WO2008004992A2 - Formulations transdermiques contenant des immunogènes du virus de hépatite c - Google Patents

Formulations transdermiques contenant des immunogènes du virus de hépatite c Download PDF

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Publication number
WO2008004992A2
WO2008004992A2 PCT/US2006/020101 US2006020101W WO2008004992A2 WO 2008004992 A2 WO2008004992 A2 WO 2008004992A2 US 2006020101 W US2006020101 W US 2006020101W WO 2008004992 A2 WO2008004992 A2 WO 2008004992A2
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WO
WIPO (PCT)
Prior art keywords
transdermal delivery
transdermal
immunogenic composition
seq
ethoxylated
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PCT/US2006/020101
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English (en)
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WO2008004992A3 (fr
Inventor
Frederick Jordan
Matti Sallberg
Original Assignee
Tripep Ab
Oryxe
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Publication of WO2008004992A2 publication Critical patent/WO2008004992A2/fr
Publication of WO2008004992A3 publication Critical patent/WO2008004992A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • A61K8/606—Nucleosides; Nucleotides; Nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/92—Oils, fats or waxes; Derivatives thereof, e.g. hydrogenation products thereof
    • A61K8/922—Oils, fats or waxes; Derivatives thereof, e.g. hydrogenation products thereof of vegetable origin
    • 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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • 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
    • A61Q19/08—Anti-ageing preparations
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • Embodiments of the present invention relate to the discovery of several formulations of a transdermal delivery composition that delivers low and high molecular weight compounds, preferably hepatitis C virus (HCV) antigens and immunogens to a subject.
  • aspects of the invention include said transdermal delivery compositions, transdermal delivery devices for providing said compositions to subjects in need thereof and methods of making and using the foregoing.
  • the skin provides a protective barrier against foreign materials and infection. In mammals this is accomplished by forming a highly insoluble protein and lipid structure on the surface of the corneocytes termed the cornified envelope (CE).
  • CE cornified envelope
  • the CE is composed of polar lipids, such as ceramides, sterols, and fatty acids, and a complicated network of cross-linked proteins; however, the cytoplasm of stratum corneum cells remains polar and aqueous.
  • the CE is extremely thin (10 microns) but provides a substantial barrier. Because of the accessibility and large area of the skin, it has long been considered a promising route for the administration of drugs, whether dermal, regional, or systemic effects are desired.
  • a topical route of drug administration is sometimes desirable because the risks and inconvenience of parenteral treatment can be avoided; the variable absorption and metabolism associated with oral treatment can be circumvented; drug administration can be continuous, thereby permitting the use of pharmacologically active agents with short biological half-lives; the gastrointestinal irritation associated with many compounds can be avoided; and cutaneous manifestations of diseases can be treated more effectively than by systemic approaches.
  • transdermal delivery compositions achieve epidermal penetration by using a skin penetration enhancing vehicle.
  • skin penetration enhancers Such compounds or mixtures of compounds are known in the art as “penetration enhancers” or “skin enhancers”.
  • skin enhancers in the literature enhance transdermal absorption, several possess certain drawbacks in that (i) some are regarded as toxic; (ii) some irritate the skin; (iii) some have a thinning effect on the skin after prolonged use; (iv) some change the intactness of the skin structure resulting in a change in the diffusability of the drug; and (v) all are incapable of delivering high molecular weight pharmaceuticals and cosmetic agents.
  • transdermal delivery compositions that deliver a wide-range of pharmaceuticals and cosmetic agents, preferably HCV antigens and immunogens.
  • the transdermal delivery composition comprises a unique formulation of penetration enhancer (an ethoxylated lipid, modified lipid, fatty acid, fatty alcohol, or fatty amine therein having 10-19 ethoxylations per molecule) or transdermal delivery enhancer (an ethoxylated compound with a multi-functional backbone) that delivers a wide range of pharmaceuticals and cosmetic agents having molecular weights of less than 100 daltons to greater than 500,000 daltons.
  • penetration enhancer an ethoxylated lipid, modified lipid, fatty acid, fatty alcohol, or fatty amine therein having 10-19 ethoxylations per molecule
  • transdermal delivery enhancer an ethoxylated compound with a multi-functional backbone
  • embodiments of the transdermal delivery composition include formulations that deliver a therapeutically effective amount of a pharmaceutical, including NSAIDs, capsaicin or Boswellin-containing pain-relief solutions, other drugs or chemicals, dyes, low and high molecular weight peptides (e.g., collagens or fragments thereof), hormones, nucleic acids, antibiotics, vaccine preparations, and immunogenic preparations, preferably HCV antigens and immunogens.
  • Methods of making the transdermal delivery compositions described herein and systems for their delivery are embodiments. Further embodiments include methods of using said compositions (e.g., the treatment and prevention of undesired human conditions or diseases or cosmetic applications).
  • aspects of the invention concern transdermal delivery compositions that comprise lipospheres.
  • the liposphere comprises an ethoxylated composition having a carbon chain length of at least 10, wherein the ethoxylated composition (e.g., a fatty acid, fatty alcohol, or fatty amine), comprises, consists of, or consists essentially of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 ethoxylations per molecule.
  • the invention also include propoxylated compositions or compositions that comprise a combination of propoxylated and ethoxylated compositions.
  • the ethoxylated or propoxylated composition is a fatty moiety, such as a fatty acid (e.g., an unsaturated fatty acid or a polyunsaturated fatty acid).
  • the fatty moiety is a fatty alcohol
  • the liposphere comprises an ethoxylated or propoxylated oil or lipid having carbon chain lengths of at least 10, wherein the ethoxylated or propoxylated oil or lipid (e.g., a nut oil, a tri-alcohol, a tri-fatty amine, a glycolipid, a spliingolipid, a glycosphingolipid, or any other modified lipid moiety), comprises, consists of, or consists essentially of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 ethoxylations per molecule.
  • the number of ethoxylations or propoxylations per molecule is the same as the number of carbons in the fatty moiety or lipid moiety.
  • the fatty moiety has a carbon chain length of at least 10, 12, 14, 16, 18, 20, 22, or 24.
  • the liposphere comprises a homogeneous mixture of an ethoxylated or propoxylated fatty moiety in some embodiments, while in other embodiments, the liposphere comprises a heterogeneous mixture of an ethoxylated or propoxylated fatty moiety.
  • transdermal delivery compositions comprised of an ethoxylated lipid moiety, such as an oil, glycolipid, sphingolipid, or glycosphingolipid.
  • an ethoxylated lipid moiety such as an oil, glycolipid, sphingolipid, or glycosphingolipid.
  • the ethoxylated oil that can be used in the formulations described herein can be a vegetable, nut, animal, or synthetic oil having at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or more ethoxylations per molecule.
  • Preferred oils include macadamia nut oil or meadowfoam (limnanthes alba).
  • the measured composition is the algebraic sum of the compositions of the species in the mix.
  • transdermal delivery enhancer refers to a molecule that comprises a multi-functional backbone having at least two reactive (R) groups.
  • the R groups on the multifunctional backbone comprise a reactive hydrogen, such as -OH, COOH, amines, sulfydryl groups, and aldehydes.
  • multifunctional backbones include trialcholols, triacids, amino acids, dipeptides, tripeptides, sugars, and other compounds such as glucosamine.
  • At least one R group is substituted with a fatty moiety, and least one reactive group is substituted with a polyethoxy or polyethoxy/polypropoxy group, wherein the polyethoxy or the polyethoxy/polypropoxy group comprises between 10 and 19 ethoxy or propoxy/ethoxy substituents, respectively.
  • the ethoxylated fatty moiety is about 0.1% to greater than 99% by weight of the transdermal delivery composition described herein.
  • the transdermal delivery composition further comprises an alcohol and/or water and/or an aqueous adjuvant.
  • the aqueous adjuvant is a plant extract from the family of Liliaceae, such as Aloe Vera.
  • Other embodiments of the invention include the transdermal delivery composition described above, wherein about 0/1% to 15% by weight or volume is alcohol or 0.1% to 15% is water or both, or wherein about 0.1% to 85% by weight or volume is water or Aloe Vera or another aqueous adjuvant.
  • Alcohol, water, and other aqueous adjuvants are not present in some formulations of the transdermal delivery composition described herein. It has been discovered that some delivered agents (e.g., steroids) are soluble and stable in ethoxylated oil in the absence of alcohol or water and some delivered agents are soluble and stable in ethoxylated oil/alcohol emulsions, ethoxylated oil/water emulsions, ethoxylated oil/alcohol/water emulsions, and ethoxylated oil/a ⁇ cohol/water ⁇ 4/oe Vera emulsions.
  • some delivered agents e.g., steroids
  • ethoxylated oil/water emulsions ethoxylated oil/alcohol/water emulsions
  • ethoxylated oil/a ⁇ cohol/water ⁇ 4/oe Vera emulsions ethoxylated oil/a ⁇ cohol/water ⁇ 4/oe Vera e
  • a particular Aloe Vera, alcohol, or water mixture was not essential to obtain a transdermal delivery composition provided that an appropriately ethoxylated oil was mixed with the delivered agent. That is, the alcohol, water, and Aloe Vera can be removed from the formulation by using a light oil (e.g., macadamia nut oil) that has been ethoxylated to approximately 10-19 ethoxylations/molecule, desirably 11-19 ethoxylations/molecule, more desirably 12-18 ethoxylations/molecule, still more desirably 13-17 ethoxylations/molecule, preferably 14 -16 ethoxylations/molecule and most preferably 15 orl ⁇ ethoxylations/molecule.
  • a light oil e.g., macadamia nut oil
  • some ethoxylated oils e.g., macadamia nut oil comprising, consisting of or consisting essentially of 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 ethoxylations/molecule
  • low and high molecular weight peptides e.g., collagen and fragments of collagen
  • amino acids in the absence of alcohol and Aloe Vera.
  • Some embodiments however, have a ratio of ethoxylated lipid: alcohol: aqueous adjuvant selected from the group consisting of 1:1:4, 1:1:14, 3:4:3, and 1:10:25.
  • the transdermal delivery compositions described herein can also include fragrances, creams, bases and other ingredients that stabilize the formulation, facilitate delivery, or protect the delivered agent from degradation (e.g., agents that inhibit DNAse, RNAse, or proteases).
  • the transdermal delivery compositions described herein are useful for the delivery of a wide variety of delivered agents.
  • the transdermal delivery composition comprises delivered agents that are hormones.
  • the delivered agent is a peptide hormone.
  • peptide hormones include oxytocin, vasopressin, melanocyte-stimulating hormone, corticortropin, lipotropin, thyrotropin, growth hormone, prolactin, luteinizing hormone, human chorionic gonadotropin, follicle stimulating hormone, corticotropin-releasing factor, gonadotropin- releasing factor, prolactin-releasing factor, prolactin-inhibiting factor, growth-hormone releasing factor, somatostatin, thyrotropin-releasing factor, calcitonin gene-related peptide, parathyroid hormone, glucagon-like peptide 1, glucose-dependent insulinotropic polypeptide, gastrin, secretin, cholecystokinin
  • the delivered agent is a non-peptide hormone.
  • hormones that are not peptide hormones useful in embodiments include thyroxine, triiodothyronine, calcitonin, estradiol, estrone, progesterone, testosterone, Cortisol, corticosterone, aldosterone, epinephrine, norepinepherine, androstiene, or calcitriol.
  • peptides such as collagen, or fragments thereof, are delivered agents in certain embodiments.
  • the delivered agent is a pharmacologically active small compound.
  • the delivered agent is an anesthetic such as articaine, procaine, tetracaine, chloroprocaine and benzocaine, novocain, mepivicaine, bupivicaine, benzocaine, or lidocaine.
  • Analgesics are delivered agents in other embodiments.
  • the delivered agent is tramadol hydrochloride, fentanyl, metamizole, morphine sulphate, ketorolac tromethamine, hydrocodone, oxycodone, morporine, loxoprofen, Capsaicin, or Boswellin.
  • NSAIDs non-steroidal anti-inflammatory drugs
  • the delivered agent is ibuprofen (2-(isobutylphenyl)-propionic acid); methotrexate (N-[4-(2, 4 diamino 6 - pteridinyl - methyl] methylamino] benzoyl)-L- glutamic acid); aspirin (acetylsalicylic acid); salicylic acid; diphenhydramine (2- (diphenylmethoxy)-NN-dimethylethylamine hydrochloride); naproxen (2- naphthaleneacetic acid, 6-methoxy-9-methyl-, sodium salt, (-)); phenylbutazone (4-butyl- l,2-diphenyl-3,5-pyrazolidinedione); sulindac-(2)-5-fluoro-2-methyl-l-[[p-)
  • methotrexate N-[4-(2, 4 diamino 6 - p
  • the delivered agent is a steroidal antiinflammatory compound, such as hydrocortisone, prednisolone, triamcinolone, or piroxicam.
  • the delivered agent is an anti-infective agent.
  • the delivered agent is an antimicrobial or antifungal agent such as amoxicillin, clavulanate potassium, itraconazole, flucanazole, erythromycin ehtysuccinate, acetyl sulfisoxazole, penicillin V, erythromycin, azithromycin, tetracycline, ciproflaxin, gentamycin sulfathiazole.
  • the delivered agent is an anti-viral compound, such as for example acyclovir, lamivudine, indinavir sulfate, stavudine, saquinavir, ritonavir, ribavirin, or hepsysls.
  • an anti-viral compound such as for example acyclovir, lamivudine, indinavir sulfate, stavudine, saquinavir, ritonavir, ribavirin, or hepsysls.
  • the delivered agent is a nucleic acid
  • the nucleic acid is an oligonucleotide consisting of cysteine and guanidine, (e.g., a CpG molecule)
  • the nucleic acid is a polynucleotide
  • the polynucleotide comprises a nucleic acid sequence that is capable of eliciting an immune response from an animal.
  • the nucleic acid comprises nucleic acid sequences from HIV, influenza A virus, hepatitis C virus, hepatitis A virus, hepatitis B virus, hantavirus, SARS, or sequences encoding members of the Inhibitor of Apoptosis family of proteins.
  • Preferred nucleic acid sequences for incorporation into a transdermal delivery system described herein include nucleic acids that encode NS3/4A, preferably a codon-optimized (human) NS3/4A sequence, as described herein (e.g., SEQ. ID. NOs.: 163, 197, nucleic acids encoding SEQ. ID.
  • nucleic acids at least, less than or equal to, or more than 50, 60, 75, 80, 90, 100, 110, 120, 130, 140, 150, 160 , 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 consecutive nucleotides in length or the complement thereof).
  • transdermal delivery compositions disclosed herein also include transdermal delivery systems that comprise adjuvants.
  • some formulations of transdermal delivery compositions comprise an immunogenic peptide or nucleic acid encoding said peptide, a vaccine, such as a DNA vaccine, polypeptide vaccine, or other vaccine, and an adjuvant, such as aluminium hydroxide, ribavirin, calcium phosphate, cytokines (such as, e.g., interleukin-12 (IL-12)), co-stimulatory molecules (such as, e.g., B7-1 (CD80) or B7-2 (CD86)), and haptens, such as dinitrophenyl (DNP), and the like.
  • a vaccine such as a DNA vaccine, polypeptide vaccine, or other vaccine
  • an adjuvant such as aluminium hydroxide, ribavirin, calcium phosphate, cytokines (such as, e.g., interleukin-12 (IL-12)), co-stimulatory molecules (
  • the delivered agent is an immune response modifier.
  • the delivered immune response modifier is, for example, an imidazoquinoline amine including, but not limited to, substituted imidazoquinoline amines.
  • the delivered agent is an amide substituted imidazoquinoline amine, a sulfonamide substituted imidazoquinoline amine, a urea substituted imidazoquinoline amine, an aryl ether substituted imidazoquinoline amine, a heterocyclic ether substituted imidazoquinoline amine, an amido ether substituted imidazoquinoline amine, a sulfonamido ether substituted imidazoquinoline amine, a urea substituted imidazoquinoline ether, a thioether substituted imidazoquinoline amine, or a 6-, 7-, 8-, or 9-aryl or heteroaryl substituted imidazoquinoline amine.
  • the delivered agent is a tetrahyd'roimidazoquinoline amine such as an amide substituted tetrahydroimidazoquinoline amine, a sulfonamide substituted tetrahydroimidazoquinoline amine, a urea substituted tetrahydroimidazoquinoline amine, an aryl ether substituted tetrahydroimidazoquinoline amine, a heterocyclic ether substituted tetrahydroimidazoquinoline amine, an amido ether substituted tetrahydroimidazoquinoline amine, a sulfonamido ether substituted tetrahydroimidazoquinoline amine, a urea substituted tetrahydroimidazoquinoline ether, or a thioether substituted tetrahydroimidazoquinoline amine.
  • a tetrahyd'roimidazoquinoline amine such as an amide substitute
  • the deleivered agent is an imidazopyridine amine such as an amide substituted imidazopyridine amine, a sulfonamide substituted imidazopyridine amine, a urea substituted imidazopyridine amine, an aryl ether substituted imidazopyridine amine, a heterocyclic ether substituted imidazopyridine amine, an amido ether substituted imidazopyridine amine, a sulfonamido ether substituted imidazopyridine amine, a urea substituted imidazopyridine ether, or a thioether substituted imidazopyridine amines.
  • an imidazopyridine amine such as an amide substituted imidazopyridine amine, a sulfonamide substituted imidazopyridine amine, a urea substituted imidazopyridine amine, an aryl ether substituted imidazopyridine amine, a heterocyclic ether substituted imidazopyridine
  • the delivered agent is a 1,2-bridged imidazoquinoline amine; 6,7- fused cycloalkylimidazopyridine amine, a idazonaphthyridine amine, a tetrahydroimidazonaphthyridine amines, an oxazoloquinoline amine, a thiazoloquinoline amine; an oxazolopyridine amine, a thiazolopyridine amine, a oxazolonaphthyridine amine, a thiazolonaphthyridine amine, a lH-imidazo dimers fused to a pyridine amine, a quinoline amine, a tetrahydroquinoline amine, a naphthyridine amine, or a tetrahydronaphthyridine amine.
