
  • the DSPE is covalently modified to include polyethylene glycol (PEG), hi one embodiment, the PEG is PEG2000.
  • the invention also provides compositions comprising water-insoluble drugs, amphiphilic peptides, amphiphilic proteins, or polynucleotides incorporated in a sterically- stabilized mixed micelle (SSMM), said SSMM encapsulated in a sterically-stabilized liposome (SSL).
  • the SSMM is composed of distearoylphosphatidylethanolamine (DSPE).
  • the SSMM comprises a bile salt and one or more lipids.
  • the SSMM is composed of distearoylphosphatidylethanolamine (DSPE) and phosphatidylcholine (PC).
  • the DSPE is covalently modified to include polyethylene glycol (PEG).
  • the PEG is PEG2000.
  • the invention also provides compositions comprising water-insoluble drugs, amphiphilic peptides, amphiphilic proteins, or polynucleotides incorporated in a sterically- stabilized crystal (SSC), said SSC encapsulated in a sterically-stabilized liposome (SSL).
  • SSC sterically- stabilized crystal
  • SSL sterically-stabilized liposome
  • the SSC is composed of distearoylphosphatidylethanolamine (DSPE).
  • DSPE is covalently modified to include polyethylene glycol (PEG).
  • the PEG is PEG2000.
  • the invention also provides compositions comprising water-insoluble drugs, amphiphilic peptides, amphiphilic proteins, or polynucleotides incorporated in a sterically- stabilized simple micelle (SSSM), said SSSM encapsulated in a liposome.
  • the liposome is a sterically-stabilized liposome (SSL), said SSL comprising at least one lipid component covalently modified to include a targeting agent.
  • the SSSM is composed of distearoylphosphatidylethanolamine (DSPE).
  • DSPE distearoylphosphatidylethanolamine
  • the DSPE is covalently modified to include polyethylene glycol (PEG), h a further embodiment, the PEG is PEG2000.
  • the SSL comprises DSPE covalently modified to include the targeting agent.
  • the targeting agent is vasoactive intestinal peptide (NIP).
  • the invention also provides compositions comprising water-insoluble drugs, amphiphilic peptides, amphiphilic proteins, or polynucleotides incorporated in a sterically- stabilized mixed micelle (SSMM), said SSMM encapsulated in a sterically-stabilized liposome (SSL), said SSL comprising at least one lipid component covalently modified to include a targeting agent.
  • the SSMM is composed of distearoylphosphatidylethanolamine (DSPE).
  • the SSMM is composed of DSPE and phosphatidylcholine (PC).
  • the SSL comprises DSPE covalently modified to include polyethylene glycol (PEG).
  • the PEG is PEG2000.
  • the targeting agent is vasoactive intestinal peptide (VIP).
  • the SSL comprises DSPE further covalently modified to include vasoactive intestinal peptide (VIP) as the targeting agent.
  • the invention further provides compositions comprising water-insoluble drugs, amphiphilic peptides, amphiphilic proteins, or polynucleotides incorporated in a sterically- stabilized crystal (SSC), said SSC encapsulated in a sterically-stabilized liposome (SSL), said SSL comprising at least one lipid component covalently modified to include a targeting agent, h one aspect, the SSC is composed of distearoylphosphatidylethanolamine (DSPE). In another aspect, the DSPE is covalently modified to include polyethylene glycol (PEG). In a further embodiment, the PEG is PEG2000. In one aspect, the SSL comprises DSPE covalently modified to include the targeting agent. In another aspect, the targeting agent is vasoactive intestinal peptide (VIP).
  • VIP vasoactive intestinal peptide
  • the invention also provides methods of preparing compositions comprising a compound selected from the group consisting of water-insoluble drugs, amphiphilic peptides, amphiphilic proteins, or polynucleotides comprising the steps of: a) incorporating the compound in sterically-stabilized simple micelles (SSSM) comprising a single type of lipid, wherein said lipid is covalently modified to include a water-soluble polymer; and b) mixing an aqueous solution containing said SSSM containing the drug with a dried lipid film under conditions that permit the lipid film to form liposomes and encapsulate the SSSM, said lipid film comprising a mixture of at least two types of lipids wherein one type of lipid in said film is covalently modified to include a water soluble polymer and resulting liposomes are sterically-stabilized liposomes (SSL).
  • the methods further comprise the step of isolating said SSL containing the drug or compound incorporated in SSSM.
  • compositions comprising a compound selected from the group consisting of water-insoluble drags, amphiphilic peptides, amphiphilic proteins, or polynucleotides comprising the steps of: a) mixing the compound and a lipid under conditions that permit compound and lipid association to form micelles, wherein said lipid is covalently modified to include a water- soluble polymer and resulting micelles are sterically-stabilized simple micelles (SSSM); and b) mixing an aqueous solution containing said SSSM containing the compound with a dried lipid film under conditions that permit the lipid film to form liposomes and encapsulate the SSSM, said lipid film comprising a mixture of at least two types of lipids wherein one type of lipid in said film is covalently modified to include a water soluble polymer and resulting liposomes are sterically-stabilized liposomes (SSL), hi one aspect, the methods further comprise the step of isolating
  • the invention provides methods of preparing compositions comprising a compound selected from the group consisting of water-insoluble drugs, amphiphilic peptides, amphiphilic proteins, or polynucleotides comprising the steps of: a) incorporating the compound in sterically-stabilized simple micelles (SSSM) comprising a single type of lipid, wherein a molar fraction of said lipid is covalently modified to include a water-soluble polymer; and b) mixing an aqueous solution containing said sterically- stabilized micelles containing the compound with a dried lipid film under conditions that permit the lipid film to form liposomes and encapsulate the SSSM, said lipid film comprising a mixture of at least two types of lipids wherein one type of lipid in said film is covalently modified to include a water soluble polymer and resulting liposomes are sterically-stabilized liposomes (SSL).
  • SSL sterically-stabilized liposomes
  • the invention also provides methods of preparing compositions comprising a compound selected from the group consisting of water-insoluble drugs, amphiphilic peptides, amphiphilic proteins, or polynucleotides comprising the steps of: a) mixing the compound and a lipid under conditions that compound and lipid association to form micelles, wherein a molar fraction of said lipid is covalently modified to include a water-soluble polymer and resulting micelles are sterically-stabilized simple micelles (SSSM) containing the drug or compound; and b) mixing an aqueous solution containing said SSSM containing the compound with a dried lipid film under conditions that permit the lipid film to form liposomes and encapsulate the SSSM, said lipid film comprising a mixture of at least two types of lipids wherein one type of lipid in said film is covalently modified to include a water soluble polymer and resulting liposomes are sterically-stabilized liposomes (SSL).
  • SSL
  • the invention provides methods of preparing compositions comprising a compound selected from the group consisting of water-insoluble drugs, amphiphilic peptides, amphiphilic proteins, or polynucleotides comprising the steps of: a) incorporating the compound in sterically-stabilized mixed micelles (SSMM) comprising at least two types of lipid, wherein a molar fraction of one type of lipid is covalently modified to include a water-soluble polymer; and b) mixing an aqueous solution containing said SSMM containing the compound with a dried lipid film under conditions that permit the lipid film to form liposomes and encapsulate the SSMM, said lipid film comprising a mixture of at least two types of lipids wherein one type of lipid in said film is covalently modified to include a water soluble polymer and resulting liposomes are sterically-stabilized liposomes (SSL).
  • the methods further comprise the step of isolating said SSL containing
  • the present invention also provides methods of preparing compositions comprising a compound selected from the group consisting of water-insoluble drugs, amphiphilic peptides, amphiphilic proteins, or polynucleotides comprising the steps of: a) mixing the compound with a combination of at least two different types of lipids under conditions that permit said compound and said lipids to form micelles, wherein one type of lipid is covalently modified to include a water-soluble polymer and resulting micelles are sterically-stabilized mixed micelles (SSMM) containing the compound; and b) mixing an aqueous solution containing said SSMM containing the compound with a dried lipid film under conditions that permit the lipid film to form liposomes and encapsulate the SSMM, said lipid film comprising a mixture of at least two types of lipids wherein one type of lipid in said film is covalently modified to include a water soluble polymer and resulting liposomes are sterically-stabilized liposomes (SSL
  • the invention also provides methods of preparing compositions comprising a compound selected from the group consisting of water-insoluble drugs, amphiphilic peptides, amphiphilic proteins, or polynucleotides comprising the steps of: a) incorporating the compound in sterically-stabilized mixed micelles (SSMM) comprising a single type of lipid, wherein a molar fraction of said lipid is covalently modified to include a water-soluble polymer; and b) mixing an aqueous solution containing said SSMM containing the compound with a dried lipid film under conditions that permit the lipid film to form liposomes and encapsulate the SSMM, said lipid film comprising a mixture of at least two types of lipids wherein one type of lipid in said film is covalently modified to include a water soluble polymer and resulting liposomes are sterically-stabilized liposomes (SSL).
  • the methods further comprise the step of isolating said SSL containing the compound incorporated in
  • the invention provides methods of preparing compositions comprising a compound selected from the group consisting of water-insoluble drugs, amphiphilic peptides, amphiphilic proteins, or polynucleotides comprising the steps of: a) incorporating the compound in sterically-stabilized mixed micelles (SSMM) comprising at least two types of lipids, wherein one type of said lipids is covalently modified to include a water-soluble polymer; and b) mixing an aqueous solution containing said SSMM containing the compound with a dried lipid film under conditions that permit the lipid film to form liposomes and encapsulate the SSMM, said lipid film comprising a mixture of at least two types of lipids wherein one type of lipid in said film is covalently modified to include a water soluble polymer and resulting liposomes are sterically-stabilized liposomes (SSL).
  • the methods further comprise the step of isolating said SSL containing the compound incorporated
  • the invention provides methods of preparing compositions comprising a compound selected from the group consisting of water-insoluble drugs, amphiphilic peptides, amphiphilic proteins, or polynucleotides comprising the steps of: a) incorporating the compound in sterically-stabilized crystals (SSC) comprising a single type of lipid, wherein said lipid is covalently modified to include a water-soluble polymer; and b) mixing an aqueous solution containing said SSC containing the compound with a dried lipid film under conditions that permit the lipid film to form liposomes and encapsulate the SSC, said lipid film comprising a mixture of at least two types of lipids wherein one type of lipid in said film is covalently modified to include a water soluble polymer and resulting liposomes are sterically-stabilized liposomes (SSL).
  • the methods further comprise the step of isolating said SSL containing the compound incorporated in SSC.
  • compositions comprising a compound selected from the group consisting of water-insoluble drugs, amphiphilic peptides, amphiphilic proteins, or polynucleotides comprising the steps of: a) mixing the compound and a lipid under conditions that permit compound and lipid association to form micelles, wherein said lipid is covalently modified to include a water- soluble polymer and resulting micelles are sterically-stabilized crystals (SSC); and b) mixing an aqueous solution containing said SSC containing the drug or compound with a dried lipid film under conditions that permit the lipid film to form liposomes and encapsulate the SSC, said lipid film comprising a mixture of at least two types of lipids wherein one type of lipid in said film is covalently modified to include a water-soluble polymer and resulting liposomes are sterically-stabilized liposomes (SSL).
  • the methods further comprise the step of isolating said
  • the present invention also provides methods of preparing compositions comprising a compound selected from the group consisting of water-insoluble drugs, amphiphilic peptides, amphiphilic proteins, or polynucleotides comprising the steps of: a) incorporating the compound in sterically-stabilized crystals (SSC) comprising a single type of lipid, wherein a molar fraction of said lipid is covalently modified to include a water-soluble polymer; and b) mixing an aqueous solution containing said SSC containing the compound with a dried lipid film under conditions that permit the lipid film to form liposomes and encapsulate the SSC, said lipid film comprising a mixture of at least two types of lipids wherein one type of lipid in said film is covalently modified to include a water soluble polymer and resulting liposomes are sterically-stabilized liposomes (SSL).
  • SSC sterically-stabilized crystals
  • the invention provides methods comprising the further step of is
  • compositions comprising a compound selected from the group consisting of water-insoluble drugs, amphiphilic peptides, amphiphilic proteins, or polynucleotides and a lipid under conditions that permit compound and lipid association to form micelles, wherein a molar fraction of said lipid is covalently modified to include a water-soluble polymer and resulting micelles are sterically-stabilized crystals (SSC) containing the compound; and b) mixing an aqueous solution containing said SSC containing the compound with a dried lipid film under conditions that permit the lipid film to form liposomes and encapsulate the SSC, said lipid film comprising a mixture of at least two types of lipids wherein one type of lipid in said film is covalently modified to include a water soluble polymer and resulting liposomes are sterically-stabilized liposomes (SSL).
