WO2003047494A2 - Reverse micelle compositions and uses thereof - Google Patents

Reverse micelle compositions and uses thereof Download PDF

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Publication number
WO2003047494A2
WO2003047494A2 PCT/US2002/038474 US0238474W WO03047494A2 WO 2003047494 A2 WO2003047494 A2 WO 2003047494A2 US 0238474 W US0238474 W US 0238474W WO 03047494 A2 WO03047494 A2 WO 03047494A2
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WO
WIPO (PCT)
Prior art keywords
reverse micelle
micelle composition
biologically active
hydrophilic
reverse
Prior art date
Application number
PCT/US2002/038474
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French (fr)
Other versions
WO2003047494A3 (en
Inventor
Panos P. Constantinides
Likan Liang
Eun-Hyun Jang
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Dor Biopharma Inc.
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Application filed by Dor Biopharma Inc. filed Critical Dor Biopharma Inc.
Priority to AU2002362040A priority Critical patent/AU2002362040A1/en
Publication of WO2003047494A2 publication Critical patent/WO2003047494A2/en
Publication of WO2003047494A3 publication Critical patent/WO2003047494A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/0291—Micelles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin

Definitions

  • the present invention relates to delivery systems for the mucosal and parenteral administration of biologically active molecules, including, but not limited to, therapeutic agents, vaccines, allergens, antigens and diagnostic agents.
  • the present invention relates to reverse micelle compositions comprising a surfactant, a hydrophilic ⁇ Q phase, and one or more biological active molecules, and methods of administering biologically active molecules to an animal utilizing said compositions.
  • the compositions of the invention promote the absorption of biologically active molecules across mucosal epithelial barriers.
  • the compositions of the invention can be used prophylactically, therapeutically, diagnostically or cosmetically.
  • Drug delivery takes a variety of forms, depending on the agent to be delivered and the administration route. The most convenient way to administer drugs into the body is by
  • a problem with systemic immunization is that it may not effectively induce mucosal immune responses, particularly production of IgA, that are important as the first defense barrier to invaded microorganisms. For this reason, it would be beneficial to provide oral vaccination, if the problems of low absorbability and instability could be overcome.
  • Q Controlled release systems for drug delivery are often designed to administer drugs to specific areas of the body. In the gastrointestinal tract it is important that the drug not be eliminated before it has had a chance to exert a localized effect or to pass into the bloodstream.
  • Enteric coated formulations have been widely used for many years to protect drugs administered orally, as well as to delay release.
  • Several microsphere formulations have been proposed as a means for oral drug delivery.
  • International Application No. PCT/US90/06433 by Enzytech describes the use of a hydrophobic protein, such as zein, to form microparticles;
  • U.S. Patent No. 4,976,968 to Steiner et al. describes the use of "proteinoids" to form microparticles;
  • European Patent Application No. 0 333 523 by the UAB Research Foundation and Southern Research Institute describes the use of synthetic
  • polymers such as polylactic acid-glycolic acid to form microspheres.
  • Particles less than ten microns in diameter can be taken up by cells in specialized areas, such as Peyer's patches and other intestinal mucosal lymphoid aggregates, located in the intestine, especially in the ileum, into the lymphatic circulation.
  • Entrapping a drug or antigen in a ⁇ microparticulate system can protect the drug or antigen from acidic and enzymatic degradation, yet still allow the drug or antigen to be administered orally, where they are taken up by the specialized uptake systems, and release the entrapped material in a sustained manner or are processed by phagocytic cells such as macrophages.
  • the entrapped material is a drug, elimination of the first-pass effect (metabolism by the liver) is highly
  • Lipid systems have been widely exploited for development of drug delivery vehicles and systems. Other lipid-based systems may be appropriate for development as mucosal
  • Candidate mucosal delivery systems may additionally incorporate absorption enhancers, such as the salicylates, bile salts and other surfactants.
  • Absorption enhancers may function to increase the permeation of peptide and protein molecules across epithelial barriers because of their interaction with the GI mucosa and concomitant opening of the
  • amphiphilic molecules are known to behave as absorption enhancers.
  • medium chain fatty acid salts and esters In addition to bile salts and salicylates, medium chain fatty acid salts and esters, and medium chain monoglycerides and di- glycerides are known to have mucosal absorption enhancing activity.
  • Absorption enhancement with these molecules is attributed to the presence of medium chain C6-C12 fatty acyl chains (6-12 carbon atoms in length), particularly those derivatized with C8-C10 fatty acids (8-10 carbon atoms in length).
  • Enhancing molecules may be involved in opening up channels or tight junctions between cells, allowing paracellular transport of co-administered molecules.
  • these molecules may act as inhibitors of intestinal efflux pumps, such as the P-glycoprotein.
  • Other strategies to improve oral delivery include mixing the therapeutic agent with protease
  • inhibitors such as aprotinin, soybean trypsin inhibitor in an attempt to limit degradation of the administered therapeutic agent.
  • This approach alone, however, has limited commercial utility due to lack of significant absorption enhancement.
  • Lipids and surfactants are differentiable from short and long chain hydrocarbons in that they are amphiphilic molecules, having both hydrophilic and hydrophobic moieties.
  • Surfactants are conveniently classified on an empirical scale known as the hydrophile- lipophile balance (HLB) which runs from about 1 to about 45 and from about 1 to about 20
  • HLB values closer to 1 represent surfactants with more lipophilic character, while HLB values that are greater than about 10 represent more hydrophilic surfactants.
  • surfactants form different kinds of aggregates. Phospholipids characteristically form bilayer membranes in water, whereas in water with a low concentration of other polar lipids, micellar structures form.
  • micelles are either spherical, typically containing 50- 100 lipid molecules, or rod-shaped or disc-shaped macrostructures. In each of these cases, the hydrocarbon tails form the interior of the micelle and polar head groups are in contact with water.
  • reverse-type micelles, or reverse micelles form.
  • the conventional micellar phase is also known as the LI phase. The reverse
  • L2 micellar phase is also known as L2.
  • water forms the internal phase and the hydrophobic tails of the lipid form the continuous phase.
  • Reverse micelles containing oil(s), surfactant(s) and an aqueous phase are also characterized as water-in-oil microemulsions (see Constantinides, P.P. Lipid Microemulsions for Improving Drug Dissolution and Oral Absorption : Physical and Biopharmaceutical Aspects, Pharm. Res. 12 (11) 1561-1572, n 1995 and references therein).
  • a number of liquid crystalline structures can also co-exist in mixtures of polar lipid and water, analogous to normal and reverse micelles, including hexagonal phases and inverse hexagonal.
  • simple reverse micelles water/amphiphile
  • microemulsion systems are ternary or quaternary
  • U.S. Patent No. 5,707,648 describes microemulsions that contain an oil phase, an aqueous phase, and a mixture of surfactants.
  • the solubilization of one phase into another in a microemulsion system is affected by a balance of attractive and repulsive forces.
  • microemulsions are thermodynamically stable, the droplets will not coalesce and precipitate over time.
  • Emulsion droplets are much larger, generally greater than a micron, while microemulsion droplets are in the 10-200 nanometer range.
  • a microemulsion can be characterized by the amount of the dispersed phase solubilized in the continuous phase.
  • Microemulsions have traditionally been formed using, in addition to the oil phase, one or more surfactants and a cosurfactant, usually short chain alcohols ⁇ e.g., ethanol or butanol), glycols ⁇ e.g., propylene glycol and polyethylene glycol), medium chain alcohols, amines, or i n acids.
  • Luteinizing hormone-releasing hormone (LHRH) agonists and analogs thereof suppress endogenous gonanotropins, causing a hypogonadal condition.
  • LHRH agonists include, but are not limited to, leuprolide, goserelin, nafarelin and histrelin.
  • GnRH gonadotropin-releasing hormone
  • LHRH agonists 2 pituitary gland, leading to long-term inhibition of gonadotropins. In men, testosterone levels are reduced to castrate levels within 14-21 days of therapy, and are reversible upon discontinuation.
  • the primary disease indications for LHRH agonists are prostate cancer, endometriosis and precocious puberty in children.
  • the use of LHRH agonists in other disease and disorder indications have been reported (Plosker G.L, Brogden R.N.,
  • the present invention provides compositions and methods for the delivery of hydrophilic molecules and other poorly absorbed water-soluble molecules to an animal.
  • the present invention relates to compositions and methods for the delivery of biologically active molecules, including hydrophilic and poorly absorbed water-soluble molecules.
  • biologically active molecules include, but are not limited to, therapeutic agents, diagnostic agents, antigens, antibodies, peptides, polypeptides, viruses, nucleic acids, growth factors, cytokines, and drugs.
  • the present invention provides stable reverse micelle compositions suitable for the mucosal delivery of biologically active molecules, such as, e.g., therapeutic agents, diagnostic agents, and antigens.
  • the reverse micelle compositions of the present invention promote the absorption 5 of biologically active molecules by mucosal tissues.
  • the reverse micelle compositions of the invention reduce the dosage of a biologically active molecule necessary to achieve a prophylactic or therapeutic effect in an animal, and thus, reduce the toxicity associated with administering higher dosages of certain biologically active molecules.
  • the reverse micelle compositions of the invention reduce the dosage of a diagnostic agent necessary to ⁇ diagnose or monitor the state of a disease or disorder in an animal.
  • the present invention provides reverse micelle compositions comprising a surfactant ⁇ e.g., a P-glycoprotein inhibitor), a hydrophilic phase, and one or more biologically active molecules.
  • the surfactant is a P-glycoprotein inhibitor.
  • the present invention also provides reverse micelle compositions comprising one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and one or more biologically active agents.
  • the present invention further provides reverse micelle compositions comprising one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and one or more biologically active molecules, wherein less than about 15%, less than 10%, less than 5%, or less than 2% by weight of the reverse micelle composition is ⁇ triester.
  • the fatty acids in the fatty acid esters of the reverse micelle composition have a length of about 6 to about 12 carbon atoms.
  • the present invention also provides reverse micelle compositions which are self-emulsifying dispersions comprising monoglycerides, diglycerides, or hydrophilic derivatives thereof, a hydrophilic phase, and one or more biological active molecules.
  • the 5 present invention encompasses reverse micelle compositions comprising monoglycerides or diglycerides or a mixture thereof, a hydrophilic phase, and one or more biologically active molecules, wherein the acyl groups of the monoglycerides or diglycerides are enriched in fatty acids having 6-12 carbon atoms.
  • the monoglycerides or diglycerides of the reverse micelle compositions of the invention may be partially derivatized with a hydrophilic moiety to provide polarity to increase water solubility.
  • the reverse micelle compositions of the invention comprise less than 15%, less than 10%, less than 5%, or less than 2% by weight of triglycerides .
  • the reverse micelle compositions of the invention are encapsulated for oral delivery in, e.g., starch or gelatin capsules.
  • the reverse micelle compositions of the invention may optionally comprise an adjuvant when the biologically active molecule being administered to an animal is an antigen.
  • the reverse micelle compositions of the ⁇ ⁇ present invention have prophylactic and therapeutic utility.
  • the reverse micelle compositions of the present invention also have utility in diagnosing and/or monitoring the state of a variety of diseases and disorders in an animal.
  • the present invention provides reverse micelle compositions comprising a surfactant, a hydrophilic phase, and one or more biologically active molecules, wherein at
  • reverse micelle compositions comprise one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and one or more biologically active molecules, wherein at least one of the biologically active molecules is a protein, polypeptide or peptide.
  • the reverse micelle compositions comprise
  • the peptides incorporated in the reverse micelle compositions of the invention have a molecular weight ranging from 500 to 10,000 daltons, more preferably from 500 to 5,000 daltons.
  • hormones ⁇ e.g., luteinizing hormone-releasing hormone (LHRH), parathyroid hormone (PTH), calcitonin, insulin, and growth hormone) or agonists
  • reverse micelle compositions comprise one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and one or more LHRH agonists.
  • LHRH agonists include, but are not limited to, leuprolide, goserelin, nafarelin and histrelin.
  • the LHRH agonist is leuprolide.
  • the reverse micelle compositions comprise one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and parathyroid hormone.
  • reverse micelle compositions comprise one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and calcitonin.
  • reverse micelle compositions comprise one or more fatty acid esters or
  • reverse micelle compositions comprise one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and human growth hormone.
  • reverse micelle compositions comprise one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and insulin. In accordance with these embodiments, the reverse micelle compositions comprise less than 15%, less than 10%, less than 5%, or less than 2% by weight of triester.
  • the present invention is based, in part, on Applicants' discovery that the bioavailability of peptide or protein drugs delivered mucosally using reverse micelle compositions comprising medium chain monoglycerides, diglycerides or a mixture thereof ⁇ e.g., monoglycerides or diglycerides with 6-12 carbon atom fatty acid side chains) and less than 10 % triglycerides are at least equivalent to that of previously known reverse micelles i Q consisting of medium chain monoglycerides, medium chain diglycerides, greater than 20 % triglycerides and other surfactants.
  • the present invention is also based, in part, on Applicants' discovery that the simple reverse micelle compositions of the invention provide for high biovailability of peptides or proteins without the need for complex water-in-oil microemulsions. Further, the present invention is based, in part, on Applicants' surprising
  • reverse micelle compositions comprising partial monoglycerides, diglycerides or ethoxylated or polyglycolized analogs thereof incorporate therapeutically effective amounts of therapeutic agents in a biphasic system which is thermodynamically stable and optically clear and transparent (the clarity of the particle being indicative of the presence of an isotropic micelle phase, such as the reverse micelle phase).
  • the present invention provides methods for the delivery of one or more biologically active molecules to an animal, said methods comprising administering to said animal a reverse micelle composition comprising a surfactant, a hydrophilic phase, and one or more biologically active molecules.
  • the present invention provides methods for the delivery of one or more biologically active molecules to an animal, said methods comprising 5 administering to said animal a reverse micelle composition comprising one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and one or more biologically active molecules.
  • such a composition comprises less than 15%, less than 10%, less than 5%, or less than 2% by weight of triester.
  • the present invention provides methods for the mucosal delivery of one or more biologically
  • .,0 active molecules to an animal, said methods comprising mucosally administering to said animal a reverse micelle composition comprising monoglycerides, diglycerides or hydrophilic derivatives thereof, a hydrophilic phase, and one or more biological active molecules.
  • the present invention provides methods for the mucosal delivery of one or more biologically active molecules to an animal, said methods comprising 5 mucosally administering to said animal a reverse micelle composition comprising monoglycerides or diglycerides or a mixture thereof, a hydrophilic phase, and one or more biologically active molecules, wherein the acyl groups of the monoglycerides or diglycerides are enriched in fatty acids having 6-8, 6-10, 6-12, 6-20, 8-10, 8-12, or 8-20 carbon atoms.
  • the monoglycerides or diglycerides may be partially derivatized with a hydrophilic moiety to provide polarity to increase water solubility.
  • the reverse micelle compositions administered to an animal comprise less than
  • the present invention provides methods for the prevention, treatment, or amelioration of one or more symptoms associated with a disease or disorder in an animal, said methods comprising administering to an animal in need thereof an effective amount of a reverse micelle composition comprising a surfactant, a hydrophilic phase, and one or more ⁇ ⁇ prophylactic or therapeutic agents useful in the prevention, treatment or amelioration of one or more symptoms associated with said disease or disorder.
  • the present invention provides methods for the prevention, treatment or amelioration of one or more symptoms associated with a disease or disorder in an animal, said methods comprising administering to an animal in need thereof an effective amount of a reverse micelle composition comprising one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and one or more prophylactic or therapeutic agents useful in the prevention, treatment or amelioration of one or more symptoms associated with said disease or disorder.
  • a reverse micelle compositions of the invention administered to an animal comprise less than 15%, less than 10%, less than 5%, or less than 2% by weight of
  • the present invention provides methods for the prevention, treatment or amelioration of one or more symptoms associated with a disease or disorder in an animal, said methods comprising mucosally administering to an animal in need thereof an effective amount of a reverse micelle composition comprising monoglycerides, diglycerides
  • the present invention provides methods for the prevention, treatment or amelioration of one or more symptoms associated with a disease or disorder in an animal, said methods comprising Q mucosally administering to an animal in need thereof an effective amount of a reverse micelle composition comprising monoglycerides or diglycerides or a mixture thereof, a hydrophilic phase, and one or more prophylactic or therapeutic agents useful in the prevention, treatment or amelioration of one or more symptoms associated with said disease or disorder, wherein the acyl groups of the monoglycerides or diglycerides are enriched in
  • the monoglycerides or diglycerides may be partially derivatized with a hydrophilic moiety to provide polarity to increase water solubility.
  • the reverse micelle compositions administered to an animal comprise less than 15%, less than 10%, less than 5%, or less than 2% by weight of triglycerides.
  • the present invention provides methods for the prevention, treatment or amelioration of one or more symptoms associated with a disease or disorder in an animal, said methods
  • the present invention provides methods for the prevention, treatment or amelioration of one or more symptoms associated with a disease or disorder in an animal, ⁇ ⁇ said methods comprising administering to said animal a reverse micelle composition comprising one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and one or more biologically active molecules, wherein at least one of the biologically active molecules is a protein, polypeptide or peptide.
  • the reverse micelle composition administered to an animal comprises less than
  • the present invention provides methods of administering hormones ⁇ e.g., luteinizing hormone- releasing hormone (LHRH), parathyroid hormone (PTH), calcitonin, insulin, and growth hormone) and agonists thereof (e.g., LHRH agonists) to an animal in need thereof to prevent, treat or ameliorate one or more symptoms associated with a disease or disorder
  • hormones e.g., luteinizing hormone- releasing hormone (LHRH), parathyroid hormone (PTH), calcitonin, insulin, and growth hormone
  • agonists thereof e.g., LHRH agonists
  • the present invention provides methods for the prevention, treatment or amelioration of one or more symptoms associated with prostate cancer, endometriosis, precocious puberty, uterine lelomyotama, fertility disorder, premenopausal breast cancer, endometiral cancer, ovarian cancer, benign prostatic hypertrophy, functional
  • a reverse micelle composition comprising one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and one
  • the present invention provides methods for the prevention, treatment or amelioration of one or more symptoms associated with hypopituitarism, hypothyroidism, human growth hormone deficiency, Gushing' s syndrome, nutritional short stature, intrauterine growth retardation, Russell Silver syndrome or achondroplasia, said methods comprising administering to an animal in need thereof an
  • a reverse micelle composition comprising one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and growth hormone, preferably human growth hormone.
  • the present invention provides methods for the prevention, treatment or amelioration of one or more symptoms associated a bone- reabsorption disease such as osteoporosis, metastatic bone cancer, osteolytic lesions with an orthopedic implant, Paget's disease, or bone loss associated with hyperparathyroidism, said methods comprising administering to an animal in need thereof an effective amount of a bone- reabsorption disease such as osteoporosis, metastatic bone cancer, osteolytic lesions with an orthopedic implant, Paget's disease, or bone loss associated with hyperparathyroidism, said methods comprising administering to an animal in need thereof an effective amount of a bone- reabsorption disease such as osteoporosis, metastatic bone cancer, osteolytic lesions with an orthopedic implant, Paget's disease, or bone loss associated with hyperparathyroidism, said methods comprising administering to an animal in need thereof an
  • the present invention provides methods for the prevention, treatment or amelioration of one or more symptoms associated with diabetes, said methods comprising administering to an animal in need thereof an effective amount of a reverse micelle ⁇ ⁇ composition comprising one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and insulin.
  • the reverse micelle compositions comprise less than 15%, less than 10%, less than 5%, or less than 2% by weight of triester.
  • the present invention provides methods for diagnosing or monitoring the state of a
  • the methods comprising administering to an animal an amount of a reverse micelle composition comprising a surfactant, a hydrophilic phase, and one or more diagnostic agents useful in the diagnosis of said disease or disorder.