  • the immune response modifier is a purine derivative, an imidazoquinoline amide derivative, a lH-imidazopyridine derivative, a benzimidazole derivatives, a derivative of a 4-aminopyrimidine fused to a five membered nitrogen containing heterocyclic ring (including adenine derivatives), a 3-.beta.-D- ribofuranosylthiazolo[4,5-d]pyri- midine derivative, or a lH-imidazopyridine derivatives.
  • the immune response modifier is ribavirin.
  • immune response modifier compounds useful as delivered agents include 2-propyl[l,3]thiazolo[4,5-c]quinolin-4-amine, 4-amino- . alpha., .alpha.-dimethyl-lH-imidazo[4,5-c]quinoline-l-ethanol, and 4-amino-2-
  • Immune response modifier compounds include N-[4-(4- amino-2-butyl- 1 H-imidazo[4,5-c] [ 1 ,5]naphthyridin-l -yl)butyl]-N'-c- yclohexylurea, 2- methyl-1 -(2-methylpropyl)- 1 H-imidazo[4,5-c] [ 1 ,5]naphthyri- din-4-amine, 1 -(2- methylpropyl)-l H-imidazo[4,5-c] [ 1 ,5]naphthyridin-4-amine-, N- ⁇ 2-[4-amino-2-
  • Resiquimod and 4-amino-2-ethoxymethyl- . alpha. , . alpha, -dimethyl- 1 H-imidazo [4,5- cjquinolin- e-1-ethanol.
  • the delivered agent is an analgesic.
  • analgesiscs include tramadol hydrochloride, fentanyl, metamizole, morphine sulphate, ketorolac tromethamine, morphine, and loxoprofen sodium.
  • the delivered agent is a migraine therapeutic, such as ergotamine, melatonin, sumatriptan, zolmitriptan, or rizatriptan.
  • the delivered agent is an imaging component, such as iohexol, technetium, TC99M, sestamibi, iomeprol, gadodiamide, oiversol, and iopromide.
  • Diagnostic contrast components such as alsactide, americium, betazole, histamine, mannitol, metyraphone, petagastrin, phentolamine, radioactive B 12, gadodiamide, gadopentetic acid, gadoteridol, perflubron are delivered agents in certain embodiments.
  • a liposphere is made by identifying a delivered agent for incorporation into a liposphere and mixing said delivered agent with an ethoxylated or propoxylated fatty moiety, ethoxylated or propoxylated lipid moiety, or ethoxylated or propoxylated multifunctional backbone, wherein said ethoxylated fatty moiety, lipid moiety, or multifunctional backbone has between 10 and 19 ethoxylations per molecule.
  • the fatty moiety, or at least one of the fatty components of the lipid moiety or multifunctional backbone has a carbon chain length of between about 10 and 24 carbon residues.
  • compositions described herein are placed into a vessel that is joined to an applicator such that the active ingredients can be easily provided to a subject.
  • Applicators include, but are not limited to, roll-ons, bottles, jars, tubes, sprayer, atomizers, brushes, swabs, gel dispensing devices, and other dispensing devices.
  • compositions comprising a transdermal delivery and a transdermal delivery device, which provides a measured amount of said transdermal delivery system. Accordingly, desired dosages of delivered agents can be delivered to a subject in need.
  • An exemplary transdermal delivery device is depicted in Figures 17-20.
  • Yet other aspects of the present invention relate to methods of delivering an amount of a transdermal delivery composition
  • methods of delivering an amount of a transdermal delivery composition comprising providing a transdermal delivery composition within a transdermal delivery device, wherein the device is designed to administer a measured amount of the transdermal delivery composition and providing a transdermal delivery composition to be administered to a subject.
  • transdermal delivery compositions Several methods of using the transdermal delivery compositions are also embodiments.
  • one approach involves a method of reducing pain or inflammation by using a transdermal delivery composition that comprises an antiinflammatory molecule (e.g., an NSAID or MSM) on a subject in need of a reduction of pain or inflammation.
  • an antiinflammatory molecule e.g., an NSAID or MSM
  • Monitoring the reduction in inflammation may also be desired as part of a rehabilitation program.
  • NSAIDs and other chemotherapeutic agents have also been shown to improve the health, welfare, or survival of subjects that have cancer or Alzheimer's disease.
  • the tendency of these compounds to cause adverse side effects such as gastrointestinal irritation liver and kidney problems renders them particularly desirable transdermal delivery agents.
  • some embodiments concern methods of using transdermal delivery compositions that comprise delivered agents (e.g., any one or combination of the NSAIDs disclosed above or other chemotherapeutic agents such as fluorouracil) to treat or prevent cancer or hyperproliferative cell disorders (e.g., basal cell carcinoma or actinic keratosis.)
  • delivered agents e.g., any one or combination of the NSAIDs disclosed above or other chemotherapeutic agents such as fluorouracil
  • cancer or hyperproliferative cell disorders e.g., basal cell carcinoma or actinic keratosis.
  • a method to improve the health, welfare, or survival of a subject that has cancer or Alzheimer's disease or a method of treating or preventing cancer or Alzheimer's disease in said subject can be conducted by using a transdermal delivery composition that comprises a COX enzyme inhibitor and providing said transdermal delivery composition to said subject.
  • transdermal delivery compositions can be used to reduce oxidative stress to cells, tissues and the body of a subject.
  • a method to improve the health, welfare, or survival of a subject that is in need of a reduction in oxidative stress to a cell, tissue, or the body as a whole involves providing to said subject a transdermal delivery composition that comprises an antioxidant such as ascorbic acid, tocopherol or tocotrienol or an anti-stress compound such as Bacocalmine (Bacopa Monniera Extract obtained from Sederma Laboratories).
  • an antioxidant such as ascorbic acid, tocopherol or tocotrienol
  • an anti-stress compound such as Bacocalmine (Bacopa Monniera Extract obtained from Sederma Laboratories).
  • transdermal delivery composition that comprises peptides that promote wound healing ⁇ e.g., peptides comprising the sequence LKEKK (SEQ. ID.
  • psoriasis or eczema are provided to a subject in need of a treatment or reduction in psoriasis or eczema or a condition associated with psoriasis or eczema ⁇ e.g., allergies) or treatment of a wound.
  • transdermal delivery composition can be used to relax the muscles of a subject.
  • a transdermal delivery composition that comprises a compound that relaxes the muscles ⁇ e.g., chlorzoxazone or ibuprofen) is provided to a subject in need of a muscle relaxant. Accordingly methods of treating or preventing muscle soreness are embodiments.
  • transdermal delivery compositions can be used to raise the levels of a hormone in a subject in need thereof.
  • a transdermal delivery composition that comprises a hormone ⁇ e.g., any one of or combination of the hormones disclosed above or derivatives or functional analogues thereof) is provided to a subject in need thereof. Accordingly methods of treating or preventing a hormone deficiency or methods of increasing the level of a hormone in a subject using one of the transdermal delivery compositions described herein are embodiments.
  • transdermal delivery composition can be used to raise the levels of a hormone, for example, growth factor in a subject in need thereof.
  • a transdermal delivery composition that comprises a growth factor ⁇ e.g., a growth factor contained in Bioserum, which is obtainable through Atrium Biotechnologies of Quebec City, Canada) is provided to a subject in need thereof.
  • a transdermal delivery composition comprising a peptide that comprises the sequence LKEKK (SEQ ID NO:1) is provided to a subject in need of an increase in a growth factor.
  • transdermal delivery composition that comprises oxytocin, vasopressin, insulin, melanocyte-stimulating hormone, corticortropin, lipotropin, thyrotropin, growth hormone, prolactin, luteinizing hormone, human chorionic gonadotropin, follicle stimulating hormone, corticotropin-releasing factor, gonadotropin- releasing factor, prolactin-releasing factor, prolactin-inhibiting factor, growth-hormone releasing factor, somatostatin, thyrotropin-releasing factor, calcitonin, calcitonin gene- related peptide, parathyroid hormone, glucagon-like peptide 1, glucose-dependent insulinotropic polypeptide, gastrin, secretin, cholecystokinin, motilin,
  • transdermal delivery compositions described herein are used to brighten the skin, reduce age spots or skin discolorations, reduce stretch marks, reduce spider veins, or add dyes, inks, (e.g., tattoo ink), perfumes, or fragrances to the skin of a subject.
  • dyes e.g., tattoo ink
  • perfumes e.g., perfumes, or fragrances
  • transdermal delivery compositions that comprise a compound that brightens the skin or reduces age spots or skin discolorations (e.g., Melaslow, a citrus-based melanin (tyrosinase) inhibitor obtainable from Revivre, Laboratories of Singapore or Etioline, a skin brightener made from an extract from the Mitracarpe leaf obtainable from Krobell, USA), or a compound that reduces stretch marks (Kayuuputih Eucalyptus Oil, obtainable from Striad Laboratories) or add dyes, inks, (e.g., tattoo ink), perfumes, or fragrances are provided to the skin of a subject.
  • a compound that brightens the skin or reduces age spots or skin discolorations e.g., Melaslow, a citrus-based melanin (tyrosinase) inhibitor obtainable from Revivre, Laboratories of Singapore or Etioline, a skin brightener made from an extract from the Mitracarpe leaf
  • Still more embodiments concern formulations of a transdermal delivery system that contain viral antigens, in particular hepatitis viral antigens and, preferably, HCV antigens (e.g., the HCV antigens encoded by SEQ. ID. NOs.: 163, 197, nucleic acids encoding SEQ. ID.
  • nucleic acids at least, less than or equal to, or more than 50, 60, 75, 80, 90, 100, 110, 120, 130, 140, 150, 160 , 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 consecutive nucleotides in length or the complement thereof).
  • transdermal delivery compositions that comprise an ethoxylated oil, for example, a macadamia nut oil of 10-19 ethoxylations/molecule (i.e., 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 ethoxylations/molecule), an aqueous adjuvant (e.g., water), optionally an alcohol, and an HCV immunogen, wherein said immunogen comprises, consists of, or consists essentially of an NS3/4A molecule (e.g., an NS3/4A molecule encoded by SEQ. ID. NOs.: 163, 197, nucleic acids encoding SEQ. ID.
  • an ethoxylated oil for example, a macadamia nut oil of 10-19 ethoxylations/molecule (i.e., 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 ethoxylations/molecule), an aqueous adjuvant (e.g., water), optionally an alcohol, and an HCV immunogen, wherein said
  • nucleic acids at least, less than or equal to, or more than 50, 60, 75, 80, 90, 100, 110, 120, 130, 140, 150, 160 , 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 consecutive nucleotides in length or the complement thereof).
  • ethoxylated oil by itself, preferably macadamia nut oil having 10-19 ethoxylations/molecule (i.e., 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 ethoxylations/molecule), has therapeutic and cosmetic properties.
  • an ethoxylated oil e.g., macadamia nut oil having 16 ethoxylations/molecule
  • an ethoxylated oil e.g., macadamia nut oil having 16 ethoxylations/molecule
  • a burn e.g., a sun burn or a skin burn obtained from overheated metal
  • some embodiments concern a transdermal delivery composition
  • a transdermal delivery composition comprising an ethoxylated oil (e.g., macadamia nut oil that was ethoxylated 10-19 ethoxylations per molecule, 11-19 per molecule, 12-18 ethoxylations per molecule, 13-17 ethoxylations per molecule, 14-16 ethoxylations per molecule, or 15 ethoxylations per molecule) and these compositions are used to reduce the appearance of stretch marks and spider veins or facilitate the recovery from burns of the skin.
  • an ethoxylated oil e.g., macadamia nut oil that was ethoxylated 10-19 ethoxylations per molecule, 11-19 per molecule, 12-18 ethoxylations per molecule, 13-17 ethoxylations per molecule, 14-16 ethoxylations per molecule, or 15 ethoxylations per molecule
  • compositions that have high molecular weight delivered agents e.g., collagens
  • methods of use of such compositions are embodiments of the invention.
  • Preferred formulations of the transdermal delivery composition comprise a collagen (natural or synthetic) or fragment thereof at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 24, 30, 40, 50, 100, 250, 500, 1000, 1500, 2000, 2500, 3000, 5000, or more amino acids in length and these compositions are used to reduce wrinkles and fine lines on a subject.
  • some embodiments concern a transdermal delivery composition
  • a transdermal delivery composition comprising an ethoxylated fatty moiety, an ethoxylated lipid (e.g., macadamia nut oil that was ethoxylated 10-19 ethoxylations per molecule, 11-19 per molecule, 12-18 ethoxylations per molecule, 13-17 ethoxylations per molecule, 14-16 ethoxylations per molecule, or 15 ethoxylations per molecule), or an ethoxylated transdermal delivery enhancer and a therapeutically effective amount of a collagen or fragment thereof (e.g., marine collagen).
  • a collagen or fragment thereof e.g., marine collagen
  • a transdermal delivery composition comprising an ethoxylated oil and collagen also contains water and/or an alcohol and/or an aqueous adjuvant such as Aloe Vera.
  • the collagen has a molecular weight less than, or equal to 6,000 daltons or greater than 6,000 daltons.
  • the collagen can have an approximate molecular weight as low as 2,000 daltons or lower. In other embodiments, the molecular weight is from about 300,000 daltons to about 500,000 daltons.
  • these transdermal delivery compositions can have a therapeutically effective amount of collagen or fragment thereof by weight or volume that is 0.1% to 85.0%.
  • the collagen can be any natural or synthetic collagen, for example, Hydrocoll EN-55, bovine collagen, human collagen, a collagen derivative, marine collagen, Solu-Coll, or Plantsol, recombinant or otherwise man made collagens or derivatives or modified versions thereof (e.g., protease resistant collagens).
  • an apparatus having a vessel joined to an applicator that houses the transdermal delivery composition containing collagen is also an embodiment and preferred applicators or dispensers include a roll-on or a sprayer.
  • some of the embodied methods concern the reduction of wrinkles and or the improvement of skin tone by using a transdermal delivery composition comprising an ethoxylated oil and a collagen and/or a fragment thereof.
  • Some formulations to be used to reduce wrinkles and improve skin tone include an ethoxylated fatty moiety, an ethoxylated lipid moiety (e.g., macadamia nut oil that was ethoxylated 10-19 ethoxylations per molecule, 11-19 per molecule, 12-18 ethoxylations per molecule, 13-17 ethoxylations per molecule, 14-16 ethoxylations per molecule, or 15 ethoxylations per molecule), or an ethoxylated transdermal delivery enhancer, and a therapeutically effective amount of a collagen or fragment thereof (e.g., marine collagen) that is at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 24, 30, or 40 amino acids in length.
  • a collagen or fragment thereof e.g.,
  • transdermal delivery composition comprising an ethoxylated oil and collagen or fragment thereof, as described above, and, optionally, water and/or an alcohol and/or an aqueous adjuvant such as Aloe Vera.
  • a method of reducing wrinkles or improving skin tone is practiced by identifying a subject in need thereof and providing said subject a transdermal delivery composition, as described herein and, optionally, monitoring the subject for restoration or improvement of skin tone and the reduction of wrinkles.
  • FIGURE 1 is a graph showing the antibody titer in H-2 d mice against NS3 as a function of time after the first intra muscular immunization.
  • Diamonds denote antibody titer in mice immunized with NS3/4A-pVAX and squares denote antibody titer in mice immunized with NS3-pVAX.
  • FIGURE 2 shows the in vivo protection conferred by one gene gun immunization of NS3/4A-pVAXl (4 ⁇ g) or MSLFl -p VAXl (4 ⁇ g). Mice were immunized with the respective plasmid and 14 days later the mice were challenged with an NS3/4A expressing SP2/0 cell line (approximately 10 6 cells/mouse). Tumor size was then measured through the skin daily following day 6 post-challenge and the data plotted.
  • FIGURE 3 shows the in vivo protection conferred by two gene gun immunizations of NS3/4A-pVAXl (4 ⁇ g) or MSLFl -p VAXl (4 ⁇ g).
  • Mice were immunized with the respective plasmid at weeks zero and week four and, 14 days after the last immunization, the mice were challenged with an NS3/4A expressing SP2/0 cell line (approximately 10 6 cells/mouse). Tumor size was then measured through the skin daily following day 6 post-challenge and the data plotted.
  • FIGURE 4 shows the in vivo protection conferred by three gene gun immunizations of NS3/4A-pVAXl (4 ⁇ g) or MSLF 1-p VAXl (4 ⁇ g).
  • Mice were immunized with the respective plasmid at weeks zero, week four, and week eight and, 14 days after the last immunization, the mice were challenged with an NS3/4A expressing SP2/0 cell line (approximately 10 6 cells/mouse). Tumor size was then measured through the skin daily following day 6 post-challenge and the data plotted.
  • FIGURE 5A is a graph showing the percentage of specific CTL- mediated lysis of SP2/0 target cells as a function of the effector to target ratio.
  • Phosphate Buffered Saline (PBS) was used as a control immunogen.
  • FIGURE 5B is a graph showing the percentage specific CTL-mediated lysis of SP2/0 target cells as a function of the effector to target ratio. Plasmid NS3/4A- pVAX was used as the immunogen.
  • FIGURE 6A is a graph showing the response of naive splenic T cells that were stimulated with peptide coated RMA-S cells. The naive splenic T cells were obtained from C57/BL6 mice.