  • the methods further comprise the step of isolating said SSL containing
  • compositions comprising a compound selected from the group consisting of water-insoluble drugs, amphiphilic peptides, amphiphilic proteins, or polynucleotides comprising the steps of: a) incorporating the compound in sterically-stabilized simple micelles (SSSM) comprising a single type of lipid, wherein a molar fraction of said lipid is covalently modified to include a water-soluble polymer; b) mixing an aqueous solution containing said SSSM containing the compound with a dried lipid film under conditions that permit the lipid film to form liposomes and encapsulate the SSSM, said lipid film comprising a mixture of at least two types of lipids wherein one type of lipid in said film is covalently modified to include a water soluble polymer and resulting liposomes are sterically-stabilized liposomes (SSL); and c) incubating said SSL with a lipid covalently modified to include a lipid covalently modified to include a
  • the present invention also provides methods of preparing compositions comprising a compound selected from the group consisting of water-insoluble drugs, amphiphilic peptides, amphiphilic proteins, or polynucleotides comprising the steps of: a) incorporating the compound in sterically-stabilized mixed micelles (SSMM) comprising a single type of lipid, wherein a molar fraction of said lipid is covalently modified to include a water-soluble polymer; b) mixing an aqueous solution containing said SSMM containing the compound with a dried lipid film under conditions that permit the lipid film to form liposomes and encapsulate the SSMM, said lipid film comprising a mixture of at least two types of lipids wherein one type of lipid in said film is covalently modified to include a water soluble polymer and resulting liposomes are sterically-stabilized liposomes (SSL); and c) incubating said SSL with a lipid covalently modified to include a targeting
  • the present invention also provides methods of preparing compositions comprising a compound selected from the group consisting of water-insoluble drugs, amphiphilic peptides, amphiphilic proteins, or polynucleotides comprising the steps of: a) incorporating the compound in sterically-stabilized crystals (SSC) comprising a single type of lipid, wherein a molar fraction of said lipid is covalently modified to include a water-soluble polymer; b) mixing an aqueous solution containing said SSC containing the drug or compound with a dried lipid film under conditions that permit the lipid film to form liposomes and encapsulate the SSC, said lipid film comprising a mixture of at least two types of lipids wherein one type of lipid in said film is covalently modified to include a water soluble polymer; and c) incubating said SSL with a lipid covalently modified to include a targeting agent under conditions that permit the lipid covalently modified to include the targeting agent to incorporate into the lip
  • the invention further provides SSMM comprising a compound selected from the group consisting of water-insoluble drugs, amphiphilic peptides, amphiphilic proteins, or polynucleotides, wherein at least one lipid component of the SSMM includes a targeting agent.
  • the targeting agent is attached to a lipid component of the SSMM.
  • the targeting agent is attached to a water-soluble moiety of a lipid component of the SSMM.
  • the targeting agent is covalently attached to the lipid component of the water soluble polymer component of the SSMM.
  • the invention further provides methods of preparing SSMM compositions comprising a targeting agent and a compound selected from the group consisting of water-insoluble drugs, amphiphilic peptides, amphiphilic proteins, or polynucleotides by any of the methods above for preparation of
  • compositions comprising water-insoluble compounds and methods for preparing the compositions.
  • compositions comprising a compound that is significantly insoluble in an aqueous environment, wherein the compound is incorporated in a micelle and said micelle encapsulated in a liposome component.
  • a "significantly insoluble” compound is one that exhibits low, negligible, or no therapeutic potential in an aqueous environment as a result of hydrophobicity.
  • compositions of the invention comprising a compound selected from the group consisting of water-insoluble drugs, amphiphilic peptides, amphiphilic proteins, or polynucleotides incorporated in a sterically-stabilized simple micelle (SSSM), a sterically- stabilized mixed micelle (SSMM), and/or a sterically-stabilized crystal (SSC). fn one aspect each of these components is then encapsulated in a sterically-stabilized liposome (SSL).
  • the SSSM, SSMM, and/or SSC is composed of distearoylphosphatidylethanolamine (DSPE).
  • the DSPE is covalently modified to include polyethylene glycol (PEG).
  • the PEG is PEG2000.
  • Sterically-stabilized mixed micelles as used herein are the second generation of sterically-stabilized simple micelles (SSSM), previously called sterically-stabilized micelles (SSM).
  • SSMM have greater solubihzation or incorporation potential for hydrophobic drugs or compounds than SSSM because they have a greater hydrophobic core due to the incorporation of PC or PE.
  • SSMM have retained all the advantages of SSSM while increasing the solubihzation or incorporation capacity of the micelle for a hydrophobic drug or compound.
  • the term SSM as used herein may therefore mean SSSM or SSMM.
  • compositions of the compositions comprise any type of lipid or lipids (at any molar fraction when applicable) modified with any water-soluble polymer at any molar ratio as known within the art. Micelle components also include all combinations of any embodiment expressly described herein.
  • the invention contemplates liposome compositions of varying sizes.
  • the compositions of the invention comprise liposomes having a mean diameter greater than 200 nm.
  • compositions of the invention comprise liposomes having a mean diameter of less than about 200 nm.
  • the liposome compositions have a mean diameter of between about 50 nm and about 200 nm, and in another aspect, the liposomes have a mean diameter of between about 100 nm and about 200 nm.
  • the invention contemplates liposome and micelle components comprising any lipid or combination of lipids known in the art to be useful for liposome and/or micelle preparation, including naturally occurring and synthetic lipids.
  • the choice of lipids is influenced by, inter alia, the desired degree of stability, the desired degree of liposome membrane fluidity, the drug or compound being incorporated into the composition.
  • compositions of the invention further comprise a liposome component in which a micelle component of the invention is encapsulated.
  • the liposome component is a sterically-stabilized liposome (SSL).
  • a SSL comprises at least one type of lipid that is covalently modified to include a water-soluble polymer.
  • the SSL comprises at least one type of lipid that is covalently modified to include a water soluble polymer and/or a targeting agent.
  • the water-soluble polymer is PEG, and in one aspect, the PEG is PEG2000.
  • the targeting agent is vasoactive intestinal peptide (VIP).
  • targeting agents include compounds which bind to proteins/receptors in target tissues such as peptides, proteins, folic acid, and monoclonal antibodies identified as specific biomarkers of a disease. These targeting agents include, but are not limited to, secretin, transtuzumab, cetuximab, and pertuzurnab.
  • amphipathic peptides which are members of the family of peptide compounds including, but not limited to, vasoactive intestinal peptide (VIP), growth hormone releasing factor (GRF), hypocretins, peptide histidine isoleucine (PHI), peptide histidine methionine (PHM), pituitary adenylate cyclase activating peptide (PACAP), gastric inhibitory hormone (GIP), hemodermin, the growth hormone releasing hormone (GHRH), sauvagine and urotensin I, secretin, glucagon, galanin, endothelin, calcitonin, ⁇ i- proteinase inhibitor, angiotensin II, corticotropin releasing factor, antibacterial peptides and proteins in general, surfactant peptides and proteins, -MSH, adrenomedullin, ANF, IGF-1, ⁇ 2 amylin, orphanin
  • VIP vasoactive intestinal peptide
  • peptides of interest that may be used as targeting agents include neuropeptides, which serve as integrative chemical messengers, conveying information from one discrete neuronal population to another. Furthermore, it is becoming evident that neuropeptides are involved in coupling transductive events from neurons to glial and to immune cells.
  • neuropeptide research encompass pain and analgesia, appetite control, inflammation, mood and affective behavior, hi addition to the neuropeptides discussed herein, other neuropeptides include, but are not limited to, helospectin I or II, neuropeptide Y (NPY), neuropeptide YY (NPYY), including neuropeptide fragments 2-36 and related fragments, ACTH, calcitonin, GAP (GnRH precursor molecule), glutamate-decarboxylase, keyhole limpet hemocyanin, leucin-enkephalin, mesotocin, methionin-enkephalin, neurotensin, peroxydase, somatostatin, substance P, vasopressin, and vasotocin.
  • the present invention contemplates the solubihzation or incorporation of compounds in a vehicle which is safe and miscible with body fluids.
  • a typical phospholipid molecule for example, phosphatidylcholine (PC) is predominantly hydrophobic in nature and cylindrical in shape. Therefore for these molecules in the presence of excess water, the thermodynamically most preferred state of aggregation is the bilayer structure.
  • These lamellar structures can grow in two dimensions until they fold to form spherical vesicles, liposomes. The size and structure of the liposomes vary according to the environmental conditions in which the bilayers are formed. These bilayers are metastable structures.
  • Micelles can be formed from typical phospholipid molecules mainly by two ways.
  • the phospholipid can be incorporated into the micelles of another amphiphilic lipid which is relatively water soluble such as bile salt (BS).
  • BS bile salt
  • This approach will result in a mixed micelle of the phospholipid and the BS.
  • the solubihzation properties of this type of phospholipid micelles are discussed in detail herein.
  • the phospholipid molecule can be chemically modified to make it more hydrophilic. This approach is usually carried out by covalent attachment of a hydrophilic polymer to the polar head group of the phospholipid molecule.
  • the polymer used for this purpose is polyethylene glycol (PEG) with molecular weight varying from 1000 to 10000.
  • a flexible and linear polymer to the small headgroup of, for example, PE and/or PC molecules, serves another purpose in addition to making the phospholipid molecules more hydrophilic. Due to the large head group, the shape of the molecule changes from cylindrical to conical, which makes it possible to aggregate as a spherical micelle. Micelles are spherical in shape and form spontaneously above the critical micelle concentration (CMC). There is always an equilibrium between the micelles and the monomers of the amphiphile.
  • CMC critical micelle concentration
  • Micelles are dynamic structures, which break and reform according to changes in aqueous media in order to maintain constant monomer concentration.
  • the preparation of PEGylated phospholipid micelles is fairly simple and the reproducibility is good.
  • mixed micelles are formed with a larger hydrophobic core.
  • Phospholipids with very short acyl chains and lyso-phospholipids with single acyl chains can also form micelles, but they are more limited with respect to drug or compound solubihzation and delivery.
  • the present invention provides improved methods of preparing biologically active micelle components comprising one or more compounds in association with a micelle inside a liposome.
  • the term "compounds” embraces polynucleotides, peptides, proteins, and enzymes in general, as well as vaccines, fragments, analogs, and modulators thereof.
  • polypeptides the invention contemplates use of both L and D forms together or independently. Where compounds of the invention exist in both cis and trans conformations, the invention comprehends use of either form alone or a combination of both forms.
  • the compositions of the invention deliver and enhance bioactivity of the compound in a manner which provides improvements in the efficacy and duration of the biological effects of the associated compounds.
  • the invention overcomes problems associated with previous liposomal formulations, such as, but not limited to, uptake by the reticuloendothehal system, degradation of the compound, or delivery of the compound in an inactive conformation.
  • micelles are sterically-stabilized simple micelles (SSSM), which are produced from a combination of lipids which includes at least one lipid component covalently bonded to a water-soluble polymer.
  • This polymer bound phospholipid is the micelle forming component.
  • Other lipids are actually incorporated in this micelle to form mixed micelles.
  • the water-soluble polymer e.g. polyethylene glycol (PEG) increases the lipid solubility to form micelles instead of vesicles in aqueous media. It also acts to sterically stabilize the resulting micelle against uptake by components of the reticuloendothehal system.
  • PEG polyethylene glycol
  • polyethylene- glycol is covalently conjugated to DSPE and used to form polymeric micelles which are then passively loaded with VIP.
  • the PEG-DSPE forms micelles with a hydrophobic core consisting of distearoyl phosphatidylethanolamine (DSPE) fatty acid chains which is surrounded by a hydrophilic "shell" formed by the PEG polymer.
  • DSPE distearoyl phosphatidylethanolamine
  • micelles are sterically-stabilized mixed micelles (SSMM), which are formed by adding a typical phospholipid like phosphatidylcholine into SSSM with the purpose of increasing the hydrophobic core.
  • SSMM have all the advantages of SSSM with superior solubihzation capacity.
  • the SSMM further comprises a targeting agent attached to a lipid component of the SSMM or to a water soluble polymer component, also attached to a lipid component.
  • micelles are sterically-stabilized crystals (SSC), which are drug particles coated by PEGylated lipids, formed after a drug concentration exceeds the solubihzation potential of the sterically-stabilized simple micelle (SSSM).
  • SSC sterically-stabilized crystals
  • SSSM simple micelle
  • the formation of SSC may be explained by the low lipid/drug ratios resulting in insufficient SSSM to solubilize or incorporate the existing drug molecules in a molecular state.
  • the terms "solubilize(d) or incorporate ⁇ )” are used interchangeably to describe the process of being dissolved or taken up.
  • liposomes are sterically-stabilized liposomes (SSL), which are polymer-coated liposomes, wherein the polymer, e.g. polyethylene glycol (PEG), is covalently conjugated to one of the phospholipids and provides a hydrophilic cloud outside the vesicle bilayer.
  • SSL sterically-stabilized liposomes
  • PEG polyethylene glycol
  • the mean vesicle diameter should be under 200 nm, with PEG at a molecular weight of approximately 2,000 Da at a concentration of 5% (9-12) [Lasic and Martin, Stealth Liposomes, CRC Press, Inc., Boca Raton, FL (1995); Woodle, et al, Biochem. Biophys. Ada 1105:193-200 (1992); Litzinger, et al, Biochem. Biophys. Ada 1190:99-107 (1994); Bedu Addo, et al, Pharm. Res. 13:718-724 (1996)].
  • the mean vesicle diameter of the SSL is not limited to 200 nm.
  • compositions of the invention include micelle or crystalline compounds wherein the water soluble polymer is polyethylene glycol (PEG).
  • medicaments of the invention include use of micelles having an average diameter of less than about 25 nm.
  • medicaments of the invention include compositions comprising micelle or crystalline components wherein the combination of lipids consists of distearoyl-phosphatidylethanolamine covalently bonded to PEG (PEG-DSPE).
  • medicaments of the invention include compositions comprising micelle or crystalline components wherein the combination of lipids consists of distearoyl-phosphatidylethanolamine covalently bonded to PEG (PEG-DSPE) and a phospholipid like phosphatidylcholine (PC).
  • PEG-DSPE distearoyl-phosphatidylethanolamine covalently bonded to PEG
  • PC phospholipid like phosphatidylcholine
  • amphipathic compounds include those characterized by having one or more - or ⁇ -helical domains in their biologically active conformation and particularly those in which polar and apolar residues are separated on opposite sides of the helix.
  • Specific amphipathic compounds useful with the invention • include any member of the vasoactive intestinal peptide (VIP)/growth hormone releasing factor (GRF) family of peptides which includes biologically active analogs thereof.