  • a reverse micelle composition comprising a surfactant, a hydrophilic phase, and one or more diagnostic agents useful in the diagnosis of said disease or disorder.
  • the present invention provides methods for diagnosing or monitoring the state of a disease or disorder, said methods comprising administering to an animal an effective amount of a
  • reverse micelle composition comprising one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and one or more diagnostic agents useful in the diagnosis said disease or disorder.
  • the present invention provides methods diagnosing or monitoring the state of a disease or disorder, said methods comprising mucosally administering an animal an effective amount of a reverse micelle
  • composition comprising monoglycerides, diglycerides or a hydrophilic derivative thereof, a hydrophilic phase, and one or more diagnostic agents useful in the diagnosis of said disease or disorder.
  • the present invention provides methods diagnosing or monitoring the state of a disease or disorder, said methods comprising mucosally administering to an animal an effective amount of a reverse micelle composition comprising
  • the monoglycerides or diglycerides may be partially derivatized with a hydrophilic moiety to provide polarity to
  • the reverse micelle compositions administered to an animal comprise less than 15%, less than 10%, less than 5%, or less than 2% by weight of triglycerides.
  • kits comprising in an appropriate container(s) reverse micelle compositions comprising a surfactant, a hydrophilic phase, and one or more biologically active molecules.
  • the present invention provides kits comprising
  • kits of the invention comprise less than 15%, less than 10%, less than 5%, or less than 2% by weight of triester.
  • kits comprising reverse micelle ⁇ compositions comprising monoglycerides, diglycerides, or hydrophilic derivatives thereof, a hydrophilic phase, and one or more biological active molecules.
  • kits comprising in an appropriate container(s) reverse micelle compositions comprising monoglycerides or diglycerides or a mixture thereof, a hydrophilic phase, and one or more biologically active molecules, wherein the acyl groups of the monoglycerides or 5 diglycerides are enriched in fatty acids having 6-12 carbon atoms.
  • the reverse micelle compositions included in the kits of the invention may comprise monoglycerides or diglycerides which are partially derivatized with a hydrophilic moiety to provide polarity to increase water solubility.
  • the reverse micelle compositions included in the kits of the invention comprise less than 15%, less than 10%, less than 5%, or less than 2% by
  • kits may be formulated in a compatible pharmaceutical carrier.
  • the kits of the invention are accompanied by instructions for administration.
  • the kits of the invention may further comprise a list of the diseases and/or disorders for which the reverse micelle compositions may be used to prevent, treat, diagnose or monitor.
  • kits comprising in one or more containers one or more reverse micelle compositions comprising a surfactant, a hydrophilic phase, and one or more biologically active molecules, wherein at least one of the biologically active molecules is a protein, polypeptide or peptide.
  • the kits of the invention comprise in one or more containers reverse micelle compositions comprising one or more
  • kits comprise less than 15%, less than 10%, less than 5%, or less than 2% by weight of triester.
  • the present invention provides kits
  • LHRH luteinizing hormone- releasing hormone
  • PTH parathyroid hormone
  • calcitonin insulin, and growth hormone
  • agonists thereof e.g., LHRH agonists
  • kits of the invention comprise in one or more containers one or more reverse micelle compositions comprising one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and one or more LHRH
  • kits of the invention comprise in one or more containers reverse micelle compositions comprising one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and growth hormone, preferably human growth hormone.
  • the kits of the invention comprise in one or more containers reverse micelle compositions comprising one or more fatty acid i Q esters or hydrophilic derivatives thereof, a hydrophilic phase, and parathyroid hormone or calcitonin.
  • the kits of the invention comprise in one or more containers reverse micelle compositions comprising one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and insulin.
  • the reverse micelle compositions included in the kits comprise less than 15%,
  • hydrophilic phase and “aqueous phase” refer to compounds which are miscible with water including, but are not limited to, water, glycerol,
  • buffer solution is defined as an aqueous solution or aqueous solution containing less than 25% of a miscible organic solvent, in which a buffer
  • buffers include, but are not limited to, acetate buffer, citrate buffer, PBS (phosphate buffered saline), TRIS (tris-(hydroxymethyl)aminomethane), HEPES (hydroxyethylpiperidme ethane sulfonic acid), sodium acetate, sodium phosphate, and TES (2-[(tris-hydroxymethyl)methyl] amino-1-ethanesulfonic acid).
  • mucosa and “mucosal” refer to a mucous tissue such as epithelium, lamina intestinal, and a layer of smooth muscle in the digestive tract.
  • Mucosal delivery refers to the administration of a composition to the mucosal tissue.
  • Mucosal delivery refers to the delivery of a
  • FIG. 1 Pharmacokinetics of human growth hormone in rats following intraduodenal administration.
  • FIG. 2. Pharmacokinetics of LHRH in rats following intraduodenal administration.
  • FIG. 3. Resistivity of various RM (Reverse Micelle) formulations containing electrolytes upon dilution with deionized water.
  • FIG. 4 Leuprolide plasma concentration profile in dogs following subcutaneous and intraduodenal administration.
  • the present invention provides compositions and methods for the delivery of hydrophilic molecules and other poorly absorbed water-soluble molecules to an animal.
  • the present invention relates to compositions and methods for the delivery of biologically active molecules, including hydrophilic and poorly absorbed water-soluble 5 molecules.
  • biologically active molecules include, but are not limited to, therapeutic agents, diagnostic agents, antigens, antibodies, peptides, polypeptides, viruses, nucleic acids, growth factors, cytokines, and drugs.
  • the reverse micelle compositions of the present invention promote the absorption of biologically active molecules by mucosal tissues.
  • the reverse micelle compositions of the invention also ⁇ reduce the dosage of a biologically active molecule necessary to achieve a prophylactic or therapeutic effect in an animal, and thus, reduce the toxicity associated with administering higher dosages of certain biologically active molecules. Further, the reverse micelle compositions of the invention reduce the dosage of a diagnostic agent necessary to diagnose or monitor the state of a disease or disorder. 5 The invention provides for reverse micelle compositions comprising a surfactant
  • the invention provides for reverse micelle compositions comprising one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and a biologically active molecule, wherein the reverse micelle compositions have less than 15%, preferably less than 10%, less than 5%, or less than 2% by weight of triester.
  • the reverse micelle compositions comprise fatty acid esters having fatty acids with a length of about 6 to about 12 carbon atoms.
  • the present invention also provides reverse micelle compositions comprising monoglycerides, diglycerides or mixtures thereof, a hydrophilic phase, and one or more biologically active molecules.
  • the reverse micelle 5 compositions of the invention may combined with one or more pharmaceutically acceptable carriers, diluents or excipients.
  • the reverse micelle compositions of the invention may be used to administer a variety of biologically active molecules to prevent, treat, or ameliorate the symptoms associated with disease or disorders.
  • the reverse micelle compositions of the invention may also be used to administer a diagnostic agent to facilitate the diagnosis of a disease or a disorder.
  • the invention provides methods for administering a biologically active molecule to an animal, said methods comprising administering to said animal an effective amount of a reverse micelle composition comprising a surfactant, a hydrophilic phase, and a biologically active molecule.
  • the present invention provides methods for administering a biologically active molecule to an animal, said methods comprising administering to said animal an effective amount of a reverse micelle composition comprising one or more fatty ⁇ acid esters or a hydrophilic derivative thereof, a hydrophilic phase and a biologically active molecule, wherein the reverse micelle compositions comprise less than about 10% by weight of triester, preferably about 5% by weight of triester.
  • the reverse micelle compositions of the invention are administered mucosally to an animal as a capsule, soft elastic gelatin-capsule, caplet, aerosol, spray, solution,
  • the reverse micelle compositions of the invention are administered to a mammal, more preferably a human to prevent, treat, diagnose or monitor a disease or disorder.
  • the invention provides for the use of a single component surfactant micelle which avoids more complex mixtures of oils and surfactants, yet still allows for suitable transepithelial transport of biologically active molecules.
  • the biologically active molecule is contained within the hydrophilic phase of a reverse micellar (L2) phase of a single type of
  • the present invention also provides reverse micelle compositions comprising of a mixture of a surfactant, an aqueous or hydrophilic phase, and one or more biologically active molecules.
  • the present invention provides reverse micelle compositions comprising a surfactant, a hydrophilic phase and one or more biologically active molecules, wherein the surfactant is a P-glycoprotein inhibitor.
  • the reverse micelle compositions of the invention comprise one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and one or more biologically active molecules.
  • such compositions comprise less than about 15%, less than 10%, less than 5%, or less than 2% by weight of triester and the fatty acid in fatty acid esters of the reverse micelle compositions have a length of about 6 to about 20
  • the reverse micelle compositions of the invention comprise monoglycerides or diglycerides or a mixture thereof, a hydrophilic phase, and one or more biologically active molecules.
  • the reverse micelle compositions of the invention comprise monoglycerides, diglycerides or mixtures thereof, a hydrophilic phase, and one or more biologically active molecules, wherein the acyl groups of the monoglycerides or diglycerides are enriched in fatty acids having 6-8, 6-10, 6-12, 8-10
  • the monoglycerides or diglycerides may be partially derivatized with a hydrophilic moiety to provide polarity to increase water solubility.
  • the reverse micelle compositions of the invention comprise less than 15%, less than 10%, less than 5%, or less than 2% by weight of triglycerides.
  • the reverse micelle compositions of the invention may comprise ⁇ ⁇ buffering, oxidizing, reducing and/or tonicity agents in the hydrophilic phase to provide adequate solubility and stability of the biologically active molecule.
  • the hydrophilic phase of the reverse micelle compositions of the invention comprises an amount from about 0 to about 70%, about 0 to about 65%, about 0 to about 50%, about 0 to about 40%, about 0 to about 30%, about 0 to about 20%,
  • the hydrophilic phase of the reverse micelle compositions of the invention comprises an amount from 0 to 70%, 0 to 65%, 0 to 50%, 0 to 40%, 0 to 30%, 0 to 20%, 5% to 70%, 5 to 65%, 5 to 50%, 5 to 40%, 5
  • the hydrophilic phase of the reverse micelle compositions of the invention comprise an amount from about 5% to about 25% by weight of the reverse micelle, more preferably 5% to 25% by weight of the reverse micelle. Examples of compounds which may be included in the hydrophilic phase of a reverse micelle composition of the invention
  • the hydrophilic phase of the reverse micelle compositions of the invention comprise water.
  • the surfactant incorporated in the reverse micelle compositions of the invention may be non-ionic in nature, that is having a neutral overall electrical charge, or ionic in nature,
  • the surfactants incorporated in the reverse micelle composition are non-ionic in nature.
  • the surfactant incorporated into a reverse micelle composition of the invention may be a single compound or a mixture of compounds.
  • the reverse micelle compositions of the invention only comprise
  • surfactants include, but are not limited to, fatty acid esters and hydrophilic analogs thereof.
  • the surfactant is one or more fatty acid esters or hydrophilic derivatives thereof.
  • the surfactant is: (1) monoglycerides or hydrophilic derivatives or analogs thereof; (2) diglycerides or hydrophilic derivatives or analogs thereof; or (3) a mixture of monoglycerides or hydrophilic derivatives or analogs thereof and diglycerides or hydrophilic derivatives or analogs thereof.
  • present invention is a P-glycoprotein inhibitor.
  • Fatty acid chain lengths of 8-10 carbon atoms enriched with monoglycerides, diglycerides and their polyoxyethylated analogs can, e.g., be derived from coconut oil by alcoholysis and transesterification reactions. In another method, these esters can be derived by direct esterification of glycerol using C8/C10 fatty acid esters in the presence of ⁇ ⁇ polyethylene glycol or ethylene oxide if so desired.
  • the following medium chain fatty acid monoglycerides, diglycerides/polyoxyethylene esters are available commercially under different names as presented in Table 1 and can be used in the reverse micelle compositions of the invention.
  • the surfactant is generally chosen from unsubstituted or partially substituted monoglycerides and diglycerides having fatty acids with a length of 6-20 carbon atoms (C6-C20), preferably 6-12 carbon atoms, most preferably 8-10 carbon atoms.
  • the most preferred substitution of the monoglycerides and diglycerides is ethoxylation or pegylation.
  • Other suitable hydrophilic analogs of monoglycerides include, but are not limited, lactic acid, acetic acid, citric acid, succinic acid, and diacetyl tartaric acid esters.
  • the surfactant has an HLB value between about 1 and about 40, preferably between about 1 and about 20, most preferably between about 5 and about 20.
  • Pegylated or polyglycolized glycerides are derived in the synthesis from a mixture of monoglycerides, diglycerides and triglycerides and polyethylene glycol (PEG) monoesters and diesters, usually with a molecular weight (MW) between 200 and 10,000 daltons, preferably between 200 and 4,000 daltons.
  • the HLB value of the polyglycolized glycerides is adjusted by the length of the PEG chain and of the length and degree of saturation of the fatty acid substitutions.
  • the surfactant is composed of C8-C10 substituted polyglycolized glycerides, having an HLB value less than 20 and preferably, between 5 and 15.
  • Reverse micelle compositions of the invention may be fabricated with a surfactant in which a biologically active molecule is functionally solubilized in the hydrophilic phase of the reverse micelle.
  • reverse micelle compositions of the invention are fabricated with chemically-modified monoglycerides or diglycerides in which the biologically active molecule is functionally solubilized in the hydrophilic phase of the reverse micelle.
  • the hydrophilic moiety of the modified glyceride is a hydrophilic group, such as polyethylene glycol of various chain lengths. Medium chain fatty acid glyceride side chains are from 6-8,
  • the characteristics of the reverse micelle compositions can be modified and adjusted according to chemical conjugation of other surfactant active groups to the glyceride backbone.
  • the resulting self-emulsifying systems are advantageous since they can be formed without the addition of oil and additional surfactant molecules.
  • the reverse micelles of the present invention may be utilized for the delivery of a wide variety of biological active molecules.
  • biologically active molecule and analogous terms refer to eukaryotic and procaryotic cells, viruses, vectors, proteins, peptides, polypeptides, nucleic acids ⁇ e.g., DNA and RNA nucleotides including, but not limited to, antisense nucleotide sequences, triple helices and nucleotide sequences encoding biologically active proteins, polypeptides or peptides), saccharides, polysaccharides, carbohydrates, lipids, glycoproteins, and combinations thereof, and synthetic organic drugs and inorganic drugs exerting a biological effect when administered to an animal.
  • biologically active molecules have an aqueous solubility of greater than 0.1 mg/ml, preferably greater than 1 mg/ml. Examples of
  • 5 biologically active molecules include, but are not limited to, anti-angiogenesis factors, antibodies ⁇ e.g., monoclonal antibodies, scFvs and Fab fragments), antigens ⁇ e.g., viral, microbial or tumor-associated antigens), growth factors, hormones, enzymes, peptides (preferably, peptides with a molecular weight (MW) from about 500 to about 10,000 daltons, more preferably with a MW from about 500 to about 5,000 daltons), drugs ⁇ e.g., ⁇ ⁇ steroids, anti-cancer drugs such as chemotherapeutic agents, antiviral agents, anti- inflammatory agents and antibiotics), insecticides, insect repellents, fertilizers, vitamins, or any other material having a biological effect.
  • the reverse micelle compositions of the invention may be engineered to contain a biologically active molecule derived from the same or different species as the recipient of the reverse micelle composition.
  • a reverse micelle composition containing a biologically active molecule derived from a human is administered to a human.
  • the reverse micelles compositions of the invention have utility for the mucosal delivery of a wide variety of vaccines and/or antigens.
  • the reverse micelles compositions of the present invention may be designed to carry a wide variety of antigens
  • HTLV-1 or HTLV-2 antigens ⁇ e.g., HTLV-1 envelope protein or an antigenic fragment thereof
  • influenza virus antigens ⁇ e.g., influenza virus hemagglutinin or an antigenic fragment thereof
  • polio virus antigens rhinovirus antigens, rabies virus antigens, vaccinia virus antigens, Epstein-Barr virus antigens, hepatitis virus antigens,
  • the reverse micelle compositions may be engineered to contain an antigen derived from any species.
  • the reverse micelle compositions of the invention may also be utilized for the mucosal delivery of a wide variety of prophylactic or therapeutic agents.
  • prophylactic agents and analogous terms refer to biologically active molecules which can be used to prevent the onset, development or progression of one or more symptoms of a disease and/or disorder.
  • therapeutic agent and analogous terms refer to biologically active molecules which can be used to treat or ameliorate one or more symptoms associated with a disease and/or a disorder.
  • therapeutic agents include, but are not limited to, chemotherapeutic agents, antibiotics, cytokines, hormones, enzymes ⁇ e.g., superoxide dismutase, asparaginase, arginase, arginine deaminase, adenosine deaminase, ribonuclease, trypsin, chymotrypsin and papain), tachykinin receptor agonists and antagonist peptides, vasoactive intestinal peptide, calcitonins, vasopressins, growth hormone releasing peptide, luteinizing hormone-releasing hormone (LHRH) agonists, fibrinogen receptor antagonists (RGD peptides see, e.g., the RGD peptide described in International Publication No. WO 93/02664) having in their sequence arginine-glycine-D-
  • chemotherapeutic agents include, but are not limited to, arabinofuranosyladenine, acylguanosine, Nordeoxyguanosine, dideoxyadenosine, ⁇ dideoxycytidine, dideoxyinosine Floxuridine, 6-mercaptopurine, doxorubicin, Daunorubicin, I-darubicin, quinidine, cisplatin, carboplatin, epirubicin, leuprolide, goserelin, nafarelin, histrelin, bicalutamide, goserelin, nafarelin, irinotecan, gemcitabine, and sargramostim.
  • the peptides described in International Publication No. WO 93/02664 are incorporated herein by reference, in particular the peptides described on pages 10-12.
  • antibiotics include, but are not limited to, aminoglycoside antibiotics
  • apramycin arbekacin, bambermycins, butirosin, dibekacin, neomycin, neomycin, undecylenate, netilmicin, paromomycin, ribostamycin, sisomicin, and spectinomycin
  • amphenicol antibiotics ⁇ e.g., azidamfenicol, chloramphenicol, florfenicol, and thiamphenicol
  • ansamycin antibiotics ⁇ e.g., rifamide and rifampin
  • carbacephems ⁇ e.g.,
  • loracarbef carbapenems ⁇ e.g., biapenem and imipenem
  • cephalosporins ⁇ e.g., cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefozopran, cefpimizole, cefpiramide, and cefpirome
  • cephamycins ⁇ e.g., cefbuperazone, cefmetazole, and cefminox
  • monobactams ⁇ e.g., aztreonam, carumonam, and tigemonam
  • oxacephems e.g., flomoxef, and moxalactam
  • penicillins e.g., amdinocillin, amdinocillin pivoxil, amoxicillin, 5 bacampicillin, benzylpenicillinic acid, benzylpenicillin sodium, epicillin, fenbenicillin
  • erythromycin azithromycin, carbomycin, clarithomycin, dirithromycin, erythromycin, and ⁇ erythromycin acistrate), amphomycin, bacitracin, capreomycin, colistin, enduracidin, enviomycin, tetracyclines (e.g., apicycline, chlortetracycline, clomocycline, and demeclocycline), 2,4-diaminopyrimidines (e.g., brodimoprim), nitrofurans (e.g., furaltadone, and furazolium chloride), quinolones and analogs (e.g., cinoxacin, ciprofloxacin, clinafloxacin, flumequine, and grepagloxacin), sulfonamides (e.g., acetyl sulfamethoxypyrazine, benzylsulfamide, noprylsulfamide,
  • cytokines include, but are not limited to, interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin 15 (IL-15), interleukin 18 (IL-18), platelet derived growth factor (PDGF),
  • Epo erythropoietin
  • EGF epidermal growth factor
  • FGF fibroblast growth factor
  • GM-CSF granulocyte macrophage stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • M-CSF macrophage colony stimulating factor
  • IFN interferon
  • hormones include, but are not limited to, luteinizing hormone releasing hormone (LHRH), growth hormone (GH), ⁇ ⁇ growth hormone releasing hormone, ACTH, somatostatin, somatotropin, somatomedin, parathyroid hormone, hypothalamic releasing factors, parathyroid hormone, insulin, glucagon, enkephalins, vasopressin, calcitonin, heparin, low molecular weight heparins, heparinoids, synthetic and natural opioids, insulin thyroid stimulating hormones, and endorphins.