  • FIGURE 6B is a graph showing the response of splenic T cells that were restimulated with peptide coated RMA-S cells. The splenic T cells were obtained from C57/BL6 mice that were provided a single 4 ⁇ g dose of MSLFl -p V AXl .
  • FIGURE 6C is a graph showing the response of splenic T cells that were restimulated with peptide coated RMA-S cells.
  • the splenic T cells were obtained from C57/BL6 mice that were provided a single 4 ⁇ g dose of NS3/4A-pVAXl .
  • FIGURE 6D is a graph showing the response of naive splenic T cells that were stimulated with peptide coated RMA-S cells.
  • the naive splenic T cells were obtained from C57/BL6 mice.
  • FIGURE 6E is a graph showing the response of splenic T cells that were restimulated with peptide coated RMA-S cells.
  • the splenic T cells were obtained from C57/BL6 mice that were provided two 4 ⁇ g doses of MSLFl -p V AXl.
  • FIGURE 6F is a graph showing the response of splenic T cells that were restimulated with peptide coated RMA-S cells.
  • the splenic T cells were obtained from C57/BL6 mice that were provided two 4 ⁇ g doses of NS3/4A-pVAXl .
  • FIGURE 6G is a graph showing the response of naive splenic T cells that were stimulated with NS3/4A expressing EL-4 cells.
  • the naive splenic T cells were obtained from C57/BL6 mice.
  • FIGURE 6H is a graph showing the response of splenic T cells that were restimulated with NS3/4A expressing EL-4 cells.
  • the splenic T cells were obtained from C57/BL6 mice that were provided a single 4 ⁇ g dose of MSLFl -p V AXl.
  • FIGURE 61 is a graph showing the response of splenic T cells that were restimulated with NS3/4A expressing EL-4 cells.
  • the splenic T cells were obtained from C57/BL6 mice that were provided a single 4 ⁇ g dose of NS3/4A-pVAXl.
  • FIGURE 6J is a graph showing the response of naive splenic T cells that were stimulated with NS3/4A expressing EL-4 cells.
  • the naive splenic T cells were obtained from C57/BL6 mice.
  • FIGURE 6K is a graph showing the response of splenic T cells that were restimulated with NS3/4A expressing EL-4 cells.
  • the splenic T cells were obtained from C57/BL6 mice that were provided two 4 ⁇ g doses of MSLFl -p V AXl.
  • FIGURE 6L is a graph showing the response of splenic T cells that were restimulated with NS3/4A expressing EL-4 cells.
  • the splenic T cells were obtained from C57/BL6 mice that were provided two 4 ⁇ g doses of NS3/4A-pVAXl .
  • FIGURE 7 is a graph showing the humoral response to 10 and lOO ⁇ g recombinant Hepatitis C virus (HCV) non structural 3 protein (NS3), as determined by mean end point titres, when a single dose of lmg of ribavirin was co-administered.
  • HCV Hepatitis C virus
  • FIGURE 8 is a graph showing the humoral response to 20 ⁇ g recombinant Hepatitis C virus (HCV) non structural 3 protein (NS3), as determined by mean end point titres, when a single dose of 0.1, 1.0, or lOmg of ribavirin was coadministered.
  • HCV Hepatitis C virus
  • NS3 non structural 3 protein
  • FIGURE 9 is a graph showing the effects of a single dose of lmg ribavirin on NS3-specific lymph node proliferative responses, as determined by in vitro recall responses.
  • FIGURE 10 shows the mean NS3-specific antibody responses primed by gene gun immunisations with 4 ⁇ g wtNS3/4A-pVAXl and coNS3/4A-pVAXl, or s.c. injection of 10 7 wtNS3/4A-SFV particles in groups of ten H-2 d mice (a). All mice were immunized at weeks zero and four. Values are given as mean end-point antibody titres ( ⁇ SD.). Also shown (b) are the IgG subclass patterns from groups of five mice immunized twice with wtNS3/4A-pVAXl given i.m., coNS3/4A-pVAXl given Lm.
  • FIGURE 11 shows a flow cytometric quantification of the precursor frequency of NS3/4A-specific CD8+ T cells using peptide-loaded H-2D b :Ig fusion protein.
  • a) the mean % NS3-specific CD8+ T cells from groups of five mice immunized twice with wtNS3-pVAXl, wtNS3/4A- ⁇ VAXl, or coNS3/4A-pVAXl using gene gun is shown.
  • a "*" sign indicates a difference of p ⁇ 0.05, and NS (not significant) indicates no statistical difference (Mann- Whitney).
  • FIGURE 12 shows the priming of in vitro detectable CTLs in H-2 b mice by gene gun immunization of the wtNS3-pVAXl, wtNS3/4A, and coNS3/4A plasmids, or s.c. injection of wtNS3/4A-SFV particles. Groups of five to 10 H-2 b mice were immunized once (a) or twice (b).
  • the percent specific lysis corresponds to the percent lysis obtained with either NS3-peptide coated RMA-S cells (upper panel in (a) and (b) or NS3/4A-expressing EL-4 cells (lower panel in a and b) minus the percent lysis obtained with unloaded or non-transfected EL-4 cells. Values have been given for effector to target (E:T) cell ratios of 60:1, 20:1 and 7:1. Each line indicates an individual mouse.
  • FIGURE 13 shows the specificity of tumor inhibiting immune responses primed by gene gun immunization (panel (a)).
  • Panels of ten C57BL/6 mice were either left untreated or were given two monthly immunizations with 4 ⁇ g of coNS3/4A-pVAXl.
  • mice were injected sub cutaneously with the parental EL-4 cell line or 10 6 NS3/4A-expressing EL-4 cells. Tumor sizes were measured through the skin at days 6, 7, 10, 11, 12, and 14 after tumour injection, hi (b) the in vivo functional effector cell population was determined in groups of 10 C57BL/6 mice immunized twice with the coNS3/4A-pVAXl plasmid using gene gun.
  • CD4+ or CD8+ T cells were depleted by administration of monoclonal antibodies one week prior to, and during, challenge with the NS3/4A- expressing EL-4 cell line.
  • Tumor sizes were measured through the skin at days 5, 6, 8, 11, 13, 14, and 15 after tumour injection. Values have been given as the mean tumor size ⁇ standard error.
  • a "**" sign indicates a statistical difference of p ⁇ 0.01
  • a "*” sign indicates a difference of p ⁇ 0.05
  • NS (not significant) indicates no statistical difference (area under the curve values compared by ANOVA).
  • FIGURE 14 shows an evaluation of the ability of different immunogens to prime HCV NS3/4A-specific rumor-inhibiting responses after a single immunization.
  • Groups of ten C57BL/6 mice were either left untreated or were given one immunization with the indicated immunogen (4 ⁇ g DNA using gene gun in (a), (b), (c), (g), and (h); 10 7 SFV particles s.c. in d; 100 ⁇ g peptide in CFA s.c. in (e); and 20 ⁇ g rNS3 in CFA s.c. in (f).
  • mice Two weeks after last immunization, mice were injected sub cutaneously with 10 6 NS3/4A-expressing EL-4 cells.
  • Tumor sizes were measured through the skin at days 6 to 19 after tumor injection. Values have been given as the mean tumor size ⁇ standard error.
  • a negative control the mean data from the group immunized with the empty pVAX plasmid by gene gun has been plotted in each graph.
  • the negative controls were non-immunized mice. Also given is the p value obtained from the statistical comparison of the control with each curve using the area under the curve and ANOVA.
  • FIGURE 15 shows the comparative efficiency of gene gun delivered wtNS3/4A-pVAXl and coNS3/4A-pVAXl plasmids in priming tumor inhibiting immune responses.
  • a "**" sign indicates a statistical difference of p ⁇ 0.01, a "*" sign indicates a difference of p ⁇ 0.05, and NS (not significant) indicates no statistical difference (area under the curve values compared by ANOVA).
  • FIGURE 16 shows the effect of therapeutic vaccination with the coNS3/4A plasmid using the gene gun.
  • Groups of ten C57BL/6 mice were inoculated with 10 6 NS3/4A-EL4 cells.
  • One group had been immunized once with 4 ⁇ g coNS3/4A DNA using a gene gun two weeks prior to challenge (positive control), one group was immunized the same way six days after tumor inoculation, and one group was immunized 12 days after tumor inoculation.
  • One group was not immunized (negative control).
  • Tumor sizes were measured through the skin at days 6, 10, 11, 12, 13, 14, 18, 19, and 20 after tumour injection. Values have been given as the mean tumor size ⁇ standard error.
  • a "**" sign indicates a statistical difference of p ⁇ 0.01, a "*" sign indicates a difference of p ⁇ 0.05, and NS (not significant) indicates no statistical difference (area under the curve values compared by ANOVA).
  • FIGURE 17A schematically depicts in an exploded state a dispenser for delivery of a transdermal drug delivery system fluid comprising a removable cartridge.
  • FIGURE 17B schematically depicts the dispenser of Figure 17A in an assembled state.
  • FIGURE 18 schematically depicts a cross section of the dispenser of Figure IB.
  • FIGURE 19A schematically depicts a cross section of the upper portion of a partially filled dosing chamber having an upper wall configured allow air to escape, but prevent fluid from escaping.
  • FIGURE 19B schematically depicts the upper portion of the dosing chamber of Figure 19 A, where the dosing chamber is full and fluid is prevented from escaping.
  • FIGURE 20A schematically depicts a cross-section of the dispenser of Figure 18, taken along the line 4, wherein the slidable member is in a first position permitting filling of the dosing chamber.
  • FIGURE 2OB schematically depicts the cross-section of Figure 2OA, wherein the slidable member is in a second position permitting delivery of the dosed fluid.
  • transdermal delivery compositions and devices for providing said compositions to a subject are described herein.
  • Embodiments of the invention can be used to transdermally deliver low or high (or both low and high) molecular weight pharmaceuticals, prophylactics, diagnostics, and cosmetic agents to a subject.
  • the transdermal delivery compositions disclosed herein are useful for the delivery of various types of compounds including but not limited to nucleic acids, peptides, modified peptides, small molecules, immunogenic preparations, and the like.
  • Preferred embodiments concern transdermal delivery compositions that comprise an NS3/4A nucleic acid sequence provided by SEQ. ID. NOs.: 163, 197, nucleic acids encoding SEQ. ID.
  • nucleic acids at least, less than or equal to, or more than 50, 60, 75, 80, 90, 100, 110, 120, 130, 140, 150, 160 , 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 consecutive nucleotides in length or the complement thereof and, optionally, ribavirin.
  • transdermal delivery compositions that can administer compounds having molecular weights greater than 6,000 daltons.
  • One embodiment for example, includes a transdermal delivery composition that can administer a therapeutically effective amount of a non-steroidal anti-inflammatory drug (NSAID).
  • NSAID non-steroidal anti-inflammatory drug
  • Still more embodiments concern transdermal delivery compositions that can administer hormones, anesthetics, collagen preparations e.g., soluble collagens, hydrolyzed collagens, and fragments of collagen), cardiovascular pharmaceutical compounds, anti-infective compounds (e.g.
  • antibiotics and antiviral compounds include diabetes-related treatments, immunogenic compositions, vaccines, immune response modifiers, enzyme inhibitors, analgesics ⁇ e.g., a formulation comprising capsaicin or Boswellin or both), migraine therapies, sedatives, imaging and contrast compounds.
  • analgesics e.g., a formulation comprising capsaicin or Boswellin or both
  • migraine therapies sedatives, imaging and contrast compounds.
  • liposphere refers to a spherical or ovoid-shaped structure comprising an ethoxylated or propoxylated fatty moiety, which contains or is associated with (e.g., joined to) a delivered agent. That is, the term “lipospheres” includes, but is not limited to, liposomes that comprise an ethoxylated or propoxylated oil, fatty acid, fatty amine, or fatty alcohol. Accordingly, the term “fatty moiety” can refer to a fatty acid, a fatty alcohol, fatty amine, or other fatty acid derivative.
  • the ethoxylated fatty moiety or lipid moiety has both hydrophobic and hydrophilic properties, in that the hydrocarbon chain of the fatty moiety or lipid moiety is hydrophobic, and the polyethoxy groups confer hydrophilicity on the molecule.
  • the preparation of propoxylated fatty moieties and lipid moieties is well known. ⁇ See, e.g., Raths et al., supra). Due to their similarity in structure, propoxylated fatty moieties and lipids will share many of the same characteristics as ethoxylated fatty moieties and lipids. Accordingly, fatty moieties and lipid moieties that are propoxylated or ethoxylated and propoxylated are contemplated penetration enhancers and transdermal delivery enhancers.
  • the number of ethoxylations is adjusted to between 10 and 19 ethoxylations per molecule to achieve optimal transdermal delivery of the delivered agent.
  • Ethoxylated fatty acids, fatty alcohols, and fatty amines are commercially available ⁇ e.g., Ethox Chemicals, LLC, Greenville, SC; A&E Connock, Ltd., Hampshire, England; Floratech, Glibert, AZ).
  • ethoxylated fatty moieties are synthesized using methods known to those skilled in the art ⁇ See, U.S. Patent No. 6,300,508 to Raths et al. ⁇ U.S. Patent No. 5,936,107 to Raths et al.) by reacting fatty moieties with ethylene oxide.
  • ethoxylated oils are prepared using a two-step process that starts with trans-esterification with added glycerol followed ethoxylation of the product of this reaction.
  • Trans-esterification is performed by any method available to those skilled in the art, such as heating an ester, such as the glycerol esters present in natural vegetable oils, in the presence of another alcohol or polyol, such as glycerol, in the presence of a catalyst.
  • Catalysts useful in the transesterification reaction include gaseous catalysts, such hydrochloric acid bubbled through the reaction mixture.
  • solid catalysts such as zinc oxide or the acetates of copper, cobalt or zinc can also be used.
  • the transesterificaiton reaction produces one or two fatty acids attached to a molecule of glycerol.
  • the ratio of mono- and di-esters can be controlled by the amount of glycerol used in the reaction (i.e. higher ratios of glycerolroil will yield more reactive -OH and fewer fatty acid moieties per molecule, and a lower ratio of glycerol: oil would give more fatty acids, as is apparent to those skilled in the art.
  • the hydroxyl groups are subsequently reacted with ethylene oxide in the presence of an appropriate catalyst, (e.g., aluminum) using methods known to those skilled in the art.
  • transdermal delivery compositions described herein comprise a penetration enhancer that includes an ethoxylated lipid moiety. It was discovered that ethoxylated lipids ⁇ e.g., ethoxylated oils) can be used as transdermal penetration enhancers in that they effectively transport low and high molecular weight compounds through the skin. It was also discovered that ethoxylated oils, by themselves, have therapeutic and cosmetic applications ⁇ e.g., the reduction of the appearance of spider veins and stretch marks or promoting expedited recovery from burns to the skin).
  • Ethoxylated lipids can be created in many ways, however, a preferred approach involves the reaction of ethylene oxide with a vegetable, nut ⁇ e.g., macadamia nut), animal, or synthetic oil.
  • the transdermal delivery composition comprises an ethoxylated oil
  • ethoxylated fatty moieties are used to fortify or supplement ethoxylated oils in some embodiments.
  • ethoxylated macadamia nut oil can be fortified with ethoxylated palmitic or oleic acid.
  • transdermal delivery enhancers disclosed herein are compounds having a multifunctional backbone.
  • the multifunctional backbone can be one of many chemical structures that have at least two reactive hydrogen residues, such that the multifunctional backbone is the basis of a transdermal delivery enhancer with least one fatty moiety and at least one polyethoxy group.
  • the reactive hydrogen residues (R) are present in -OH, COOH, SH, and NH 2 , groups.
  • the polyethoxy group has the structure:
  • n is between 10 and 19 per molecule of transdermal delivery enhancer to possess superior transdermal delivery properties.
  • the fatty moiety component of the t ⁇ ansdermal delivery enhancer has a carbon chain of at least 10 carbon residues.
  • the chain length of the fatty moiety can be for example 10, 12, 14, 16, 18, 20, 22, or 24 residues.
  • the fatty moiety may be saturated, unsaturated, or polyunsaturated.
  • the multifunctional backbone has at least three reactive groups.
  • the reactive groups can be homogeneous.
  • the multifunctional backbone is a tri-alcohol comprising three —OH groups, such as 1, 2, 3-butanetriol, 1, 2, 4 butantetriol, pyrogallol (1, 2, 3-benezentriol), hydroxyquinol (1, 2, 4-benzenetriol), trimethyololpropane, 1, 2, 6-hexanetriol and the like.
  • tri-acids comprising three carboxylate groups, such as hemi-mellitic acid, trimellitic acid, trimesic acid, nitrilotriacetic acid, and the like.
  • Those skilled in the art will appreciate that other tricarboxylic acids are suitable as multifunctional backbones.
  • Alternative multifunctional backbones have heterogeneous reactive groups, e.g., a combination of at least two different reactive groups (e.g., a COOH group and an NH 2 group).
  • a COOH group and an NH 2 group For example, amino acids such as glutamic acid, aspartic acid, cysteine, glutamine, serine, threonine, tryrosine, and lysine have three reactive groups and are suitable as multifunctional backbones.
  • di- and tri-peptides will have three or more reactive groups and are thus suitable as multifunctional backbones.
  • Triethanolamine, diethanolamine, dimethylolurea, and glucosamine are other exemplary multifunctional backbones with heterogeneous reactive groups.
  • Simple carbohydrates are small straight-chain aldehydes and ketones with several hydroxyl groups, usually one on each carbon except the functional group. Due to the presence of the multiple -OH groups on carbohydrates such as tetroses, pentoses, hexoses, and so forth, these compounds are another source of multifunctional backbones useful as components of transdermal delivery enhancers.
  • Exemplary carbohydrates that are useful components of transdermal delivery enhancers include glucose, mannose, fructose, ribose, xylose, threose, erythrose, and the like.