  • VIP vasoactive intestinal peptide
  • GRF growth hormone releasing factor
  • the mammalian and non-mammalian VIP/GRF family of peptides includes functional analogs of VIP and GRF, peptide histidine isoleucine (PHI), peptide histidine methionine (PHM), growth hormone releasing factor (GRF), hypocretins, pituitary adenylate cyclase activating peptide (PACAP), secretin, and glucagon.
  • PHI peptide histidine isoleucine
  • PLM peptide histidine methionine
  • GRF growth hormone releasing factor
  • hypocretins ese histidine methionine
  • PACAP pituitary adenylate cyclase activating peptide
  • secretin and glucagon.
  • glucagon peptide histidine isoleucine
  • PLM peptide histidine methionine
  • GRF growth hormone releasing factor
  • hypocretins peptide histidine methionine
  • the invention also contemplates the use of other neuropeptides including neuropeptide Y (NPY), neuropeptide YY (NPYY), ACTH, calcitonin, GAP (GnRH precursor molecule), glutamate-decarboxylase, GnRH / GL , keyhole limpet hemocyanin, leucin-enkephalin, mesotocin, methionin-enkephalin, neurotensin, peroxydase, somatostatin, substance P, vasopressin, and vasotocin.
  • the invention also contemplates modulators having enhanced bioactivity in association with micelles prepared by a method of the invention.
  • a particularly preferred peptide for use according to the invention is VIP.
  • micelles according to the invention are characterized by an average diameter of less than about 20 to 50 nm.
  • the biologically active peptide products of the invention may be utilized in a wide variety of therapeutic, diagnostic, cosmetic and organ, tissue and cell preservative uses wherein it is desired to deliver a high level of compound or to detect targeted delivery of the micelle component as will be described below.
  • compositions of the invention include those wherein the polynucleotide, peptide, protein, enzyme, vaccine, fragment, analog, and or modulator thereof has an activity selected from the group consisting of anti-oxidant activity, anti-pain, anti-inflammatory, wound healing activity, anti-microbial, antibronchospasm, metabolic activity, anti-cancer activity, cardiovascular activity, antiglaucoma activity, anti-apoptosis, anti-wrinkling activity, cryopreservation, and anti-aging activity.
  • Compositions of the invention include cosmetic, therapeutic and diagnostic compositions.
  • the composition further comprises a detectable label selected from the group consisting of a fluorescent label, a radioactive label, a dye, a gas, and a compound which enhances radiographic, magnetic resonance, and ultrasound imaging.
  • the invention also provides a method of treating and use of components of the invention in the production of a medicament for the treatment of a pathology selected from the group consisting of immune disorders, inflammatory conditions, cancer, Hashimoto's thyroiditis, pernicious anemia, Addison's disease, diabetes, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, dermatomyositis, multiple sclerosis, myasthenia gravis, Reiter's syndrome, Graves disease, inflammatory bowel disease, osteoarthritis, rheumatoid arthritis, asthma, allergies, inflammatory neuropathies (Guillain Barre, • inflammatory polyneuropathies), vasculitis (Wegener's granulomatosus, polyarteritis nodosa), and rare disorders such as polymyalgia rheumatica, temporal arteritis, Sjogren's syndrome, Bechet's disease, Churg-Str
  • the invention provides a method of preventing VIP -induced hypotension comprising the step of administering to an individual an amount of a sterically- stabilized composition comprising a micelle or crystalline component effective to treat a target pathology, said sterically-stabilized micelle or crystalline component prepared by any one of the methods herein.
  • the present invention provides improved methods of preparing biologically active micelle components in liposomes comprising compounds in association with a micelle.
  • the invention also provides methods for preparing sterically-stabilized crystalline components in liposomes comprising compounds that are insoluble in an aqueous solution.
  • the compounds of the invention are prepared alone or in combination with a targeting compound. It is preferred that the targeting compound is an amphipathic compound that assumes a more favorable biological conformation.
  • the preferred amphipathic compounds are characterized by having hydrophilic and hydrophobic domains segregated to the extent that the hydrophobic domain is capable of associating within the micellar core.
  • Compounds of the invention preferably attain a biologically active conformation in association with or within the micelle.
  • More biologically active conformations are those in which the desired compound is most likely to be capable of effecting its normal biological activity, for example, through receptor or ligand recognition and binding, and comparison of biological activity is made with respect to the compound in association with the micelle or crystalline component of the invention compared to the compound in an aqueous solution or environment.
  • Compounds of the invention may be characterized by having one or more discrete ⁇ - or ⁇ - helical domains which segregate the hydrophobic and hydrophilic domains.
  • Preferred compounds of the invention are members of the VIP/GRF peptide family. While biologically active compounds are associated with the micelle core, the association is not irreversible and the compound may be released either quickly or over time from association with the micelle, depending on properties of the micelle and the compound.
  • any compound that is insoluble in as aqueous solution can be incorporated into crystalline component.
  • the insoluble compounds associate in the hydrophobic core of the associated lipids to the extent that the insoluble compound crystallizes. While the invention contemplates the use of any insoluble compound to produce the crystalline components, preferred compounds are normally insoluble anti-cancer agents, antifungal agents, sedatives, and steroidal compounds.
  • the insoluble compounds are selected from the group consisting of paclitaxel (Taxol®), betulinic acid, doxorubicin, amphotericin B, diazepam, nystatin, propofol, testosterone, estrogen, prednisolone, prednisone, 2,3 mercaptopropanol, and progesterone.
  • VIP vasoactive intestinal peptide
  • GRF growth hormone releasing factor
  • PHI peptide histidine isoleucine
  • PLM peptide histidine methionine
  • PACAP pituitary adenylate cyclase activating peptide
  • GHRH gastric inhibitory hormone
  • GHRH the growth hormone releasing hormone
  • sauvagine and urotensin I secretin, glucagon, galanin, endothelin, calcitonin, ⁇ rproteinase inhibitor, angiotensin II, corticotropin releasing factor, antibacterial peptides and proteins in general, surfactant peptides and proteins, ⁇ - MSH, adrenolmedullin, ANF, IGF-1, oc2 amylin, orphanin,
  • neuropeptides of interest include neuropeptides, which serve as integrative chemical messengers, conveying information from one discrete neuronal population to another. Furthermore, it is becoming evident that neuropeptides are involved in coupling transductive events from neurons to glial and to immune cells. Major areas of neuropeptide research encompass pain and analgesia, appetite control, inflammation, mood and affective behavior.
  • neuropeptides include, but are not limited to, helospectin I or II, neuropeptide Y (NPY), neuropeptide YY (NPYY), including neuropeptide fragments 2-36 and related fragments, ACTH, calcitonin, GAP (GnRH precursor molecule), glutamate-decarboxylase, keyhole limpet hemocyanin, leucin- enkephalin, mesotocin, methionin-enkephalin, neurotensin, peroxydase, somatostatin, substance P, vasopressin, and vasotocin.
  • Micelle components according to the invention may be produced from combinations of lipid materials well known and routinely utilized in the art to produce micelles and including at least one lipid component covalently bonded to a water-soluble polymer.
  • Lipids may include relatively rigid varieties, such as sphingomyelin, or fluid types, such as phospholipids having unsaturated acyl chains.
  • the lipid materials may be selected by those of skill in the art in order that the circulation time of the micelles be balanced with the drug release rate. To make full use of the power of these micelles in drug or compound delivery, a key challenge is to prevent the leakage of the drug or compound from the micelle to a level significantly less than the plasma distribution rate.
  • Polymers of the invention may thus include any compounds known and routinely utilized in the art of sterically-stabilized liposome (SSL) technology and technologies which are useful for increasing circulatory half-life for proteins, including for example polyvinyl alcohol, polylactic acid, polyglycolic acid, polyvinylpyrrolidone, polyacrylamide, polyglycerol, polyaxozlines, or synthetic lipids with polymeric headgroups.
  • SSL sterically-stabilized liposome
  • the most preferred polymer of the invention is PEG at a molecular weight between 1000 and 5000.
  • Preferred lipids for producing micelles according to the invention include distearoyl-phosphatidylethanolamine covalently bonded to PEG (PEG-DSPE) alone or in further combination with phosphatidylcholine (PC), and phosphatidylglycerol (PG) in further combination with cholesterol (Choi) and/or calmodulin.
  • Methods of the invention for preparation of sterically-stabilized micelle components or sterically-stabilized crystalline components can be carried using various techniques.
  • micelle components are mixed in an organic solvent and the solvent is removed using either evaporation or lyophilization. Removal of the organic solvent results in a lipid film, or cake, which is subsequently hydrated using an aqueous solution to permit formation of micelles.
  • the resulting micelles are mixed with an amphipathic compound of the invention whereby the amphipathic compound associates with the micelle and assumes a more favorable biologically active conformation.
  • one or more lipids are mixed in an aqueous solution after which the lipids spontaneously form micelles.
  • the resulting micelles are mixed with an amphipathic compound which associates with the micelle components and assumes a more favorable biologically active conformation.
  • Preparing micelle components by this method is particularly amenable for large scale and safer preparation and requires a considerable shorter time frame than methods previously described. The procedure is inherently safer in that use of organic solvents is eliminated.
  • one or more lipid compounds are mixed in an organic solvent with one or more insoluble compounds.
  • the organic solvent is removed either by evaporation or lyophilization to provide a film, or cake.
  • the resulting film, or cake is then hydrated by introduction of an aqueous solution.
  • the insoluble compound associates within the hydrophobic core of the lipid structure and is solubilized or re-crystallizes.
  • the solubilized compound or crystalline component is mixed with a targeting compound which, as described above for preparation of micelle components of the invention, associates with the crystalline component in a more favorable biologically active conformation.
  • Crystalline components of the invention provide advantages in that they, like sterically-stabilized micelle components, are able to evade the RES. More importantly, the crystalline components of the invention permit administration of higher concentrations of the insoluble compound in a small volume, preferable in a size less than 300 nm. The crystalline components also provide a method wherein insoluble compounds, which are normally difficult to effectively administer because of their inherent insolubility, can be effectively administered to a mammal in need thereof.
  • the micelle and crystalline components in liposomes produced according to the methods of the invention are characterized by improved stability and biological activity and are useful in a variety of therapeutic, diagnostic and/or cosmetic applications.
  • the invention comprehends a composition comprising a biologically active micelle component wherein said biologically active amphipathic compound has anti-oxidant activity, anti-aging, anti- rinkle formation or wound healing capacity.
  • Compositions of this type may be of cosmetic or therapeutic nature.
  • the preferred cosmetic composition includes a biologically active member of the VlP/glucagon/secretin family of peptides including peptide fragments and analogs.
  • the invention also provides an oral controlled release preparation for the treatment of a gastrointestinal disorder wherein said preparative method further comprises the step of encapsulating the biologically active micelle or crystalline component in an enteric coated capsule.
  • the micelle or crystalline component may be encapsulated in a gelatin capsule.
  • the oral controlled release preparation is useful in a variety of gastrointestinal disorders including those selected from the group consisting of inflammatory bowel disease, chronic constipation, Hirschprung's disease, achalasia, infantile hypertrophic pyloric stenosis, and ulcers.
  • micelles of the invention particularly those micelles containing a member of the VIP/GRF family of proteins, peptides and fragments, analogs, and modulators, include asthma, chronic obstruction pulmonary disease, arthritis, lupus erythematosus, Alzheimer's disease, cerebral palsy, stroke, glaucoma, acute food impaction, scleroderma, rhinitis, systemic and pulmonary hypertension, psoriasis, baldness, autism, multiple sclerosis, eneuresis, Parkinson's disease, amyotrophic lateral sclerosis, and AIDS-associated dementias, impotence and female arousal sexual dysfunction.
  • asthma chronic obstruction pulmonary disease
  • arthritis lupus erythematosus
  • Alzheimer's disease cerebral palsy
  • stroke glaucoma
  • acute food impaction scleroderma
  • rhinitis systemic and pulmonary hypertension
  • psoriasis baldness
  • autism multiple sclerosis
  • the preferred oral preparation includes a biologically active member of the VIP/glucagon/secretin or IL-2 family of peptides including peptide fragments and analogs.
  • Micelle preparations comprising a biologically active member of the VIP/glucagon/secretin or IL-2 family of peptides including peptide fragments and analogs are also a promising therapeutic agent for conditions such as asthma, chronic obstruction pulmonary disease, systemic and pulmonary hypertension, scleroderma, cystic fibrosis, bronchiectasis, myocardial ischemia, impotence and baldness.
  • Still other indications include decreased sperm/ova motility, decreased mucociliary clearance, Kartagener's syndrome, increased inflammatory cell migration and activation, increased secretion of mucin, decreased chloride ion secretion (often associated with cystic fibrosis), vasoconstriction, vascular obstruction to an organ or tissue (often associated with sickle vaso-occlusive crisis), constipation, impotence and female sexual arousal dysfunction.
  • the invention further provides methods for cosmetic use and preserving a bodily organ, tissue, or cell type for storage and transplantation or fertilization in a recipient comprising the step of incubating said organ, tissue, or cell in a micelle composition comprising a member of the VIP/glucagon/secretin or IL-2 family of peptides including peptide fragments and analogs.
  • micelle and crystalline components in liposomes prepared with or without associated amphipathic compounds can be used to improve viability of cells, tissues, and organs that are stored cryogenically.
  • cells are contacted with a micelle component of the invention prior to cryogenic storage either alone, or in the presence of other storage compounds, e.g., dimethylsulfoxide (DMSO), sucrose, glycerol, or ethylene glycol, well known and routinely used in the art.