  • a reverse micelle composition of the invention comprises LHRH or an analog thereof.
  • a reverse micelle composition of the invention comprises LHRH agonists such as leuprolide, goserelin, nafarelin and histrelin.
  • a reverse micelle composition of the invention comprises parathyroid hormone or calcitonin.
  • a reverse micelle composition of the invention comprises insulin.
  • a reverse micelle composition of the invention comprises human growth hormone or an analog thereof.
  • the amount of the peptides incorporated into the reverse micelle compositions is determined by their solubility in the hydrophilic phase.
  • composition is from about 0.05 to about 100 mg/ml, more preferably from about 0.05 to about 50 mg/ml, and most preferably from about 0.05 to about 10 mg/ml.
  • biologically active molecule is also used herein to include diagnostic agents.
  • diagnostic agents include, but are not limited to, radio-opaque compounds, magnetic compounds, fluorescent compounds, radioactive
  • 3 Q compounds and other contrast agents used with ultrasound, x-rays, fluorescence, MRI, CT, and other techniques known to those skilled in the art. Formulation of these materials is typically critical for effective delivery, detection sensitivity, targeting to an intended site, and for improved comfort to the patient.
  • the reverse micelle compositions are particularly suitable for delivery through mucosal tissue or epithelia. Accordingly, the reverse micelle compositions of the present invention are preferrably administered by those routes which optimize uptake by mucosa, for example, oral, sublingual, buccal, rectal and intranasal routes of administration. However, topical, transdermal and parenteral delivery may also be used.
  • the most preferred route of administration of the reverse micelle composition is oral administration.
  • the reverse micelle compositions of the invention can be delivered orally in the form of tablets, capsules, cachets, gelcaps, solutions, suspensions and the like.
  • the reverse micelle compositions of the invention can be delivered orally in the form of tablets, capsules, cachets, gelcaps, solutions, suspensions and the like.
  • Oral dosage forms may further fabricated to release the biologically active molecules at different regions of the GI tract, such as the small intestine and the colon, and in a time-dependent manner.
  • the dosage unit form of the reverse micelle composition comprising an antigen is a capsule, it can contain, in addition to the material of the above type, a liquid carrier or adjuvant. If administered topically the reverse micelles will typically be administered in the form of an ointment, cream or transdermal patch. If administered intranasally the reverse micelle composition will typically be administered in an aerosol form, spray, mist or in the form of drops.
  • the reverse micelle compositions of the present invention are suitable for administration to animals, in particular domestic animals and birds, and more particularly humans.
  • domestic animals such as dogs and cats, as well as domesticated herds, cattle, sheep, pigs and the like may be treated or vaccinated with the reverse micelle compositions of the present invention.
  • the reverse micelle compositions of the present invention are administered to humans.
  • a reverse micelle composition of the present invention comprising two or more biologically active molecules may be administered to an animal in need thereof.
  • the biologically active molecules incorporated in the reverse micelle compositions of the invention act together additively or synergistically to achieve the desired biological effect.
  • two or more reverse micelle compositions containing the same biologically active molecule may be administered to an animal in need thereof simultaneously or separately.
  • two or more reverse micelle compositions comprising one or more different biologically active molecules may be administered to an animal in need thereof simultaneously or separately.
  • Reverse micelle compositions are generally provided in a hermetically sealed container such as an ampule or sachet, and stored at room temperature or 4°C.
  • the reverse micelle compositions of the invention may be provided in the form of tablets, capsules, cachets, gelcaps, solutions, suspensions and the like.
  • the reverse micelle compositions of the invention may further be lyophilized into a fine powder which can be distributed in the form of a capsule or other suitable dosage form.
  • the maximum amount of water that can be used in the reverse micelles in a capsule depends on the type and property of the capsules. Reverse micelles having lower water contents are normally more compatible with gelatin capsules.
  • reverse micelles used in LiCaps gelatin capsules from CAPSUGEL have a water content by weigh of 0 to about 70%, about 0 to about 65%, about Q 0 to about 50%, about 0 to about 40%, about 0 to about 30%, about 0 to about 20%, about 0 to about 15%, about 0 to about 10%>, about 5 to about 70%, about 5 to about 60%, about 5 to about 50%, about 5 to about 40%, about 5 to about 30%, about 5 to about 20% about 5 to about 15%, or about 5 to about 10%.
  • reverse micelles used in LiCaps gelatin capsules from CAPSUGEL have a water content by weight of about 0 to about 40%.
  • reverse micelles used in LiCaps gelatin capsules from CAPSUGEL have a water content by weigh of about 0 to about 30%. In a more preferred embodiment, reverse micelles used in LiCaps gelatin capsules from CAPSUGEL have a water content by weigh of about 0 to about 20%. In a most preferred embodiment, reverse micelles used in LiCaps gelatin capsules from CAPSUGEL have a ⁇ water content by weigh of about 0 to about 15%>.
  • the compatibility of the reverse micelles with capsules can be changed by a modifier, for example a gelling agent, in the reverse micelles to reduce the interaction of water with the inner capsule wall, by applying coatings on the inner surface of the capsules, or by changing the storage conditions such as lowering the storage temperature.
  • the effective amount the amount of the composition sufficient to result in a desired therapeutic effect, such as treatment, prevention or amelioration of one or more symptoms of a disease or disorder
  • the dosage of the reverse micelle composition is from Q about 0.1 to about 1000 mg/kg, more preferably from about 0.1 to about 100 mg/kg, and most preferably from about 0.1 to about 50 mg/kg.
  • Such dosages can be determined by a skilled physician using standard techniques.
  • the reverse micelle compositions can be used to prevent, treat or diagnose many diseases and/or disorders including, but not limited to, cancer, infectious diseases, and 5 immune disorders (e.g., autoimmune disorders, asthma, and allergies).
  • diseases and/or disorders including, but not limited to, cancer, infectious diseases, and 5 immune disorders (e.g., autoimmune disorders, asthma, and allergies).
  • the present invention provides methods for the prevention, treatment or amelioration of one or more symptoms associated with prostate cancer, endometriosis, precocious puberty, uterine lelomyotama, fertility disorder, premenopausal breast cancer, endometiral cancer, ovarian cancer, benign prostatic hypertrophy, functional bowel disease, cluster headache, premenstrual syndrome, idiopathic hirsuitism, hirsuitism second to polycycstic ovarian disease, adenomyosis, Meniere's disease, sickle cell anaemia
  • the present invention provides methods for the prevention, treatment or amelioration of one or more symptoms associated with ⁇ ⁇ hypopituitarism, hypothyroidism, human growth hormone deficiency, Gushing' s syndrome, nutritional short stature, intrauterine growth retardation, Russell Silver syndrome or achondroplasia, said methods comprising administering to an animal in need thereof an effective amount of a reverse micelle composition comprising one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and growth hormone, preferably
  • the present invention provides methods for the prevention, treatment or amelioration of one or more symptoms associated a bone- reabsorption disease such as osteoporosis, metastatic bone cancer, osteolytic lesions with an orthopedic implant, Paget's disease, or bone loss associated with hyperparathyroidism, said methods comprising administering to an animal in need thereof an effective amount of a bone- reabsorption disease such as osteoporosis, metastatic bone cancer, osteolytic lesions with an orthopedic implant, Paget's disease, or bone loss associated with hyperparathyroidism, said methods comprising administering to an animal in need thereof an effective amount of a bone- reabsorption disease such as osteoporosis, metastatic bone cancer, osteolytic lesions with an orthopedic implant, Paget's disease, or bone loss associated with hyperparathyroidism, said methods comprising administering to an animal in need thereof an effective amount of a bone- reabsorption disease such as osteoporosis, metastatic bone cancer, osteolytic lesions with an orthopedic implant, Paget's disease, or bone loss
  • reverse micelle composition comprising one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and parathyroid hormone or calcitonin.
  • the present invention provides methods for the prevention, treatment or amelioration of one or more symptoms associated with diabetes, said methods comprising administering to an animal in need thereof an effective amount of a reverse micelle
  • composition comprising one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and insulin.
  • the reverse micelle compositions comprise less than 15%, less than 10%, less than 5%, or less than 2% by weight of triester.
  • the reverse micelle compositions of the invention can be administered in any order.
  • a reverse micelle composition of the invention comprising a chemotherapeutic agent can be administered to an animal with cancer in combination with radiation therapy.
  • the reverse micelle compositions comprise one or more antigens for use as vaccines.
  • the vaccine formulations of the invention comprise a reverse micelle composition of the invention.
  • Suitable preparations of vaccines formulations include, but are not limited to, liquid solutions or suspensions; solid forms such as capsules
  • the active immunogenic ingredients incorporated into the vaccine formulations of the invention are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • the vaccine preparation may also include minor amounts of ⁇ ⁇ auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine.
  • Antigens may be formulated into the vaccine as neutral or salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids, such as, for example,
  • hydrochloric or phosphoric acids or organic acids such as acetic, oxalic, tartaric, maleic, and the like.
  • Salts formed with free carboxyl groups may also be derived from inorganic bases, such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.
  • the vaccine formulations of the invention comprise an effective amount of a reverse micelle composition and a pharmaceutically acceptable carrier or excipient.
  • Pharmaceutically acceptable carriers are well known in the art and include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, and combinations thereof.
  • a physiologically acceptable carrier is a physiologically acceptable carrier.
  • the carrier used in the vaccine formulation is preferably sterile and the formulation should suit the mode of administration.
  • the vaccine formulations of the invention can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • 3 ⁇ vaccine formulation can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
  • Vaccine formulations for oral administration can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
  • the vaccine formulations are provided in a hermetically sealed container
  • the vaccine formulations are generally stored at room temperature or 4°C prior to use.
  • the vaccine formulations of the invention may be lyophilized to form a fine powder which can be distributed in the form of a capsule or other suitable dosage form.
  • the vaccine formulations of the invention may be multivalent or univalent. Many methods may be used to introduce the vaccine formulations of the invention; these include, but are not limited to, oral, intradermal, intramuscular, intraperitoneal, intravenous,
  • the patient to which the vaccine is administered is preferably an animal, more preferably a mammal, most preferably a human.
  • the precise dose of vaccine formulation to be employed will depend on the route of administration, and the nature of the patient, and should be decided according to the ⁇ ⁇ judgment of the practitioner and each patient's circumstances according to standard clinical techniques.
  • An effective immunizing amount is that amount sufficient to produce an immune response to the antigen in the host to which the vaccine preparation is administered.
  • purified antigens as vaccine formulations can be carried out by standard methods.
  • the purified protein(s) should be adjusted to an appropriate
  • Suitable adjuvants may include, but are not limited to: mineral gels, e.g., aluminum hydroxide; surface active substances such as lysolecithin or pluronic polyols; polyanions; peptides; oil emulsions; alum, Lipid A and derivatives of Lipid A (e.g., monophosphoryl lipid A (MPLA)), cytokines, N-acetyl-murmyl-L-threonyl-D-isoglutamine
  • MPLA monophosphoryl lipid A
  • microbial toxins e.g., cholera toxin and heat labile toxin
  • Effective doses (immunizing amounts) of the vaccines of the invention may also be provided.
  • the present invention thus provides a method of immunizing an animal, or treating or preventing various diseases or disorders in an animal, comprising administering to the animal an effective immunizing dose of a vaccine formulation of the present invention.
  • kits comprising in an appropriate container(s) a reverse micelle composition comprising a surfactant, a hydrophilic phase, and one or more biologically active molecules.
  • the present invention also provides kits comprising reverse micelle compositions comprising in an appropriate container(s) one or more fatty acid esters 35 or a hydrophilic derivative thereof, a hydrophilic phase, and one or more biologically active agents.
  • kits comprise in an appropriate container(s) reverse micelle compositions comprising monoglycerides, diglycerides, or a hydrophilic derivative thereof, a hydrophilic phase, and one or more biologically active molecules.
  • kits comprise in an appropriate container(s) reverse micelle compositions comprising monoglycerides, diglycerides, or a hydrophilic derivative thereof, a hydrophilic phase, and different biologically active molecules.
  • kits comprise reverse
  • the reverse micelle compositions included in the kits of the invention comprise less than 15%, less than 10%, less than 5%, or less than 2% by weight of triglycerides.
  • the reverse micelle compositions of the invention may be formulated in a compatible pharmaceutical carrier.
  • the reverse micelle compositions of the invention may be formulated in a compatible pharmaceutical carrier.
  • the kits of the invention are
  • kits of the invention may also comprise a list of the diseases and/or disorders for which the compositions may be used to prevent, treat, diagnose or monitor.
  • Antibodies generated against an antigen by immunization of an animal e.g., a mouse, rat, rabbit, monkey, etc.
  • a reverse micelle composition of the invention comprising an antigen are useful in diagnostic immunoassays, passive immune therapy, and generation of antiidiotypic antibodies. 5
  • the generated antibodies may be isolated by standard techniques known in the art ⁇ e.g., inmunoaffinity chromatography, centrifugation, precipitation, etc.) and used in diagnostic immunoassays.
  • the antibodies may also be used to monitor treatment and/or disease progression. Any immunoassay system known in the art may be used for this purpose including, but not limited to, competitive and noncompetitive assay systems using
  • the reverse micelle compositions of the invention can also be used to produce antibodies for use in passive immunotherapy, in which short-term protection of an animal is achieved by the administration of a pre-formed antibody directed against a heterologous antigen.
  • the antibodies generated by the reverse micelle compositions of the invention can also be used in the production of antiidiotypic antibody.
  • the antiidiotypic antibody can then in turn be used for immunization, in order to produce a subpopulation of antibodies that bind the initial antigen of the pathogenic microorganism (Jerne et al. Ann. Immunol. 125c:373, 1974; Jerne et al. EMBO J. 1:234, 1982).
  • the amount of immunogen to be used and the immunization schedule will be determined by a physician skilled in the at and will be administered by reference to the immune response and antibody titers of the animal.
  • a solution of human growth hormone (“hGH”) was prepared by dissolving purified human growth hormone in water.
  • a reverse micelle was obtained by mixing the aqueous solution of human growth hormone with Acconon CC-12 in a 5:95 ratio (water to surfactant). Subsequently, the mixture was mixed by vortexing and a transparent dispersion was obtained, indicative of stable reverse micelle formation. It will be appreciated by the skilled artisan that any mixing method known in the art that can disperse two phases can be used to prepare the reverse micelle compositions of the invention. The reverse micelle was stable upon storage at 4° C and room temperature.
  • Sprague-Dawley rats (approximately weighing 120 grams each) were catheterized surgically with jugular and duodenal catheters. Each group of rats, 3-5 animals per group, received 600 micrograms of human growth hormone in Acconon CC-12 reverse micelle formulation or control formulations consisting of aqueous growth hormone or Acconon CC- 12 water reverse micelle. Blood samples were obtained from the jugular catheter at the 0,
  • Plasma samples were obtained from the collected blood specimens and analyzed for presence of hGH by an enzyme-linked immunosorbent assay (ELISA) (Alexon-Trend, BioCheck).
  • ELISA enzyme-linked immunosorbent assay
  • the assay system utilizes polyclonal sheep anti-hGH for solid phase (microwells) immobilization, and mouse monoclonal anti-hGH in the antibody-enzyme (horseradish peroxidase) conjugate solution.
  • the test serum or formulation sample was allowed to react simultaneously with the coated and conjugated antibodies, resulting in the hGH molecule being sandwiched between the solid phase and enzyme-linked antibodies. After a 45-minute incubation at room
  • HORMONE AGONIST REVERSE MICELLES This example demonstrates that a higher percentage of bioavailability of luteinizing hormone releasing hormone (LHRH) is achieved when LHRH is administered intraduodenally in a reverse micelle formulation than when LHRH is administered in a water-in-oil emulsion formulation.
  • a water-in-oil microemulsion was prepared
  • Captex 355 2 grams of Capmul MCM, and 1 gram of polyoxyethylated(20) sorbitan oleate (Tween 80) and mixed until a transparent microemulsion was obtained.
  • the final composition of the microemulsion is 60% Captex, 20%) Capmul MCM, 10%
  • Sprague-Dawley rats (approximately weighing 120 grams each) were catheterized surgically with jugular and duodenal catheters. Each group of rats, 3-5 animals per group,
  • Plasma samples were analyzed by a competitive ELISA assay as follows. Plastic 96 well plates were coated with anti-rabbit immunoglobulins followed by addition of rabbit anti-LHRH and biotinylated-LHRH with sample dilutions. Binding of biotinylated LHRH was assayed by development with HRP-avidin and color development with TMB (tetra-methyl-benzidine). Pharmacokinetic parameters were calculated from the
  • ID intraduodenal administration
  • SC subcutaneous administration
  • AUCinf Area Under Curve from time 0 to Infinity
  • n number of rats. * PK data with RM-A12 was reproduced via four independent experiments.
  • Leuprolide concentration in reverse micelle (RM) formulations was measured by reversed phase HPLC using a Hewlett Packard 1050 system equipped with a Waters C-18 4.6x250mm 5 ⁇ m column, a Supelguard Discovery C-18 2 x 4.0mm guard column and UV Diode array detection at 220nm.
  • Mobile phase A was 0.1% (v/v) trifluoroacetic acid (TFA)
  • Sprague-Dawley rats (approximately weighing 120 grams each) were catheterized 3 surgically with jugular and duodenal catheters. Each group of rats, 4-5 animals per group, were given 0.4 - 3.6 mg/kg of leuprolide either as free in solution or incorporated in micelles. As further control, several groups of animals were given a solution of free leuprolide subcutaneously. Blood samples were collected at 0, 20, 40, 60, 90, 120, and 240 minutes following administration of leuprolide or controls. The time 0 blood collection was 3 obtained approximately 15 minutes before administration of leuprolide formulations.
  • the amount of leuprolide released into rat serum or leuprolide in formulation was determined by using competitive enzyme-linked immunosorbent assay (ELISA) (Peninsula Laboratories, Inc).
  • ELISA enzyme-linked immunosorbent assay
  • the assay system utilizes goat anti-rabbit Ig G for solid phase (microwells) immobilization.
  • the test serum or formulation sample was allowed to react simultaneously with the coated antibody, rabbit anti-leuprolide and biotinylated leuprolide.
  • the biotinylated leuprolide competes for the antibody binding sites with standard or the unknown sample leuprolide.
  • ID intraduodenal administration
  • AUCinf Area Under Curve from time 0 to Infinity
  • the intraduodenal bioavailability of leuprolide from a solution formulation is very low (about 0.2%) whereas the intraduodenal bioavailability from the RM-S is about 22 to about 39%.
  • Beagle dogs (3 female and 3 male weighing 8 - 10 kg each) were catheterized surgically with duodenal catheters. For each treatment, the dogs were given 0.4 - 2.4 mg/kg of leuprolide either in solution or incorporated in micelles. The treatments were administered via the duodenal catheter. For the positive control, the dogs were given a solution of free leuprolide by subcutaneous injection. Blood samples were collected from cephalic catheters or the jugular vein at 0, 15, 30, 45, 60, 90, 120, 240, 360, 480, and 1440 minutes following administration of leuprolide or controls. Leuprolide concentration levels are illustrated in the standard curves in Figure 4. The time 0 blood collection was obtained approximately 10 minutes before administration of leuprolide formulations.