  • Sugar alcohols such as sorbitol, mannitol, xylitol, erythritol, petaerythritol, and inositol are useful components of transdermal delivery enhancers.
  • the ethoxylated fatty moiety, ethoxylated lipid moiety, or ethoxylated multifunctional backbone encapsulates the delivered agent in a sphere-like composition, forming a "liposphere" that exhibits greatly enhanced transdermal delivery properties.
  • Each of the disclosed transdermal delivery compositions can contain additional compounds such as alcohols, nonionic solubilizers or emulsifiers. In some compositions, these compounds are added to improve the solubility of the delivered agent or effectiveness or fluidity of the liposphere, penetration enhancer, or transdermal delivery enhancer.
  • Suitable hydrophilic components include, but are not limited to, ethylene glycol, propylene glycol, dimethyl sulfoxide (DMSO), dimethyl polysiloxane (DMPX), oleic acid, caprylic acid, isopropyl alcohol, 1-octanol, ethanol (denatured or anhydrous), and other pharmaceutical grade or absolute alcohols.
  • transdermal delivery compositions comprise an aqueous adjuvant.
  • Aqueous adjuvants include, but are not limited to, water (distilled, deionized, filtered, or otherwise prepared), Aloe Vera juice, and other plant extracts such as chlorophyll or Spirulina.
  • hydrophobic/hydrophilic component comprising an ethoxylated fatty moiety (e.g., palmitoleic acid, oleic acid, or palmitic acid) or an ethoxylated oil (e.g., macadamia nut oil, coconut oil, eucalyptus oil, synthetic oils, castor oil, glycerol, corn oil, jojoba oil, or emu oil) and may contain a hydrophilic component comprising an alcohol, a nonionic solubilizer, or an emulsif ⁇ er (e.g., isopropyl alcohol) and/or, optionally, an aqueous adjuvant, such as water and/or Aloe Vera extract.
  • an ethoxylated fatty moiety e.g., palmitoleic acid, oleic acid, or palmitic acid
  • an ethoxylated oil e.g., macadamia nut oil, coconut oil, euca
  • transdermal delivery compositions of the invention can also be components of a transdermal delivery composition of the invention including fragrance, creams, ointments, colorings, and other compounds so long as the added component does not deleteriously affect transdermal delivery of the delivered agent.
  • the Aloe Vera which allows for transdermal delivery of high molecular weight delivered agents, including collagen having an average molecular weight greater than 6,000 daltons, can be removed from transdermal delivery compositions comprising a light oil (e.g., macadamia nut oil) that has been ethoxylated to the range of 10 - 19 ethoxylations/molecule.
  • Formulations lacking Aloe Vera provide the unexpected benefit of efficient transdermal delivery, uniform application and quick penetration making these formulations superior to formulations that contain A Io e Vera.
  • formulations of transdermal delivery compositions that lack alcohol provide the unexpected benefit of efficient transdermal delivery, uniform application, and quick penetration without the drying or irritation brought about by the alcohol. Additionally, formulations lacking water or other aqueous adjuvants provide efficient transdermal delivery while maintaining the highest possible concentration of delivered agent and, also, provide for quick penetration without the skin-drying effects seen with some formulations that contain alcohol.
  • a molecule or a mixture of molecules (e.g., a pharmaceutical, chemical, or cosmetic agent) that are delivered to the body using an embodiment of a transdermal delivery composition are termed "delivered agents".
  • a delivered agent that can be administered to the body using an embodiment of the invention can include, for example, a protein or peptide, a sugar, a nucleic acid, a chemical, a lipid, or derivatives of the same.
  • Desirable delivered agents include, but are not limited to, glycoproteins, enzymes, genes, nucleic acids, peptides, drugs, and ceramides.
  • Preferred delivered agents include NSAIDS, collagens or fragments thereof, capsaicin, and Boswellin.
  • a transdermal delivery composition comprises a combination of any two of the aforementioned delivered agents.
  • Other delivered agents include, for example, hormones, anti-inflammatory drugs, anesthetics, analgesics, sedatives, migraine therapies, cardiovascular pharmaceuticals, anti-infective agents, diabetes-related therapies, vaccines, imaging agents, contrast agents, glucosamine, chondroitin sulfate, MSM, perfumes, melasin, nicotine, nicotine analogs, peptides, amino acids, nucleic acids, and peptidomimetics.
  • a transdermal delivery composition is prepared by mixing an ethoxylated fatty moiety with a delivered agent.
  • a transdermal delivery composition is prepared by mixing a hydrophilic component with a hydrophobic component and an aqueous adjuvant.
  • the delivered agent can be solubilized in either the ethoxylated oil, a hydrophobic, hydrophilic, or aqueous adjuvant or water prior to mixing.
  • heat can be applied to help coalesce the mixture.
  • the temperature is not raised above 40°C.
  • transdermal delivery compositions are within the scope of aspects of the invention, hi embodiments wherein the transdermal delivery composition includes an aqueous adjuvant, in further embodiments, the formulation comprises a ratio of hydrophilic component :hydrophobic component: aqueous adjuvant of 3:4:3.
  • the amount of delivered agent that is incorporated into the penetration enhancer depends on the compound, desired dosage, and application.
  • the amount of delivered agent in a particular formulation can be expressed in terms of percentage by weight, percentage by volume, or concentration.
  • a transdermal delivery composition comprising an NSAID, capsaicin, Boswellin or any combination thereof is provided to a patient in need of treatment, such as for relief of pain and/or inflammation.
  • a patient can be contacted with the transdermal delivery composition and treatment continued for a time sufficient to reduce pain or inflammation or inhibit the progress of disease, hi other embodiments, a transdermal delivery system comprising a nucleic acid that encodes an HCV antigen (e.g., SEQ. ID. NOs.: 163, 197, nucleic acids encoding SEQ. ID.
  • nucleic acids at least, less than or equal to, or more than 50, 60, 75, 80, 90, 100, 110, 120, 130, 140, 150, 160 , 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 consecutive nucleotides in length or the complement thereof) is provided to a subject in need of an immune response to HCV so as to promote an immune response in said subject to the virus.
  • aspects of the invention concern methods of inducing an immune response in a subject to HCV comprising providing a transdermal delivery system, as described herein, further comprising an NS3/4A nucleic acid of SEQ. ID. NOs.: 163, 197, nucleic acids encoding SEQ. ID.
  • nucleic acids at least, less than or equal to, or more than 50, 60, 75, 80, 90, 100, 110, 120, 130, 140, 150, 160 , 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 consecutive nucleotides in length or the complement thereof to said subject.
  • a method of reducing wrinkles, removing age spots, and increasing skin tightness and flexibility is provided.
  • a transdermal delivery composition comprising a collagen or fragment thereof or melaslow or other skin brightening agent is provided to a patient in need, the patient is contacted with the transdermal delivery composition, and treatment is continued for a time sufficient to restore a desired skin tone (e.g., reduce wrinkles, age spots, or restore skin brightness, tightness and flexibility), hi the disclosure below, there is provided a description of several of the delivered agents that can be incorporated into the transdermal delivery compositions described herein.
  • transdermal delivery compositions described herein Many different delivered agents can be incorporated into the various transdermal delivery compositions described herein. While the transdermal delivery of molecules having a molecular weight in the vicinity of 6000 daltons has been reported, it has not been possible, until the present invention, to administer molecules of greater size transdermally. (See U.S. Pat. No. 5,614,212 to D'Angelo et ah).
  • the described embodiments can be organized according to their ability to deliver a low or high molecular weight delivered agent.
  • Low molecular weight molecules e.g., a molecule having a molecular weight less than 6,000 daltons
  • high molecular weight molecules e.g., a molecule having a molecular weight greater than 6,000 daltons
  • a transdermal delivery composition described herein provides a therapeutically, prophylactically, diagnostically, or cosmetically beneficial amount of a delivered agent having a molecular weight of 50 daltons to less than 6,000 daltons.
  • a transdermal delivery composition described herein provides a therapeutically, prophylactically, diagnostically, or cosmetically beneficial amount of a delivered agent having a molecular weight of 50 daltons to 2,000,000 daltons or less. That is, a transdermal delivery composition described herein, preferably, provides a delivered agent having a molecular weight of less than or equal to or greater than 50, 100, 200, 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 5,500, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000, 28,000, 29,000, 30,000, 31,000, 32,000, 33,000, 34,000, 35,000, 36,000, 37,000, 38,000, 39,000, 40,000, 41,000, 42,000, 43,000, 44,000, 45,000,
  • a low molecular weight compound e.g., a pain relieving substance or mixture of pain relieving substances
  • the delivered agent can be, for example, any one or more of a number of compounds, including non-steroidal antiinflammatory drugs (NSAIDs) that are frequently administered systemically.
  • NSAIDs non-steroidal antiinflammatory drugs
  • ibuprofen (2-(isobutylphenyl)-propionic acid); methotrexate (N-[4-(2, 4 diamino 6 - pteridinyl - methyl] methylamino] benzoyl)-L-glutamic acid); aspirin (acetylsalicylic acid); salicylic acid; diphenhydramine (2-(diphenylmethoxy)-NN-dimethylethylamine hydrochloride); naproxen (2-naphthaleneacetic acid, 6-methoxy-9-methyl-, sodium salt, (- )); phenylbutazone (4-butyl-l,2-diphenyl-3,5-pyrazolidinedione); sulindac-(2)-5-fluoro-2- methyl-l-[[p-(methylsulfinyl)phenyl]methylene-]-lH-indene-3-acetic acid; diflunisal (2',4' 3
  • transdermal delivery compositions described herein which contain NSAIDs, desirably comprise an amount of the compound that is therapeutically beneficial for the treatment or prevention of disease or inflammation.
  • NSAIDs Several studies have determined an appropriate dose of an NSAID for a given treatment or condition. (See e.g., Woodin, RN, August: 26-33 (1993) and Amadio et al, Postgrduate Medicine, 93(4):73-97 (1993)). The maximum recommended daily dose for several NSAIDs is listed in TABLE 1.
  • a sufficient amount of NSAID can be incorporated into a transdermal delivery composition described herein such that a therapeutically effective amount of NSAID is effectively delivered to, a subject.
  • a therapeutically effective amount of NSAID is about 800mg/dose.
  • a 30 ml bottle containing a transdermal delivery system formulation and ibuprofen can contain 24 grams of ibuprofen such that 800mg of ibuprofen is provided in each 1.0 ml.
  • transdermal delivery compositions described herein can provide a delivered agent in a site-specific manner, a lower total dose of therapeutic agent, as compared to the amounts provided systemically, will provide therapeutic benefit. Additionally, greater therapeutic benefit can be gained by using a transdermal delivery composition described herein because a greater concentration of therapeutic agent (e.g., an NSAID) can be provided to the particular site of inflammation. That is, in contrast to systemic administration, which applies the same concentration of therapeutic to all regions of the body, a transdermal delivery composition can site-specifically provide the therapeutic agent and, thereby, provide a much greater regional concentration of the agent than if the same amount of therapeutic were administered systemically.
  • therapeutic agent e.g., an NSAID
  • Additional embodiments include a transdermal delivery composition that provides a pain relieving mixture comprising capsaicin (e.g., oleoresin capsicum) or Boswellin or both.
  • capsaicin e.g., oleoresin capsicum
  • Boswellin the pain relieving mixture
  • Capsaicin (8-methyl-N-vanillyl-6-nonenamide) the pungent component of paprika and peppers, is a potent analgesic.
  • Capsaicin produces a level of analgesia comparable to morphine, yet it is not antagonized by classical narcotic antagonists such as naloxone. Further, it effectively prevents the development of cutaneous hyperalgesia, but appears to have minimal effects on normal pain responses at moderate doses. At high doses capsaicin also exerts analgesic activity in classical models of deep pain, elevating the pain threshold above the normal value. Capsaicin can be readily obtained by the ethanol extraction of the fruit of Capsicum frutescens or Capsicum annum. Capsaicin and analogs of capsaicin are available commercially from a variety of suppliers, and can also be prepared synthetically by published methods. Aspects of the invention encompass the use of synthetic and natural capsaicin, capsaicin derivatives, and capsaicin analogs.
  • capsaicin used in several desirable embodiments is oleoresin capsicum.
  • Oleoresin capsicum contains primarily capsaicin, dihydrocapsaicin, nordihydrocapsaicin, homocapsaicin, and homodihydrocapsaicin.
  • capsaicin collectively refers to all forms of capsaicin, capsicum, and derivatives or modifications thereof. The pungency of these five compounds, expressed in Scoville units, is provided in TABLE 2.
  • the transdermal delivery compositions that are formulated to contain capsaicin desirably comprise by weight or volume 0.01% to 1.0% capsaicin or 1.0% to 10% oleoresin capsicum.
  • Preferred amounts of this delivered agent include by weight or volume 0.02% to 0.75% capsaicin or 2.0% to 7.0% oleoresin capsicum.
  • the transdermal delivery compositions that contain capsaicin can comprise by weight or volume less than or equal to 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, 0.05%, 0.055%, 0.06%, 0.065%, 0.07%, 0.075%, 0.08%, 0.085%, 0.09%, 0.095%, 0.1%, 0.15%, 0.175%, 0.2%, 0.225%, 0.25%, 0.275%, 0.3%, 0.325%, 0.35%, 0.375%, 0.4%, 0.425%, 0.45%, 0.475%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, and 1.0% capsaicin.
  • the transdermal delivery compositions of that contain capsaicin can also comprise an amount of capsaicin by weight or volume that is greater than 1.0%, such as 1.2%, 1.5%, 1.8%, 2.0%, 2.2%, 2.5%, 2.8%, 3.0%, 3.5%, 4.0%, 4.5%, and 5.0%.
  • the transdermal delivery compositions that contain oleoresin capsicum can comprise an amount of oleoresin capsicum less than 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 11.0%, 12.0%, and 13.0%.
  • Boswellin also known as Frankincense, is an herbal extract of a tree of the Boswellia family. Boswellin can be obtained, for example, from Boswellia thurifera, Boswellia carteri, Boswellia sacra, and Boswellia serrata. There are many ways to extract Boswellin and Boswellin gum resin and boswellic acids are obtainable from several commercial suppliers (a 65% solution of Boswellic acid is obtainable from Nature's Plus). Some suppliers also provide creams and pills having Boswellin with and without capsaicin and other ingredients.
  • Embodiments of the invention comprise Boswellin and the term "Boswellin” collectively refers to Frankincense, an extract from one or more members of the Boswellia family, Boswellic acid, synthetic Boswellin, or modified or derivatized Boswellin.
  • the transdermal delivery compositions that contain Boswellin desirably comprise 0.1% to 10% Boswellin by weight or volume. Preferred amounts of this delivered agent include 1.0% to 5.0% Boswellin by weight.
  • the transdermal delivery compositions that contain Boswellin can comprise by weight or volume less than or equal to 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1.0%, 1.1%, 1.15%, 1.2%, 1.25%, 1.3%, 1.35%, 1.4%, 1.45%, 1.5%, 1.55%, 1.6%, 1.65%, 1.7%, 1.75%, 1.8%, 1.85%, 1.9%, 1.95%, and 2.0%, 2.1%, 2.15%, 2.2%, 2.25%, 2.3%, 2.35%, 2.4%, 2.45%, 2.5%, 2.55%, 2.6%, 2.65%, 2.7%, 2.75%, 2.
  • the transdermal delivery compositions that contain Boswellin can also comprise amounts of Boswellin by weight that are greater than 5.0%, such as 5.5%, 5.7%, 6.0%, 6.5%%, 6.7%, 7.0%, 7.5%, 7.7%, 8.0%, 8.5%, 8.7%, 9.0%, 9.5%, 9.7%, and 10.0% or greater.
  • Boswellin from different sources can be combined to compose the Boswellin component of an embodiment. For example, in one embodiment an extract from Boswellia thurifera is combined with an extract from Boswellia serrata.
  • Additional embodiments of the invention comprise a transdermal delivery composition that can administer a pain relieving solution comprising two or more members selected from the group consisting of NSAIDs, capsacin, and Boswellin.
  • the transdermal delivery compositions that include two or more members selected from the group consisting of NSAIDs, capsacin, and Boswellin desirably comprise an amount of delivered agent that can be included in a delivered agent having an NSAID, capsaicin, or Boswellin by itself.
  • the amount of NSAID that can be used can be an amount recommended in the literature (See e.g., Woodin, RN, August: 26-33 (1993) and Amadio, et al., Postgrduate Medicine, 93(4):73-97 (1993)), or an amount listed in TABLE 1.
  • the transdermal delivery composition can comprise by weight or volume less than or equal to 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, 0.05%, 0.055%, 0.06%, 0.065%, 0.07%, 0.075%, 0.08%, 0.085%, 0.09%, 0.095%, 0.1%, 0.15%, 0.175%, 0.2%, 0.225%, 0.25%, 0.275%, 0.3%, 0.325%, 0.35%, 0.375%, 0.4%, 0.425%, 0.45%, 0.475%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, and 1.0% capsaicin or less than 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%
  • the delivery system can comprise by weight or volume less than or equal to 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1.0%, 1.1%, 1.15%, 1.2%, 1.25%, 1.3%, 1.35%, 1.4%, 1.45%, 1.5%, 1.55%, 1.6%, 1.65%, 1.7%, 1.75%, 1.8%, 1.85%, 1.9%, 1.95%, 2.0%, 2.1%, 2.15%, 2.2%, 2.25%, 2.3%, 2.35%, 2.4%, 2.45%, 2.5%, 2.55%, 2.6%, 2.65%, 2.7%, 2.75%, 2.8%, 2.85%, 2.9%, 2.95%, 3.0%, 3.1%, 3.15%, 3.2%, 3.25%, 3.3%, 3.35%, 3.4%, 3.45%, 3.5%, 3.55%, 3.6%
  • tramadol hydrochloride, fentanyl, metamizole, morphine sulphate, ketorolac tromethamine, hydrocodone, oxycodone, morphine and loxoprofen sodium are delivered agents in certain embodiments.