  • DMSO dimethylsulfoxide
  • sucrose sucrose
  • glycerol glycerol
  • ethylene glycol ethylene glycol
  • the invention further provides methods of administering a compound to a target tissue comprising the steps of: preparing biologically active micelle or crystalline components in lipsomes comprising a biologically active amphipathic compound in association with micelle or crystalline components in liposomes according to the methods of the invention and administering a therapeutically effective amount of the micelle or crystalline components in liposomes to said target tissue.
  • the micelle or crystalline components in liposomes of the invention maybe administered intravenously, intraarterially, intranasally, such as by aerosol administration, nebulization, inhalation, or insufflation, intratracheally, intra-articularly, orally, transdermally, subcutaneously, topically onto mucous membranes, such as, but not limited to, oral mucosa, lower gastrointestinal mucosa and conjunctiva, and directly onto target tissues.
  • Methods of administration for amphipathic compounds are equally amenable to administration of compounds that are insoluble in aqueous solutions.
  • Biologically active compounds in therapeutic methods can be administered at significantly reduced dosage levels as compared to administration of the compound alone, particularly wherein the compound has a particularly short half life or lowered bioactivity in circulation.
  • VIP in association with micelle or crystalline components in liposomes can be expected to exhibit enhanced and prolonged bioactivity in comparison to VIP administered alone.
  • the micelle or crystalline components in liposomes must be tested in order to determine a biologically effective amount required to achieve the same result affected by the compound administered by conventional means.
  • Another aspect of the invention is the means for preventing VlP-induced hypotension.
  • One of the deleterious effects of VIP administration has been the resulting hypotension brought on by vasodilation.
  • Hypotension is an abnormal condition in which the blood pressure is lower than 90/60 or is low enough to cause symptoms or interfere with well-being. Blood pressure is normally above 90/60 mmHg (millimeters of mercury). When the blood pressure is too low there is inadequate blood flow to the heart, brain, and other vital organs.
  • VIP administered in a micelle or crystalline component in liposomes provides a means for limiting VIP-induced hypotension.
  • An exemplary regimen in the treatment for example, of autism, multiple sclerosis, eneuresis, Parkinson's disease, amyotrophic lateral sclerosis, and AIDS-associated dementias, would include administration of from 0.001 mg/kg body weight to about 1000 mg/kg, from about 0.01 mg/kg to about 100 mg/kg, from about 0.1 mg/kg to about 100 mg/kg, about 1.0 mg/kg to about 50 mg/kg, or from about 1 mg/kg to about 20 mg/kg,. given in daily doses or in equivalent doses at longer or shorter intervals, e.g., every other day, twice weekly, weekly, monthly, semi-annually, or even twice or three times daily.
  • dosages may be measured in international units (IU) ranging from about 0.001 IU kg body weight to about 1000 IU/kg, from about 0.01 IU/kg to about 100 IU/kg, from about 0.1 IU/kg to about 100 IU /kg, from about 1 IU/kg to about 100 IU/kg, from about 1 IU/kg to about 50 IU/kg, or from about 1 IU/kg to about 20 IU/kg.
  • Administration may be oral, intravenous, subcutaneous, intranasal, inhalation, transdermal, transmucosal, or by any other route discussed herein.
  • association of a biologically active amphipathic or insoluble compound with SSM or SSC component, respectively, of the invention would be expected to increase the magnitude of the biological effects of the compound from about 50 to 100% over the effects observed following administration of the compound alone. Likewise, association with SSM or SSC of the invention would be expected to invoke a longer lasting biological effect.
  • the invention further provides improved diagnostic compositions comprising biologically active micelle components and methods for their use comprising the steps of: preparing a biologically active micelle component comprising a biologically active amphipathic compound in association with a micelle prepared according to the methods of the invention; administering a diagnostically effective amount of the micelle component to a target tissue or organ; and detecting uptake or interaction of the micelle component at the target tissue or organ.
  • the target tissue is a tumor.
  • the micelle component is detectably labeled with a label selected from the group including a radioactive label, a fluorescent label, a non-fluorescent label, a dye, a gas, or a compound which enhances radiographic, magnetic resonance, and ultrasound imaging (MRI) which label is detected at the target tissue.
  • a label selected from the group including a radioactive label, a fluorescent label, a non-fluorescent label, a dye, a gas, or a compound which enhances radiographic, magnetic resonance, and ultrasound imaging (MRI) which label is detected at the target tissue.
  • MRI ultrasound imaging
  • the invention also provides use of a biologically active micelle component comprising a biologically active amphipathic compound and produced according to methods of the invention for the treatment of inflammation, chronic obstruction pulmonary disease, increased secretion of mucin, acute food impaction, rhinitis, Kartagener's syndrome, cystic fibrosis, bronchi ectasis, hypertension, allergy, Alzheimer's disease, cerebral palsy sleep disorder, stroke, atherosclerosis, inflammatory bowel disorder, chronic constipation, Hirschprung's disease, achalasia, infantile hypertrophic pyloric stenosis, ulcers, to enhance or decrease cell proliferation, prevent apoptosis, to promote wound healing in a body organ or tissue, and to prevent cell, organ, tissue rejection, autism, multiple sclerosis, eneuresis, Parkinson's disease, amyotrophic lateral sclerosis, and AIDS-associated dementias, impotence and female arousal sexual dysfunction.
  • a biologically active micelle component compris
  • neuropeptides including a member of the VIP/glucagon/secretin or IL-2 family of peptides including peptide fragments and analogs.
  • an endogenously expressed member of the VIP/glucagon/secretin or IL-2 family of peptides including peptide fragments and analogs may be biologically inactive (or partially inactivated). Because the circulating peptide is inactive, and its effects not realized, additional peptide is continually produced to achieve the desired effect.
  • VIP receptors are rendered dysfunctional to the extent that native VIP cannot interact, whereas VIP in a micelle composition of the invention is able to either recognize or interact with the modified receptor, or able to affect its biological activity through a non-receptor mediated pathway.
  • Sterically-stabilized crystalline components of the invention are particularly useful for administration of anti-cancer agents.
  • crystalline components of the invention comprising paclitaxel (Taxol®) as the insoluble compound and VIP as the targeting agent can be targeted to breast cancer cells and tissue which are known to express higher levels of VIP receptor than normal breast cells and tissue, or express receptors with higher affinity of binding for VIP.
  • Paclitaxel (Taxol®) has been shown to selectively kill breast cancer cells.
  • Cosmetic uses for the micelle and crystalline components of the invention include anti-aging, anti-wrinkling, and antioxidant activities, as well as use as a sunscreen.
  • lipids L- -egg yolk phosphatidylcholine type V-E in chloroform : methanol (9:1) (Lot # 34H8395, and 75H8368), L- ⁇ -egg yolk phosphatidyl-D- o.-Glycerol in chloroform : methanol (98:2) (Lot # 72H8431, and 85H8395), and cholesterol (Lot #60H0476) from Sigma Chemical Co. (St. Louis, MO). Di-Palmitoyl-phosphatidyl choline (Lot #LP-04-01-l 12-187) from Sygenal Ltd. (Switzerland).
  • PEG-DSPE in lyophilized powder form (Lot # 180PHG2PK-26) from Avanti Polar Lipids Inc. (Alabaster, AL).
  • Inflammation refers to a localized, protective response elicited by injury or destruction of tissues, which serves to destroy, dilute or wall off (sequester) both the injurious agent and the injured tissue. Inflammation is notably associated with influx of leukocytes and or neutrophil chemotaxis.
  • Inflammation may result from infection with pathogenic organisms and viruses and from noninfectious means such as trauma or reperfusion following myocardial infarction or stroke, immune response to foreign antigen, and autoimmune responses. Accordingly, inflammatory disorders amenable to the invention encompass disorders associated with reactions of the specific defense system as well as with reactions of the non-specific defense system.
  • the term "specific defense system” refers to the component of the immune system that reacts to the presence of specific antigens. Examples of inflammation resulting from a response of the specific defense system include the classical response to foreign antigens, autoimmune diseases, and delayed type hypersensitivity response mediated by T-cells. Chronic inflammatory diseases, the rejection of solid transplanted tissue and organs, e.g., kidney and bone marrow transplants, and graft versus host disease (GVHD), are further examples of inflammatory reactions of the specific defense system.
  • GVHD graft versus host disease
  • non-specific defense system refers to inflammatory disorders that are mediated by leukocytes that are incapable of immunological memory (e.g., granulocytes, macrophages).
  • inflammation that result, at least in part, from a reaction of the non-specific defense system include inflammation associated with conditions such as adult (acute) respiratory distress syndrome (ARDS) or multiple organ injury syndromes; reperfusion injury; acute glomeralonephritis; reactive arthritis; dermatoses with acute inflammatory components; acute purulent meningitis or other central nervous system inflammatory disorders such as 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 glomeralonephritis reactive arthritis
  • dermatoses with acute inflammatory components acute purulent meningitis or other central nervous system inflammatory disorders such as stroke; thermal injury; inflammatory bowel disease; granulocyte transfusion associated syndromes;
  • Autoimmune disease refers to any group of disorders in which tissue injury is associated with humoral or cell-mediated responses to the body's own constituents.
  • Allergic disease refers to any symptoms, tissue damage, or loss of tissue function resulting from allergy.
  • Arthritic disease refers to any disease that is characterized by inflammatory lesions of the joints attributable to a variety of etiologies.
  • Dermatis refers to any of a large family of diseases of the skin that are characterized by inflammation of the skin attributable to a variety of etiologies.
  • Transplant rejection refers to any immune reaction directed against grafted tissue (including organs or cells (e.g., bone marrow), characterized by a loss of function of the grafted and surrounding tissues, pain, swelling, leukocytosis, and thrombocytopenia.
  • the therapeutic methods of the present invention include methods for the amelioration of disorders associated with inflammatory cell activation.
  • “Inflammatory cell activation” refers to the induction by a stimulus (including, but not limited to, cytokines, antigens or auto-antibodies) of a proliferative cellular response, the production of soluble mediators (including but not limited to cytokines, oxygen radicals, enzymes, prostanoids, or vasoactive amines), or cell surface expression of new or increased numbers of mediators (including, but not limited to, major histocompatability antigens or cell adhesion molecules) in inflammatory cells (including but not limited to monocytes, macrophages, T lymphocytes, B lymphocytes, granulocytes (polymo ⁇ honuclear leukocytes including neutrophils, basophils, and eosinophils), mast cells, dendritic cells, Langerhans cells, and endothelial cells). It will be appreciated by persons skilled in the art that the activ
  • the present invention enables methods of treating various diseases associated with or characterized by inflammation, for example, arthritic diseases such as rheumatoid arthritis, osteoarthritis, gouty arthritis, spondylitis; Behcet disease; sepsis, septic shock, endotoxic shock, gram negative sepsis, gram positive sepsis, and toxic shock syndrome; multiple organ injury syndrome secondary to septicemia, trauma, or hemorrhage; ophthalmic disorders such as allergic conjunctivitis, vernal conjunctivitis, uveitis, and thyroid-associated ophthahnopathy; eosinophilic granuloma; pulmonary or respiratory disorders such as asthma, chronic bronchitis, allergic rhinitis, ARDS, chronic pulmonary inflammatory disease (e.g., chronic obstructive pulmonary disease), silicosis, pulmonary sarcoidosis, pleurisy, alveolitis, vasculitis, pneumonia
  • Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft versus host disease and autoimmune inflammatory eye disease.
  • Such a protein (or antagonists thereof, including antibodies) of the present invention may also to be useful in the treatment of allergic reactions and conditions (e.g., anaphylaxis, serum sickness, drug reactions, food allergies, insect venom allergies, mastocytosis, allergic rhinitis, hypersensitivity pneumonitis, urticaria, angioedema, eczema, atopic dermatitis, allergic contact dermatitis, erythema multiforme, Stevens Johnson syndrome, allergic conjunctivitis, atopic keratoconjunctivitis, venereal keratoconjunctivitis, giant papillary conjunctivitis and contact allergies), such as asthma (particularly allergic asthma) or other respiratory problems.
  • allergic reactions and conditions e.g., anaphylaxis, serum sickness, drug reactions, food allergies, insect venom allergies, mastocytosis, allergic rhinitis, hypersensitivity pneumonitis, urticaria, angioedema, e
  • the present invention also provides methods of treating cancer in an animal, comprising administering to the animal an effective amount of a compound that inhibits DNA-PK activity.
  • the invention is further directed to methods of inhibiting cancer cell growth, including processes of cellular proliferation, invasiveness, and metastasis in biological systems.
  • Methods include use of a compound of the invention as an inhibitor of cancer cell growth.
  • the methods are employed to inhibit or reduce cancer cell growth, invasiveness, metastasis, or tumor incidence in living animals, such as mammals.
  • Methods of the invention are also readily adaptable for use in assay systems, e.g., assaying cancer cell growth and properties thereof, as well as identifying compounds that affect cancer cell growth.
  • Compounds of the invention are possess one or more desirable but unexpected combinations of properties, including increased activity and/or solubility, and reduction of negative side effects. These compounds have been found to inliibit cancer growth, including proliferation, invasiveness, and metastasis, thereby rendering them particularly desirable for the treatment of cancer.
  • compounds of the invention exhibit cancer-inhibitory properties at concentrations that appear to be substantially free of side effects. These compounds are therefore useful for extended treatment protocols, where the use of conventional chemotherapeutic compounds can exhibit undesirable side effects.
  • the coadministration of a compound of the invention with another, more toxic, chemotherapeutic agent can achieve beneficial inhibition of a cancer, while effectively reducing the toxic side effects in the patient.
  • the properties of hydrophilicity and hydrophobicity of the compounds of the invention are well balanced, thereby enhancing their utility for both in vitro and especially in vivo uses, while other compounds lacking such balance are of substantially less utility.