  • the amount of leuprolide released into dog serum was determined by using a competitive enzyme-linked immunosorbent assay (ELISA) from Peninsula Laboratories, Inc. which utilizes goat anti- rabbit IgG for solid phase (microwell) immobilization.
  • ELISA enzyme-linked immunosorbent assay
  • the test serum was allowed to react simultaneously with the coated antibody, rabbit anti-leuprolide and biotinylated leuprolide. After two-hour incubation at room temperature, unbound biotinylated peptide was removed by washing, and streptavidin-conjugated horseradish peroxidase (SA-HRP) was added and allowed to bind the immobilized rabbit anti-leuprolide/ biotinylated leuprolide complex.
  • SA-HRP streptavidin-conjugated horseradish peroxidase
  • TMB 3,3'5,5'-Tetramethylbenzidine dihydrochloride
  • Reverse micelles that are compatible with gelatin capsules were prepared by mixing an excipient, for example, Softigen 767 or Labrasol, with a leuprolide solution in buffer at an appropriate ratio, for example, 85:15 w/w. Based on the weight of the animals, an appropriate amount of reverse micelle containing leuprolide was placed into the body part of a capsule of suitable size (for example, size 00 or 13 gelatin capsules) and the cap was then put in place and locked. The capsule was sealed by applying a few microliters of an alcoholic solution (for example, 1 :1 isopropanol- water solution) and allowing the solution to dry.
  • an alcoholic solution for example, 1 :1 isopropanol- water solution
  • Enteric coating of a reverse micelle capsule was performed using techniques known to others in the art. (Enteric Coating of Hard Gelatine Capsules Application of EUDRAGIT® L 30 D-55, Rohm Pharma Polymers Application Note 4.1.9.4) A modified coating method and a modified coating solution were also used. Specifically, Eudragit L30D-55 (10.56 g) was diluted with 7.9 g of distilled water and mixed with 0.63 g of triethyl citrate. To this, 0.134 g of 30% Tween 80 was added. The resulting suspension was stirred for 30 minutes.
  • the coating solution was either used as is, or the pH of the coating solution was adjusted to a desired level (for example, pH 5.5) so that the coated capsule would have a better dissolution profile in the duodenum.
  • a dipping method was used for the coating of the capsules.
  • the capsules (especially the size 13 capsules) were held by a multi-port valve- controlled vacuum suction device, which was developed by the same authors, during the 5 coating processes. Alternatively, the capsules were coated using a coater.
  • Dissolution test using a continued in situ monitoring system Dissolution of the capsules was tested using a USP test method on a dissolution test station (VanKel VK 7000).
  • USP Physical Tests ⁇ 711> Dissolution and ⁇ 724> Drug ⁇ Release Automation of the sampling and measurement was achieved by using a continued in situ monitoring system developed by the applicants.
  • a HPLC solvent inlet filter was attached to the inlet of a sample intake tube inserted into the dissolution test vessel.
  • the intake tube was connected to a HPLC pump which was directly connected to one or more detectors such as diol array detectors, RI detectors, fluorescent detectors, UV j 5 detectors, and so forth, and combinations of such, in a HPLC system.
  • the enteric coated reverse micelle capsules were stable in the acid stage for at least 2 hours as indicated in the dissolugrams by the relatively unchanged UV 220, 280 and 540 nm signals. In buffer stage, the enteric coated capsules dissolved within 10 minutes according to the dissolugrams.
  • Model compounds with special UV absorption wavelengths such as dimethylaminopyridine (280 nm), Bacto Methylene Blue (340 nm) or fluorescent compounds were also used replacing leuprolide in the reverse micelle in capsules for the
  • FITC-Dextran 4,000 daltons, 10,000 daltons, or 20,000 daltons were dissolved in water at different ratios of water to surfactant.
  • Stable micelles were formed at up to 50% total water phase as determined by the quenching of FITC fluorescence and obtained a transparent dispersion. Fluorescence of FITC were retrieved by dilution of the stable reverse micelles into excess water, indicating extrusion of the water- soluble dextran from the surfactant phase.
  • RMs Reverse micelles
  • conductivity or resistivity measurements can be used to confirm the reverse micellar structure.
  • RMs were prepared by mixing 15% (w/w) IX phosphate-buffered saline (PBS) with 85% (w/w) corresponding excipients. Subsequently, deionized water was added incrementally to up to 90% total content of water by weight. The resistivity of solution was measured incrementally using Millicell-ERS electrode system (Millipore). The resistivity 5 of deionized water was exceed measurable range of electrode (> 19.99KOhms), of tested excipients (0%> water) were about 15.5KOhms and of lx PBS was 2-3 Ohms.
  • RM containing 15% PBS still showed high resistance due to the fact that the most of electrolytes are remained in core of reverse micellar structure within the continuous phase of excipients.
  • RM systems using different surfactants exhibited similar pattern in the drop of resistivity as 0 increasing % water. Sharp drop in resistivity indicated release of encapsulated electrolytes from the core of the RM system into medium upon dilution with non-conductive deionized water. The resistivity reached plateau (at about 15 Ohms) after exceeding 40% total water content in the system. This implies that the reverse micelle system (L2 phase) has been converted into regular micelle (LI phase) after this point where the continuous phase is 5 aqueous (water).

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Abstract

The invention relates to reverse micelle compositions and method s for drug delivery suitable for promoting the transmucosal absorption of biologically active molecules, especially drugs with poor intrinsic bioavailability, such as peptides, proteins, vaccines, and nucleic acids. The delivery system of this invention preferably comprises fatty acid esters and their hydrophilic derivatives that associate with water and othe polar solvents to form stable reverse micelles. When administered as pharmaceutical to mucosal surfaces following oral or intrasanal administration, biologically active molecules principally solubilized in the hydrophilic phase are protected from digestion by mucosal enzymes and other mucosal degradative processes and are taken up by absorptive cell mechanisms and reach appropriate body compartments. The reverse micelle compositions described in this invention may comprise monoglycerides and/or their transesterified products containing C6-C12 fatty acids chains, wherein the transester groups consist of hydrophilic moieties.

Description

REVERSE MICELLE COMPOSITIONS AND USES THEREOF
This application claims the benefit of U.S. provisional application nos. 60/336,471, filed December 3, 2001; 60/354,720, filed February 5, 2002; and 60/377,674, filed May 3, 2002, each of which is incorporated herein by reference in its entirety.
5 1. FIELD OF THE INVENTION
The present invention relates to delivery systems for the mucosal and parenteral administration of biologically active molecules, including, but not limited to, therapeutic agents, vaccines, allergens, antigens and diagnostic agents. In particular, the present invention relates to reverse micelle compositions comprising a surfactant, a hydrophilic ι Q phase, and one or more biological active molecules, and methods of administering biologically active molecules to an animal utilizing said compositions. The compositions of the invention promote the absorption of biologically active molecules across mucosal epithelial barriers. The compositions of the invention can be used prophylactically, therapeutically, diagnostically or cosmetically.
15
2. BACKGROUND OF THE INVENTION
2.1. Drug Delivery
Drug delivery takes a variety of forms, depending on the agent to be delivered and the administration route. The most convenient way to administer drugs into the body is by
2Q oral administration. However, many drugs, in particular proteins and peptides, are poorly absorbed and unstable during passage through the gastrointestinal (GI) tract. The administration of these drugs is generally performed through parenteral injection. Although oral vaccination is more convenient, vaccines are generally given through injection. This is particularly true with killed or peptidic vaccines, because of their low
25 absorbability and instability in the GI tract. A problem with systemic immunization is that it may not effectively induce mucosal immune responses, particularly production of IgA, that are important as the first defense barrier to invaded microorganisms. For this reason, it would be beneficial to provide oral vaccination, if the problems of low absorbability and instability could be overcome. Q Controlled release systems for drug delivery are often designed to administer drugs to specific areas of the body. In the gastrointestinal tract it is important that the drug not be eliminated before it has had a chance to exert a localized effect or to pass into the bloodstream.
Enteric coated formulations have been widely used for many years to protect drugs administered orally, as well as to delay release. Several microsphere formulations have been proposed as a means for oral drug delivery. For example, International Application No. PCT/US90/06433 by Enzytech describes the use of a hydrophobic protein, such as zein, to form microparticles; U.S. Patent No. 4,976,968 to Steiner et al. describes the use of "proteinoids" to form microparticles; and European Patent Application No. 0 333 523 by the UAB Research Foundation and Southern Research Institute describes the use of synthetic
5 polymers such as polylactic acid-glycolic acid to form microspheres.
Particles less than ten microns in diameter, such as the microparticles of European Patent Application No. 0 333 523 can be taken up by cells in specialized areas, such as Peyer's patches and other intestinal mucosal lymphoid aggregates, located in the intestine, especially in the ileum, into the lymphatic circulation. Entrapping a drug or antigen in a ι microparticulate system can protect the drug or antigen from acidic and enzymatic degradation, yet still allow the drug or antigen to be administered orally, where they are taken up by the specialized uptake systems, and release the entrapped material in a sustained manner or are processed by phagocytic cells such as macrophages. When the entrapped material is a drug, elimination of the first-pass effect (metabolism by the liver) is highly
15 advantageous.
2.2 Mucosal Delivery
Lipid systems have been widely exploited for development of drug delivery vehicles and systems. Other lipid-based systems may be appropriate for development as mucosal
2 delivery vehicles.
Candidate mucosal delivery systems may additionally incorporate absorption enhancers, such as the salicylates, bile salts and other surfactants. Absorption enhancers may function to increase the permeation of peptide and protein molecules across epithelial barriers because of their interaction with the GI mucosa and concomitant opening of the
25 tight junctions. A wide variety of amphiphilic molecules are known to behave as absorption enhancers. In addition to bile salts and salicylates, medium chain fatty acid salts and esters, and medium chain monoglycerides and di- glycerides are known to have mucosal absorption enhancing activity. Absorption enhancement with these molecules is attributed to the presence of medium chain C6-C12 fatty acyl chains (6-12 carbon atoms in length), particularly those derivatized with C8-C10 fatty acids (8-10 carbon atoms in length). Enhancing molecules may be involved in opening up channels or tight junctions between cells, allowing paracellular transport of co-administered molecules. Furthermore, these molecules may act as inhibitors of intestinal efflux pumps, such as the P-glycoprotein. Other strategies to improve oral delivery include mixing the therapeutic agent with protease
2 inhibitors, such as aprotinin, soybean trypsin inhibitor in an attempt to limit degradation of the administered therapeutic agent. This approach alone, however, has limited commercial utility due to lack of significant absorption enhancement.
Enhanced absorption of protein therapeutic agents across mucosal membranes has also been pursued by using amphiphilic agents that modify the globular nature of protein molecules as described in U.S. Patent No. 6,245,359. In this case, it is thought that
5 increased penetration across membranes is due to a reversible interaction of the amphiphile with a macromolecule such that the hydrodynamic radius of the molecule is altered enough to penetrate paracellularly.
Each of these strategies has the intent of protecting macromolecules from degradation and promoting the interaction of molecules with absorptive cells in mucosal i Q tissues.
Lipids and surfactants are differentiable from short and long chain hydrocarbons in that they are amphiphilic molecules, having both hydrophilic and hydrophobic moieties. Surfactants are conveniently classified on an empirical scale known as the hydrophile- lipophile balance (HLB) which runs from about 1 to about 45 and from about 1 to about 20
1 for non-ionic surfactants. HLB values closer to 1 represent surfactants with more lipophilic character, while HLB values that are greater than about 10 represent more hydrophilic surfactants. In contact with water, surfactants form different kinds of aggregates. Phospholipids characteristically form bilayer membranes in water, whereas in water with a low concentration of other polar lipids, micellar structures form. Depending on the
2ø concentration of polar lipid in water, micelles are either spherical, typically containing 50- 100 lipid molecules, or rod-shaped or disc-shaped macrostructures. In each of these cases, the hydrocarbon tails form the interior of the micelle and polar head groups are in contact with water. At higher concentration of polar lipid in water, reverse-type micelles, or reverse micelles form. The conventional micellar phase is also known as the LI phase. The reverse
2 micellar phase is also known as L2. In the L2 phase, water forms the internal phase and the hydrophobic tails of the lipid form the continuous phase. Reverse micelles containing oil(s), surfactant(s) and an aqueous phase are also characterized as water-in-oil microemulsions (see Constantinides, P.P. Lipid Microemulsions for Improving Drug Dissolution and Oral Absorption : Physical and Biopharmaceutical Aspects, Pharm. Res. 12 (11) 1561-1572, n 1995 and references therein). In addition, a number of liquid crystalline structures can also co-exist in mixtures of polar lipid and water, analogous to normal and reverse micelles, including hexagonal phases and inverse hexagonal. Traditionally, simple reverse micelles (water/amphiphile) have not been used in mucosal drug delivery systems.
In contrast to reverse micelles, microemulsion systems are ternary or quaternary
05 systems typically formed from an oil phase, a surfactant, and water. For example, U.S. Patent No. 5,707,648 describes microemulsions that contain an oil phase, an aqueous phase, and a mixture of surfactants. The solubilization of one phase into another in a microemulsion system is affected by a balance of attractive and repulsive forces. As microemulsions are thermodynamically stable, the droplets will not coalesce and precipitate over time. Emulsion droplets are much larger, generally greater than a micron, while microemulsion droplets are in the 10-200 nanometer range. The interface of emulsion
5 droplets can be considered as a monolayer of surfactant. A microemulsion can be characterized by the amount of the dispersed phase solubilized in the continuous phase. Microemulsions have traditionally been formed using, in addition to the oil phase, one or more surfactants and a cosurfactant, usually short chain alcohols {e.g., ethanol or butanol), glycols {e.g., propylene glycol and polyethylene glycol), medium chain alcohols, amines, or i n acids.
2.3 Luteinizing Hormone-Releasing Hormone Agonists Luteinizing hormone-releasing hormone (LHRH) agonists and analogs thereof suppress endogenous gonanotropins, causing a hypogonadal condition. Examples of LHRH agonists include, but are not limited to, leuprolide, goserelin, nafarelin and histrelin. Each
15 of these agonists are synthetic analogs of naturally occurring gonadotropin-releasing hormone (GnRH) which has the following amino acid sequence: p-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2, MW = 1182. The modifications to the natural compound result in increased potency and a longer half-life than that of the native peptide. Chronic administration of LHRH agonists exerts constant stimulation of the
2 pituitary gland, leading to long-term inhibition of gonadotropins. In men, testosterone levels are reduced to castrate levels within 14-21 days of therapy, and are reversible upon discontinuation. The primary disease indications for LHRH agonists are prostate cancer, endometriosis and precocious puberty in children. In addition, the use of LHRH agonists in other disease and disorder indications have been reported (Plosker G.L, Brogden R.N.,
2 Leuprorelin. A review of its pharmacology and therapeutic use in prostatic cancer, endometriosis and other sex hormo re-related disorders. Drugs 1994, 48(6): 930-967). These indications include uterine lelomyomata, fertility disorders, premenopausal breast cancer, endometrial cancer, ovarian cancer, benign prostatic hypertrophy, functional bowel disease, cluster headache, premenstrual syndrome, idiopathic hirsutism or hirsutism second to polycystic ovarian disease, adenomyosis, Meniere's disease, sickle cell anaemia associated priapism and catamental pneumothorax.
No oral dosage forms of LHRH or any of its agonists are available due to the very low oral bioavailability of these molecules (<1%). Thus, there remains a need in the art for any drug delivery approaches that enhance the intestinal absorption and oral bioavailability
3 of these molecules in a patient in need thereof. Citation or identification of any reference in this section, or any section of this application shall not be construed as an admission that such reference is available as prior art to the present invention.
3. SUMMARY OF THE INVENTION The present invention provides compositions and methods for the delivery of hydrophilic molecules and other poorly absorbed water-soluble molecules to an animal. In particular, the present invention relates to compositions and methods for the delivery of biologically active molecules, including hydrophilic and poorly absorbed water-soluble molecules. In accordance with the present invention, biologically active molecules include, but are not limited to, therapeutic agents, diagnostic agents, antigens, antibodies, peptides, polypeptides, viruses, nucleic acids, growth factors, cytokines, and drugs. The present invention provides stable reverse micelle compositions suitable for the mucosal delivery of biologically active molecules, such as, e.g., therapeutic agents, diagnostic agents, and antigens. The reverse micelle compositions of the present invention promote the absorption 5 of biologically active molecules by mucosal tissues. The reverse micelle compositions of the invention reduce the dosage of a biologically active molecule necessary to achieve a prophylactic or therapeutic effect in an animal, and thus, reduce the toxicity associated with administering higher dosages of certain biologically active molecules. Further, the reverse micelle compositions of the invention reduce the dosage of a diagnostic agent necessary to ø diagnose or monitor the state of a disease or disorder in an animal.
The present invention provides reverse micelle compositions comprising a surfactant {e.g., a P-glycoprotein inhibitor), a hydrophilic phase, and one or more biologically active molecules. In one embodiment, the surfactant is a P-glycoprotein inhibitor. The present invention also provides reverse micelle compositions comprising one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and one or more biologically active agents. The present invention further provides reverse micelle compositions comprising one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and one or more biologically active molecules, wherein less than about 15%, less than 10%, less than 5%, or less than 2% by weight of the reverse micelle composition is ø triester. Preferably, the fatty acids in the fatty acid esters of the reverse micelle composition have a length of about 6 to about 12 carbon atoms.
The present invention also provides reverse micelle compositions which are self-emulsifying dispersions comprising monoglycerides, diglycerides, or hydrophilic derivatives thereof, a hydrophilic phase, and one or more biological active molecules. The 5 present invention encompasses reverse micelle compositions comprising monoglycerides or diglycerides or a mixture thereof, a hydrophilic phase, and one or more biologically active molecules, wherein the acyl groups of the monoglycerides or diglycerides are enriched in fatty acids having 6-12 carbon atoms. The monoglycerides or diglycerides of the reverse micelle compositions of the invention may be partially derivatized with a hydrophilic moiety to provide polarity to increase water solubility. Preferably, the reverse micelle compositions of the invention comprise less than 15%, less than 10%, less than 5%, or less than 2% by weight of triglycerides .
Optionally, the reverse micelle compositions of the invention are encapsulated for oral delivery in, e.g., starch or gelatin capsules. Further, the reverse micelle compositions of the invention may optionally comprise an adjuvant when the biologically active molecule being administered to an animal is an antigen. The reverse micelle compositions of the ι ø present invention have prophylactic and therapeutic utility. The reverse micelle compositions of the present invention also have utility in diagnosing and/or monitoring the state of a variety of diseases and disorders in an animal.
The present invention provides reverse micelle compositions comprising a surfactant, a hydrophilic phase, and one or more biologically active molecules, wherein at
1 5 least one of the biologically active molecules is a protein, polypeptide or peptide. In a specific embodiment, reverse micelle compositions comprise one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and one or more biologically active molecules, wherein at least one of the biologically active molecules is a protein, polypeptide or peptide. In accordance with this embodiment, the reverse micelle compositions comprise
2ø less than 15%, less than 10%, less than 5%, or less than 2% by weight of triester.