  • Steroidal anti-inflammatory compounds are also useful delivered agents in the transdermal delivery compositions described herein.
  • hydrocortisone, prednisolone, triamcinolone, and priroxicam are delivered agents in certain embodiments.
  • Local anesthetics are low molecular weight compounds that are useful as delivered agents in the transdermal delivery compositions described herein.
  • the transdermal delivery compositions disclosed herein are particularly useful in the context of local anesthetics, where a local, concentrated dose of a delivered agent is desirable.
  • Embodiments of the transdermal delivery compositions include local anesthetics, such as articaine, procaine, tetracaine, chloroprocaine and benzocaine, novocain, mepivicaine, bupivicaine, benzocaine, and lidocaine, and the like.
  • the maximum single dose for local anesthetic solutions is somewhere between 70 mg to 500 mg, depending upon the age and health of the patient.
  • Antibiotics are compounds that either kill bacterial or fungal cells, or prevent them from multiplying.
  • Several antibiotics are known to those skilled in the art and are delivered agents in certain embodiments of the transdermal delivery compositions, including but not limited to amoxicillin, clavulanate potassium, itraconazole, acyclovir, fluconazole, terbinafme hydrochloride, erythromycin ethylsuccinate, acetyl sulf ⁇ soxazole, penicillin V, cephalexin, erythromycin, azithromycin, tetracycline, ciproflaxin, gentamycin, sulfathiazole, nitrofurantoin, norfloxacin, flumequine, and ibafloxacin, metronidazole, and nystatin.
  • acyclovir acyclovir
  • lamivudine indinavir sulfate
  • stavudine stavudine
  • humates medium molecular weight delivered agents
  • synthetic humates are medium molecular weight compounds (1,000 to 100,000 daltons), which are known to be strong antiviral and antimicrobial medicaments. (See International Application Publication No. WO 9834629 to Laub). Hepsyls are generally characterized as polymeric phenolic materials comprised of conjugated aromatic systems to which are attached hydroxyl, carboxyl, and other covalently bound functional groups.
  • a transdermal delivery composition that can provide hepsyls to cells of the body has several pharmaceutical uses, including but not limited to, treatment of topical bacterial and viral infections.
  • a transdermal delivery system that can provide a medium molecular weight compound (e.g., a form of hepsyl) to cells of the body.
  • a medium molecular weight compound e.g., a form of hepsyl
  • many different medium molecular weight compounds can be provided using an embodiment of a transdermal delivery composition described herein and the use of a medium molecular weight hepsyl as a delivered agent is intended to demonstrate that embodiments of the invention can deliver many medium molecular weight compounds to cells of the body.
  • amino acids, peptides, nucleotides, nucleosides, and nucleic acids are transdermally delivered to cells in the body using an embodiment of the transdermal delivery composition described herein. That is, any amino acid or peptide having at least, less than, more than, or equal to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 75, 100, 125, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 7000, or 10,000 amino acids can be incorporated into a transdermal delivery composition described herein and said delivered agent can be delivered to cells in the body shortly after application of the composition.
  • Non-limiting examples of peptide hormones that are delivered agents in certain embodiments include oxytocin (SEQ ID NO:2), vasopressin (SEQ ID NO:3), melanocyte-stimulating hormone (SEQ ID NO:4 (alpha) SEQ ID NO:5 (beta) SEQ ID NO:6 (gamma)), corticortropin (SEQ ID NO:7), lipotropin (SEQ ID NO:8 (beta) SEQ ID NO:9 (gamma)), thyrotropin (SEQ ID NO: 10), growth hormone (SEQ ID NO:1), prolactin (SEQ ID NO: 11), luteinizing hormone (SEQ ID NO: 12), human chorionic gonadotropin (available from SIGMA- Aldrich, St.
  • glucagon SEQ ID NO:35
  • placental lactogen SEQ ID NO:37
  • relaxin SEQ ID NO:38
  • inhibin A SEQ ID NO:39
  • Inhibin B SEQ ID NO:40
  • endorphins e.g., SEQ ID NO:41
  • angiotensin II SEQ ID NO:42
  • atrial natriuretic peptide SEQ ID NO:201
  • embodiments of the invention include Cortisol (available from SIGMA Aldrich, St. Louis, MO, Cat. No. H3160), corticosterone (available from SIGMA Aldrich, St. Louis, MO, Cat. No. C27840), aldosterone (available from SIGMA Aldrich, St. Louis, MO, Cat. No. 05521), epinephrine (available from SIGMA Aldrich, St. Louis, MO, Cat. No. 02252), noepinephrine (available from SIGMA Aldrich, St. Louis, MO, Cat. No.
  • nucleic acid immunogens and/or vaccines and therapies are known in the art and are useful as delivered agents in embodiments of the transdermal delivery compositions disclosed herein.
  • nucleic acid immunogens that induce an immune response both humoral and cellular
  • DNA vaccines for several viruses, as well as for tumors are known.
  • nucleic acid immunogens contain essential regulatory elements such that upon administration to a host, the immunogen is able to direct host cellular machinery to produce translation products encoded by the respective delivered nucleic acids.
  • HIV Human Immunodeficiency Virus
  • compositions comprising the nucleic acid encoding the HIV Nef gene, fragments thereof, or variants that are optimized for efficacy as vaccines in humans, are capable of inducing a cellular immune response in a host.
  • the HIV Nef protein has been shown to promote viral replication.
  • DNA sequences comprising the Nef sequence including the sequences of SEQ ID NOs:52, 53, and 54 are known to be capable of inducing a cellular immune response in individuals.
  • WO 04/050856 discloses that DNA vaccines comprising the nucleic acid sequences and variants of HIV g ⁇ l20 (SEQ ID NOs:153, 154, 155, 156) and a codon-optimized nucleic acid encoding HIV-I Gag (SEQ ID NO: 152) are capable of inducing antibody and humoral immune responses. Nucleic acids encoding HIV-I Gag and variants thereof have also been shown to induce an immune response when administered to a host (Qui et al, 2000, J. Virology. 74(13):5997- 6005). Any of the above sequences from HIV are useful delivered agents for the transdermal delivery compositions disclosed herein.
  • Influenza A is the causative agent of the flu in humans. Flu epidemics cause morbidity and mortality worldwide, and each year in the USA alone more than 200,000 patients are admitted to hospitals because of influenza and there are approximately 36,000 influenza-related deaths. Immunogens directed against Influenza A generally comprise attenuated strains of the virus.
  • WO 04/060720 teaches that a DNA vaccine comprising nucleic acids of sequence SEQ ID NO:51 are capable of inducing a cellular immune response against Influenza virus A.
  • HCV nucleic acid-based immunogens and vaccines for the hepatitis viruses, such as hepatitis C, hepatitis B and hepatitis A.
  • HCVgpl The amino acid sequence encoded by the complete coding sequence of the prototype HCV-I genome (HCVgpl) is provided (SEQ ID NO: 128).
  • Houghton et al. U.S.S.N. 2002/0002272 disclose nucleic acids that encode several portions of HCVgpl that are capable of inducing a humoral immune response.
  • nucleic acids encoding the HCV E2 envelope protein or portions thereof (SEQ ID NOs:129, 130, 131, 132), or nucleic acids encoding both HCVE1/E2 envelope proteins (SEQ ID NOs:133, 134) were capable of eliciting an immune response.
  • Schiver et al. International Pub. No. WO 01/43693 disclose other nucleic acid sequences from HCV that elicit protective immune responses, including the sequences of SEQ ID NO's:52, 53, 54.
  • Preferred delivered agents for use in the transdermal formulations described herein include HCV antigens provided by the peptides encoded by the NS3/4A sequences described herein ⁇ see e.g., Example 10). That is, several embodiments concern a transdermal delivery system, as described herein, and an NS3/4A nucleic acid of SEQ. ID. NOs.: 163, 197, nucleic acids encoding SEQ. ID.
  • nucleic acids at least, less than or equal to, or more than 50, 60, 75, 80, 90,- 100, 110, 120, 130, 140, 150, 160 , 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 consecutive nucleotides in length or the complement thereof.
  • Embodiments of the present invention also contemplate sequences from HBV, such as nucleic acids that encode HBV core antigen (SEQ ID NO: 135); HBVsAg (GenbankTM Accession No. ARl 41190), and the like. Additionally, nucleic acid sequences from the HAV genome (GenbankTM Accession No. NC_001489) are contemplated as delivered agents.
  • nucleic acid-based immunogens and vaccines against viral pathogens have been described in the art, such as vaccines comprising nucleic acids from Hantavirus.
  • Hantavirus is the causative agent of Hantavirus Pulmonary Syndrome (HPS), a form of adult respiratory disease syndrome that is potentially fatal in humans.
  • HPS Hantavirus Pulmonary Syndrome
  • WO 04/058808 discloses sequences (SEQ ID NOs: 126, 127) that are useful delivered agents.
  • Chen International Pub. No. WO 04/110483 discloses several amino acid sequences, (SEQ ID NOs:147, 148, 149 150), the encoding nucleic acid sequences of which are useful as delivered agents for vaccines SARS.
  • Vaccines and immunogens comprising nucleic acids that encode a member of the Inhibitor of Apoptosis (IAP) family of proteins are also useful in the context of cancer treatment.
  • IAP Inhibitor of Apoptosis
  • Xiang et al. International Publication No. WO 04/099389 teach DNA vaccines comprising sequences encoding members of the Inhibitor of Apoptosis (IAP) family of proteins, such as nucleic acids encoding the sequences of SEQ ID NO's:136, 137, 138, and 139. These sequences are also useful as delivered agents in one or more of the transdermal delivery systems described herein for the purposes of anti-tumor therapy.
  • Immune response modifiers are compounds that act on the immune system by inducing and/or suppressing cytokine biosynthesis. IRMs possess potent immunostimulating activity including, but not limited to, antiviral and antitumor activity, and can also down-regulate other aspects of the immune response, for example shifting the immune response away from a TH 2 immune response, which is useful for treating a wide range of TH 2 mediated diseases. IRMs can also be used to modulate humoral immunity by stimulating antibody production by B cells. Some IRMs are small organic compounds having a molecular weight under about 1000 daltons, preferably under about 500 daltons.
  • some IRMs can be immunomodulatory or immunostimulatory, depending on how the forumaltion is provided to a subject. For example, it was determined that daily ribavirin therapy induces an immunomodulatory shift in THl and TH2 responses, however, if ribavirin were provided to a subject in combination with or co-administered with a viral antigen, the ribavirin has an immunostimulatory or adjuvant effect in that the total antibody response is elevated.
  • transdermal formulations comprising ribavirin can be provided to a subject on a daily regimen (e.g., when an immunomodualtory effect is desired) or said transdermal formulation comprising ribavirin can also comprise a viral antigen (e.g., SEQ. ID. NOs.: 163, 197, nucleic acids encoding SEQ. ID.
  • a viral antigen e.g., SEQ. ID. NOs.: 163, 197, nucleic acids encoding SEQ. ID.
  • transdermal formulation comprising ribavirin
  • a transdermal formulation comprising one or more of the nucleic acids described herein (preferably, an NS3/4A sequence of SEQ. ID.
  • Examples of classes of small molecule IRM compounds include, but are not limited to, compounds having a 2-aminopyridine fused to a five-membered nitrogen-containing heterocyclic ring.
  • Such compounds include, for example, imidazoquinoline amines including, but not limited to, substituted imidazoquinoline amines such as, for example, amide substituted imidazoquinoline amines, sulfonamide substituted imidazoquinoline amines, urea substituted imidazoquinoline amines, aryl ether substituted imidazoquinoline amines, heterocyclic ether substituted imidazoquinoline amines, amido ether substituted imidazoquinoline amines, sulfonamido ether substituted imidazoquinoline amines, urea substituted imidazoquinoline ethers, thioether substituted imidazoquinoline amines, and 6-, 7-, 8-, or 9-aryl or heteroaryl substituted imidazoquinoline
  • IRMs small molecule IRMs said to induce interferon (among other things), include purine derivatives (such as those described in U.S. Pat. Nos. 6,376,501, and 6,028,076), imidazoquinoline amide derivatives (such as those described in U.S. Pat. No. 6,069,149), lH-imidazopyridine derivatives (such as those described in Japanese Patent Application No. 9-255926), benzimidazole derivatives (such as those described in U.S. Pat. No. 6,387,938), derivatives of a 4-aminopyrimidine fused to a five membered nitrogen containing heterocyclic ring (such as adenine derivatives described in U.S. Pat. Nos.
  • Examples of small molecule IRMs that comprise a 4-aminopyrimidine fused to a five-membered nitrogen-containing heterocyclic ring include adenine derivatives (such as those described in U.S. Pat. Nos. 6,376,501; 6,028,076 and 6,329,381; and in International Publication No. WO 02/08595).
  • IRM compounds include 2- propyl[l,3]thiazolo[4,5-c]quinolin-4-amine, which is considered predominantly a TLR 8 agonist (and not a substantial TLR 7 agonist), 4-amino-. alpha., . alpha. -dimethyl- IH- imidazo[4,5-c]quinoline-l-ethanol, which is considered predominantly a TLR 7 agonist (and not a substantial TLR 8 agonist), and 4-amino-2-(ethoxymethyl)-alpha,alpha.- dimethyl-6,7,8,9-tetrahydro-lH-imidazo[4,5-c]quinolines-l-ethanol, which is a TLR 7 and TLR 8 agonist.
  • 4-amino-alpha,alpha-dimethyl-lH-imidazo[4,5-c]quinoline-l-ethanol has beneficial characteristics, including that it has a much lower CNS effect when delivered systemically compared to imiquimod.
  • IRM compounds include, e.g., N-[4-(4-amino-2-butyl-lH-imidazo[4,5-c][l,5]naphthyridin-l-yl)butyl]-N'- cyclohexylurea, 2-methyl-l-(2-methylpropyl)-lH-imidazo[4,5-c][l,5]naphthyridin-4- amine, l-(2-methylpropyl)-lH-imidazo[4,5-c][l,5]naphthyridin-4-amine, N- ⁇ 2-[4-amino- 2-(ethoxymethyl)- 1 H-imidazo[4,5-c]quinolin-l -yl]-l , 1 - dimethylethyl ⁇ methanesulfonamide, N-[4-(4-amino-2-ethyl-lH-imidazo[4,5-c]quinol
  • Resiquimod 4-amino-2-ethoxymethyl- . alpha. , . alpha.-dimethyl- 1 H-imidazo [4,5 - c]quinoline-l-ethanol, may also be used in certain situations where a combination TLR 7 and TLR 8 agonist is desired.
  • IRMs include large biological molecules such as oligonucleotide sequences.
  • Some IRM oligonucleotide sequences contain cytosine-guanine dinucleotides (CpG) and are described, for example, in U.S. Pat. Nos. 6,194,388; 6,207,646; 6,239,116; 6,339,068; and 6,406,705.
  • CpG-containing oligonucleotides can include synthetic immunomodulatory structural motifs such as those described, for example, in U.S. Pat. Nos. 6,426,334 and 6,476,000.
  • Other IRM nucleotide sequences lack CpG and are described, for example, in International Patent Publication No. WO 00/75304.
  • IRMs are delivered agents in embodiments of the transdermal delivery compositions of the present invention.
  • Embodiments of the invention are also useful for delivery of compounds used to facilitate imaging of tissues and organs within the body.
  • imaging methods commonly used include Xray, CT scans, ultrasound, and magnetic resonance imaging.
  • Various compounds are administered to individuals that facilitate the imaging process.
  • diagnostic or contrast components useful in imaging methods now known or later discovered include iohexol, technetium, Tc99M, sestamibi, iomeprol, gadodiamide, oiversol, iopromide, alsactide, americium, betazole, histamine, mannitol, metyraphone, petagastrin, phentolamine, radioactive B 12, gadodiamide, gadopentetic acid, gadoteridol, or perflubron as delivered agents.
  • glycoproteins are high molecular weight compounds, which are generally characterized as conjugated proteins containing one or more heterosaccharides as prosthetic groups.
  • the heterosaccharides are usually branched but have a relatively low number of sugar residues, lack a serially repeating unit, and are covalently bound to a polypeptide chain.
  • glycoproteins are found in the body.
  • glycoproteins are glycoproteins
  • the substances that fill the intercellular spaces e.g., extracellular matrix proteins
  • glycoproteins the substances that fill the intercellular spaces
  • the compounds that compose collagens, proteoglycans, mucopolysaccharides, glycosaminoglycans, and ground substance are glycoproteins.
  • a delivery system that can administer glycoproteins to cells of the body has several pharmaceutical and cosmetic uses, including but not limited to, the restoration of skin elasticity and firmness (e.g., the reduction in the appearance of fine lines and wrinkles by transdermal delivery of collagen) and the restoration of flexible and strong joints (e.g., water retention in joints can be increased by transdermal delivery of proteoglycans).
  • a transdermal delivery composition that can administer a high molecular weight compound (e.g., a form of collagen or fragment thereof) to cells of the body.
  • a high molecular weight compound e.g., a form of collagen or fragment thereof
  • many different high molecular weight compounds can be administered by using an embodiment of a transdermal delivery composition of the invention and the use of a high molecular weight collagen as a delivered agent is intended to demonstrate that embodiments of the invention can deliver many high molecular weight compounds to cells of the body.