  • compounds of the invention have an appropriate degree of solubility in aqueous media which permits abso ⁇ tion and bioavailability in the body, while also having a degree of solubility in lipids which permits the compounds to traverse the cell membrane to a putative site of action.
  • compounds of the invention are maximally effective when they can be delivered to the site of the tumor and they enter the tumor cells.
  • the cancers treatable by methods of the present invention preferably occur in mammals.
  • Mammals include, for example, humans and other primates, as well as pet or companion animals such as dogs and cats, laboratory animals such as rats, mice and rabbits, and farm animals such as horses, pigs, sheep, and cattle.
  • Tumors or neoplasms include growths of tissue cells in which the multiplication of the cells is uncontrolled and progressive. Some such growths are benign, but others are termed “malignant” and may lead to death of the organism. Malignant neoplasms or “cancers” are distinguished from benign growths in that, in addition to exhibiting aggressive cellular proliferation, they may invade surrounding tissues and metastasize. Moreover, malignant neoplasms are characterized in that they show a greater loss of differentiation (greater "dedifferentiation"), and of their organization relative to one another and their surrounding tissues. This property is also called “anaplasia.” Neoplasms treatable by the present invention also include solid tumors, i.e., carcinomas and sarcomas.
  • Carcinomas include those malignant neoplasms derived from epithelial cells which infiltrate (invade) the surrounding tissues and give rise to metastases.
  • Adenocarcinomas are carcinomas derived from glandular tissue, or which form recognizable glandular structures.
  • Another broad category or cancers includes sarcomas, which are tumors whose cells are embedded in a fibrillar or homogeneous substance like embryonic connective tissue.
  • the invention also enables treatment of cancers of the myeloid or lymphoid systems, including leukemias, lymphomas and other cancers that typically do not present as a tumor mass, but are distributed in the vascular or lymphoreticular systems.
  • the type of cancer or tumor cells amenable to treatment according to the invention include, for example, ACTH-producing tumor, acute lymphocytic leukemia, acute nonlymphocytic leukemia, cancer of the adrenal cortex, bladder cancer, brain cancer, breast cancer, cervical cancer, chronic lymphocytic leukemia, chronic myelocytic leukemia, colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer, esophageal cancer, Ewing's sarcoma, gallbladder cancer, hairy cell leukemia, head and neck cancer, Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, liver cancer, lung cancer (small and non-small cell), malignant peritoneal effusion, malignant pleural effusion, melanoma, mesothelioma, multiple myeloma, neuroblastoma, glioma, non-Hodgkin's lymphoma, osteosarcoma, ova
  • the invention is particularly illustrated herein in reference to treatment of certain types of experimentally defined cancers, hi these illustrative treatments, standard state-of- the-art in vitro and in vivo models have been used. These methods can be used to identify agents that can be expected to be efficacious in in vivo treatment regimens. However, it will be understood that the method of the invention is not limited to the treatment of these tumor types, but extends to any solid tumor derived from any organ system. Cancers whose invasiveness or metastasis is associated with DNA-PK expression or activity are especially susceptible to being inhibited or even induced to regress by means of the invention.
  • the invention further relates to radiosensitizing tumor cells.
  • radiationosensitizer is defined as a molecule, preferably a low molecular weight molecule, administered to animals in therapeutically effective amounts to increase the sensitivity of the cells to be radiosensitized to electromagnetic radiation and/or to promote the treatment of diseases that are treatable with electromagnetic radiation.
  • Diseases that are treatable with electromagnetic radiation include neoplastic diseases, benign and malignant tumors, and cancerous cells.
  • Electromagnetic radiation treatment of other diseases not listed herein is also contemplated by the present invention.
  • the terms "electromagnetic radiation” and “radiation” as used herein include, but are not limited to, radiation having the wavelength of 10-20 to 100 meters.
  • Preferred embodiments of the present invention employ the electromagnetic radiation of: gamma-radiation (10-20 to 10-13 m), X-ray radiation (10-12 to 10-9 m), ultraviolet light (10 nm to 400 nm), visible light (400 nm to 700 nm), infrared radiation (700 nm to 1.0 mm), and microwave radiation (1 mm to 30 cm).
  • Radiosensitizers are known to increase the sensitivity of cancerous cells to the toxic effects of electromagnetic radiation.
  • hypoxic cell radiosensitizers e.g., 2-nitroimidazole compounds, and benzotriazine dioxide compounds
  • non-hypoxic cell radiosensitizers e.g., halogenated pyrimidines
  • various other potential mechanisms of action have been hypothesized for radiosensitizers in the treatment of disease.
  • radiosensitizers activated by the electromagnetic radiation of X-rays.
  • X-ray activated radiosensitizers include, but are not limited to, the following: metronidazole, misonidazole, desmethylmisonidazole, pimonidazole, etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233, EO9, RB 6145, nicotinamide, 5-bromodeoxyuridine (BUdR), 5-iododeoxyuridine (IUdR), bromodeoxycytidine, fluorodeoxyuridine (FUdR), hydroxyurea, cisplatin, and therapeutically effective analogs and derivatives of the same.
  • Photodynamic therapy (PDT) of cancers employs visible light as the radiation activator of the sensitizing agent.
  • photodynamic radiosensitizers include the following, but are not limited to: hematopo ⁇ hyrin derivatives, Photofrin(r), benzopo ⁇ hyrin derivatives, NPe6, tin etiopo ⁇ hyrin (SnET2), pheoborbide-a, bacteriochlorophyll-a, naphthalocyanines, phthalocyanines, zinc phthalocyanine, and therapeutically effective analogs and derivatives of the same.
  • Radiosensitizers may be administered in conjunction with a therapeutically effective amount of one or more other compounds, including but not limited to: compounds that promote the inco ⁇ oration of radiosensitizers to the target cells; compounds that control the flow of therapeutics, nutrients, and/or oxygen to the target cells; chemotherapeutic agents that act on the tumor with or without additional radiation; or other therapeutically effective compounds for treating cancer or other disease.
  • radiosensitizers examples include, but are not limited to: 5- fluorouracil (5-FU), leucovorin, 5(-amino-5(-deoxythymidine, oxygen, carbogen, red cell transfusions, perfluorocarbons (e.g., Fluosol(r)-DA), 2,3-DPG, BW12C, calcium channel blockers, pentoxyfylline, anti-angiogenesis compounds, hydralazine, and L-BSO.
  • chemotherapeutic agents that may be used in conjunction with radiosensitizers include, but are not limited to: adriamycin, camptothecin, carboplatin, cisplatin, daunorubicin, doxorubicin, interferon (alpha, beta, and gamma), interleukin 2, irinotecan, docetaxel, paclitaxel, topotecan, and therapeutically effective analogs and derivatives of the same.
  • the invention can also be practiced by including with a compound of the invention another anti-cancer chemotherapeutic agent, such as any conventional chemotherapeutic agent.
  • another anti-cancer chemotherapeutic agent such as any conventional chemotherapeutic agent.
  • the combination of the tetracycline compound with such other agents can potentiate the chemotherapeutic protocol.
  • Numerous chemotherapeutic protocols will present themselves in the mind of the skilled practitioner as being capable of inco ⁇ oration into the method of the invention.
  • Any chemotherapeutic agent can be used, including alkylating agents, antimetabolites, hormones and antagonists, radioisotopes, as well as natural products.
  • the compound of the invention can be administered with antibiotics such as doxorubicin and other anthracycline analogs, nitrogen mustards such as cyclophosphamide, pyrimidine analogs such as 5-fluorouracil, cisplatin, hydroxyurea, paclitaxel (Taxol®) and its natural and synthetic derivatives, and the like.
  • antibiotics such as doxorubicin and other anthracycline analogs
  • nitrogen mustards such as cyclophosphamide
  • pyrimidine analogs such as 5-fluorouracil, cisplatin, hydroxyurea, paclitaxel (Taxol®) and its natural and synthetic derivatives, and the like.
  • the compound in the case of mixed tumors, such as adenocarcinoma of the breast, where the tumors include gonadotropin- dependent and gonadotropin-independent cells, the compound can be administered in conjunction with leuprolide or goserelin (syn
  • antineoplastic protocols include the use of a tetracycline compound with another treatment modality, e.g., surgery, radiation, etc., also referred to herein as "adjunct antineoplastic modalities.”
  • another treatment modality e.g., surgery, radiation, etc.
  • the method of the invention can be employed with such conventional regimens with the benefit of reducing side effects and enhancing efficacy.
  • compositions are within the scope of the present invention. Such pharmaceutical compositions may comprise a therapeutically effective amount of a composition of the invention alone or in admixture with a pharmaceutically or physiologically acceptable formulation agent selected for suitability with the mode of administration. Pharmaceutical compositions may comprise a therapeutically effective amount of one or more compositions in admixture with a pharmaceutically or physiologically acceptable formulation agent selected for suitability with the mode of administration.
  • the pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adso ⁇ tion or penetration of the composition.
  • Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen sulfite); buffers (such as borate, bicarbonate, Tris HC1, citrates, phosphates, other organic acids); bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta cyclodextrin or hydroxypropyl beta cyclodextrin); fillers; monosaccharides; disaccharides and other carbohydrates (such as glucose, mannose, or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring; flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such
  • compositions can be selected for parenteral delivery. Alternatively, the compositions may be selected for inhalation or for delivery through the digestive tract, such as orally.
  • the preparation of such pharmaceutically acceptable compositions is within the skill of the art.
  • a pharmaceutical composition may be formulated for inhalation.
  • a product of the invention may be lyophihzed or formulated as a dry powder for inhalation.
  • Pharmaceutical composition inhalation solutions may also be formulated with a propellant for aerosol delivery.
  • solutions may be nebulized. Pulmonary administration is further described in PCT Application No. PCT US 94/001875, which describes pulmonary delivery of chemically modified proteins.
  • compositions of the invention which are administered in this fashion can be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules.
  • a capsule may be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre systemic degradation is minimized.
  • Additional agents can be included to facilitate abso ⁇ tion of the products of the invention. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be employed.
  • compositions of the invention may involve an effective quantity of compositions of the invention in a mixture with nontoxic excipients which are suitable for the manufacture of tablets.
  • excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.
  • compositions will be evident to those skilled in the art, including formulations involving compositions of the invention in sustained or controlled delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery means, such as liposome carriers, bio erodible microparticles or porous beads and depot injections, are also known to those skilled in the art.
  • the pharmaceutical composition to be used for in vivo administration typically must be sterile. This may be accomplished by filtration through sterile filtration membranes. Where the composition is lyophihzed, sterilization using this method may be conducted either prior to or following lyophilization and reconstitution.
  • the composition for parenteral administration may be stored in lyophihzed fo ⁇ n or in solution.
  • parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • the pharmaceutical composition may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or a dehydrated or lyophihzed powder.
  • Such formulations may be stored either in a ready to use form or in a form (e.g., lyophihzed) requiring reconstitution prior to administration.
  • An effective amount of a pharmaceutical composition to be employed therapeutically will depend, for example, upon the therapeutic context and objectives.
  • One skilled in the art will appreciate that the appropriate dosage levels for treatment will thus vary depending, in part, upon the molecule delivered, the indication for which the composition is being used, the route of administration, and the size (body weight, body surface or organ size) and condition (the age and general health) of the patient. Accordingly, the clinician may titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.
  • a typical dosage may range from about 0.1 mg/kg to up to about 100 mg/kg or more, depending on the factors mentioned above. In other embodiments, the dosage may range from 0.1 mg/kg up to about 100 mg/kg; or 1 mg/kg up to about 100 mg/kg; or 5 mg/kg up to about 100 mg kg.
  • the frequency of dosing will depend upon the pharmacokinetic parameters of the composition in the formulation used. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect.
  • the composition may therefore be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. Appropriate dosages may be ascertained through use of appropriate dose response data.
  • the route of administration of the pharmaceutical composition is in accord with known methods, e.g. orally, through injection by intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intra ocular, intraarterial, intraportal, or intralesional routes, by sustained release systems or by implantation devices.
  • the compositions may be administered by bolus injection or continuously by infusion, or by implantation device.
  • the composition may be administered locally via implantation of a membrane, sponge, or another appropriate material on to which the desired molecule has been absorbed or encapsulated.
  • a membrane, sponge, or another appropriate material on to which the desired molecule has been absorbed or encapsulated may be used.
  • the device may be implanted into any suitable tissue or organ, and delivery of the desired molecule may be via diffusion, timed release bolus, or continuous administration.
  • compositions in an ex vivo manner.
  • cells, tissues, or organs that have been removed from the patient are exposed to pharmaceutical compositions after which the cells, tissues and/or organs are subsequently implanted back into the patient.
  • compositions of the invention can be introduced for treatment into a mammal by other modes, such as but not limited to, intra-articular, intra-tumor, cerebrospinal, intra-arterial, infra-peritoneal, intra- rectal and colon, intra-lesion, topical, subconjunctival, infra-bladder, infra-vaginal, epidural, infracostal, intra-dermal, inhalation, transdermal, trans-serosal, intra-buccal, oral, infra-nasal, infra-muscular, dissolution in the mouth or other body cavities, instillation to the airway, insuflation through the airway, injection into vessels, tumors, organ and the like, and injection or deposition into cavities in the body of a mammal.