Preferably, the peptides incorporated in the reverse micelle compositions of the invention have a molecular weight ranging from 500 to 10,000 daltons, more preferably from 500 to 5,000 daltons. In particular, hormones {e.g., luteinizing hormone-releasing hormone (LHRH), parathyroid hormone (PTH), calcitonin, insulin, and growth hormone) or agonists
2 thereof {e.g. , LHRH agonists) may be incorporated into the reverse micelles of the invention. In a preferred embodiment, reverse micelle compositions comprise one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and one or more LHRH agonists. Examples of LHRH agonists include, but are not limited to, leuprolide, goserelin, nafarelin and histrelin. Preferably, the LHRH agonist is leuprolide. oø In one embodiment, the reverse micelle compositions comprise one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and parathyroid hormone. In another embodiment, reverse micelle compositions comprise one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and calcitonin. In another embodiment, reverse micelle compositions comprise one or more fatty acid esters or
35 hydrophilic derivatives thereof, a hydrophilic phase, and heparin, preferably low molecular weight heparin. In another embodiment, reverse micelle compositions comprise one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and human growth hormone. In another embodiment, reverse micelle compositions comprise one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and insulin. In accordance with these embodiments, the reverse micelle compositions comprise less than 15%, less than 10%, less than 5%, or less than 2% by weight of triester. The present invention is based, in part, on Applicants' discovery that the bioavailability of peptide or protein drugs delivered mucosally using reverse micelle compositions comprising medium chain monoglycerides, diglycerides or a mixture thereof {e.g., monoglycerides or diglycerides with 6-12 carbon atom fatty acid side chains) and less than 10 % triglycerides are at least equivalent to that of previously known reverse micelles i Q consisting of medium chain monoglycerides, medium chain diglycerides, greater than 20 % triglycerides and other surfactants. The present invention is also based, in part, on Applicants' discovery that the simple reverse micelle compositions of the invention provide for high biovailability of peptides or proteins without the need for complex water-in-oil microemulsions. Further, the present invention is based, in part, on Applicants' surprising
1 discovery that reverse micelle compositions comprising partial monoglycerides, diglycerides or ethoxylated or polyglycolized analogs thereof incorporate therapeutically effective amounts of therapeutic agents in a biphasic system which is thermodynamically stable and optically clear and transparent (the clarity of the particle being indicative of the presence of an isotropic micelle phase, such as the reverse micelle phase).
2ø The present invention provides methods for the delivery of one or more biologically active molecules to an animal, said methods comprising administering to said animal a reverse micelle composition comprising a surfactant, a hydrophilic phase, and one or more biologically active molecules. In particular, the present invention provides methods for the delivery of one or more biologically active molecules to an animal, said methods comprising 5 administering to said animal a reverse micelle composition comprising one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and one or more biologically active molecules. Preferably, such a composition comprises less than 15%, less than 10%, less than 5%, or less than 2% by weight of triester. In a specific embodiment, the present invention provides methods for the mucosal delivery of one or more biologically
.,0 active molecules to an animal, said methods comprising mucosally administering to said animal a reverse micelle composition comprising monoglycerides, diglycerides or hydrophilic derivatives thereof, a hydrophilic phase, and one or more biological active molecules. In another embodiment, the present invention provides methods for the mucosal delivery of one or more biologically active molecules to an animal, said methods comprising 5 mucosally administering to said animal a reverse micelle composition comprising monoglycerides or diglycerides or a mixture thereof, a hydrophilic phase, and one or more biologically active molecules, wherein the acyl groups of the monoglycerides or diglycerides are enriched in fatty acids having 6-8, 6-10, 6-12, 6-20, 8-10, 8-12, or 8-20 carbon atoms. In accordance with this embodiment, the monoglycerides or diglycerides may be partially derivatized with a hydrophilic moiety to provide polarity to increase water solubility. Preferably, the reverse micelle compositions administered to an animal comprise less than
5 15%, less than 10%, less than 5%, or less than 2% by weight of triglycerides.
The present invention provides methods for the prevention, treatment, or amelioration of one or more symptoms associated with a disease or disorder in an animal, said methods comprising administering to an animal in need thereof an effective amount of a reverse micelle composition comprising a surfactant, a hydrophilic phase, and one or more ι ø prophylactic or therapeutic agents useful in the prevention, treatment or amelioration of one or more symptoms associated with said disease or disorder. In particular, the present invention provides methods for the prevention, treatment or amelioration of one or more symptoms associated with a disease or disorder in an animal, said methods comprising administering to an animal in need thereof an effective amount of a reverse micelle composition comprising one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and one or more prophylactic or therapeutic agents useful in the prevention, treatment or amelioration of one or more symptoms associated with said disease or disorder. Preferably, the reverse micelle compositions of the invention administered to an animal comprise less than 15%, less than 10%, less than 5%, or less than 2% by weight of
2Q triesters.
In a specific embodiment, the present invention provides methods for the prevention, treatment or amelioration of one or more symptoms associated with a disease or disorder in an animal, said methods comprising mucosally administering to an animal in need thereof an effective amount of a reverse micelle composition comprising monoglycerides, diglycerides
25 or hydrophilic derivatives thereof, a hydrophilic phase, and one or more prophylactic or therapeutic agents useful in the prevention, treatment or amelioration of one or more symptoms associated with said disease or disorder. In another embodiment, the present invention provides methods for the prevention, treatment or amelioration of one or more symptoms associated with a disease or disorder in an animal, said methods comprising Q mucosally administering to an animal in need thereof an effective amount of a reverse micelle composition comprising monoglycerides or diglycerides or a mixture thereof, a hydrophilic phase, and one or more prophylactic or therapeutic agents useful in the prevention, treatment or amelioration of one or more symptoms associated with said disease or disorder, wherein the acyl groups of the monoglycerides or diglycerides are enriched in
3 fatty acids having 6-8, 6-10, 6-12, 8-10, or 8-12 carbon atoms. In accordance with this embodiment, the monoglycerides or diglycerides may be partially derivatized with a hydrophilic moiety to provide polarity to increase water solubility. Preferably, the reverse micelle compositions administered to an animal comprise less than 15%, less than 10%, less than 5%, or less than 2% by weight of triglycerides.
The present invention provides methods for the prevention, treatment or amelioration of one or more symptoms associated with a disease or disorder in an animal, said methods
5 comprising administering to said animal a reverse micelle composition comprising a surfactant, a hydrophilic phase, and one or more biologically active molecules, wherein at least one of the biologically active molecules is a protein, polypeptide or peptide. In a specific embodiment, the present invention provides methods for the prevention, treatment or amelioration of one or more symptoms associated with a disease or disorder in an animal, ι ø said methods comprising administering to said animal a reverse micelle composition comprising one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and one or more biologically active molecules, wherein at least one of the biologically active molecules is a protein, polypeptide or peptide. In accordance with this embodiment, the reverse micelle composition administered to an animal comprises less than
15 15%), less than 10%, less than 5%, or less than 2% by weight of triester. In particular, the present invention provides methods of administering hormones {e.g., luteinizing hormone- releasing hormone (LHRH), parathyroid hormone (PTH), calcitonin, insulin, and growth hormone) and agonists thereof (e.g., LHRH agonists) to an animal in need thereof to prevent, treat or ameliorate one or more symptoms associated with a disease or disorder
2ø utilizing the reverse micelle compositions of the invention.
In a specific embodiment, the present invention provides methods for the prevention, treatment or amelioration of one or more symptoms associated with prostate cancer, endometriosis, precocious puberty, uterine lelomyotama, fertility disorder, premenopausal breast cancer, endometiral cancer, ovarian cancer, benign prostatic hypertrophy, functional
25 bowel disease, cluster headache, premenstrual syndrome, idiopathic hirsuitism, hirsuitism second to polycycstic ovarian disease, adenomyosis, Meniere's disease, sickle cell anaemia associated priapism or catamental pneumothorax, said methods comprising administering to an animal in need thereof an effective amount of a reverse micelle composition comprising one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and one
3 or more LHRH agonists. In another embodiment, the present invention provides methods for the prevention, treatment or amelioration of one or more symptoms associated with hypopituitarism, hypothyroidism, human growth hormone deficiency, Gushing' s syndrome, nutritional short stature, intrauterine growth retardation, Russell Silver syndrome or achondroplasia, said methods comprising administering to an animal in need thereof an
35 effective amount of a reverse micelle composition comprising one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and growth hormone, preferably human growth hormone. In another embodiment, the present invention provides methods for the prevention, treatment or amelioration of one or more symptoms associated a bone- reabsorption disease such as osteoporosis, metastatic bone cancer, osteolytic lesions with an orthopedic implant, Paget's disease, or bone loss associated with hyperparathyroidism, said methods comprising administering to an animal in need thereof an effective amount of a
5 reverse micelle composition comprising one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and parathyroid hormone or calcitonin. In another embodiment, the present invention provides methods for the prevention, treatment or amelioration of one or more symptoms associated with diabetes, said methods comprising administering to an animal in need thereof an effective amount of a reverse micelle ι ø composition comprising one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and insulin. In accordance with these embodiments, the reverse micelle compositions comprise less than 15%, less than 10%, less than 5%, or less than 2% by weight of triester.
The present invention provides methods for diagnosing or monitoring the state of a
15 disease or disorder, said methods comprising administering to an animal an amount of a reverse micelle composition comprising a surfactant, a hydrophilic phase, and one or more diagnostic agents useful in the diagnosis of said disease or disorder. In particular, the present invention provides methods for diagnosing or monitoring the state of a disease or disorder, said methods comprising administering to an animal an effective amount of a
2ø reverse micelle composition comprising one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and one or more diagnostic agents useful in the diagnosis said disease or disorder. In a specific embodiment, the present invention provides methods diagnosing or monitoring the state of a disease or disorder, said methods comprising mucosally administering an animal an effective amount of a reverse micelle
25 composition comprising monoglycerides, diglycerides or a hydrophilic derivative thereof, a hydrophilic phase, and one or more diagnostic agents useful in the diagnosis of said disease or disorder. In another embodiment, the present invention provides methods diagnosing or monitoring the state of a disease or disorder, said methods comprising mucosally administering to an animal an effective amount of a reverse micelle composition comprising
3ø monoglycerides or diglycerides or a mixture thereof, a hydrophilic phase, and one or more diagnostic agents useful in the diagnosis of said disease or disorder, wherein the acyl groups of the monoglycerides or diglycerides are enriched in fatty acids having 6-8, 6-10, 6-12, 8-10, or 8-12 carbon atoms. In accordance with this embodiment, the monoglycerides or diglycerides may be partially derivatized with a hydrophilic moiety to provide polarity to
35 increase water solubility. Preferably, the reverse micelle compositions administered to an animal comprise less than 15%, less than 10%, less than 5%, or less than 2% by weight of triglycerides.
The present invention provides kits comprising in an appropriate container(s) reverse micelle compositions comprising a surfactant, a hydrophilic phase, and one or more biologically active molecules. In particular, the present invention provides kits comprising
5 in an appropriate container(s) reverse micelle compositions comprising one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and one or more biologically active agents. Preferably, the reverse micelle compositions included in the kits of the invention comprise less than 15%, less than 10%, less than 5%, or less than 2% by weight of triester. The present invention also provides kits comprising reverse micelle ι compositions comprising monoglycerides, diglycerides, or hydrophilic derivatives thereof, a hydrophilic phase, and one or more biological active molecules. The present invention further provides kits comprising in an appropriate container(s) reverse micelle compositions comprising monoglycerides or diglycerides or a mixture thereof, a hydrophilic phase, and one or more biologically active molecules, wherein the acyl groups of the monoglycerides or 5 diglycerides are enriched in fatty acids having 6-12 carbon atoms. The reverse micelle compositions included in the kits of the invention may comprise monoglycerides or diglycerides which are partially derivatized with a hydrophilic moiety to provide polarity to increase water solubility. Preferably, the reverse micelle compositions included in the kits of the invention comprise less than 15%, less than 10%, less than 5%, or less than 2% by
2ø weight of triglycerides. The reverse micelle compositions of the invention included in kits may be formulated in a compatible pharmaceutical carrier. Preferably, the kits of the invention are accompanied by instructions for administration. The kits of the invention may further comprise a list of the diseases and/or disorders for which the reverse micelle compositions may be used to prevent, treat, diagnose or monitor.
25 The present invention provides kits comprising in one or more containers one or more reverse micelle compositions comprising a surfactant, a hydrophilic phase, and one or more biologically active molecules, wherein at least one of the biologically active molecules is a protein, polypeptide or peptide. In a specific embodiment, the kits of the invention comprise in one or more containers reverse micelle compositions comprising one or more
3 fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and one or more biologically active molecules, wherein at least one of the biologically active molecules is a protein, polypeptide or peptide. In accordance with this embodiment, the reverse micelle compositions included in the kits comprise less than 15%, less than 10%, less than 5%, or less than 2% by weight of triester. In particular, the present invention provides kits
3 comprising reverse micelle compositions comprising hormones {e.g., luteinizing hormone- releasing hormone (LHRH), parathyroid hormone (PTH), calcitonin, insulin, and growth hormone) or agonists thereof (e.g., LHRH agonists).
In a preferred embodiment, the kits of the invention comprise in one or more containers one or more reverse micelle compositions comprising one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and one or more LHRH
5 agonists. In another preferred embodiment, the kits of the invention comprise in one or more containers reverse micelle compositions comprising one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and growth hormone, preferably human growth hormone. In another preferred embodiment, the kits of the invention comprise in one or more containers reverse micelle compositions comprising one or more fatty acid i Q esters or hydrophilic derivatives thereof, a hydrophilic phase, and parathyroid hormone or calcitonin. In yet another preferred embodiment, the kits of the invention comprise in one or more containers reverse micelle compositions comprising one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and insulin. In accordance with these embodiments, the reverse micelle compositions included in the kits comprise less than 15%,
15 less than 10%), less than 5%, or less than 2% by weight of triester.
3.1 Definitions
As used herein the terms "hydrophilic phase" and "aqueous phase" refer to compounds which are miscible with water including, but are not limited to, water, glycerol,
2ø sorbitol, mannitol, propylene glycol, ethylene glycol, polyethylene glycol, buffering agents, tonicity agents, oxidizing agents, reducing agents, antimicrobial agents, preservatives, other stabilizing agents, or mixtures thereof.
As used herein, the term "buffer solution" is defined as an aqueous solution or aqueous solution containing less than 25% of a miscible organic solvent, in which a buffer
25 has been added to control the pH of the solution. Examples of suitable buffers include, but are not limited to, acetate buffer, citrate buffer, PBS (phosphate buffered saline), TRIS (tris-(hydroxymethyl)aminomethane), HEPES (hydroxyethylpiperidme ethane sulfonic acid), sodium acetate, sodium phosphate, and TES (2-[(tris-hydroxymethyl)methyl] amino-1-ethanesulfonic acid).
30 As used herein, the terms "mucosa" and "mucosal" refer to a mucous tissue such as epithelium, lamina propria, and a layer of smooth muscle in the digestive tract. "Mucosal delivery", "mucosal administration" and analogous terms as used herein refer to the administration of a composition to the mucosal tissue. Mucosal delivery", "mucosal administration" and analogous terms include, but are not limited to, the delivery of a
35 composition the through bronchi, gingival, lingual, nasal, oral, vaginal, rectal, and intestinal mucosal tissue. 4. BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. Pharmacokinetics of human growth hormone in rats following intraduodenal administration.
FIG. 2. Pharmacokinetics of LHRH in rats following intraduodenal administration. FIG. 3. Resistivity of various RM (Reverse Micelle) formulations containing electrolytes upon dilution with deionized water.
FIG. 4 Leuprolide plasma concentration profile in dogs following subcutaneous and intraduodenal administration.
0 5. DETAILED DESCRIPTION OF THE INVENTION
The present invention provides compositions and methods for the delivery of hydrophilic molecules and other poorly absorbed water-soluble molecules to an animal. In particular, the present invention relates to compositions and methods for the delivery of biologically active molecules, including hydrophilic and poorly absorbed water-soluble 5 molecules. In accordance with the present invention, biologically active molecules include, but are not limited to, therapeutic agents, diagnostic agents, antigens, antibodies, peptides, polypeptides, viruses, nucleic acids, growth factors, cytokines, and drugs. The reverse micelle compositions of the present invention promote the absorption of biologically active molecules by mucosal tissues. The reverse micelle compositions of the invention also ø reduce the dosage of a biologically active molecule necessary to achieve a prophylactic or therapeutic effect in an animal, and thus, reduce the toxicity associated with administering higher dosages of certain biologically active molecules. Further, the reverse micelle compositions of the invention reduce the dosage of a diagnostic agent necessary to diagnose or monitor the state of a disease or disorder. 5 The invention provides for reverse micelle compositions comprising a surfactant
{e.g., a P-glycoprotein inhibitor), a hydrophilic phase and one or more biologically active molecules. In particular, the invention provides for reverse micelle compositions comprising one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and a biologically active molecule, wherein the reverse micelle compositions have less than 15%, preferably less than 10%, less than 5%, or less than 2% by weight of triester. Preferably, the reverse micelle compositions comprise fatty acid esters having fatty acids with a length of about 6 to about 12 carbon atoms. The present invention also provides reverse micelle compositions comprising monoglycerides, diglycerides or mixtures thereof, a hydrophilic phase, and one or more biologically active molecules. Optionally, the reverse micelle 5 compositions of the invention may combined with one or more pharmaceutically acceptable carriers, diluents or excipients. The reverse micelle compositions of the invention may be used to administer a variety of biologically active molecules to prevent, treat, or ameliorate the symptoms associated with disease or disorders. The reverse micelle compositions of the invention may also be used to administer a diagnostic agent to facilitate the diagnosis of a disease or a disorder. The invention provides methods for administering a biologically active molecule to an animal, said methods comprising administering to said animal an effective amount of a reverse micelle composition comprising a surfactant, a hydrophilic phase, and a biologically active molecule. In particular, the present invention provides methods for administering a biologically active molecule to an animal, said methods comprising administering to said animal an effective amount of a reverse micelle composition comprising one or more fatty ι acid esters or a hydrophilic derivative thereof, a hydrophilic phase and a biologically active molecule, wherein the reverse micelle compositions comprise less than about 10% by weight of triester, preferably about 5% by weight of triester. In a preferred embodiment of the invention, the reverse micelle compositions of the invention are administered mucosally to an animal as a capsule, soft elastic gelatin-capsule, caplet, aerosol, spray, solution,
15 suspension, emulsion, cachet, tablet, capsule, soft elastic gelatin capsule, aerosol, powder or granule. Preferably, the reverse micelle compositions of the invention are administered to a mammal, more preferably a human to prevent, treat, diagnose or monitor a disease or disorder.
2ø 5.1 Reverse Micelles
The invention provides for the use of a single component surfactant micelle which avoids more complex mixtures of oils and surfactants, yet still allows for suitable transepithelial transport of biologically active molecules. The biologically active molecule is contained within the hydrophilic phase of a reverse micellar (L2) phase of a single type of
25 surfactant or polar lipid. In particular, the present invention also provides reverse micelle compositions comprising of a mixture of a surfactant, an aqueous or hydrophilic phase, and one or more biologically active molecules. In one embodiment, the present invention provides reverse micelle compositions comprising a surfactant, a hydrophilic phase and one or more biologically active molecules, wherein the surfactant is a P-glycoprotein inhibitor. 0 In another embodiment, the reverse micelle compositions of the invention comprise one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and one or more biologically active molecules. Preferably, such compositions comprise less than about 15%, less than 10%, less than 5%, or less than 2% by weight of triester and the fatty acid in fatty acid esters of the reverse micelle compositions have a length of about 6 to about 20
3 carbon atoms, more preferably about 6 to about 12 carbon atoms, most preferably about 8 to about 10. In a specific embodiment, the reverse micelle compositions of the invention comprise monoglycerides or diglycerides or a mixture thereof, a hydrophilic phase, and one or more biologically active molecules. In a preferred embodiment, the reverse micelle compositions of the invention comprise monoglycerides, diglycerides or mixtures thereof, a hydrophilic phase, and one or more biologically active molecules, wherein the acyl groups of the monoglycerides or diglycerides are enriched in fatty acids having 6-8, 6-10, 6-12, 8-10
5 or 8-12 carbon atoms. In accordance with this embodiment, the monoglycerides or diglycerides may be partially derivatized with a hydrophilic moiety to provide polarity to increase water solubility. Preferably, the reverse micelle compositions of the invention comprise less than 15%, less than 10%, less than 5%, or less than 2% by weight of triglycerides. Optionally, the reverse micelle compositions of the invention may comprise ι ø buffering, oxidizing, reducing and/or tonicity agents in the hydrophilic phase to provide adequate solubility and stability of the biologically active molecule.