  • Collagens exist in many forms and can be isolated from a number of sources. Additionally, several forms of collagen can be obtained commercially (e.g., Brooks Industries Inc., New Jersey). Many low molecular weight collagens can be made, for example, by hydrolysis. Several transdermal delivery compositions of the invention can deliver collagens having molecular weights below 6,000 daltons. Additionally, several high molecular weight collagens exist. Some are isolated from animal or plant sources and some are synthesized or produced through techniques common in molecular biology. Several transdermal delivery compositions of the invention can deliver collagens having molecular weights of 1,000 daltons to greater than 2,000,000 daltons.
  • embodiments of the transdermal delivery compositions can deliver collagens having molecular weights of less than or equal to or greater than 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 5,500, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000, 28,000, 29,000, 30,000, 31,000, 32,000, 33,000, 34,000, 35,000, 36,000, 37,000, 38,000, 39,000, 40,000, 41,000, 42,000, 43,000, 44,000, 45,000, 46,000, 47,000, 48,000, 49,000, 50,000, 51,000, 52,000, 53,000, 54,000, 55,000, 56,000, 57,000, 58,000, 59,000, 60,000, 61,000, 62,000, 63,000, 64,000, 65,000, 66,000, 67,000, 68,000, 69,000, 70,000,
  • the commercially available collagen "Hydrocoll EN-55" was provided as the delivered agent and was delivered to cells of a test subject. This form of collagen is hydrolyzed collagen and has a molecular weight of 2,000 daltons.
  • the commercially available "Ichtyocollagene” or marine collagen was provided as the delivered agent and was delivered to a test subject. This form of soluble collagen has a molecular weight of greater than 100,000 daltons.
  • the commercially available collagen "Solu-Coll” was provided as the delivered agent and was delivered to cells of a test subject.
  • This form of collagen is a soluble collagen having a molecular weight of 300,000 daltons.
  • An additional embodiment includes the commercially available collagen "Plantsol", which is obtained from yeast and has a molecular weight of 500,000 daltons. This collagen was also provided as a delivered agent and was delivered to cells of a test subject.
  • the transdermal delivery compositions that contain a form of collagen or fragment thereof desirably comprise by weight or volume between 0.1% to 85.0% of the delivered agent depending on the type and form of the collagen, its solubility, and the intended application. That is, some transdermal delivery compositions comprise by weight or volume less than or equal to or greater than 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1.0%, 1.25%, 1.5%, 1.75%, 2.0%, 2.25%, 2.5%, 2.75%, 3.0%, 3.25%, 3.5%, 3.75%, 4.0%,.
  • embodiments having Hydrocoll-EN55 can comprise by weight or volume less than or equal to or greater than 1.0%, 1.25%, 1.5%, 1.75%, 2.0%, 2.25%, 2.5%, 2.75%, 3.0%, 3.25%, 3.5%, 3.75%, 4.0%,. 4.25%, 4.5%, 4.75%, 5.0%, 5.25%, 5.5%, 5.75%, 6.0%, 6.25%, 6.5%, 6.75%, 7.0%, 7.25%, 7.5%, 7.75%, 8.0% 8.25%, 8.5%, 8.75%, 9.0%, 9.25%, 9.5%, 9.75%, 10.0%, 10.25%, 10.5%, 10.75%, 11.0%,.
  • Embodiments having marine collagen can comprise by weight or volume less than or equal to or greater than 1.0%, 1.25%, 1.5%, 1.75%, 2.0%, 2.25%, 2.5%, 2.75%, 3.0%, 3.25%, 3.5%, 3.75%, 4.0%,. 4.25%, 4.5%, 4.75%, 5.0%, 5.25%, 5.5%, 5.75%, 6.0%, 6.25%, 6.5%, 6.75%, 7.0%, 7.25%, 7.5%, 7.75%, 8.0% 8.25%, 8.5%, 8.75%, 9.0%, 9.25%, 9.5%, 9.75%, 10.0%, 10.25%, 10.5%, 10.75%, 11.0%,.
  • transdermal delivery compositions that contain Solu-Coll can comprise by weight or volume less than or equal to or greater than 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1.0%, 1.1%, 1.15%, 1.2%, 1.25%, 1.3%, 1.35%, 1.4%, 1.45%, 1.5%, 1.55%, 1.6%, 1.65%, 1.7%, 1.75%, 1.8%, 1.85%, 1.9%, 1.95%, or 2.0% Solu-Coll.
  • transdermal delivery compositions that contain Plantsol can comprise by weight or volume less than or equal to or greater than 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1.0%, 1.1%, 1.15%, 1.2%, 1.25%, 1.3%, 1.35%, 1.4%, 1.45%, 1.5%, 1.55%, 1.6%, 1.65%, 1.7%, 1.75%, 1.8%, 1.85%, 1.9%, 1.95%, 2.0%, 2.1%, 2.15%, 2.2%, 2.25%, 2.3%, 2.35%, 2.4%, 2.45%, 2.5%, 2.55%, 2.6%, 2.65%, 2.7%, 2.75%, 2.8%, 2.85%, 2.9%, 2.95%, 3.0%, 3.1%, 3.15%, 3.2%, 3.25%, 3.3%, 3.35%, 3.4%, 3.45%, 3.5%, 3.55%, 3.6%, 3.
  • a transdermal delivery composition that can provide a collagen solution comprising two or more forms of collagen (e.g., Hydro-Coll EN-55, marine collagen, Solu-coll, or Plantsol) is provided.
  • the transdermal delivery compositions that include two or more forms of collagen desirably comprise an amount of delivered agent that can be included in a delivered agent having the specific type of collagen by itself.
  • the amount of Hydro-Coll EN55 in the transdermal delivery composition can comprise by weight or volume less than or equal to or greater than 1.0%, 1.25%, 1.5%, 1.75%, 2.0%, 2.25%, 2.5%, 2.75%, 3.0%, 3.25%, 3.5%, 3.75%, 4.0%,. 4.25%, 4.5%, 4.75%, 5.0%, 5.25%, 5.5%, 5.75%, 6.0%, 6.25%, 6.5%, 6.75%, 7.0%, 7.25%, 7.5%, 7.75%, 8.0% 8.25%, 8.5%, 8.75%, 9.0%, 9.25%, 9.5%, 9.75%, 10.0%, 10.25%, 10.5%, 10.75%, 11.0%,.
  • the amount of marine collagen in the delivery system can comprise by weight or volume less than or equal to or greater than 1.0%, 1.25%, 1.5%, 1.75%, 2.0%, 2.25%, 2.5%, 2.75%, 3.0%, 3.25%, 3.5%, 3.75%, 4.0%,. 4.25%, 4.5%, 4.75%, 5.0%, 5.25%, 5.5%, 5.75%, 6.0%, 6.25%, 6.5%, 6.75%, 7.0%, 7.25%, 7.5%, 7.75%, 8.0% 8.25%, 8.5%, 8.75%, 9.0%, 9.25%, 9.5%, 9.75%, 10.0%, 10.25%, 10.5%, 10.75%, 11.0%,.
  • the amount of Solu-coll in the delivery system can comprise by weight or volume less than or equal to or greater than 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1.0%, 1.1%, 1.15%, 1.2%, 1.25%, 1.3%, 1.35%, 1.4%, 1.45%, 1.5%, 1.55%, 1.6%, 1.65%, 1.7%, 1.75%, 1.8%, 1.85%, 1.9%, 1.95%, or 2.0% or Solu-Coll.
  • the amount of Plantsol in the delivery system can comprise by weight or volume less than or equal to or greater than 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1.0%, 1.1%, 1.15%, 1.2%, 1.25%, 1.3%, 1.35%, 1.4%, 1.45%, 1.5%, 1.55%, 1.6%, 1.65%, 1.7%, 1.75%, 1.8%, 1.85%, 1.9%, 1.95%, 2.0%, 2.1%, 2.15%, 2.2%, 2.25%, 2.3%, 2.35%, 2.4%, 2.45%, 2.5%, 2.55%, 2.6%, 2.65%, 2.7%, 2.75%, 2.8%, 2.85%, 2.9%, 2.95%, 3.0%, 3.1%, 3.15%, 3.2%, 3.25%, 3.3%, 3.35%, 3.4%, 3.45%, 3.5%, 3.5%, 3.5%, 3.5%, 3.2%, 3.2
  • modified or stabilized collagens or collagen derivatives are contemplated for use in some of the embodiments described herein.
  • Particularly preferred are collagens that are resistant to proteases.
  • Recombinant engineering can be used to generate collagens or fragments thereof that lack protease cleavage sites for example.
  • Resistant collagens or fragments thereof can also be prepared by incorporating D-amino acids in synthetically prepared collagens or fragments thereof.
  • Cross-linked collagens can also be used. ⁇ See e.g., Charulatha, Biomaterials Feb;24(5):759-67 (2003)).
  • amidated collagen or collagen fragments can be prepared using synthetic chemistry and these collagen derivatives can be mixed with an ethoxylated oil with or without water or alcohol so as to form a transdermal delivery composition containing collagen.
  • synthetic, recombinant, or cross-linked collagens are known to those of skill in the art and many are commercially available.
  • protease resistant fragments of collagen can be prepared and isolated using conventional techniques.
  • marine collagen, procollagen, or collagen obtained from human placenta is incubated with bovine serum, pepsin, or bacterial collagenase for one hour and the preparation is then separated by gel electrophoresis, size exclusion, reverse phase, or ionic exchange chromatography ⁇ e.g., FPLC or HPLC).
  • protease resistant fragments of collagen ⁇ e.g., 15 kDa or 3OkDa; see e.g., Tasab et a!., JBC 277(38):35007 (2002) or 38kDa see e.g., Odermatt et al, Biochem J. May l;211(2):295-302 (1983) both of which are herein expressly incorporated by reference in their entireties) are separated from the hydrolytic products and these fragments are isolated from the column and concentrated ⁇ e.g., centric ⁇ n filters) or lyophilized using conventional techniques. The protease resistant fragments of collagen are then incorporated into a transdermal delivery composition, as described herein. Alternatively, the protease resistant domain of collagen can be prepared synthetically or obtained commercially ⁇ e.g., pepsinized collagens can also be obtained from Chemicon of Temecula, CA).
  • An additional delivered agent that can be included in a transdermal delivery composition is Etioline (Sederma or Croda of Parsippany, New Jersey).
  • Etioline is a tyrosinase inhibitor made from the extract Mitracarpe and bearberry that effectively whitens the skin.
  • Formulations of a transdermal delivery composition described herein containing Etioline ⁇ e.g., at 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%) are also embodiments of the invention.
  • Another skin brightening or whitening formulation of a transdermal delivery composition comprises Melaslow (Sederma of Parsippany, New Jersey). Melaslow is an extract made from Citrus reticulate Blanco var. Unshiu. Melaslow is also an inhibitor of melanogenesis and formulations of a transdermal delivery composition described herein containing Melaslow (e.g., at 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%) are also embodiments of the invention.
  • Melaslow Sederma of Parsippany, New Jersey
  • Melaslow is an extract made from Citrus reticulate Blanco var. Unshiu. Melaslow is also an inhibitor of melanogenesis and formulations of a transdermal delivery composition described herein containing Melaslow (e.g., at 1%, 2%
  • Matrixyl is a compound comprising the peptide KTTKS (SEQ. ID. NO:2), which has been shown to stimulate collagen synthesis. See Katayama et al., J. Biol. Chem. 268, 9941 (1993). Formulations of a transdermal delivery composition described herein containing Matrixyl or the peptide KTTKS (SEQ. ID.
  • a penetration enhancer included in many embodiments of the invention is comprised of two components — a hydrophobic component and a hydrophilic component.
  • the hydrophobic component comprises a polyether compound, such as an ethoxylated fatty moiety, preferably, an ethoxylated oil, such as vegetable, nut, synthetic, or animal oil, which has the ability to reduce the surface tension of materials that are dissolved into it.
  • ethoxylated oil such as ethoxylated macadamia nut oil
  • ethoxylated macadamia nut oil is a mixture of various fatty acids, fatty alcohols, and fatty amines
  • the components of the oil may have varying amounts of ethoxylation. Accordingly, measurements of ethoxylation/molecule (e.g., 16 ethoxylations/molecule) are an average of the amount of ethoxylation present on the components of the oil rather than on any specific component itself.
  • ethoxylated oils can be obtained or created from, for example, macadamia nut oil, meadowfoam, castor oil, jojoba oil, corn oil, sunflower oil, sesame oil, and emu oil. Many of these oils are commercially available from Floratech of Gilbert, Arizona or other suppliers. Alternatively, ethoxylated oils can be prepared by reacting the oil with ethylene oxide. Pure carrier oils that are suitable for ethoxylation so as to create a penetration enhancer for use with the transdermal delivery compositions described herein are included in TABLES 3-17 and can be obtained from Esoteric oils Pty. Ltd., Pretoria South Africa.
  • TABLES 3-17 also list the component fatty acids of these oils, all of which are individually suitable for ethoxylation and incorporation into an embodiment of a transdermal delivery composition. That is, it is contemplated that ethoxylated fatty acids, ethoxylated fatty alcohols, and ethoxylated fatty amines, in particular ethoxylated fatty acids, ethoxylated fatty alcohols, and ethoxylated fatty amines that contain 12, 13, 14, 15, 16, 17, 18, or 19 ethoxylations are suitable penetration enhancers for use in the transdermal delivery compositions described herein. These ethoxylated oil components can be used individually as penetration enhancers or as supplements to other penetration enhancers (e.g., ethoxylated macadamia nut oil).
  • Linolenic C18 3 2.0 % max
  • an ethoxylated oil comprises a molar ratio of ethylene oxide:oil of 35:1.
  • a 99% pure ethylene oxide/castor oil having such characteristics can be obtained commercially (BASF) or such an ethoxylated compound can be synthesized using conventional techniques.
  • the ethoxylated oil is itself the penetration enhancer. That is, it has been discovered that oils that have been ethoxylated 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 ethoxylations/molecule are sufficiently hydrophobic and sufficiently hydrophilic to allow for transdermal delivery of a variety of delivered agents without water, alcohol, or an aqueous adjuvant.
  • the ethoxylated oil can comprise at least 20-25 ethoxylations per molecule .or more, preferably, the ethoxylated lipid comprises less than 20 ethoxylations per molecule, e.g., 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 ethoxylations per molecule.
  • ethoxylated oil e.g., macadamia nut oil containing approximately 16 ethoxylations/molecule
  • a transdermal delivery composition that contain Aloe Vera and an oil with 20-30 ethoxylations/molecule are not as effective as formulations of a transdermal delivery composition that contain an oil with 10-19 ethoxylations/molecule (e.g., 16 ethoxylations/molecule) but lacking Aloe Vera and alcohol.
  • transdermal delivery composition composed of macadamia nut oil (16 ethoxylations/molecule) and water as compared with a transdermal delivery composition composed of castor oil (25 ethoxylations/molecule), water, alcohol, and Aloe Vera, for example.
  • Desirable compounds often found in ethoxylated oils that are beneficial for some embodiments and methods described herein are glycerol-polyethylene glycol ricinoleate, the fatty esters of polyethylene glycol, polyethylene glycol, and ethoxylated glycerol.
  • Some of these desirable compounds exhibit hydrophilic properties and the hydrophilic-lipophilic balance (HLB) is preferably maintained between 10 and 18.
  • HLB hydrophilic-lipophilic balance
  • some of the components of the oils in the table above and related fatty acids, fatty alcohols, and fatty amines can be ethoxylated and used as a penetration enhancer or to enhance another penetration enhancer ⁇ e.g., ethoxylated macadamia nut oil).
  • some embodiments comprise a penetration enhancer that consists of, consists essentially of, or comprises ethoxylated palmitoleic acid, ethoxylated oleic acid, ethoxylated gondoic acid, or ethoxylated erucic acid.
  • These compounds can be prepared synthetically or isolated or purified from oils that contain large quantities of these fatty acids and the synthesized, isolated, or purified fatty acids can then be reacted with ethylene oxide.
  • a transdermal delivery composition of the invention can comprise a penetration enhancer that contains, for example, ethoxylated palmitoleic acid, ethoxylated oleic acid, ethoxylated gondoic acid, or ethoxylated erucic acid, wherein the amount of one or more of the fatty acids is at least, less than, more than, or an amount equal to 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1.0%, 1.25%, 1.5%, 1.75%, 2.0%, 2.25%, 2.5%, 2.75%, 3.0%, 3.25%, 3.5%, 3.75%, 4.0%,.
  • a penetration enhancer that contains, for example, ethoxylated palmitoleic acid, ethoxylated oleic acid, ethoxylated gondo
  • ethoxylated compound 85.5%, 86%, 86.5%, 87%, 87.5%, 88%, 88.5%, 89%, 89.5%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 100% of the total fatty acid content in the composition.
  • more than one ethoxylated compound is added or another hydrophobic compound is added ⁇ e.g., Y-Ling-Y-Lang oil; Young Living Essential Oils, Lehl, Utah)) to balance or enhance the penetration enhancer.
  • Preferred embodiments include ethoxylated macadamia nut oil that has been supplemented with ethoxylated palmitoleic acid, ethoxylated oleic acid, ethoxylated gondoic acid, or ethoxylated erucic acid.
  • the amount of ethoxylated lipid(s) in the delivery system can vary.
  • delivery systems of the invention can comprise between 0.1% and 99% by weight or volume ethoxylated compound(s).
  • embodiments of the invention can comprise by weight or volume at least, less than, or equal to or greater than 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1.0%, 1.25%, 1.5%, 1.75%, 2.0%, 2.25%, 2.5%, 2.75%, 3.0%, 3.25%, 3.5%, 3.75%, 4.0%,.
  • the hydrophilic component of the penetration enhancer can comprise an alcohol, a non-ionic sohibilizer, or an emulsifier.