  • modes such as but not limited to, intra-articular, intra-tumor, cerebrospinal, intra-arterial, infra-peritoneal, intra
  • compositions of the invention can be used for the treatment of cerebro vascular ischemia, erectile dysfunction, female sexual arousal dysfunction, motor neuron disease, neuropathy, pain, depression, anxiety disorders, brain trauma, sepsis, septic shock, shock, adult respiratory distress syndrome, meconium aspiration, infantile respiratory distress syndrome, memory impairments, dementia, cognitive disorder, autism, central nervous system disease (such as Parkinson's disease, Alzheimer's disease), migraine, cerebral palsy, neurodegenerative diseases, stroke, hypertension, pulmonary hypertension, portal hypertension, ischemic heart disease, arthritis, osteoarthritis, gouty arthritis, crystal-induced arthritis, snoring, arteritis, rhinitis, psoriasis, radiation-induced tissue injury, septicemia, exocrine pancreatic insufficiency, pancreatitis, spondyloarthropathies, hypersensitivity, anaphylaxis, encephalopathy,
  • Bile salt/Phospholipid (BS/PL) mixed micelle components of the invention are Bile salt/Phospholipid (BS/PL) mixed micelle components of the invention
  • Bile salts being amphiphilic, self associate in aqueous media above critical micelle concentration (CMC) to form small globular aggregates termed primary micelles that contain 5-10 BS molecules [Ekwall et al., In: Proceedings of the 2 n International Congress of Surface Activity, London, 1957, pp. 357].
  • CMC critical micelle concentration
  • BS are surface-active agents that naturally exist in the body mostly in the gastrointestinal system and their function is to solubilize the dietary fat and cholesterol for abso ⁇ tion.
  • the solubility of BS in water varies according to the number of hydroxyl groups in the molecule. Trihydroxy BS, such as sodium cholate, are more soluble than dihydroxy BS, such as sodium deoxycholate.
  • Dihydroxy BS have lower CMC and therefore have higher solubihzation potential for water-insoluble drugs or compounds than trihydroxy BS.
  • BS are shown to be toxic mainly to the cell membrane, however, due to their detergent action [Coleman et al., Biochem. Soc. Trans. 3:747-748 (1975)].
  • BS form mixed micelles with phosphatidylcholines, glycerides, and fatty acids in the intestine [Tso, Adv. Lipid Res. 21:143-186 (1985)].
  • BS phospholipid (PL) mixed micelles as a vehicle for solubilizing or inco ⁇ orating water insoluble compounds has resulted in less deleterious effects than BS alone [Teelmann et al., Arzneinstoff-Forschung 34:1517-1523 (1984)].
  • BS/PL mixed micelles in the presence of a water-insoluble drug or compound are usually prepared by coprecipitation.
  • the ingredients are mixed in organic solvent and . evaporated under a stream of nitrogen and then dried under vacuum to a constant dried weight.
  • the dried film is reconstituted with buffer and the solution is flushed with nitrogen, sealed, and equilibrated at room temperature. To remove unsolubilized drug or compound, the solution is centrifuged.
  • BS/PL mixed micelles have the advantages of small size ( ⁇ 5nM) and ease of preparation over liposome formulation. Another advantage of the BS/PL systems, over simple surfactant micelles for delivering water-insoluble drugs or amphiphilic compounds, is the avoidance of precipitation of the drug or compound upon aqueous dilution. It has been shown that BS-egg PL mixed micelles spontaneously form liposomes upon aqueous dilution beyond the mixed micellar boundary [Son et al, Biochimica et Biophysica Ada. 981.288-294 (1989)].
  • the micelle-to-vesicle transition prevents the precipitation of drug or compound upon aqueous dilution because lipophilic drugs or compounds spontaneously associate with the phospholipid bilayer during this transition.
  • BS-egg PC mixed micelles were therefore studied as a novel delivery vehicle for water-insoluble drugs and amphiphilic compounds, including teniposide [Alkan-Onyuksel et al., Pharm. Res. 9:1556-1562 (1992)], paclitaxel [Alkan-Onyuksel et al., Pharm. Res. 11 :206-212 (1994)], and some steroidal drugs [S. Ayd, ' Ph.D.
  • mice to vesicle transition is an important property to be considered for the BS/PL mixed micellar systems.
  • BS total lipid concentration
  • PL total lipid concentration
  • This phenomenon in BS/PL mixed micelles may lead to differing solubihzation capacity for the various mo ⁇ hological forms.
  • Mixed micelles represent a dynamic system where BS monomers are in rapid dynamic equilibrium between the micellar and aqueous phases.
  • the aggregate structure changes from a mixture of spherical mixed and simple micelles, to elongated mixed micelles, and finally to vesicles. It is important to know at which point on the MTVT curve the BS/PL solubihzation system exists, hi the first range, the BS concentration is relatively high; therefore, simple BS micelles, in addition to mixed micelles, co-exist.
  • rod-like particles are difficult to reproduce due to a sha ⁇ change in size with dilution, and they form metastable structures with varying drug and/or amphiphilic compound inco ⁇ oration or solubilization/inco ⁇ oration capacity. Dilution beyond this point causes the formation of mixed BS/PL vesicles. The size of these vesicles decreases with dilution due to a smaller number of BS being inco ⁇ orated into the bilayer at high water content. Finally, small unilamellar PL vesicles with constant size and structure are formed at points near the end of the curve.
  • Critical lipid concentration was determined to be the ideal composition for a mixed BS/PL system for drug and/or amphiphilic compound inco ⁇ oration or solubihzation.
  • CLC Critical lipid concentration
  • spherical aggregates have well-defined size and mo ⁇ hology, and are relatively less toxic with high solubihzation potential, hi order to increase the total lipid in a system with predominantly spherical mixed micelles, the macroscopic molar ratios of PL and BS need to be adjusted. There is a linear relationship between the macroscopic BS/PL molar ratio and the total lipid concentration [S. Ayd, Ph.D. Thesis, supra (1997)]. Solubilization in BS/PL mixed micelles
  • BS/PL mixed micelles were shown to be greater than that of BS simple micelles for a number of drugs and compounds, such as teniposide [Alkan- Onyuksel et al, Pharm. Res. 9:1556-1562 (1992)], paclitaxel [Alkan-Onyuksel et al, Pharm Res. 11: 206-212 (1994)], tetrazepam [Hammad et al, Eur. J. Pharm. Sci. 7: 49-55 (1998)], diazepam [Rosoff et al, J. Pharm. 6:137-146 (1980)], vitamin Kl [Nagata et al, J. Pharm.
  • a molar ratio of lecithin to glycolic acid of 1:1 was found to be optimal because the lytic detergent-like properties of glycolic acid were completely inhibited by lecithin, without losing their capacity to solubilize water-insoluble compounds [Coleman et al, Biochem. Soc. Trans. 4:244 (1976); Coleman et al, Biochim. Biophys. Ada. 436:38-44 (1976)].
  • the ratio of PL to BS used for the inco ⁇ oration or solubilization of drugs and/or amphiphilic compounds varies depending on the compound being inco ⁇ orated or solubilized.
  • the ratio of phospholipid to glycolic acid can be as high as 1.3 : 1 , while for compounds like vitamin E that can partly replace PL, the ratio can be as low as 0.5:1 [Teelmann et al, supra (1984)].
  • Solubility improvement for steroidal drugs and compounds in BS/PL mixed micelles has been studied'and compared to BS micelles for deoxycorticosterone, progesterone, corticosterone, testosterone, estrone, and deoxycorticosterone acetate [S. Ayd, Ph.D. Thesis, supra (1997); Cai et al, J. Pharm. Sci. 86:372-377 (1997)].
  • These studies generally showed increased solubilities for mixed micelles compared to BS for progesterone and deoxycorticosterone. This may be due to the relatively large core size and the presence of acyl chains of the PL in the core of mixed micelles resulting in a different core environment than the simple micelles.
  • solubilization potential of the BS/PL mixed micelles were examined, and it was found that the type of PL is important for the solubilization capacity of micelles [Alkan-Onyuksel et al, supra (1992)].
  • Increased solubility of teniposide in soy-PC containing mixed micelles was seen, in comparison with egg-PC containing mixed micelles. This increase in solubility was attributed to the increased motility in the micellar interior due to higher unsaturation in the case of soy-PC [Alkan-Onyuksel et al., supra (1992)].
  • the BS polarity did not significantly change the solubilization potential of the micelles [Alkan-Onyuksel et al, supra (1994)]. This is hypothesized to be due to the smaller molecular weight of teniposide, relative to paclitaxel, and therefore may fit better into mixed micelles and interact with the BS with hydrophobic interactions. Moreover, the aggregation number of the dihydroxy micelles is greater, resulting in bigger micelles which allow more teniposide to be solubilized. An increase in teniposide solubility was .
  • the invention provide use of mixed micelles of BS/PL, which upon aqueous dilution form liposomes to overcome the shelf instability of liposomes.
  • Mixed micelles are also not stable as a solution and hence the preparations were freeze-dried.
  • Upon reconstitution, a clear solution without the precipitation of the drug was obtained [Alkan- Onyuksel et al, supra (1994)].
  • the drug teniposide did not precipitate out once the mixed micellar system was diluted and liposomes were formed [Son et al, PDA J. Pharm. Sci. Technol 50:89-93 (1996)].
  • the precipitation of the drug during shelf storage is overcome by freeze-drying.
  • SGC-soy PC (SPC) mixed micelles are better than sodium deoxycholate-soy PC mixed micelles in stabilizing tetrazepam. This has been explained by SGC-SPC mixed micelles interfering with degradation kinetics and hence displaying better stabilizing effects under both aerobic and anaerobic conditions [Hammad et al., supra (1998)]. Thus, solubilization capacity and stability of BS/PL mixed micelles are governed by the molar ratio of BS/PL, total lipid concentration, type of phospholipid and type of BS.
  • the solubilization potential of the mixed micellar solution is enhanced through an enlargement in the hydrophobic core of mixed micelles and/or an increase in the number of micelles caused by an increase in total lipid or decrease in CMC of BS. Solubilization is further enhanced by encapsulation in a sterically-stabilized liposome (SSL).
  • SSL sterically-stabilized liposome
  • BS/PL mixed micelles The toxicity of BS/PL mixed micelles was examined using both undecomposed and artificially decomposed mixed micelles on rabbits [Teelmann et al, supra (1984)].
  • the dose of diazepam used for these studies was 5 to 50 times the anticipated clinical dose, lOmg drug in 2.0mL of mixed micellar solution, administered daily up to four weeks. No teratogenic or mutagenic potential was seen when studies were conducted in rabbits [Teelmann et al, supra (1984)].
  • Acute toxicities studies were also performed in mice and rats with single intravenous bolus injections of both undecomposed and decomposed solutions revealing good tolerance [Teelmann et al, supra (1984)].
  • Lethal dose was estimated to be as high as 5 to 6 mL/Kg while the anticipated clinical dose is about 0.04 to 0.08 mL/Kg for a patient with a body weight of about 50Kg.
  • formulation was well tolerated. Only when comparatively high doses were given (30-50 times clinical injection volume) vomiting, slightly elevated liver enzymes, and slightly increased liver weights were observed.
  • decomposed fo ⁇ nulation corresponding to 40 to 60mg lysolecithin/Kg body weight, effects such as hemolysis and liver enzyme elevations were observed [Teelmann et al, supra (1984)].
  • SSSM Sterically-stabilized simple micelles
  • CMC CMC
  • mM surfactant micelles
  • these micelles are more stable upon dilution than typical surfactant micelles, most probably due to the strong interaction between the acyl chains in the core region.
  • the relative stability of SSSM upon dilution is also partly attributed to their much lower CMC compared to other surfactants [Weissig et al, Pharm. Res. 15:1552-1556 (1998)].
  • HPLC- based gel chromatography was used to determine the stability of these micelles. Analysis of serial dilutions of DSPE-PEG 5000 with inco ⁇ orated soy trypsin inhibitor (STI) showed that micelles were still present at a concentration of 140nM, together with unimers [Weissig et al, supra (1998)].
  • STI soy trypsin inhibitor
  • SSSM that contained vasoactive intestinal peptide (VIP) were diluted 10-fold, and were analyzed using HPLC-based size exclusion chromatography. It was still observed, however, that most of the VIP was associated with the micelles, confirming that SSSM are relatively stable upon dilution [Sethi et al, In: J. Pharm. Sci. Suppl, Toronto, Canada (2002)].
  • the stability of DSPE-PEG 2000 and 5000 in blood serum was also demonstrated by incubating the micelles with fetal calf serum at room temperature for 48 hours [Torchilin, J Control. Release 73:137-172 (2001)].
  • a targeting moiety such as a peptide can be conjugated to the PEG polymer of the lipid to allow the micelles for active targeting.
  • VIP vasoactive intestinal peptide
  • DSPE distearoyl phosphatidylethanolamine
  • PEG MW 3500
  • nmunomicelles were prepared by the covalent attachment of specific ligands, like monoclonal anti-nucleosome antibody to PEG-diacyllipid micelles via micelle inco ⁇ orated p-nitrophenylcarbonyl-PEG-PE, and suggested as potential carriers for targeted delivery of poorly soluble drugs [Rammohan et al, In: Proc. Int'l. Symp. Control Ret. Bioact. Mater. 28 #5167 (2001)].
  • SSMM are the next generation of SSSM. They are formed by adding a typical phospholipid like phosphatidylcholine into SSSM with the pu ⁇ ose of increasing the hydrophobic core. They are prepared by coprecipitation of the drug and/or amphiphilic compounds with the PEGylated phospholipid and phospholipid, as described before for BS/PL mixed micelles. These types of mixed micelles have all the advantages of sterically- stabilized simple micelles with superior solubilization capacity.
  • SSMM were initially observed during the preparation of sterically-stabilized liposomes where PEGylated lipids are incubated with classical liposomes to form SSL.
  • the transition from lamellar phase to micellar phase occurred due to the solubilization of liposomal lipids in the PEGylated lipid micelles.