In a specific embodiment, the hydrophilic phase of the reverse micelle compositions of the invention comprises an amount from about 0 to about 70%, about 0 to about 65%, about 0 to about 50%, about 0 to about 40%, about 0 to about 30%, about 0 to about 20%,
15 about 5% to about 70%, about 5 to about 65%, about 5 to about 50%, about 5 to about 40%, about 5 to about 30%, about 5 to about 20%, about 5% to about 15%, or about 5% to about 10% by weight of the reverse micelle. In another embodiment, the hydrophilic phase of the reverse micelle compositions of the invention comprises an amount from 0 to 70%, 0 to 65%, 0 to 50%, 0 to 40%, 0 to 30%, 0 to 20%, 5% to 70%, 5 to 65%, 5 to 50%, 5 to 40%, 5
2 to 30%, 5 to 20%, 5% to 15%, or 5% to 10% by weight of the reverse micelle. In apreferred embodiment, the hydrophilic phase of the reverse micelle compositions of the invention comprise an amount from about 5% to about 25% by weight of the reverse micelle, more preferably 5% to 25% by weight of the reverse micelle. Examples of compounds which may be included in the hydrophilic phase of a reverse micelle composition of the invention
25 include, but are not limited to, water, glycerol, sorbitol, mannitol, propylene glycol, ethylene glycol and polyethylene glycol or mixtures thereof. In a preferred embodiment, the hydrophilic phase of the reverse micelle compositions of the invention comprise water.
The surfactant incorporated in the reverse micelle compositions of the invention may be non-ionic in nature, that is having a neutral overall electrical charge, or ionic in nature,
3 that is being positively or negatively charged without affecting the stability of the compositions. In a preferred embodiment of the invention, the surfactants incorporated in the reverse micelle composition are non-ionic in nature. The surfactant incorporated into a reverse micelle composition of the invention may be a single compound or a mixture of compounds. Preferably, the reverse micelle compositions of the invention only comprise
35 one surfactant. Examples of surfactants include, but are not limited to, fatty acid esters and hydrophilic analogs thereof. In a preferred embodiment, the surfactant is one or more fatty acid esters or hydrophilic derivatives thereof. Preferably, the surfactant is: (1) monoglycerides or hydrophilic derivatives or analogs thereof; (2) diglycerides or hydrophilic derivatives or analogs thereof; or (3) a mixture of monoglycerides or hydrophilic derivatives or analogs thereof and diglycerides or hydrophilic derivatives or analogs thereof. In a specific embodiment, the surfactant incorporated in the reverse micelle compositions of the
5 present invention is a P-glycoprotein inhibitor.
Fatty acid chain lengths of 8-10 carbon atoms enriched with monoglycerides, diglycerides and their polyoxyethylated analogs can, e.g., be derived from coconut oil by alcoholysis and transesterification reactions. In another method, these esters can be derived by direct esterification of glycerol using C8/C10 fatty acid esters in the presence of ι ø polyethylene glycol or ethylene oxide if so desired. The following medium chain fatty acid monoglycerides, diglycerides/polyoxyethylene esters are available commercially under different names as presented in Table 1 and can be used in the reverse micelle compositions of the invention.
15
Table 1. Medium chain fatty acid glycerol/polyoxyethylene esters
20
25
30
35
Figure imgf000018_0001
Figure imgf000019_0001
For stable reverse micelle formation, the surfactant is generally chosen from unsubstituted or partially substituted monoglycerides and diglycerides having fatty acids with a length of 6-20 carbon atoms (C6-C20), preferably 6-12 carbon atoms, most preferably 8-10 carbon atoms. The most preferred substitution of the monoglycerides and diglycerides is ethoxylation or pegylation. Other suitable hydrophilic analogs of monoglycerides include, but are not limited, lactic acid, acetic acid, citric acid, succinic acid, and diacetyl tartaric acid esters. Typically the surfactant has an HLB value between about 1 and about 40, preferably between about 1 and about 20, most preferably between about 5 and about 20. Pegylated or polyglycolized glycerides are derived in the synthesis from a mixture of monoglycerides, diglycerides and triglycerides and polyethylene glycol (PEG) monoesters and diesters, usually with a molecular weight (MW) between 200 and 10,000 daltons, preferably between 200 and 4,000 daltons. The HLB value of the polyglycolized glycerides is adjusted by the length of the PEG chain and of the length and degree of saturation of the fatty acid substitutions. In a preferred embodiment of this invention, the surfactant is composed of C8-C10 substituted polyglycolized glycerides, having an HLB value less than 20 and preferably, between 5 and 15.
Reverse micelle compositions of the invention may be fabricated with a surfactant in which a biologically active molecule is functionally solubilized in the hydrophilic phase of the reverse micelle. Preferably, reverse micelle compositions of the invention are fabricated with chemically-modified monoglycerides or diglycerides in which the biologically active molecule is functionally solubilized in the hydrophilic phase of the reverse micelle. The hydrophilic moiety of the modified glyceride is a hydrophilic group, such as polyethylene glycol of various chain lengths. Medium chain fatty acid glyceride side chains are from 6-8,
6-10, 6-12, 6-20, 8-10, 8-12, or 8-20 carbons in length. The characteristics of the reverse micelle compositions can be modified and adjusted according to chemical conjugation of other surfactant active groups to the glyceride backbone. The resulting self-emulsifying systems are advantageous since they can be formed without the addition of oil and additional surfactant molecules.
5.2. Biologically Active Molecules
The reverse micelles of the present invention may be utilized for the delivery of a wide variety of biological active molecules. As used herein, the term "biologically active molecule" and analogous terms refer to eukaryotic and procaryotic cells, viruses, vectors, proteins, peptides, polypeptides, nucleic acids {e.g., DNA and RNA nucleotides including, but not limited to, antisense nucleotide sequences, triple helices and nucleotide sequences encoding biologically active proteins, polypeptides or peptides), saccharides, polysaccharides, carbohydrates, lipids, glycoproteins, and combinations thereof, and synthetic organic drugs and inorganic drugs exerting a biological effect when administered to an animal. In a preferred embodiment, biologically active molecules have an aqueous solubility of greater than 0.1 mg/ml, preferably greater than 1 mg/ml. Examples of
5 biologically active molecules include, but are not limited to, anti-angiogenesis factors, antibodies {e.g., monoclonal antibodies, scFvs and Fab fragments), antigens {e.g., viral, microbial or tumor-associated antigens), growth factors, hormones, enzymes, peptides (preferably, peptides with a molecular weight (MW) from about 500 to about 10,000 daltons, more preferably with a MW from about 500 to about 5,000 daltons), drugs {e.g., ι ø steroids, anti-cancer drugs such as chemotherapeutic agents, antiviral agents, anti- inflammatory agents and antibiotics), insecticides, insect repellents, fertilizers, vitamins, or any other material having a biological effect. The reverse micelle compositions of the invention may be engineered to contain a biologically active molecule derived from the same or different species as the recipient of the reverse micelle composition. Thus, in a
15 preferred embodiment, a reverse micelle composition containing a biologically active molecule derived from a human is administered to a human.
The reverse micelles compositions of the invention have utility for the mucosal delivery of a wide variety of vaccines and/or antigens. For example, the reverse micelles compositions of the present invention may be designed to carry a wide variety of antigens
2 including, but not limited to, diphtheria toxoid, tetanus toxoid, ospA antigen from Lyme disease bacterium, HTLV-1 or HTLV-2 antigens {e.g., HTLV-1 envelope protein or an antigenic fragment thereof), influenza virus antigens {e.g., influenza virus hemagglutinin or an antigenic fragment thereof), polio virus antigens, rhinovirus antigens, rabies virus antigens, vaccinia virus antigens, Epstein-Barr virus antigens, hepatitis virus antigens,
25 HIV-1 and HIV-2 antigens {e.g., glycoprotein 120 or fragment thereof), and herpes virus antigens. The reverse micelle compositions may be engineered to contain an antigen derived from any species.
The reverse micelle compositions of the invention may also be utilized for the mucosal delivery of a wide variety of prophylactic or therapeutic agents. As used herein, the term
3ø "prophylactic agents" and analogous terms refer to biologically active molecules which can be used to prevent the onset, development or progression of one or more symptoms of a disease and/or disorder. As used herein, the term "therapeutic agent" and analogous terms refer to biologically active molecules which can be used to treat or ameliorate one or more symptoms associated with a disease and/or a disorder. Examples of therapeutic agents include, but are not limited to, chemotherapeutic agents, antibiotics, cytokines, hormones, enzymes {e.g., superoxide dismutase, asparaginase, arginase, arginine deaminase, adenosine deaminase, ribonuclease, trypsin, chymotrypsin and papain), tachykinin receptor agonists and antagonist peptides, vasoactive intestinal peptide, calcitonins, vasopressins, growth hormone releasing peptide, luteinizing hormone-releasing hormone (LHRH) agonists, fibrinogen receptor antagonists (RGD peptides see, e.g., the RGD peptide described in International Publication No. WO 93/02664) having in their sequence arginine-glycine-D-
5 aspartic acid), fertility drugs, antiviral agents {e.g., ddl, AZT, ddC, acyclovir and the like), antibacterial agents, antifungal agents, and DNA and RNA nucleotides (including antisense nucleotide sequences, triple helices, and nucleotide sequences encoding peptides,. polypeptides or proteins). Examples of chemotherapeutic agents include, but are not limited to, arabinofuranosyladenine, acylguanosine, Nordeoxyguanosine, dideoxyadenosine, ι dideoxycytidine, dideoxyinosine Floxuridine, 6-mercaptopurine, doxorubicin, Daunorubicin, I-darubicin, quinidine, cisplatin, carboplatin, epirubicin, leuprolide, goserelin, nafarelin, histrelin, bicalutamide, goserelin, nafarelin, irinotecan, gemcitabine, and sargramostim. The peptides described in International Publication No. WO 93/02664 are incorporated herein by reference, in particular the peptides described on pages 10-12.
15 Examples of antibiotics include, but are not limited to, aminoglycoside antibiotics
{e.g., apramycin, arbekacin, bambermycins, butirosin, dibekacin, neomycin, neomycin, undecylenate, netilmicin, paromomycin, ribostamycin, sisomicin, and spectinomycin), amphenicol antibiotics {e.g., azidamfenicol, chloramphenicol, florfenicol, and thiamphenicol), ansamycin antibiotics {e.g., rifamide and rifampin), carbacephems {e.g.,
2 loracarbef), carbapenems {e.g., biapenem and imipenem), cephalosporins {e.g., cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefozopran, cefpimizole, cefpiramide, and cefpirome), cephamycins {e.g., cefbuperazone, cefmetazole, and cefminox), monobactams {e.g., aztreonam, carumonam, and tigemonam), oxacephems (e.g., flomoxef, and moxalactam), penicillins (e.g., amdinocillin, amdinocillin pivoxil, amoxicillin, 5 bacampicillin, benzylpenicillinic acid, benzylpenicillin sodium, epicillin, fenbenicillin, floxacillin, penamccillin, penethamate hydriodide, penicillin o-benethamine, penicillin 0, penicillin V, penicillin V benzathine, penicillin V hydrabamine, penimepicycline, and phencihicillin potassium), lincosamides {e.g., clindamycin, and lincomycin), macrolides (e.g. azithromycin, carbomycin, clarithomycin, dirithromycin, erythromycin, and ø erythromycin acistrate), amphomycin, bacitracin, capreomycin, colistin, enduracidin, enviomycin, tetracyclines (e.g., apicycline, chlortetracycline, clomocycline, and demeclocycline), 2,4-diaminopyrimidines (e.g., brodimoprim), nitrofurans (e.g., furaltadone, and furazolium chloride), quinolones and analogs (e.g., cinoxacin, ciprofloxacin, clinafloxacin, flumequine, and grepagloxacin), sulfonamides (e.g., acetyl sulfamethoxypyrazine, benzylsulfamide, noprylsulfamide, phthalylsulfacetamide, sulfachrysoidine, and sulfacytine), sulfones (e.g., diathymosulfone, glucosulfone sodium, and solasulfone), cycloserine, mupirocin and tuberin.
Examples of cytokines include, but are not limited to, interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin 15 (IL-15), interleukin 18 (IL-18), platelet derived growth factor (PDGF),
5 erythropoietin (Epo), epidermal growth factor (EGF), fibroblast growth factor (FGF), granulocyte macrophage stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), prolactin, and interferon (IFN, e.g., IFN-alpha, IFN-beta, and IFN-gamma). Examples of hormones include, but are not limited to, luteinizing hormone releasing hormone (LHRH), growth hormone (GH), ι ø growth hormone releasing hormone, ACTH, somatostatin, somatotropin, somatomedin, parathyroid hormone, hypothalamic releasing factors, parathyroid hormone, insulin, glucagon, enkephalins, vasopressin, calcitonin, heparin, low molecular weight heparins, heparinoids, synthetic and natural opioids, insulin thyroid stimulating hormones, and endorphins.
1 In a preferred embodiment, a reverse micelle composition of the invention comprises LHRH or an analog thereof. In another preferred embodiment, a reverse micelle composition of the invention comprises LHRH agonists such as leuprolide, goserelin, nafarelin and histrelin. In another preferred embodiment, a reverse micelle composition of the invention comprises parathyroid hormone or calcitonin. In another preferred
2 embodiment, a reverse micelle composition of the invention comprises insulin. In a still further preferred embodiment, a reverse micelle composition of the invention comprises human growth hormone or an analog thereof. The amount of the peptides incorporated into the reverse micelle compositions is determined by their solubility in the hydrophilic phase. Preferably, the amount of biologically active molecule included in the reverse micelle
25 composition is from about 0.05 to about 100 mg/ml, more preferably from about 0.05 to about 50 mg/ml, and most preferably from about 0.05 to about 10 mg/ml.
For ease of reference, the term "biologically active molecule" is also used herein to include diagnostic agents. Examples of diagnostic agents include, but are not limited to, radio-opaque compounds, magnetic compounds, fluorescent compounds, radioactive
3Q compounds, and other contrast agents used with ultrasound, x-rays, fluorescence, MRI, CT, and other techniques known to those skilled in the art. Formulation of these materials is typically critical for effective delivery, detection sensitivity, targeting to an intended site, and for improved comfort to the patient.
35 5.3. Modes of Administering Reverse
Micelle Compositions to an Animal
The reverse micelle compositions are particularly suitable for delivery through mucosal tissue or epithelia. Accordingly, the reverse micelle compositions of the present invention are preferrably administered by those routes which optimize uptake by mucosa, for example, oral, sublingual, buccal, rectal and intranasal routes of administration. However, topical, transdermal and parenteral delivery may also be used. The most preferred route of administration of the reverse micelle composition is oral administration. The reverse micelle compositions of the invention can be delivered orally in the form of tablets, capsules, cachets, gelcaps, solutions, suspensions and the like. The reverse micelle compositions of the invention can be delivered orally in the form of tablets, capsules, cachets, gelcaps, solutions, suspensions and the like. Oral dosage forms may further fabricated to release the biologically active molecules at different regions of the GI tract, such as the small intestine and the colon, and in a time-dependent manner. When the dosage unit form of the reverse micelle composition comprising an antigen is a capsule, it can contain, in addition to the material of the above type, a liquid carrier or adjuvant. If administered topically the reverse micelles will typically be administered in the form of an ointment, cream or transdermal patch. If administered intranasally the reverse micelle composition will typically be administered in an aerosol form, spray, mist or in the form of drops. Suitable formulations can be found in Remington's Pharmaceutical Sciences, 16th and 18th Eds., Mack Publishing, Easton, PA (1980 and 1990), and Introduction to Pharmaceutical Dosage Forms, 4th Edition, Lea & Febiger, Philadelphia (1985), each of which is incorporated herein by reference.
The reverse micelle compositions of the present invention are suitable for administration to animals, in particular domestic animals and birds, and more particularly humans. For example, domestic animals such as dogs and cats, as well as domesticated herds, cattle, sheep, pigs and the like may be treated or vaccinated with the reverse micelle compositions of the present invention. In a preferred embodiment, the reverse micelle compositions of the present invention are administered to humans.
In one embodiment, a reverse micelle composition of the present invention comprising two or more biologically active molecules may be administered to an animal in need thereof. Preferably, the biologically active molecules incorporated in the reverse micelle compositions of the invention act together additively or synergistically to achieve the desired biological effect. In another embodiment, two or more reverse micelle compositions containing the same biologically active molecule may be administered to an animal in need thereof simultaneously or separately. In another embodiment, two or more reverse micelle compositions comprising one or more different biologically active molecules may be administered to an animal in need thereof simultaneously or separately. Reverse micelle compositions are generally provided in a hermetically sealed container such as an ampule or sachet, and stored at room temperature or 4°C. The reverse micelle compositions of the invention may be provided in the form of tablets, capsules, cachets, gelcaps, solutions, suspensions and the like. The reverse micelle compositions of the invention may further be lyophilized into a fine powder which can be distributed in the form of a capsule or other suitable dosage form. The maximum amount of water that can be used in the reverse micelles in a capsule depends on the type and property of the capsules. Reverse micelles having lower water contents are normally more compatible with gelatin capsules. In a specific embodiment, reverse micelles used in LiCaps gelatin capsules from CAPSUGEL have a water content by weigh of 0 to about 70%, about 0 to about 65%, about Q 0 to about 50%, about 0 to about 40%, about 0 to about 30%, about 0 to about 20%, about 0 to about 15%, about 0 to about 10%>, about 5 to about 70%, about 5 to about 60%, about 5 to about 50%, about 5 to about 40%, about 5 to about 30%, about 5 to about 20% about 5 to about 15%, or about 5 to about 10%. In a preferred embodiment, reverse micelles used in LiCaps gelatin capsules from CAPSUGEL have a water content by weight of about 0 to about 40%. In another preferred embodiment, reverse micelles used in LiCaps gelatin capsules from CAPSUGEL have a water content by weigh of about 0 to about 30%. In a more preferred embodiment, reverse micelles used in LiCaps gelatin capsules from CAPSUGEL have a water content by weigh of about 0 to about 20%. In a most preferred embodiment, reverse micelles used in LiCaps gelatin capsules from CAPSUGEL have a ø water content by weigh of about 0 to about 15%>. The compatibility of the reverse micelles with capsules can be changed by a modifier, for example a gelling agent, in the reverse micelles to reduce the interaction of water with the inner capsule wall, by applying coatings on the inner surface of the capsules, or by changing the storage conditions such as lowering the storage temperature. 5 Dosages {i.e., the effective amount — the amount of the composition sufficient to result in a desired therapeutic effect, such as treatment, prevention or amelioration of one or more symptoms of a disease or disorder) of the reverse micelle compositions will vary depending on the individual patient, the mode of administration, and the type and severity of the disease or disorder. Preferably, the dosage of the reverse micelle composition is from Q about 0.1 to about 1000 mg/kg, more preferably from about 0.1 to about 100 mg/kg, and most preferably from about 0.1 to about 50 mg/kg. Such dosages can be determined by a skilled physician using standard techniques.
The reverse micelle compositions can be used to prevent, treat or diagnose many diseases and/or disorders including, but not limited to, cancer, infectious diseases, and 5 immune disorders (e.g., autoimmune disorders, asthma, and allergies).