  • Compounds such as ethylene glycol, propylene glycol, dimethyl sulfoxide (DMSO), dimethyl polysiloxane (DMPX), oleic acid, caprylic acid, isopropyl alcohol, 1-octanol, ethanol (denatured or anhydrous), and other pharmaceutical grade or absolute alcohols with the exception of methanol can be used.
  • Preferred embodiments comprise an alcohol (e.g., absolute isopropyl alcohol), which is commercially available.
  • the amount of hydrophilic component in the penetration enhancer depends on the type of the delivered agent and the intended application.
  • the hydrophilic component of a penetration enhancer of the invention can comprise between 0.1% and 50% by weight or volume. That is, a delivery system of the invention can comprise by weight or volume at least, less than or equal to or greater than 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1.0%, 1.25%, 1.5%, 1.75%, 2.0%, 2.25%, 2.5%, 2.75%, 3.0%, 3.25%, 3.5%, 3.75%, 4.0%,.
  • transdermal delivery compositions described herein can comprise an aqueous adjuvant.
  • aqueous adjuvants in particular, Aloe Vera, which can enhance the delivery of both low and high molecular weight molecules to the skin cells of the body.
  • aqueous adjuvant such as Aloe Vera juice or water or both.
  • Aloe refers to the genus of South African plants of the Liliaceae family, of which the Aloe barbadensis plant is a species. Aloe is an intricate plant, which contains many biologically active substances. (Cohen, et al. in Wound Healing/Biochemical and Clinical Aspects, 1st ed. WB Saunders, Philadelphia (1992)). Over 300 species of Aloe are known, most of which are indigenous to Africa.
  • Aloe products have been used in dermatological applications for the treatment of burns, sores and other wounds. These uses have stimulated a great deal of research in identifying compounds from Aloe plants that have clinical activity, especially anti-inflammatory activity. (See e.g., Grindlay and Reynolds (1986) J. of Ethnopharmacology 16:117-151; Hart, et al.
  • Aloe Vera a term used to describe the extract obtained from processing the entire leaf, isolated from the Aloe Vera species of Aloe, can be used as a vehicle for delivering hydrocortisone, estradiol, and testosterone propionate.
  • Davis U.S. Pat. No. 5,708,308
  • one embodiment of "Aloe Vera” can be prepared by "whole-leaf processing" of the whole leaf of the Aloe barbadensis plant. Briefly, whole leaves obtained from the Aloe barbadensis plant are ground, filtered, treated with cellulase (optional) and activated carbon and lyophilized. The lyophilized powder is then reconstituted with water prior to use.
  • Aloe Vera can be obtained commercially through Aloe Laboratories, for example.
  • the Aloe Vera is made as follows. First, the leaves are manually harvested. Next, the leaves are washed with water and the thorns on both ends are cut. The leaves are then hand-filleted so as to extract the inner part of the leaf. The inner gel is passed through a grinder and separator to remove fiber from the gel. Then the gel is put into a pasteurizing tank where L-Ascorbic Acid (Vitamin C) and preservatives are added. The gel is pasteurized at 85°C for 30 minutes.
  • L-Ascorbic Acid Vitamin C
  • Aloe Vera After pasteurization, the gel is put into a holding tank for about one or two days, after which the gel is sent through a Vi micron filter. Finally, the gel is cooled down through a heat exchanger and stored in a steamed, sanitized and clean 55 gallon drum.
  • Absolute Aloe Vera (100% pure) can also be obtained from commercial suppliers (Lily of the Desert, Irving, Texas).
  • Aloe Vera juice, prepared from gel fillet has an approximate molecular weight of 200,000 to 1,400,000 daltons.
  • Whole leaf Aloe Vera gel has a molecular weight of 200,000 to 3,000,000 depending on the purity of the preparation.
  • the embodiments of the invention having Aloe Vera comprise Aloe Vera juice
  • other extracts from a member of the Liliaceae family can be used (e.g., an extract from another Aloe species).
  • Transdermal delivery compositions having Aloe Vera can comprise between 0.1% to 85.0% by weight or volume Aloe Vera. That is, embodiments of the invention can comprise by weight or volume at least, less than or equal to or greater than 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1.0%, 1.25%, 1.5%, 1.75%, 2.0%, 2.25%, 2.5%, 2.75%, 3.0%, 3.25%, 3.5%, 3.75%, 4.0%,.
  • the amount of water in the delivery system generally depends on the amount of other reagents (e.g., delivered agent, penetration enhancer, and other aqueous adjuvants or fillers). Although water is used as the sole aqueous adjuvant in some embodiments, preferred embodiments use enough water to make the total volume of a particular preparation of a delivery system such that the desired concentrations of reagents in the penetration enhancer, aqueous adjuvant, and delivered agent are achieved. Suitable forms of water are deionized, distilled, filtered or otherwise purified. Clearly, however, any form of water can be used as an aqueous adjuvant.
  • Transdermal delivery compositions having water can comprise between 0.1% to 85.0% by weight or volume water. That is, embodiments of the invention can comprise by weight or volume at least, less than or equal to or greater than 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1.0%, 1.25%, 1.5%, 1.75%, 2.0%, 2.25%, 2.5%, 2.75%, 3.0%, 3.25%, 3.5%, 3.75%, 4.0%,.
  • transdermal delivery compositions are prepared by combining an ethoxylated fatty moiety or a penetration enhancer with a delivered agent and, optionally, an aqueous adjuvant.
  • a delivered agent can be solubilized in either the hydrophobic or hydrophilic components of the penetration enhancer.
  • the delivered agent readily dissolves in the ethoxylated oil without water, alcohol, or an aqueous adjuvant.
  • the delivered agent e.g., an NSAID or collagen or fragments thereof
  • the delivered agent readily dissolves in water, which is then mixed with the ethoxylated oil.
  • some delivered agents can be solubilized in the aqueous adjuvant prior to mixing with the penetration enhancer.
  • the pH of the mixture is maintained between 3 and 11 and preferably between 5 and 9.
  • the pH of the solution is desirably maintained at less than, more than, at least, or equal to 3.0, 3.25, 3.5, 3.75, 4.0, 4.25, 4.5, 4.75, 5.0, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7.0, 7.25, 7.5, 7.75, 8.0, 8.25, 8.5, 8.75, 9.0, 9.25, 9.5, 9.75, 10.0, 10.25, 10.5, 10.75, or 11.0.
  • a magnetic stir plate and bar can be used, however, the speed of stirring is preferably minimized so as not to drive air into the mixture and/or destroy the delivered agent (e.g., when the delivered agent is a peptide or a protein).
  • a rocker can be used to bring components of the delivery system together.
  • Heat can also be applied to help coalesce the mixture but desirably, the temperature is not raised above 40 0 C so that labile aqueous adjuvants or labile delivered agents are not degraded.
  • other components such as fragrances and colors are added or the delivery system is incorporated into a cream or ointment or a device for applying the delivery system.
  • the ratio of hydrophilic component:hydrophobic component: aqueous adjuvant is desirably 3:4:3, but preferred formulations comprise 1 :1:4, 1:1 :14, and 1:10:25.
  • a sufficient amount of delivered agent to suit the intended purpose is incorporated into the delivery system.
  • the amount of delivered agent that is incorporated into the penetration enhancer depends on the compound, desired dosage, and application.
  • the transdermal delivery composition is made by providing an ethoxylated oil, mixing the ethoxylated oil with an alcohol, non-ionic solubilizer, or emulsifier so as to form a penetration enhancer, mixing the penetration enhancer with an aqueous adjuvant (e.g., an extract from a plant of the Liliaeacae family), and mixing the penetration enhancer and aqueous adjuvant with a delivered agent and thereby making the transdermal delivery composition.
  • an a transdermal delivery composition comprising a pain relief solution is manufactured as follows.
  • the transdermal delivery compositions having a form of Hepsyl as a delivered agent desirably are comprised by weight or volume of between 0.005% to 12.0% Hepsyl, depending on the type of Hepsyl, its solubility, and the intended application.
  • Hepsyl CGA 1501K., and Hepsyl RA 150K can be comprised by weight or volume of 0.01-2 grams of Hepsyl delivered agent, 0-50 mL of hydrophobic penetration enhancers (e.g., ethoxylated castor oil, jojoba oil, etc.), 0-50 mL of hydrophilic penetration enhancers, nonionic solubilizers, or emulsif ⁇ ers (e.g., isopropyl. alcohol, DMSO, etc.), and 0-50 mL of aqueous adjuvant (e.g., water, Aloe Vera extract, etc.).
  • hydrophobic penetration enhancers e.g., ethoxylated castor oil, jojoba oil, etc.
  • hydrophilic penetration enhancers e.g., nonionic solubilizers, or emulsif ⁇ ers
  • emulsif ⁇ ers e.g., isopropyl. alcohol, DMSO
  • a particularly desirable embodiment of the invention is comprised of 0.1-0.5 gram of Hepsyl, 5-10 mL of ethoxylated castor oil, 5-10 mL of isopropyl alcohol, and 5-10 mL of Aloe Vera extract.
  • other delivered agents can be incorporated into a transdermal delivery composition.
  • Formulations of transdermal delivery compositions having collagens are described in the examples. The following section describes several therapeutic, prophylactic and cosmetic applications.
  • transdermal delivery compositions are suitable for treatment of subjects either as a preventive measure (e.g., to avoid pain or skin disorders) or as a therapeutic to treat subjects already afflicted with skin disorders or who are suffering pain.
  • most drugs, chemicals, and cosmetic agents that can be incorporated into a pharmaceutical or cosmetic can be formulated into a transdermal delivery composition of the invention. Because the various formulations of transdermal delivery composition described herein have a considerable range in hydrophobic and hydrophilic character, most drugs, chemicals, and cosmetic preparations can be incorporated therein. That is, by adjusting the amount of ethoxylation, alcohol, and water in a particular formulation most pharmaceutical and cosmetic agents are solubilized in a transdermal delivery composition with little effort.
  • transdermal delivery compositions described herein can deliver a wide range of materials of both high and low molecular weight to skin cells, the utility of the transdermal delivery compositions described herein is incredibly broad.
  • the aspects of the invention that follow are for exemplary purposes only, and one of skill in the art can readily appreciate the wide spread applicability of a transdermal delivery composition described herein and the incorporation of other delivered agents into a formulation of transdermal delivery composition is straight forward.
  • a method of treatment or prevention of inflammation, pain, or human diseases comprises using a transdermal delivery composition described herein that has been formulated with an NSAID.
  • delivered agents such as NSAIDs, capsaicin, and Boswellin interfere and/or inhibit cyclooxygenase enzymes (COX-I and COX-2), they provide a therapeutically beneficial treatment for cancer and Alzheimer's disease when administered by a transdermal delivery composition described herein.
  • COX-I and COX-2 cyclooxygenase enzymes
  • a transdermal delivery composition comprising a delivered agent that is effective at reducing pain or inflammation (e.g., NSAIDS, capsaicin, Boswellin, or any combination thereof) is administered to a subject in need and the reduction in pain or inflammation is monitored.
  • a transdermal delivery composition comprising a delivered agent that inhibits a COX enzyme (e.g., NSAIDS, capsaicin, Boswellin, or any combination thereof).
  • the transdermal delivery composition is preferably applied to the skin at a region of inflammation or an area associated with pain or the particular condition and treatment is continued for a sufficient time to reduce inflammation, pain, or inhibit the progress of the disease. Typically, pain and inflammation will be reduced in 5-20 minutes after application. Cancer and Alzheimer's disease can be inhibited or prevented with prolonged use.
  • restoring skin tone an approach to reduce wrinkles and increase skin tightness and flexibility (collectively referred to as "restoring skin tone”) is provided.
  • a transdermal delivery composition comprising a form of collagen or fragment thereof as a delivered agent is provided and contacted with the skin of a subject in need of treatment.
  • a subject in need of skin tone restoration is identified, a transdermal delivery composition comprising collagen or a fragment thereof is administered to the subject, and the restoration of the skin tone is monitored. Identification of a person in need of skin restoration can be based solely on visible inspection and the desire to have tight, smooth, and flexible skin. Treatment with the delivery system is continued until a desired skin tone is achieved.
  • the form of collagen in the delivered agent can be from various sources and can have many different molecular weights, as detailed above.
  • high molecular weight natural collagens are used, however, recombinant collagens, modified collagens, protease resistant collagens, and fragments thereof may be used with some of the transdermal delivery compositions described herein.
  • the transdermal delivery compositions described herein can be processed in accordance with conventional pharmacological, veterinary and cosmetological methods to produce medicinal, veterinary, and cosmetic agents for administration to animals and humans in need thereof (e.g., mammals including humans, dogs, cats, horses, cattle, and other companion or farm animals).
  • the transdermal delivery compositions described herein can be incorporated into a pharmaceutical or cosmetic product with or without modification.
  • the compositions of the invention can be employed in admixture with conventional excipients, e.g., pharmaceutically acceptable organic or inorganic carrier substances suitable for topical application that do not deleteriously react with the molecules that assemble the delivery system.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, coloring, aromatic substances and the like that do not deleteriously react with the active compounds. They can also be combined where desired with other active agents.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, coloring, aromatic substances and the like that do not deleteriously react with the active compounds. They can also be combined where desired with other active agents.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, coloring, aromatic substances and the like that do not deleteriously react with the active compounds. They can also be combined where desired with other active agents.
  • Embodiments described herein can be made according to good manufacturing processes (e.g., certified GMP), can be approved by a governmental body, such as the Food and Drug Administration, and may have indicia that indicates that said compositions were manufactured GMP or were approved by a governmental body, with or without structure-function indicia (e.
  • the effective dose and method of administration of a transdermal delivery system formulation can vary based on the individual patient and the stage of the disease, as well as other factors known to those of skill in the art. Although several doses of delivered agents have been indicated above, the therapeutic efficacy and toxicity of such compounds in a delivery system of the invention can be determined by standard pharmaceutical or cosmetological procedures with experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. Pharmaceutical and cosmetological compositions that exhibit large therapeutic indices are preferred.
  • the data obtained from animal studies is used in formulating a range of dosages for human use.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • the exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors that may be taken into account include the severity of the disease state, age, weight and gender of the patient; diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Short acting compositions are administered daily whereas long acting pharmaceutical compositions are administered every 2, 3 to 4 days, every week, or once every two weeks. Depending on half-life and clearance rate of the particular formulation, the pharmaceutical compositions of the invention are administered once, twice, three, four, five, six, seven, eight, nine, ten or more times per day.
  • Routes of administration of the delivery systems of the invention are primarily topical, although it is desired to administer some embodiments to cells that reside in deep skin layers. Topical administration is accomplished via a topically applied cream, gel, rinse, etc. containing a delivery system of the invention.
  • Compositions of delivery system-containing compounds suitable for topical application include, but are not limited to, physiologically acceptable ointments, creams, rinses, and gels.
  • the transdermal delivery composition is incorporated into a device that facilitates application.
  • the embodied compositions generally have a vessel joined to an applicator, wherein a transdermal delivery composition of the invention is incorporated in the vessel.
  • Some devices for example, facilitate delivery by encouraging vaporization of the mixture.
  • These apparatus have a transdermal delivery composition of the invention incorporated in a vessel that is joined to an applicator such as a sprayer (e.g., a pump-driven sprayer).
  • a sprayer e.g., a pump-driven sprayer
  • These embodiments can also comprise a propellant for driving the incorporated transdermal delivery composition out of the vessel.
  • Other apparatus can be designed to allow for a more focused application.
  • a device that facilitates a focused application of a transdermal delivery composition of the invention can have a roll-on or swab-like applicator joined to the vessel that houses the transdermal delivery composition.
  • Several devices that facilitate the administration of a delivery system of the invention have a wide range of cosmetic or therapeutic applications.
  • An exemplary transdermal delivery device is described in the section that follows.
  • the transdermal delivery composition is provided in a single dose application containing a pre-measured amount of a delivered agent.
  • septum sealed vials with or without an applicator e.g., a swab
  • a pre-measured amount of transdermal delivery composition e.g., 0.5ml
  • a pre-measured amount of a delivered agent e.g. 400mg of ibuprofen, 0.6mg marine collagen, or Ig of aspirin
  • a delivered agent e.g. 400mg of ibuprofen, 0.6mg marine collagen, or Ig of aspirin
  • FIGs 17A and 17B show an exemplary embodiment of a dispenser 100.
  • the dispenser 100 in which the dispenser 100 is shown in an exploded state, the dispenser 100 comprises a removable cartridge 102 and a body portion 104.
  • a latch member 106 on the body portion 104 is shown in an unsecured state, permitting the insertion and removal of the removable cartridge 102.
  • the latch member 106 is slidable between the unsecured position as shown and a secured position 108, shown in shadow, in which the insertion and/or removal of a removable cartridge 102 is inhibited.
  • a slidable latch member 106 is shown in this embodiment, it will be understood that any method of securing the removable cartridge 102 to the body portion 104 can be used.
  • a pin attached to the body portion 104 could engage an aperture on the removable cartridge 102.
  • the body portion can be designed such that a snug fit is formed without any need for additional securing methods.
  • Transparent portion 103 permits the user to view the amount of fluid remaining in removable cartridge 102.
  • transparent portion 105 allows the user to see the amount of fluid to be dosed.
  • Figure 17B shows the dispenser 100 in an assembled state, where the removable cartridge 102 has been inserted into the body portion 104.
  • the latch member 106 has been moved to the secured position 108 shown in shadow in Figure 17 A.
  • FIG 18 schematically depicts a cross-section of the dispenser 100 of Figure 17, in an assembled state.
  • the removable cartridge 104 comprises a fluid reservoir 210, which is configured to hold the therapeutic drag delivery fluid.
  • the removable cartridge 104 further comprises a movable upper wall 212, which forms the upper wall of the fluid reservoir 210.