  • This bilayer to micelle transition was studied by several techniques including nuclear magnetic resonance (NMR), X-Ray diffraction, electron spin resonance (ESR) and cryo transmission electron microscopy [Edwards et al, Biophys. J. 73: 258-266 (1997)].
  • micellisation is complete at ⁇ 40 and -30 mol% of the polymer lipids in DMPC/DMPE- PEG 2000 andDSPC/DSPE-PEG 2000 dispersions, respectively.
  • Conversion of lamellae to micellar system is regulated by tensile strength of the phospholipid matrix. It has been hypothesized that due to the lateral acyl chain packing of the gel-phase bilayers of DSPC leads to restricted diffusional motion of DSPE-PEG 2000 within the DSPC bilayer, resulting in a more effective formation of mixed micelles. Loosened packing and higher fluidity favor mixing of DMPC-PEG 2000 with DMPC and delay micelle formation [Belsito et al, supra (2001)]..
  • micellar phase to lamellar phase occurs at lower concentrations of PEGylated lipid as the PEG chain length increases [Ashok et al., In: Amer. Assoc. Pharmaceut. Philosophs, Denver (2001) (unpublished data)]. All the above data collectively suggest that longer acyl chain length PL form mixed micelles at lower concentrations, and the longer the PEG chain length the less the PEGylated PL needed to form spherically mixed micelles.
  • the size of the SSMM measured by-light scattering, shows a slight reduction in the diameter as compared to SSSM (16.8 ⁇ 0.3 nM and 13.2 ⁇ 2.4 nM for SSMM and SSSM with PEGylated lipid : DSPE-PEG 2000) [Ashok et al, supra (2001)]. This is explained by the possible transition in the conformation of the PEG chain from tuft to mushroom form upon addition of PC due to separation of PEG lipid from each other.
  • Tamoxifen was inco ⁇ orated with 100% efficiency for all ratios of 1 : 10 to 1 : 1 of tamoxifen:DSPE-PEG 5000 [Lukyanov et al, supra (2001)].
  • Significantly improved solubility of DSPE-PEG micelles over PC/TC mixed micelles for progesterone (54 ⁇ g/mL vs. 4.9 ⁇ g/mL per mM of total lipid concentration) and paclitaxel (63.7 ⁇ g/mL vs. 18.4 ⁇ g/mL per mM of total lipid concentration) was recently reported [L. Peng, Phospholipid micelles as carriers for water insoluble drugs. Ph.D. thesis. University of Illinois at Chicago, Chicago (1999)].
  • SSC sterically- stabilized crystals
  • DSPE-PEG being amphiphilic in nature, covers the surface of the drug particles, reducing surface tension and preventing further growth of crystals. Upon centrifugation, larger crystals were removed while smaller crystals covered with phospholipid SSC remained suspended in the supernatant [Krishnadas et al, Pharm. Res. (submitted, 2002)].
  • SSSM have been shown in some cases to be a better delivery system for some hydrophobic/amphiphilic drugs or compounds compared to liposomes.
  • Dequalinium a topical antimicrobial agent
  • This drug which is also being investigated as an anticarcinoma drug, is limited in its applications due to its poor solubility.
  • dequalinium was inco ⁇ orated into liposomes and micelles of DSPE-PEG 5000 [Weissig et al, supra (1998)]. Dequalinium was inco ⁇ orated into 50- lOOnM liposomes, made by probe sonication, up to 50 mol%.
  • solubilization is the total lipid concentration of the system.
  • Paclitaxel solubilization increased linearly along with DSPE-PEG 2000 concentration [Krishnadas et al, supra (2000)].
  • the total lipid concentration was limited, however, by the number of micelles that can exist freely in a given volume of aqueous environment. Above a certain concentration, micelles will interact with each other, and the viscosity of the system will increase significantly.
  • the invention therefore provides SSMM which are designed to provide specific targeting of a compound through use of a targeting agent attached to a lipid component of the SSMM, either through direct attachment to a lipid component of the SSMM, for example by covalent attached at the polar head of the lipid, or through attachment to a polymer component of covalently modified lipid component of the SSMM.
  • a targeting agent attached to a lipid component of the SSMM, either through direct attachment to a lipid component of the SSMM, for example by covalent attached at the polar head of the lipid, or through attachment to a polymer component of covalently modified lipid component of the SSMM.
  • the inco ⁇ oration or solubilization and stability of drugs or compounds in SSSM depends on the type of drug, PEG chain length, and the amount of lipid and drug or compound added to the system.
  • micelles with attached targeting agent improve targeted delivery of water-insoluble drugs and amphiphilic compounds.
  • the solubilization potential of SSMM prepared using DSPE-PEG 2000 and egg PC at a molar ratio of 90:10 at a total lipid concentration of 5mM was tested for a water-insoluble anticancer drug, paclitaxel.
  • Drug and/or amphiphilic compound solubilization or inco ⁇ oration improved significantly when compared to SSSM and BS/PL mixed micelles.
  • cytotoxicity of the drug in this new SSMM formulation when tested on MCF-7 breast cancer cells, showed similar anti-cancer activity for paclitaxel in SSSM or dimethyl sulfoxide (DMSO) [Krishnadas et al., supra (2002); Krishnadas et al, In: Proc. Int'l. Symp. Control. Rel Bioact. Mater., Seoul (2002)].
  • DMSO dimethyl sulfoxide
  • solubilization of drugs or compounds in SSMM should be dependent on the molar ratio of soluble lipid (DSPE-PEG) and insoluble lipid (PC).
  • DSPE-PEG soluble lipid
  • PC insoluble lipid
  • Different ratios of DSPE-PEG 2000:PC were studied at a constant total lipid concentration to solubilize paclitaxel [Krishnadas et al, supra (2002)].
  • the system was homogenous; a single micellar peak existed as detected by light scattering [Krishnadas et al, supra (2002)].
  • the PC content was increased to 15 , 20, or 25%, however, heterogeneous systems with multiple peaks were detected by quasi-elastic light scattering. The heterogeneity is probably due to the micellar to lamellar phase transition occurring at higher PC content [Krishnadas et al, supra (2002)].
  • the optimum inco ⁇ oration or solubility of a drug or amphiphilic compound in SSMM will vary according to the PC/DSPE-PEG ratios, the length of the PEG chain, and the drug or compound properties. For example, a drug or compound inco ⁇ orated in the palisade region will be more affected by the PEG chain length than a drug or compound, which is inco ⁇ orated in the core.
  • BS/PL mixed micelles have shown increased solubilization of water- insoluble drugs or compounds, they are limited by non-reproducibility on changing ratios and total lipid concentration. Also, the BS toxicity can still be an issue. These systems have been overshadowed by the emergence of the polymer based micellar systems.
  • SSSM formed from PEGylated PL have high in vitro and in vivo stability as a result of low CMCs in micromolar range, and increased circulation half-life, making them one of the leaders among the micellar drug or compound delivery systems being studied. They have shown good solubilization inco ⁇ oration potential for a variety of drugs and/or compounds. In addition, they are also being explored for targeted delivery both by passive and active mechanisms.
  • the most promising system for the solubilization of hydrophobic drugs or compounds among the three phospholipid micelles covered herein may be SSMM due to their superior solubilization/inco ⁇ oration potential and acceptable stability.
  • the size of the micelles governs its application in therapy.
  • micelles In the presence of leaky vasculature, as in tumors, micelles extravasate and accumulate to a larger than liposomes in solid tumors, like Lewis lung carcinoma [Weissig et al., supra (1998)].
  • Soybean trypsin inhibitor inco ⁇ orated DSPE-PEG 5000 micelles and PEG-liposomes due to their longevity and slow uptake in the liver and spleen, demonstrated improved accumulation in tumor (subcutaneous Lewis lung carcinoma) in mice.
  • SSL sterically- stabilized liposomes
  • micelles accumulate in a tumor much faster than liposomes, probably due to their smaller size.
  • the invention contemplates the use of SSSM in SSL, SSMM in SSL, and SSC in SSL to overcome the phenomenon of multidrug resistance.
  • the development of drug resistance during cancer chemotherapy results in reduced responsiveness to prolonged delivery of chemotherapeutic drugs.
  • One mechanism of resistance [Bradley et al, Biochim. Biophys. Ada 48:87-128 (1988); Moscow et al, J. Natl. Cancer Inst. 80:14-20 (1988)] is due to the enhanced expression of a membrane pump, which is due to the overexpression of a membrane glycoprotein, P-glycoprotein (PGP).
  • PGP is encoded by the mdrl gene [Ueda et al, Proc. Natl. Acad. Sci.
  • SSC Sterically-stabilized crystals
  • Sterically-stabilized crystalline components comprise one or more biologically active compounds which are insoluble in an aqueous solution. They are derived by dissolving in an organic solvent said biologically active compound(s) and one or more lipids wherein at least one lipid is conjugated to a water-soluble polymer; removing the organic solvent to leave a dry film; liydrating the dry film with an aqueous solution; and forming a sterically-stabilized crystalline component.
  • insoluble is defined according to the U.S. Pharmacopoeia National Formulary [USP 23, 1995, page 10], as requiring 10,000 or more parts of solvent for 1 part of solute.
  • the crystalline component is essentially a micelle-encased aggregate of the insoluble compound which is densely packed and crystallized.
  • Sterically-stabilized crystalline components are amenable to the use of any compound that is insoluble in an aqueous solution.
  • Preferred insoluble compounds include, but are not limited to, progesterone, testosterone, estrogen, prednisolone, prednisone, 2,3 mercaptopropanol, amphotericin B, betulinic acid, camptothecin, diazepam, nystatin, propofol, cyclosporin A, doxorubicin, and paclitaxel (Taxol®), and tetramethyl NDGA .
  • any targeting compound that assumes or maintains a biologically active conformation when in association with the sterically-stabilized crystalline component can be used, hi a preferred embodiment, any of the amphipathic compounds as described above are utilized, hi another preferred embodiment, the targeting compound is a chemical entity that binds to a molecule inside or outside the cell, including but not limited to, peptides, proteins, antibodies, folate, transferring, sugars, and mucopolysaccharides.
  • the targeting compound is VIP or other member of the VIP/GRF family or proteins.
  • the targeting compound can be helospectin I or II or any member of the neuropeptide family such as neuropeptide Y (NPY), neuropeptide YY (NPYY), including neuropeptide fragments 2-36 and related fragments, ACTH, calcitonin, GAP (GnRH precursor molecule), glutamate- decarboxylase, keyhole limpet hemocyanin, leucin-enkephalin, mesotocin, methionin- enkephalin, neurotensin, peroxydase, somatostatin, substance P, vasopressin, and vasotocin.
  • NPY neuropeptide Y
  • NPYYY neuropeptide YY
  • neuropeptide fragments 2-36 and related fragments ACTH
  • GAP GnRH precursor molecule
  • glutamate- decarboxylase keyhole limpet hemocyanin
  • leucin-enkephalin mesotocin
  • methionin- enkephalin me
  • This example describes the preparation of aqueous dispersions of paclitaxel.
  • Paclitaxel solubilized in poly(ethylene glycol-2000)-conjugated distearoyl phosphatidylethanolamine (PEG(2000)-DSPE) and paclitaxel-mixed micelles were prepared by coprecipitation. Briefly, for simple micelles, paclitaxel and PEG(2000)-DSPE, in a molar ratio of 0.16 was dissolved in methanol. The solvent was then removed by vacuum rotary evaporation under a stream of argon to form a dry film. This dry film was further dried under vacuum overnight to remove any traces of remaining solvent. The dried film was rehydrated with isotonic 0.01M HEPES buffer, pH 7.4.
  • the solution was then flushed with argon, sealed and equilibrated for 12 h at room temperature.
  • the unsolubilized excess paclitaxel was removed by centrifugation at 13,000 x g for 5 min to obtain a clear dispersion.
  • the maximum solubility of paclitaxel in the absence of crystal formation was determined in simple micelles of PEG(2000)-DSPE by keeping the phospholipid concentration fixed at 5 mM and systematically reducing the drug concentration (Drug:phospholipid, molar ratios, 0.076, 0.078, 0.082, 0.088) until a single homogenous system was determined as confirmed by a single peak by size analysis.
  • SSMM sterically-stabilized mixed micelles
  • EPC egg phosphatidylcholine
  • SSSM or SSMM sterically-stabilized micelles
  • Particle size distribution and mean diameter of the prepared aqueous dispersions of paclitaxel were determined by quasi-elastic light scattering using a NICOMP 380 Submicron Particle Sizer (Santa Barbara, CA) using a Helium-Neon laser at 632.8 nm. Data was analyzed in terms of volume and intensity weighted distributions.
  • the aqueous dispersions of paclitaxel with PEG(2000)-DSPE indicated the presence of two populations by quasi-elastic light scattering analysis.
  • SSC sterically-stabilized crystals
  • Paclitaxel being hydrophobic, in the absence of sufficient PEG(2000)-DSPE may favor this structure.
  • Paclitaxel has been shown to undergo concentration dependent self-aggregation possibly due to hydrogen bonded stacking. The formation of this second species of lipid-coated crystals is currently being explored as a potential delivery system, which is not reported in this paper. This current study focuses on only sterically-stabilized mixed and simple micelles as improved lipid based delivery systems. Therefore, the amount of paclitaxel added to the PEG(2000)-DSPE during preparation was then reduced systematically until only a single micellar species was observed by light scattering particle size analysis with a mean diameter of 15 ⁇ 1.0 nm.
  • the mo ⁇ hology of paclitaxel in the presence and absence of PEG(2000)-DSPE was visualized by Transmission Electron Microscopy (TEM) using negative staining.