In a specific embodiment, the present invention provides methods for the prevention, treatment or amelioration of one or more symptoms associated with prostate cancer, endometriosis, precocious puberty, uterine lelomyotama, fertility disorder, premenopausal breast cancer, endometiral cancer, ovarian cancer, benign prostatic hypertrophy, functional bowel disease, cluster headache, premenstrual syndrome, idiopathic hirsuitism, hirsuitism second to polycycstic ovarian disease, adenomyosis, Meniere's disease, sickle cell anaemia
5 associated priapism or catamental pneumothorax, said methods comprising administering to an animal in need thereof an effective amount of a reverse micelle composition comprising one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and one or more LHRH agonists. In another embodiment, the present invention provides methods for the prevention, treatment or amelioration of one or more symptoms associated with ι ø hypopituitarism, hypothyroidism, human growth hormone deficiency, Gushing' s syndrome, nutritional short stature, intrauterine growth retardation, Russell Silver syndrome or achondroplasia, said methods comprising administering to an animal in need thereof an effective amount of a reverse micelle composition comprising one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and growth hormone, preferably
15 human growth hormone. In another embodiment, the present invention provides methods for the prevention, treatment or amelioration of one or more symptoms associated a bone- reabsorption disease such as osteoporosis, metastatic bone cancer, osteolytic lesions with an orthopedic implant, Paget's disease, or bone loss associated with hyperparathyroidism, said methods comprising administering to an animal in need thereof an effective amount of a
2ø reverse micelle composition comprising one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and parathyroid hormone or calcitonin. In another embodiment, the present invention provides methods for the prevention, treatment or amelioration of one or more symptoms associated with diabetes, said methods comprising administering to an animal in need thereof an effective amount of a reverse micelle
25 composition comprising one or more fatty acid esters or hydrophilic derivatives thereof, a hydrophilic phase, and insulin. In accordance with these embodiments, the reverse micelle compositions comprise less than 15%, less than 10%, less than 5%, or less than 2% by weight of triester.
The reverse micelle compositions of the invention can be administered in
3 combination with any known or currently used treatment for the jprevention, treatment or amelioration of one or more symptoms associated with a particular disease or disorder. For example, a reverse micelle composition of the invention comprising a chemotherapeutic agent can be administered to an animal with cancer in combination with radiation therapy.
35 5.4. Vaccine Formulations
In certain embodiments, the reverse micelle compositions comprise one or more antigens for use as vaccines. The vaccine formulations of the invention comprise a reverse micelle composition of the invention. Suitable preparations of vaccines formulations include, but are not limited to, liquid solutions or suspensions; solid forms such as capsules
5 and tablets, and liquids for injections. The active immunogenic ingredients incorporated into the vaccine formulations of the invention are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine preparation may also include minor amounts of ι ø auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine.
Antigens may be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids, such as, for example,
15 hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, maleic, and the like. Salts formed with free carboxyl groups may also be derived from inorganic bases, such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.
2ø The vaccine formulations of the invention comprise an effective amount of a reverse micelle composition and a pharmaceutically acceptable carrier or excipient. Pharmaceutically acceptable carriers are well known in the art and include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, and combinations thereof. One example of such an acceptable carrier is a physiologically
25 balanced salt solution containing one or more agents such as stabilized, hydrolyzed proteins, lactose, etc., which serve to prevent degradation of the vaccine formulation. The carrier used in the vaccine formulation is preferably sterile and the formulation should suit the mode of administration. The vaccine formulations of the invention, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The
3ø vaccine formulation can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. Vaccine formulations for oral administration can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
Generally, the vaccine formulations are provided in a hermetically sealed container
35 such as an ampule or a sachet. The vaccine formulations are generally stored at room temperature or 4°C prior to use. The vaccine formulations of the invention may be lyophilized to form a fine powder which can be distributed in the form of a capsule or other suitable dosage form.
The vaccine formulations of the invention may be multivalent or univalent. Many methods may be used to introduce the vaccine formulations of the invention; these include, but are not limited to, oral, intradermal, intramuscular, intraperitoneal, intravenous,
5 subcutaneous, intranasal, topical, rectal, and via scarification (scratching through the top layers of skin, e.g. , using a bifurcated needle). The patient to which the vaccine is administered is preferably an animal, more preferably a mammal, most preferably a human. The precise dose of vaccine formulation to be employed will depend on the route of administration, and the nature of the patient, and should be decided according to the ι ø judgment of the practitioner and each patient's circumstances according to standard clinical techniques. An effective immunizing amount is that amount sufficient to produce an immune response to the antigen in the host to which the vaccine preparation is administered.
Use of purified antigens as vaccine formulations can be carried out by standard methods. For example, the purified protein(s) should be adjusted to an appropriate
15 concentration, formulated with any suitable vaccine adjuvant and encapsulated within the reverse micelle. Suitable adjuvants may include, but are not limited to: mineral gels, e.g., aluminum hydroxide; surface active substances such as lysolecithin or pluronic polyols; polyanions; peptides; oil emulsions; alum, Lipid A and derivatives of Lipid A (e.g., monophosphoryl lipid A (MPLA)), cytokines, N-acetyl-murmyl-L-threonyl-D-isoglutamine
2ø (thr-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamine,
N-acetylmuramyl-L-isoglutaminyl-L-alanine-2( ,2'-dipalmitoyl-sn-glycero-3-hydroxyphosp horyloxy)-ethylamine, saponins, and microbial toxins (e.g., cholera toxin and heat labile toxin) and genetically altered derivatives thereof.
Effective doses (immunizing amounts) of the vaccines of the invention may also be
25 extrapolated from dose-response curves derived from animal model test systems.
The present invention thus provides a method of immunizing an animal, or treating or preventing various diseases or disorders in an animal, comprising administering to the animal an effective immunizing dose of a vaccine formulation of the present invention.
3ø 5.5. Kits
The present invention provides kits comprising in an appropriate container(s) a reverse micelle composition comprising a surfactant, a hydrophilic phase, and one or more biologically active molecules. The present invention also provides kits comprising reverse micelle compositions comprising in an appropriate container(s) one or more fatty acid esters 35 or a hydrophilic derivative thereof, a hydrophilic phase, and one or more biologically active agents. In one embodiment, kits comprise in an appropriate container(s) reverse micelle compositions comprising monoglycerides, diglycerides, or a hydrophilic derivative thereof, a hydrophilic phase, and one or more biologically active molecules. In another embodiment, kits comprise in an appropriate container(s) reverse micelle compositions comprising monoglycerides, diglycerides, or a hydrophilic derivative thereof, a hydrophilic phase, and different biologically active molecules. In another embodiment, kits comprise reverse
5 micelle compositions comprising monoglycerides or diglycerides or a mixture thereof, a hydrophilic phase, and one or more biologically active molecules, wherein the acyl groups of the monoglycerides or diglycerides are enriched in fatty acids having 6-12 carbon atoms. In accordance with this embodiment, the monoglycerides or diglycerides may be partially derivatized with a hydrophilic moiety to provide polarity to increase water solubility. ι ø Preferably, the reverse micelle compositions included in the kits of the invention comprise less than 15%, less than 10%, less than 5%, or less than 2% by weight of triglycerides. The reverse micelle compositions of the invention may be formulated in a compatible pharmaceutical carrier. The reverse micelle compositions of the invention may be formulated in a compatible pharmaceutical carrier. Preferably, the kits of the invention are
1 packaged with instructions for methods of administering a reverse micelle composition of the invention to an animal. The kits of the invention may also comprise a list of the diseases and/or disorders for which the compositions may be used to prevent, treat, diagnose or monitor.
2ø 5.6. Use of Antibodies Generated by the Reverse
Micelle Compositions of the Invention
Antibodies generated against an antigen by immunization of an animal (e.g., a mouse, rat, rabbit, monkey, etc.) with a reverse micelle composition of the invention comprising an antigen are useful in diagnostic immunoassays, passive immune therapy, and generation of antiidiotypic antibodies. 5
The generated antibodies may be isolated by standard techniques known in the art {e.g., inmunoaffinity chromatography, centrifugation, precipitation, etc.) and used in diagnostic immunoassays. The antibodies may also be used to monitor treatment and/or disease progression. Any immunoassay system known in the art may be used for this purpose including, but not limited to, competitive and noncompetitive assay systems using
30 techniques such as radioimmunoassays, ELISAs (enzyme-linked immunosorbent assays),
"sandwich" immunoassays, precipitin reactions, gel-diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays and immunoelectrophoresis assays. 5
The reverse micelle compositions of the invention can also be used to produce antibodies for use in passive immunotherapy, in which short-term protection of an animal is achieved by the administration of a pre-formed antibody directed against a heterologous antigen.
The antibodies generated by the reverse micelle compositions of the invention can also be used in the production of antiidiotypic antibody. The antiidiotypic antibody can then in turn be used for immunization, in order to produce a subpopulation of antibodies that bind the initial antigen of the pathogenic microorganism (Jerne et al. Ann. Immunol. 125c:373, 1974; Jerne et al. EMBO J. 1:234, 1982).
In immunization procedures, the amount of immunogen to be used and the immunization schedule will be determined by a physician skilled in the at and will be administered by reference to the immune response and antibody titers of the animal.
The following examples are presented by way of illustration and not by way of limitation of the scope of the invention.
6. EXAMPLE: REVERSE MICELLES CONTAINING
HUMAN GROWTH HORMONE
This example demonstrates that a reverse micelles increase the bioavailability of agents.
Preparation of Reverse Micelles
A solution of human growth hormone ("hGH") was prepared by dissolving purified human growth hormone in water. A reverse micelle was obtained by mixing the aqueous solution of human growth hormone with Acconon CC-12 in a 5:95 ratio (water to surfactant). Subsequently, the mixture was mixed by vortexing and a transparent dispersion was obtained, indicative of stable reverse micelle formation. It will be appreciated by the skilled artisan that any mixing method known in the art that can disperse two phases can be used to prepare the reverse micelle compositions of the invention. The reverse micelle was stable upon storage at 4° C and room temperature.
Bioavailability of hGH Following Intraduodenal Administration in Rats.
Sprague-Dawley rats (approximately weighing 120 grams each) were catheterized surgically with jugular and duodenal catheters. Each group of rats, 3-5 animals per group, received 600 micrograms of human growth hormone in Acconon CC-12 reverse micelle formulation or control formulations consisting of aqueous growth hormone or Acconon CC- 12 water reverse micelle. Blood samples were obtained from the jugular catheter at the 0,
20, 40, 60, 90, 120, and 240 minutes after administration. Plasma samples were obtained from the collected blood specimens and analyzed for presence of hGH by an enzyme-linked immunosorbent assay (ELISA) (Alexon-Trend, BioCheck). The assay system utilizes polyclonal sheep anti-hGH for solid phase (microwells) immobilization, and mouse monoclonal anti-hGH in the antibody-enzyme (horseradish peroxidase) conjugate solution. The test serum or formulation sample was allowed to react simultaneously with the coated and conjugated antibodies, resulting in the hGH molecule being sandwiched between the solid phase and enzyme-linked antibodies. After a 45-minute incubation at room
5 temperature, the sample well was washed to remove unbound enzyme labeled antibody. A solution of 3,3',5,5'-Tetramethylbenzidine (TMB) was added and incubated for 15 minutes, resulting in the development of a blue color. The addition of Stop Solution stops the reaction and converts the color to yellow. The intensity of the yellow color is directly proportional to the concentration of hGH in the sample. As shown in FIG.l, the reverse ι ø micelle compositions promoted the transport of hGH, whereas no absorption was observed with control compositions.
7. EXAMPLE: LUTEINIZING HORMONE RELEASING
HORMONE AGONIST REVERSE MICELLES This example demonstrates that a higher percentage of bioavailability of luteinizing hormone releasing hormone (LHRH) is achieved when LHRH is administered intraduodenally in a reverse micelle formulation than when LHRH is administered in a water-in-oil emulsion formulation.
2ø Preparation of Reverse Micelles
LHRH is a peptide hormone secreted by the hypothalamus (see, e.g., U.S. Patent No. 4,234,571) with the following amino acid sequence: p-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 (MW = 1182 daltons; SEQ ID NO:l) 25 LHRH was obtained by synthesis (Polypeptide Labs) as the acetate salt. LHRH was dissolved in acetate buffer (pH 5.1) and prepared in a variety of vehicles as follows, a. RM-A12. 1.5 grams of a solution of LHRH was added directly to 8.5 grams of Acconon CC-12 and mixed by vortexing until a transparent dispersion was obtained. 3 b. RM-C. Capmul MCM was mixed in a 7:2 ratio with cremophor. To 9 grams of the surfactant mixture, 1 gram of a solution of LHRH was added and mixed by vortexing until the dispersion was transparent, c. RM-L: 1.5 grams of a solution of LHRH was added to 8.5 grams of Labrasol and mixed well until a clear dispersion was obtained. 35 d. RM-A8: 1.5 grams of a solution of LHRH was added directly to 8.5 grams of
Acconon MC-8 and mixed by vortexing until a transparent dispersion was obtained. e. RM-A6: 1.5 grams of a solution of LHRH was added directly to 8.5 grams of
Acconon CC-6 and mixed by vortexing until a transparent dispersion was obtained. f. RM-S : 1.5 grams of a solution of LHRH was added directly to 8.5 grams of 5 Softigen 767 and mixed by vortexing until a transparent dispersion was obtained.
A water-in-oil microemulsion was prepared
ι ø g. W/O ME: 1 gram of a solution of LHRH was added to a mixture of 6 grams of
Captex 355, 2 grams of Capmul MCM, and 1 gram of polyoxyethylated(20) sorbitan oleate (Tween 80) and mixed until a transparent microemulsion was obtained. The final composition of the microemulsion is 60% Captex, 20%) Capmul MCM, 10%
15 Tween-80, and 10% acetate buffer incorporating LHRH.
Pharmacokinetics of LHRH in Rats
Sprague-Dawley rats (approximately weighing 120 grams each) were catheterized surgically with jugular and duodenal catheters. Each group of rats, 3-5 animals per group,
2ø were given a dose 2.4 mg/kg of LHRH either as free in solution or incorporated in micelles. As further control, several groups of animals were given a solution of free LHRH subcutaneously at a dose of 0.4 mg/kg of body weight. Blood samples were collected at 0, 20, 40, 60, 90, 120, and 240 minutes following administration of LHRH or controls. The time 0 blood collection was obtained approximately 15 minutes before administration of
25 LHRH formulations. Plasma samples were analyzed by a competitive ELISA assay as follows. Plastic 96 well plates were coated with anti-rabbit immunoglobulins followed by addition of rabbit anti-LHRH and biotinylated-LHRH with sample dilutions. Binding of biotinylated LHRH was assayed by development with HRP-avidin and color development with TMB (tetra-methyl-benzidine). Pharmacokinetic parameters were calculated from the
3 data using WinNonLin software (Pharsight). Water-in-oil microemulsions promoted some absorption, but bioavailability was less than any reverse micelle composition (Table 2, FIG. 2). Reverse micelle compositions comprising LHRH in Acconon or Softigen type surfactants administered intraduodenally promoted approximately 10% bioavailability relative to subcutaneous injections of aqueous solution of LHRH. The percent absolute
3 bioavailability obtained from Capmul MCM and Labrasol reverse micelles was about 5% (Table 2). Table 2. Pharmacokinetic Parameters of LHRH in Rats.
Figure imgf000032_0001
ID = intraduodenal administration; SC = subcutaneous administration; AUCinf = Area Under Curve from time 0 to Infinity; n = number of rats. * PK data with RM-A12 was reproduced via four independent experiments.
8. EXAMPLE: LEUPROLIDE REVERSE MICELLES
This example demonstrates that a higher absolute bioavailability of leuprolide is achieved when leuprolide is administered intraduodenally in a reverse micelle formulation than when leuprolide is administered intraduodenally as a solution formulation in acetate buffer. Preparation of Softigen containing Reverse Micelles (RM-S^)
Leuprolide is a LHRH agonist which suppresses endogenous gonanotropins, causing a hypogonadal condition with the following amino acid structure: p-Glu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHC2H5 (MW = 1209 daltons; SEQ ID NO:2).
Leuprolide (acetate salt, Polypeptide Labs) was dissolved in an appropriate buffer (for 5 example, 0.1 M sodium acetate) at an appropriate pH (for example, pH = 6.0). 1.5 grams of the leuprolide solution was added to 8.5 grams of Softigen 767 and vortex mixed until the dispersion was clear and transparent.
Preparation of Labrasol containing Reverse Micelles (RM-D ι ø Leuprolide was dissolved in an appropriate buffer (for example, 0.1 M sodium acetate) at an appropriate pH (for example, pH = 6.0). 1.5 grams of the leuprolide solution was added to 8.5 grams of Labrasol and vortex mixed until the dispersion was clear and transparent.
15 Quantitation of Leuprolide in Reverse Micelle Formulations by HPLC
Leuprolide concentration in reverse micelle (RM) formulations was measured by reversed phase HPLC using a Hewlett Packard 1050 system equipped with a Waters C-18 4.6x250mm 5μm column, a Supelguard Discovery C-18 2 x 4.0mm guard column and UV Diode array detection at 220nm. Mobile phase A was 0.1% (v/v) trifluoroacetic acid (TFA)
2Q in deionized water and mobile phase B was 0.1 % (v/v) TFA in HPLC grade acetonitrile. The run condition was 10-40% B gradient in 25 minutes at a flow rate of 1.5 ml/min. The column was reconditioned with an isocratic hold of 10%> B at 0.5ml/min for 5 minutes and at 1.5ml/min for additional 10 to 15 minutes. The retention time of leuprolide in this system was between 9 and 14 minutes. The concentration was calculated by using area under curve
2 of the sample peak against a standard curve of 0.01 to 0.8 mg/ml leuprolide acetate versus corresponding area under the curve.
Pharmacokinetics of Leuprolide in Rats
Sprague-Dawley rats (approximately weighing 120 grams each) were catheterized 3 surgically with jugular and duodenal catheters. Each group of rats, 4-5 animals per group, were given 0.4 - 3.6 mg/kg of leuprolide either as free in solution or incorporated in micelles. As further control, several groups of animals were given a solution of free leuprolide subcutaneously. Blood samples were collected at 0, 20, 40, 60, 90, 120, and 240 minutes following administration of leuprolide or controls. The time 0 blood collection was 3 obtained approximately 15 minutes before administration of leuprolide formulations. The amount of leuprolide released into rat serum or leuprolide in formulation was determined by using competitive enzyme-linked immunosorbent assay (ELISA) (Peninsula Laboratories, Inc). The assay system utilizes goat anti-rabbit Ig G for solid phase (microwells) immobilization. The test serum or formulation sample was allowed to react simultaneously with the coated antibody, rabbit anti-leuprolide and biotinylated leuprolide. The biotinylated leuprolide competes for the antibody binding sites with standard or the unknown sample leuprolide. After a two-hour (or overnight) incubation at room temperature, unbound biotinylated peptide was removed by washing, and streptavidin- conjugated horseradish peroxidase (SA-HRP) was added and allowed to bind the immobilized rabbit anti-leuprolide/ biotinylated leuprolide complex. After washing away excess SA-HRP, 3,3',5,5'-Tetramethylbenzidine (TMB) was added and incubated for 15 ø minutes, resulting in the development of a blue color. The addition of Stop Solution stops the reaction and converts the color to yellow. The intensity of the yellow color depends on the quantity of biotinylated leuprolide bound to the immobilized antibody. When more sample leuprolide competes for the limited antibody, less biotinylated leuprolide/S A-HRP can be immobilized, and less color is produced by the substrate. 5
Table 3 . Pharmacokinetic parameters of Leuprolide in Rats (mean ± SD).
0
5
0
5
Figure imgf000034_0001
Figure imgf000035_0001
*RM-S = Softigen 767 reverse micelle
*RM-L = Labrasol reverse micelle
ID = intraduodenal administration; AUCinf = Area Under Curve from time 0 to Infinity;
SC = subcutaneous; n = number of rats.