  • TEM Transmission Electron Microscopy
  • a drop of the prepared paclitaxel dispersion with PEG(2000)-DSPE (molar ratio, 0.16) and without PEG(2000)-DSPE was placed on a carbon coated copper grid and stained with 1% phosphotungstic acid. After air-drying for 2-3 minutes it was then viewed under an electron microscope (JEOL 100CX) and photographed.
  • JEOL 100CX electron microscope
  • the paclitaxel crystals were visualized in buffer in the presence (drug lipid molar ratio 0.16) and absence of PEG(2000)-DSPE before and after centrifugation.
  • PEG(2000)- DSPE molecules being amphiphilic, accumulate at the surface of these hydrophobic paclitaxel particles and reduces the surface tension between the drug and buffer interface. This coating not only prevents further growth of the crystals but also prevents particle- particle interaction by providing a steric barrier. On centrifugation larger particles are removed from the system to give a clear solution. However the smaller phospholipid coated crystals remain suspended in solution. This situation is not observed with dispersions in the absence of phospholipid most probably due to the growth of paclitaxel particles to a size that precipitates on centrifugation.
  • the amounts of paclitaxel solubilized or inco ⁇ orated both in SSSM and SSMM was determined by RP-HPLC.
  • the clear aqueous dispersion was diluted with methanol. 20 ⁇ l of each sample was injected at least three times into a ⁇ Bondapak C-18 column, 3.9 mm X 30 cm (Waters, Milford, MA) equipped with a Cl 8 column guard. The column was eluted with acetonitrile/water (60:40) at 1.0 ml/min (Waters 600). Detection was by UV abso ⁇ tion measurement at 227 nm (Waters 490). Peak areas were calculated by interfacing the detector to an electronic integrator (Hewlett Packard). The drug concentration was calculated from standard curves. The assay was linear over the tested concentration range and there was no interference of the phospholipid with the assay.
  • mixed micelles at a molar ratio of 90:10 was chosen and studied further as this was the only system that was found to be homogenous and contained only spherical mixed micelles at the conditions studied.
  • Five mM PEG(2000)- DSPE was found to solubilize 319 ⁇ 1.6 ⁇ g/ml paclitaxel as simple micelles alone without the formation of SSC.
  • a significantly higher amount of paclitaxel 475.6 ⁇ 8.96 ⁇ g/ml
  • Egg-PC is a hydrophobic phospholipid.
  • P-SSSM paclitaxel-SSSM
  • P-SSMM paclitaxel-SSMM
  • MCF-7 ATCC # HTB-22
  • HTB-22 human mammary gland adenocarcinoma
  • MCF-7 was maintained in RPMI-1640 medium containing 10% fetal bovine serum and 1.0% antibiotics (penicillin and streptomycin), in a 5% carbon dioxide humidified atmosphere at 37°C.
  • a 10% dimethyl sulfoxide (DMSO) solution of paclitaxel was also tested as a control.
  • Drug-free simple micelles (SSSM) and mixed micelles (SSMM) in 0.01M HEPES buffer, pH 7.4 were also prepared at the same concentrations as the test solution and were used as controls. All the samples were prepared and tested in triplicate.
  • the cells were fixed to the plates by adding lOO ⁇ l/well of cold 20% trichloroacetic acid (TCA) and incubating for 1 hr at 4°C. The plates were then washed, air-dried and stained with lOO ⁇ l/well of 0.4% sulforhodamine B in 1% acetic acid for 30 min. The plates were then washed with 1% acetic acid, rinsed and 10 mM Tris buffer (200 ⁇ l/well) added. The optical density was then read at 515nm. The optical density readings obtained for the solvent controls were used to define 100% growth after correcting for the value obtained for the zero day control. These values were then expressed as a % survival and ED 50 values calculated using non-linear regression analysis (percent survival versus concentration).
  • TCA trichloroacetic acid
  • Tests were then performed to determine if the interaction between the drug and/or amphiphilic compound and the lipids in the formulation affected the bioactivity of the drug or compound .
  • the formulations were tested against MCF-7, a human breast cancer cell line.
  • Phospholipid micellar formulations had similar cytotoxic activities towards cultured MCF-7 cells as paclitaxel dissolved in 10 % DMSO. This indicates that paclitaxel, in simple and mixed micelles, is readily available to interact with cancer cells and retain its anti-mitotic potency. Moreover, paclitaxel activity was not altered by the presence of lipids.
  • paclitaxel is an effective anticancer drug, it is not cancer cell-specific which can lead to adverse reactions. This example discusses the targeted delivery of paclitaxel to breast cancer cells.
  • VPAC vasoactive intestinal peptide
  • VIP was conjugated to the distal end of distearoyl-phosphatidylethanolamine- conjugated poly(ethylene glycol) (DSPE-PEG- VIP) modified with n-hydroxy succinamide (Dagar et al, J Control Release. (2001) 74:129-134). Both the D-form (synthesized) and the L-form (naturally-occurring) of VIP are used in this conjugation process.
  • Paclitaxel approximately lmg/ml
  • P-SSMM sterically-stabilized mixed micelles
  • the prepared P-SSMM were passively loaded in the aqueous compartment of sterically-stabilized liposomes (P-SSL). Insertion of DSPE-PEG- VIP into liposomes was carried out with incubation under predetermined optimized conditions (37°C, 2h). P-SSL was characterized for size, paclitaxel, phospholipid, and VIP content and in vitro cytotoxicity to cultured MCF-7 breast cancer cells. The average diameter of P-SSL was 130 ⁇ 26 nm. Encapsulation capacity of the liposomes was high with a drug to lipid mole percent ratio of 1.2. Approximately 200 molecules of VIP per P-SSL were inserted into each liposome. Paclitaxel encapsulated in this carrier, with the surface modified by VIP, was cytotoxic to MCF-7 cells, indicating that the multiple forms of encapsulation proved effective in the delivery of chemotherapeutic drugs to breast cancer cells.
  • paclitaxel is an effective anticancer drug, it presents several formulation challenges due to its water-insolubility.
  • This example discusses the in vivo efficacy of intravenous administration of P-SSMM in rats with carcinogen-induced breast cancer. It also looked at the solubility of P-SSMM.
  • P-SSMM were prepared by solubilizing paclitaxel at lmg/mL, which is below its saturation solubility [Krishnadas et al, Pharm Res. 20:294-299 (2003)].
  • the solubilized molecular state of paclitaxel was, studied by circular dichroism (CD) [Campbell et al, J. Pharm. Sci. 90:1091-1104 (2001)].
  • CD circular dichroism
  • QELS quasi- elastic light scattering
  • targeting agents are covalently attached to a lipid in the SSMM.
  • Targeting agents (T) are conjugated to the distal end of distearoyl-phosphatidylethanolamine-conjugated poly(ethylene glycol) (DSPE-PEG-T) modified with n-hydroxy succinamide as described by Dagar et al [supra, (2001)].
  • DSPE-PEG-T distearoyl-phosphatidylethanolamine-conjugated poly(ethylene glycol)
  • targeting agents are also attached directly to the polar head group of a lipid component of the SSMM. Accordingly, various types of SSMM comprising targeting agents are contemplated.
  • a water-insoluble drug or amphiphilic compound (designated D) is inco ⁇ orated in sterically-stabilized mixed micelles (D-SSMM) as previously described [Krishnadas et al, supra (2002) Seoul, Korea.] wherein one lipid component of the SSMM is attached to the targeting agent either through covalent or non-covalent modification, while the second lipid component of the SSMM is modified to include the water-soluble polymer.
  • one lipid component of the SSMM is modified to include the water-soluble polymer, and the water-soluble polymer is further modified to include the targeting agent.
  • all or a molar fraction of one lipid component of the SSMM includes either the targeting agent and/or the water-soluble polymer.

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Abstract

L'invention concerne des micelles dans des compositions liposomiques pour l'administration de médicaments insolubles dans l'eau et de composés amphiphiles, des procédés de préparation desdites compositions, ainsi que des procédés de traitement de différentes maladies et de différents troubles au moyen desdites compositions de l'invention.
PCT/US2003/018686 2002-06-12 2003-06-12 Micelles phospholipidiques dans des liposomes, servant d'agents de solubilisation pour des composes insolubles dans l'eau WO2003105765A2 (fr)

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006079216A1 (fr) * 2005-01-28 2006-08-03 Bc Cancer Agency Compositions liposomales pour administration parenterale d'agents
WO2006110862A2 (fr) * 2005-04-12 2006-10-19 Wisconsin Alumni Research Foundation Composition micellaire polymere et medicament contenu dans celle-ci
WO2008089771A1 (fr) * 2007-01-24 2008-07-31 Syddansk Universitet Assemblage de membranes lipidiques régulé par adn
WO2009042114A2 (fr) 2007-09-21 2009-04-02 The Johns Hopkins University Dérivés de phénazine et leurs utilisations
WO2010042997A1 (fr) * 2008-10-17 2010-04-22 Vectus Biosystems Pty Limited Compositions et procédés destinés au traitement de troubles rénaux
US20120294945A1 (en) * 2011-05-16 2012-11-22 Postech Academy-Industry Foundation Drug delivery system using hyaluronic acid-peptide conjugate micelle
US8383136B2 (en) 2009-09-25 2013-02-26 Wisconsin Alumni Research Foundation Micelle encapsulation of therapeutic agents
WO2016167730A1 (fr) * 2015-04-13 2016-10-20 Özen Oğuz Aslan Nanomicelles permettant le traitement du cancer
EP3349729A4 (fr) * 2015-09-14 2019-06-26 VGSK Technologies, Inc. Support stériquement stabilisé pour agents thérapeutiques sous-cutanés, sublinguaux et oraux, compositions et procédés pour le traitement d'un mammifère
CN114073675A (zh) * 2020-08-10 2022-02-22 复旦大学 一种丙泊酚混合胶束及其制备方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6217886B1 (en) * 1997-07-14 2001-04-17 The Board Of Trustees Of The University Of Illinois Materials and methods for making improved micelle compositions
US6322810B1 (en) * 1997-07-14 2001-11-27 Hayat Alkan-Onyuksel Materials and methods for making improved micelle compositions

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6217886B1 (en) * 1997-07-14 2001-04-17 The Board Of Trustees Of The University Of Illinois Materials and methods for making improved micelle compositions
US6322810B1 (en) * 1997-07-14 2001-11-27 Hayat Alkan-Onyuksel Materials and methods for making improved micelle compositions

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006079216A1 (fr) * 2005-01-28 2006-08-03 Bc Cancer Agency Compositions liposomales pour administration parenterale d'agents
US8173167B2 (en) 2005-04-12 2012-05-08 Wisconsin Alumni Research Foundation Micelle composition of polymer and passenger drug
WO2006110862A2 (fr) * 2005-04-12 2006-10-19 Wisconsin Alumni Research Foundation Composition micellaire polymere et medicament contenu dans celle-ci
WO2006110862A3 (fr) * 2005-04-12 2007-07-05 Wisconsin Alumni Res Found Composition micellaire polymere et medicament contenu dans celle-ci
US9107814B2 (en) 2005-04-12 2015-08-18 Wisconsin Alumni Research Foundation Micelle composition of polymer and passenger drug
WO2008089771A1 (fr) * 2007-01-24 2008-07-31 Syddansk Universitet Assemblage de membranes lipidiques régulé par adn
WO2009042114A2 (fr) 2007-09-21 2009-04-02 The Johns Hopkins University Dérivés de phénazine et leurs utilisations
WO2010042997A1 (fr) * 2008-10-17 2010-04-22 Vectus Biosystems Pty Limited Compositions et procédés destinés au traitement de troubles rénaux
AU2009304595B2 (en) * 2008-10-17 2015-02-05 Vectus Biosystems Pty Limited Compositions and methods for treatment of kidney disorders
KR101671406B1 (ko) * 2008-10-17 2016-11-01 벡투스 바이오시스템즈 리미티드 신장 장애 치료용 조성물 및 방법
US9238054B2 (en) 2008-10-17 2016-01-19 Vectus Biosystems Pty Limited Treatment of kidney fibrosis with VIP fragments
US8569235B2 (en) 2008-10-17 2013-10-29 Vectus Biosystems Pty Limited Treatment of kidney disorders with VIP fragments
KR20110095266A (ko) * 2008-10-17 2011-08-24 벡투스 바이오시스템즈 피티와이 리미티드 신장 장애 치료용 조성물 및 방법
US8858965B2 (en) 2009-09-25 2014-10-14 Wisconsin Alumni Research Foundation Micelle encapsulation of a combination of therapeutic agents
US8529917B2 (en) 2009-09-25 2013-09-10 Wisconsin Alumni Research Foundation Micelle encapsulation of a combination of therapeutic agents
US8383136B2 (en) 2009-09-25 2013-02-26 Wisconsin Alumni Research Foundation Micelle encapsulation of therapeutic agents
US8895069B2 (en) * 2011-05-16 2014-11-25 Postech Academy-Industry Foundation Drug delivery system using hyaluronic acid-peptide conjugate micelle
US20120294945A1 (en) * 2011-05-16 2012-11-22 Postech Academy-Industry Foundation Drug delivery system using hyaluronic acid-peptide conjugate micelle
WO2016167730A1 (fr) * 2015-04-13 2016-10-20 Özen Oğuz Aslan Nanomicelles permettant le traitement du cancer
EP3349729A4 (fr) * 2015-09-14 2019-06-26 VGSK Technologies, Inc. Support stériquement stabilisé pour agents thérapeutiques sous-cutanés, sublinguaux et oraux, compositions et procédés pour le traitement d'un mammifère
CN114073675A (zh) * 2020-08-10 2022-02-22 复旦大学 一种丙泊酚混合胶束及其制备方法

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