As can be seen from the data in Table 3, the intraduodenal bioavailability of leuprolide from a solution formulation (0.1 M acetate pH 6.0) is very low (about 0.2%) whereas the intraduodenal bioavailability from the RM-S is about 22 to about 39%.
Essentially the same bioavailability was obtained in a repeated study with RM-S using 2.4 mg/kg dose of leuprolide, emphasizing the consistency and reproducibility in the absorption data. The bioavailability of leuprolide was dose dependent. That is to say that the higher the dose the higher the Cmax and AUC and the lower the percent absolute bioavailability (Table 3) .
Pharmacokinetics of Leuprolide in Dogs
Beagle dogs (3 female and 3 male weighing 8 - 10 kg each) were catheterized surgically with duodenal catheters. For each treatment, the dogs were given 0.4 - 2.4 mg/kg of leuprolide either in solution or incorporated in micelles. The treatments were administered via the duodenal catheter. For the positive control, the dogs were given a solution of free leuprolide by subcutaneous injection. Blood samples were collected from cephalic catheters or the jugular vein at 0, 15, 30, 45, 60, 90, 120, 240, 360, 480, and 1440 minutes following administration of leuprolide or controls. Leuprolide concentration levels are illustrated in the standard curves in Figure 4. The time 0 blood collection was obtained approximately 10 minutes before administration of leuprolide formulations. The amount of leuprolide released into dog serum was determined by using a competitive enzyme-linked immunosorbent assay (ELISA) from Peninsula Laboratories, Inc. which utilizes goat anti- rabbit IgG for solid phase (microwell) immobilization. The test serum was allowed to react simultaneously with the coated antibody, rabbit anti-leuprolide and biotinylated leuprolide. After two-hour incubation at room temperature, unbound biotinylated peptide was removed by washing, and streptavidin-conjugated horseradish peroxidase (SA-HRP) was added and allowed to bind the immobilized rabbit anti-leuprolide/ biotinylated leuprolide complex. After washing away excess SA-HRP, 3,3'5,5'-Tetramethylbenzidine dihydrochloride (TMB) was added and incubated for 15 minutes, resulting in the development of a blue color. The addition of stop solution stops the reaction and converts the color to yellow. Leuprolide in the samples was quantified by non-linear regression analysis of the standard curve and sample optical density values. Pharmacokinetic parameters are calculated using a pharmacokinetic software with a noncompartmental model.
Table 4 . Pharmacokinetic parameters of Leuprolide in Dogs (mean ± SD)
Figure imgf000036_0001
*RM-S = Softigen 767 reverse micelle *RM-L = Labrasol reverse micelle
**Doses of excipients were at least 13 times less than those used in the rat studies in Table 3 ID = intraduodenal administration; AUCinf = Area Under Curve from time 0 to Infinity; SC = subcutaneous; n = number of rats
9. EXAMPLE: REVERSE MICELLE CAPSULES
Preparation of Reverse Micelle Capsules
Reverse micelles that are compatible with gelatin capsules were prepared by mixing an excipient, for example, Softigen 767 or Labrasol, with a leuprolide solution in buffer at an appropriate ratio, for example, 85:15 w/w. Based on the weight of the animals, an appropriate amount of reverse micelle containing leuprolide was placed into the body part of a capsule of suitable size (for example, size 00 or 13 gelatin capsules) and the cap was then put in place and locked. The capsule was sealed by applying a few microliters of an alcoholic solution (for example, 1 :1 isopropanol- water solution) and allowing the solution to dry.
Enteric Coating of Reverse Micelle Capsules
Enteric coating of a reverse micelle capsule was performed using techniques known to others in the art. (Enteric Coating of Hard Gelatine Capsules Application of EUDRAGIT® L 30 D-55, Rohm Pharma Polymers Application Note 4.1.9.4) A modified coating method and a modified coating solution were also used. Specifically, Eudragit L30D-55 (10.56 g) was diluted with 7.9 g of distilled water and mixed with 0.63 g of triethyl citrate. To this, 0.134 g of 30% Tween 80 was added. The resulting suspension was stirred for 30 minutes. The coating solution was either used as is, or the pH of the coating solution was adjusted to a desired level (for example, pH 5.5) so that the coated capsule would have a better dissolution profile in the duodenum. A dipping method was used for the coating of the capsules. The capsules (especially the size 13 capsules) were held by a multi-port valve- controlled vacuum suction device, which was developed by the same authors, during the 5 coating processes. Alternatively, the capsules were coated using a coater.
Dissolution test using a continued in situ monitoring system Dissolution of the capsules was tested using a USP test method on a dissolution test station (VanKel VK 7000). (USP Physical Tests <711> Dissolution and <724> Drug ι Release) Automation of the sampling and measurement was achieved by using a continued in situ monitoring system developed by the applicants. Specifically, a HPLC solvent inlet filter was attached to the inlet of a sample intake tube inserted into the dissolution test vessel. The intake tube was connected to a HPLC pump which was directly connected to one or more detectors such as diol array detectors, RI detectors, fluorescent detectors, UV j5 detectors, and so forth, and combinations of such, in a HPLC system. The out-flow from the detector was directed back into the test vessel by another tube. Signals from the detectors such as UV absoφtion at multiple wavelengths of choice and UV spectra of the sample were continuously collected and stored by the HPLC system and data was analyzed using the HPLC software. Plots of the signals against time, termed here as "Dissolugrams", represent
2ø the dissolution profiles of the coating material, the capsule (220 nm) and the release of the reverse micelle (220 and 540 nm) and Leuprolide (280 nm). The enteric coated reverse micelle capsules were stable in the acid stage for at least 2 hours as indicated in the dissolugrams by the relatively unchanged UV 220, 280 and 540 nm signals. In buffer stage, the enteric coated capsules dissolved within 10 minutes according to the dissolugrams. The
25 non-coated reverse micelle capsules dissolved within 3 minutes in the acid stage according to the dissolugrams.
Model compounds with special UV absorption wavelengths such as dimethylaminopyridine (280 nm), Bacto Methylene Blue (340 nm) or fluorescent compounds were also used replacing leuprolide in the reverse micelle in capsules for the
3ø dissolution tests.
10. EXAMPLE: DEMONSTRATION OF THE FORMATION OF
REVERSE MICELLES USING DIFFERENT MOLECULAR SIZE AMORPHOUS DEXTRANS
Several fluorescently labeled dextrans of varying molecular weights were used to
35 form reverse micelles. Either FITC-Dextran 4,000 daltons, 10,000 daltons, or 20,000 daltons were dissolved in water at different ratios of water to surfactant. Stable micelles were formed at up to 50% total water phase as determined by the quenching of FITC fluorescence and obtained a transparent dispersion. Fluorescence of FITC were retrieved by dilution of the stable reverse micelles into excess water, indicating extrusion of the water- soluble dextran from the surfactant phase.
11. EXAMPLE: DEMONSTRATION OF THE FORMATION OF
REVERSE MICELLES BY ELECTRICAL RESISTANACE MEASUREMENTS
Reverse micelles (RMs) where the internal or dispersed phase is aqueous are expected to exhibit low electrical conductance or high resistance. Thus, conductivity or resistivity measurements can be used to confirm the reverse micellar structure. 0 To demonstrate this point the following experiment was conducted:
Various RMs were prepared by mixing 15% (w/w) IX phosphate-buffered saline (PBS) with 85% (w/w) corresponding excipients. Subsequently, deionized water was added incrementally to up to 90% total content of water by weight. The resistivity of solution was measured incrementally using Millicell-ERS electrode system (Millipore). The resistivity 5 of deionized water was exceed measurable range of electrode (> 19.99KOhms), of tested excipients (0%> water) were about 15.5KOhms and of lx PBS was 2-3 Ohms. RM containing 15% PBS still showed high resistance due to the fact that the most of electrolytes are remained in core of reverse micellar structure within the continuous phase of excipients. RM systems using different surfactants exhibited similar pattern in the drop of resistivity as 0 increasing % water. Sharp drop in resistivity indicated release of encapsulated electrolytes from the core of the RM system into medium upon dilution with non-conductive deionized water. The resistivity reached plateau (at about 15 Ohms) after exceeding 40% total water content in the system. This implies that the reverse micelle system (L2 phase) has been converted into regular micelle (LI phase) after this point where the continuous phase is 5 aqueous (water).
12. EXAMPLE: DEMONSTRATION OF THE ORAL
BIOAVAILABILITY OF RHODAMINE DEXTRAN IN REVERSE MICELLE ø Two rhodamine dextran containing reverse micelles were prepared separately by vortex mixing Softigen 767 (8.5 g) and a rhodamine dextran solution (1.5 ml, Mw. = 3,000 Da). Sprague-Dawley rats (approximately weighing 120 grams each) were catheterized surgically with jugular and duodenal catheters. Groups of 5 rats were given separately 1.2 mg/kg of a rhodamine dextran (Mw. 3,000 Da) in reverse micelles through the duodenal 5 catheter, or 0.4 mg/kg of a rhodamine dextran (Mw. 3,000 Da) in solution subcutaneously. Blood samples were collected at 0, 20, 40, 60, 90, 120, and 240 minutes following the administration of the test articles. The time 0 blood collection was obtained approximately 15 minutes before administration of the test articles. The amount of rhodamine dextran was determined by measuring the fluorescent intensity of the samples on fluorescent plate reader. Percent bioavailability was calculated from AUC of the rhodamine dextran plasma level in the groups received reverse micelle formulations against the AUC of the corresponding rhodamine dextran plasma level in the groups received control solutions. The bioavailability of the rhodamine dextran (Mw. 3,000 Da) was thus determined to be 18%. (Table 5)
Table 5 . Pharmacokinetic Parameters of Rhodamine Dextran (Mw 3000 Da) in Rats (mean
± SD)
Figure imgf000039_0001
The present invention is not to be limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.
All patents, patent applications and non-patent publications cited herein are incorporated by reference in their entirety to the same extent as if each individual patent, patent application or non-patent publication was specifically and individually indicated to be incoφorated herein by reference.

Claims

What is claimed is:
1. A reverse micelle composition comprising:
(a) a surfactant;
(b) a hydrophilic phase; and
5 (c) a biologically active molecule, wherein the reverse micelle composition comprises less than about 10% by weight of triester.
2. The reverse micelle composition of claim 1 , wherein the reverse micelle ι ø composition comprises less than about 5% by weight of triester.
3. The reverse micelle composition of claim 1 , wherein the surfactant comprises one or more fatty acid esters or hydrophilic derivatives thereof.
15 4. The reverse micelle composition of claim 3, wherein the fatty acid esters have a length of about 6 to about 12 carbon atoms.
5. The reverse micelle composition of claim 1, wherein surfactant comprises a monoglyceride, diglyceride or mixture thereof.
20
6. The reverse micelle composition of claim 1 , wherein the surfactant comprises a monoglyceride or a hydrophilic derivative or analog thereof.
7. The reverse micelle composition of claim 1 , wherein the surfactant 25 comprises a diglyceride or a hydrophilic derivative or analog thereof.
8. The reverse micelle composition of claim 1 , wherein the surfactant comprises a monoglyceride or diglyceride mixture.
3 9. The reverse micelle composition of claim 3, wherein at least one of the fatty acids is derivatized by a polyoxyethylene polymer.
10. The reverse micelle composition of claim 6, wherein the surfactant further comprises a diglyceride or a hydrophilic derivative or analog thereof.
35
11. The reverse micelle composition of claim 10, wherein the polyoxyethylene polymer has a molecular weight of about 200 to about 10,000 daltons.
12. The reverse micelle composition of claim 11 , wherein the polyoxyethylene polymer has a molecular weight of about 200 to about 4,000 daltons.
13. The reverse composition of claim 6, wherein the monoglyceride is derivatized by a polyoxyethylene polymer.
14. The reverse composition of claim 7, wherein the diglyceride is derivatized by a polyoxyethylene polymer. 0
15. The reverse micelle composition of claim 3, wherein at least one of the fatty acid esters is derivatized with acetic acid, citric acid, lactic acid, succinic acid, tartaric acid or mixtures thereof.
16. The reverse micelle composition of claim 3 , wherein at least one of the fatty acid esters is a caprylic acid or capric acid.
17. The reverse micelle composition of claim 1 , wherein the hydrophilic phase comprises, water, glycerol, sorbitol, mannitol, propylene glycol, ethylene glycol, ø polyethylene glycol or mixtures thereof.
18. The reverse micelle composition of claim 17, wherein the hydrophilic phase further comprises a buffering agent, a tonicity agent, an oxidizing agent, a reducing agent, an antimicrobial agent, a preservative, a stabilizing agent, or a mixture thereof. 5
19. The reverse micelle composition of claim 1, wherein the hydrophilic phase is present in an amount from about 0 to about 70% by weight.
20. The reverse micelle composition of claim 19, wherein the hydrophilic phase 0 is present in an amount from about 0 to about 50% by weight.
21. The reverse micelle composition of claim 20, wherein the hydrophilic phase is present in an amount from about 5 to 25% by weight.
5 22. The reverse micelle composition of claim 3 , wherein the fatty acid esters have an HLB of about 1 to about 20.
23. The reverse micelle composition of claim 22, wherein the fatty acid ester has an HLB of about 5 to about 15.
24. The reverse micelle composition of claim 1 , wherein the biologically active molecule is a prophylactic or therapeutic agent.
5
25. The reverse micelle composition of claim 1, wherein the biologically active molecule is a diagnostic agent.
26. The reverse micelle composition of claim 24, wherein the therapeutic agent is ι ø a LHRH or an analog thereof.
27. The reverse micelle composition of claim 24, wherein the therapeutic agent is a LHRH agonist or an analog thereof.
1 28. The reverse micelle composition of claim 27, wherein the LHRH agonist is leuprolide, goserelin, nafarelin or histrelin.
29. The reverse micelle composition of claim 1 , wherein the biologically active molecule is parathyroid hormone or an analog thereof.
20
30. The reverse micelle composition of claim 1 , wherein the biologically active molecule is calcitonin or an analog thereof.
31. The reverse micelle composition of claim 1 , wherein the biologically active 25 molecule is a low molecular weight heparin.
32. The reverse micelle composition of claim 1 , wherein the biologically active molecule is insulin.
30 33. The reverse micelle composition of claim 1 , wherein the biologically active molecule is a human growth hormone or an analog thereof.
34. The reverse micelle composition of claim 1 , wherein the biologically active molecule is a nucleic acid.
35
35. The reverse micelle composition of claim 1, wherein the concentration of the biologically active molecule is more than 0.1 mg/ml soluble in the hydrophilic phase.
36. The reverse micelle composition of claim 35, wherein the concentration of the biologically active molecule is more than 1 mg/ml soluble in the hydrophilic phase.
37. The reverse micelle composition of claim 1, wherein the concentration of the biologically active molecule is from 0.05 to 100 mg/ml.
38. A method for delivering a biologically active molecule to an animal, said method comprising administering to said animal a reverse micelle composition comprising: ι ø (a) a surfactant;
(b) a hydrophilic phase; and
(c) a biologically active molecule, wherein the reverse micelle composition comprises less than 10% by weight of triester.
-> 5
39. A method for preventing, treating or ameliorating one or more symptoms associated with a disease or disorder in an animal, said method comprising administering to an animal in need thereof an effective amount of a reverse micelle composition comprising:
(a) a surfactant;
(b) a hydrophilic phase; and
2ø (c) a prophylactic or therapeutic agent, wherein the reverse micelle composition comprises less than 10% by weight of triester.
40. A method for diagnosing a disease or disorder in an animal, said method comprising administering to said animal an effective amount of a reverse micelle
25 composition comprising:
(a) a surfactant;
(b) a hydrophilic phase; and
(c) a diagnostic agent, wherein the reverse micelle composition comprises less than 10% by weight of triester.
30
41. The method of claim 38, 39 or 40, wherein the reverse micelle composition comprises less than 5% be weight of triester.
42. The method of claim 38, 39 or 40, wherein the surfactant comprises one or 35 more fatty acid esters or a hydrophilic derivative thereof.
43. The method of claim 38, 39 or 40, wherein the surfactant comprises a monoglyceride or a hydrophilic derivative or analog thereof.
44. The method of claim 38, 39 or 40, wherein the surfactant comprises a diglyceride or a hydrophilic derivative or analog thereof.
5
45. The method of claim 38, 39 or 40, wherein the surfactant comprises a monoglyceride, diglyceride or a mixture thereof.
46. The method of claim 43, wherein the surfactant further comprises a ι ø diglyceride or a hydrophilic derivative or analog thereof.
47. The method of claim 38, 39 or 40, wherein said reverse micelle composition is administered mucosally.
5 48. The method of claim 47, wherein said reverse micelle composition is administered as a capsule, soft elastic gelatin capsule, caplet, aerosol, spray, solution, suspension, emulsion, cachet, tablet, capsule, soft elastic gelatin capsule, aerosol, powder or granule.
0 49. The method of claim 38, 39 or 40, wherein said animal is a human.
50. The method of claim 38, 39 or 40, wherein the reverse micelle composition is administered orally.
5 51. The method of claim 39, wherein the therapeutic agent is a LHRH or a
LHRH agonist.
52. The method of claim 38 wherein the biologically active molecule is a human growth hormone or an analog thereof.
30
53. The method of claim 51 , wherein the LHRH agonist is leuprolide, goserelin, nafarelin or histrelin.
54. The method of claim 38, wherein the biologically active molecule is insulin.
35
55. The method of claim 38, wherein the biologically active molecule is parathyroid hormone.
56. The method of claim 38, wherein the biologically active molecule is calcitonin or an analog thereof.
57. The method of claim 38, wherein the biologically active molecule is a low molecular weight heparin.
58. The method of claim 39, wherein the therapeutic agent is human growth hormone or an analog thereof. 0
59. The method of claim 39, wherein the therapeutic agent is insulin.
60. The method of claim 39, wherein the therapeutic agent is parathyroid hormone or an analog thereof. 5
61. The method of claim 39, wherein the therapeutic agent is calcitonin or an analog thereof.
62. The method of claim 39, wherein the therapeutic agent is a low molecular ø weight heparin.
63. The method of claim 51 , wherein the disease or disorder is prostate cancer, endometriosis, precocious puberty, uterine lelomyotama, fertility disorder, premenopausal breast cancer, endometiral cancer, ovarian cancer, benign prostatic hypertrophy, functional 5 bowel disease, cluster headache, premenstrual syndrome, idiopathic hirsuitism, hirsuitism second to polycycstic ovarian disease, adenomyosis, Meniere's disease, sickle cell anaemia associated priapism or catamental pneumothorax..
64. The method of claim 58, wherein the disease or disorder is hypopituitarism, hypothyroidism, human growth hormone deficiency, Gushing' s syndrome, nutritional short stature, intrauterine growth retardation, Russell Silver syndrome or achondroplasia.
65. The method of claim 59, wherein the disease or disorder is diabetes.
5 66. The method of claim 60 or 61 , wherein the disease or disorder is a bone-reabsorption disease.
67. The method of claim 66, wherein the bone-reabsorption disease is osteoporosis, metastatic bone cancer, osteolytic lesions with an orthopedic implant, Paget's disease, or bone loss associated with hyperparathyroidism.
68. The method of claim 72, wherein the disease or disorder is related to blood clotting.
69. The reverse micelle composition of claim 1 , wherein the surfactant is a P- glycoprotein inhibitor.
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CN106983720A (en) * 2017-05-19 2017-07-28 浙江工业大学 A kind of reverse micelle includes the preparation method of insulin system
WO2019086603A1 (en) 2017-11-03 2019-05-09 Interna Technologies B.V. Mirna molecule, equivalent, antagomir, or source thereof for treating and/or diagnosing a condition and/or a disease associated with neuronal deficiency or for neuronal (re)generation
CN111012758A (en) * 2019-12-31 2020-04-17 浙江工业大学 Preparation method of nattokinase microcapsule

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