  • the movable upper wall is displaceable in at least the downward direction.
  • the removable cartridge 102 also includes a one-way valve 214, such as a check valve, located at the bottom of the removable cartridge, which is in fluid communication with the fluid reservoir 210.
  • a one-way valve 214 such as a check valve
  • the body portion 102 of the dispenser 100 includes a dosing chamber 220.
  • the dosing chamber 220 When the dispenser 100 is in an assembled space, the dosing chamber 220 is in fluid communication with the valve 214 of the removable cartridge via an aperture 222 in the dosing chamber aligned with the valve 214.
  • the upper wall of the dosing chamber 220 is formed by the lower surface of a movable member 224, alternately referred to as a dosing member.
  • movable member 224 comprises a threaded aperture through which a threaded portion 232 portion of shaft 230 extends. Stop members 234a and 234b are located at the upper and lower ends, respectively, of the shaft 230.
  • a non-threaded portion 236 of shaft 230 extends through an aperture in the top of the body portion 104, and wide sections 238a and 238b of shaft 230 constrain vertical translation of the shaft 230 with respect to the body portion 102.
  • a knob 239 at the top of the shaft 220 facilitates rotation by a user.
  • the size of the dosing chamber 220 can be adjusted by rotating the knob 239, causing rotation of the shaft 230.
  • the movable member 224 cannot rotate along with the shaft 230.
  • the rotational movement of the shaft therefore results in vertical translation of the movable member 224, changing the volume of the dosing chamber 220.
  • the movable member reaches one of stop members 234a,b, the rotational movement of the shaft 230 will be inhibited.
  • the movable member 224 may comprise a ring of partially deformable material (not shown), such as a rubberized material, around the edges of the movable member which come in contact with the walls of the dosing chamber, in order to facilitate a tight seal between the edges of the movable member and the walls of the dosing chamber, so as to prevent undesired leakage along the sides of the movable member.
  • partially deformable material such as a rubberized material
  • the lower end of the dosing chamber 220 comprises a sloped surface 240, and an aperture 242 in the wall of the dosing chamber.
  • This aperture 242 preferably extends to the bottom of the dosing chamber at least one point along the bottom surface of the dosing chamber, such that all fluid in the dosing chamber 220 can flow out of the aperture 242.
  • the body portion 104 further comprises a plunger 250 having an upper end 252 and a lower end 254 configured to engage the movable upper wall of the removable cartridge 102.
  • the plunger 250 extends through an aperture in the top of the body portion 104.
  • the plunger 250 is preferably biased to return to a position in which the upper surface of the removable cartridge is not engaged. This may be done, for example, via a spring 256 connecting the body portion 104 and the plunger 250. As will be discussed in greater detail below, it may be desirable to permit the user to control the timing of the return to the initial position.
  • Locking member 257 is operably connected to a release button 258 on the exterior of the dispenser 100. Engaging the release button rotates locking member 257 about pivot point 259, and permits the return of the plunger to its original position. When the release button is disengaged, the bias of locking member 257 returns it to the position shown in Figure 2.
  • the body portion 104 further comprises a slidable member 260 which is movable between a first position in which the slidable member 260 inhibits fluid flow out of the dosing chamber 220 through the aperture 242, and a second position in which the slidable member 250 inhibits fluid flow from the removable cartridge 102 to the dosing chamber 220 via aperture 222.
  • the slidable member 260 is in the first position when the plunger is in a depressed position, and the second position when the plunger retracts to an undepresed position. This may be accomplished, for example, via spring 262, which connects the plunger 250 to the slidable member 262.
  • the spring 262 holds the slidable member 260 within a slot 264, located below the sloped surface 240 which forms the bottom of the chamber. As the pressure increases, the sliding member is prevented from flexing away from the dosing chamber by tabs 266. Thus, when the sliding member 260 is in the first position, shown in Figure 2, the dosing chamber can be filled and fluid will not leak out. Fluid is permitted to flow into the dosing chamber due to the shape of sliding member 260, discussed in greater detail with respect to Figure 20. When the plunger is moved to an undepressed position, the slidable member will be pushed upward to the second position, where the flow of fluid through the aperture 242 is permitted.
  • the applicator consists of an ellipsoidal applicator 280 mounted on pins 280a and 280b which extend at least partially into the applicator 280 along the axis of the applicator.
  • Applicator 280 thus provides a roll-on applicator, such that once the therapeutic fluid is released from the dosing chamber after the release button 258 is pressed, the fluid will flow downwards onto applicator 280.
  • the applicator can then be placed in contact with the skin of the user, and the dispenser moved to cause the applicator to roll across the skin of the patient, applying the desired dose of the therapeutic fluid to the patient.
  • alternate non-invasive applicators can be used in place of the roll-on applicator.
  • These alternate non-invasive applicators may include, but are not limited to, an absorbent applicator tip, such as a sponge, or an applicator surface having perforations through which the therapeutic fluid can flow.
  • the dosing chamber will be partially filled with fluid when the plunger is depressed, but the air in the dosing chamber will not be permitted to escape, and will therefore be compressed in the dosing chamber.
  • the valve 214 may be designed to close when a certain pressure has been reached. Taking into account this pressure, and the volume of the trapped air at that pressure, accurate dosing can be obtained by accounting for the volume of the trapped air in the dosing chamber.
  • the movable member 224 may comprise a mechanism for allowing air to exit the dosing chamber without permitting fluid.
  • An exemplary system for doing so is shown in Figures 19A and 19B.
  • the movable member 324 includes an aperture 370.
  • a sphere 372, which is buoyant relative to the fluid 376, which will be used, is suspended within a track 374, which permits movement of the sphere 372 upward to engage the aperture 370, forming a seal, but inhibits movement of the sphere 372 downward below a level necessary to allows air to flow over the sphere 372 and out through the aperture 370.
  • FIG 19B shows the dosing chamber full of fluid 376.
  • the buoyant sphere 372 is lifted as the fluid level rises within the dosing chamber. Because the sphere 372 is kept level with the fluid, almost all of the air is allowed to escape, but the fluid cannot escape through the aperture once the sphere engages the aperture.
  • the sphere 372 may advantageously be formed of a partially deformable material, to facilitate the forming of a seal between the sphere and the movable member 324. Bouancy of the sphere 372 may be achieved through selection of an appropriate material, or through the use of a hollow sphere, in order to increase buoyancy.
  • Figure 2OA depicts a portion of the cross section of the dispenser 100 of Figure 18, taken along line 4 of Figure 18.
  • Figure 2OA depicts an embodiment in which the slidable member 260, shown partially in shadow where it is locate behind other features, is in a first position in which the slidable member does not inhibit the flow of fluid from the fluid reservoir 210 through the aperture 222 into the dosing chamber 220 (for simplicity, the valve 214 is not depicted, but would be in line with aperture 222). This is due to the design of the slidable member 260 such that it is substantially L-shaped.
  • the slidable member 260 has been moved to a second position in which the slidable member 260 obstructs the flow of fluid through aperture 222 into dosing chamber 220, but permits the flow of fluid from dosing chamber 220 through aperture 242, and downward to applicator 280 (not shown). Because the slidable member 260 is operably connected via spring 262 to plunger 250, the slidable member 260 is in the first position when the plunger is depressed and fluid is being dispensed into the dosing chamber, and in the second position when the plunger returns to its original position after the release button is depressed.
  • the lower, thicker portion of the slidable member 260 desirably has sufficient height that at intermediate positions of the slidable member, both of the apertures 222, 242 are completely occluded. Thus, no additional fluid beyond what is already in the dosing chamber will be dispensed.
  • the dispenser may not include a fluid reservoir contained within a removable cartridge, but may instead be a disposable dispenser without a replaceable cartridge.
  • the volume of the dosing chamber need not be adjustable by the user. Such an embodiment may be advantageous in situations where precise dosing is required, or where regular fixed doses are required.
  • the user actuatable knob which controls the size of the dosing chamber need not be fixed directly to the rotatable shaft, but may instead be operably connected to the rotatable shaft via a gear or a series of gears, so as to facilitate either rapid adjustment of the dosing volume or very precise adjustment of the dosing volume, depending on the relative properties of the gears.
  • one or more of the operably connected features need not be mechanically connected, as described and depicted above. For instance, electrical connections between features and electrical actuators, such as servo motors, stepper motors, or hydraulics, can be used to replace the mechanical interconnections described above.
  • knob 239 could be replaced by two buttons, electrically connected to a motor, one of which causes the motor to drive the rotatable shaft in one direction, and the other of which causes the motor to drive the rotatable shaft in the other direction.
  • the plunger could be replaced by a plunger which is electronically actuatable at the push of a button.
  • a pressure sensor within the dosing chamber could be used to release the plunger once a sufficient pressure has been reached.
  • Example 1 describes a clinical study that was performed to evaluate the efficacy of a transdermal delivery composition that comprised capsaicin.
  • a transdermal delivery composition of the invention can administer a therapeutically effective amount of a low molecular weight delivered agent (e.g., 0.225% oleoresin capsicum).
  • a transdermal delivery composition of the invention comprising 0.225% capsaicin (“EPRS") as compared to a commercially available cream comprising Boswellin, 10% methyl salicylate, and 0.25% capsaicin. (Nature's Herbs).
  • the two pain relief preparations were tested on six subjects who suffer from degenerative arthritis, debilitating back pain, and/ or bursitis. For the first five days of the study, the subjects applied the commercially available cream three times a day.
  • a transdermal delivery composition of the invention can administer a therapeutically effective amount of a low and high molecular weight delivered agent (e.g., a low and high molecular weight collagens).
  • a clinical study was performed to evaluate the effectiveness of several transdermal delivery compositions comprising various penetration enhancers, aqueous adjuvants, and collagen delivered agents.
  • the various transdermal delivery compositions that were evaluated are provided in TABLE 18.
  • TABLE 18 Of the formulations that were originally screened, three were extensively evaluated by ten subjects (three men and seven women) in a single blind study.
  • the formulations analyzed in the single blind study are indicated in TABLE 18 by a dagger. That is, the three different formulations ("Pl", "P2", and "F4") were evaluated.
  • the Pl formulation comprised approximately 0.73% to 1.46% SoIu- CoIl, a soluble collagen having a molecular weight of 300,000 daltons.
  • the P2 formulation comprised approximately 1.43% to 2.86% Plantsol, a plant collagen obtained from yeast having a molecular weight of 500,000 daltons.
  • the F4 formulation comprised approximately 11.0% of HydroColl EN-55, a hydrolyzed collagen having a molecular weight of 2,000 daltons.
  • the evaluation of the Pl, P2, and F4 formulations was as follows. Left, right, and center mug-shot photographs were taken with a Pentax camera having a zoom 60X lens and Kodak-Gold 100 film before beginning the study.
  • each subject was given a bottle having a formulation of transdermal delivery composition and was instructed to apply the solution to the right side of the face and neck, leaving the left side untreated, twice daily for 15 days.
  • the F4 formulation was tested first and the application was carried out after showering or washing and before application of any other product to the treated area of the face.
  • three mug-shot photographs were again taken, the subjects recorded their observations on the effectiveness of the formulation in a questionnaire, and a 7 day period without application of a collagen product provided.
  • the questionnaire requested the subject to assign a score (e.g., a numerical value that represents effectiveness) on characteristics of the transdermal delivery composition formulation.
  • Characteristics that were evaluated included tackiness, odor, marketability, and overall effectiveness of the formulation, as well as, whether the formulation tightened the skin, decreased lines, conditioned or softened the skin, and had any negative side-effects.
  • the scale for the scoring was 1-10, with 1 being the worst rating and 10 being the best rating.
  • IPA Absolute isopropyl alcohol (Orange County Chemical, Santa Ana, California) Plantsol - Yeast extract collagen (Brooks Industries Inc., Code No. 06485) EN-55 - hydrolyzed bovine collagen (Brooks Industries Inc., Code No. 01000) SoluColl - soluble collagen (Brooks Industries Inc., Code No. 01029) DMPX - dimethyl polysiloxane (5 centistokes) (Sigma) YYO - Y-ling-Y-lang oil (Young Living Essential Oils, Lehl, Utah) ID - Identification number
  • Skin barrier function can be analyzed by examining the diffusion of fluorescent and colored proteins and dextrans of various molecular weights ("markers") across the skin of nude mice or swine. Swine skin is preferred for many studies because it is inexpensive, can be maintained at -20°C, and responds similarly to human skin. Prior to use, frozen swine skin is thawed, hair is removed, and subcutaneous adipose tissue is dissected away. Preferably, a thickness of skin that resembles the thickness of human skin is obtained so as to prepare a membrane that accurately reflects the thickness of the barrier layer. A dermatome can be pushed across the surface of the skin so as to remove any residual dermis and prepare a skin preparation that accurately reflects human skin.
  • markers molecular weights
  • Elevation of temperature can also be used to loosen the bond between the dermis and the epidermis of hairless skin. Accordingly, the excised skin is placed on a hot plate or in heated water for 2 minutes at a temperature of approximately 50°C - 60°C and the dermis is removed by blunt dissection. Chemical approaches (e.g., 2M salt solutions) have also been used to separate the dermis from the epidermis of young rodents.
  • the buffer is isotonic, for example a normal saline solution or an isotonic buffered solution. More physiological buffers, which contain reagents that can be metabolized by the skin, can also be used. (See e.g., Collier et al., Toxicol. Appl. Pharmacol. 99:522-533 (1989)).
  • markers with molecular weight from 1,000 daltons to 2,000,000 daltons are commercially available and can be used to analyze the transdermal delivery compositions of the invention.
  • different colored protein markers having a wide range of molecular weights (6,500 to 205,000 daltons) and FITC conjugated protein markers ⁇ e.g., FITC conjugated markers from 6,500 to 205,000 daltons) are available from Sigma (C3437, M0163, G7279, A2065, A2190, C1311, T9416, L8151, and A2315).
  • high molecular weight FITC conjugated dextrans e.g., 250,000, 500,000, and 2,000,000 daltons
  • FD250S, FD500S, and FD2000S are examples of polyethylene glycol-cosaccharide
  • swine skin preparations obtained as described above, are treated with a delivery system lacking a delivered agent and control swine skin preparations are treated with water.
  • the skin is contacted with a ImM solution of a marker with a known molecular weight suspended in Ringer's solution (pH 7.4) at 37 0 C.
  • the skin is frozen and sliced at a thickness of 5 ⁇ m.
  • the sections are counter stained with 5 ⁇ g/ml propidium and, if the marker is FITC conjugated, the sections are analyzed by fluoresence microscopy. If the marker is a colored marker, diffusion of the marker can be determined by light microscope.
  • the marker will be retained in the upper layers of the stratum corneum in the skin but the skin treated with the delivery system will be found to have the dye distributed throughout the stratum corneum and any dermal layer that remains.
  • modifications of the experiments described above can be performed by using a delivery system comprising various molecular weight markers. Accordingly, skin preparations are treated with the delivery system comprising one or more markers and control skin preparations are treated with water. After one hour, the skin is frozen and sliced at a thickness of 5 ⁇ m. The sections can be counter stained with 5 ⁇ g/ml propidium iodide and can be analyzed by fiuoresence microscopy (e.g., when a fluorescent marker is used) or alternatively, the sections are analyzed under a light microscope. The marker will be retained in the upper layers of the stratum corneum in the skin but the skin treated with the delivery system will be found to have the dye distributed throughout the stratum corneum and any dermal layer that remains.
  • the transdermal water loss (TEWL) of penetration enhancer-treated skin preparations can be compared to that of untreated skin preparations. Accordingly, skin preparations are obtained, as described above, and are treated with a delivery system of the invention lacking a delivered agent (e.g., a penetration enhancer). Control skin preparations are untreated.
  • a delivery system of the invention lacking a delivered agent (e.g., a penetration enhancer).
  • Control skin preparations are untreated.
  • an evaporimeter is used to analyze the skin preparation.
  • the Courage and Khazaka Tewameter TM210 an open chamber system with two humidity and temperature sensors, can be used to measure the water evaporation gradient at the surface of the skin. The parameters for calibrating the instrument and use of the instrument is described in Barel and Clarys Skin Pharmacol. 8: 186-195 (1995) and the manufacturer's instructions.
  • TEWL will be low.
  • TEWL in penetration enhancer-treated skin preparations will be significantly greater.
  • skin barrier function can be analyzed by examining the percutaneous absorption of labeled markers (e.g., radiolabeled, fluorescently labeled, or colored) across skin preparations in a diffusion chamber.
  • labeled markers e.g., radiolabeled, fluorescently labeled, or colored
  • Delivery systems of the invention having various molecular weight markers, for example, the proteins and dextrans described above, are administered to swine skin preparations. Swine skin preparations are mounted in side-by-side diffusion chambers and are allowed to stabilize at 37°C with various formulations of penetration enhancer.
  • Donor and receiver fluid volumes are 1.5ml. After 1 hour of incubation, a labeled marker is added to the epidermal donor fluid to yield a final concentration that reflects an amount that would be applied to the skin in an embodiment of the invention.
  • receiver fluid Five hundred microliters of receiver fluid is removed at various time points, an equal volume of penetration enhancer is added to the system. The aliquot of receiver fluid removed is then analyzed for the presence of the labeled marker (e.g., fluorescent detection, spectroscopy, or scintillation counting).
  • the labeled marker e.g., fluorescent detection, spectroscopy, or scintillation counting.
  • Control swine skin preparations are equilibrated in Ringer's solution (pH 7.4) at 37°C; the same concentration of labeled marker as used in the experimental group is applied to the donor fluid after one hour of equilibration; and 500 ⁇ l of receiver fluid is analyzed for the presence of the marker. In the experimental group, the steady-state flux of labeled marker in the skin will be significantly greater than that of the control group.
  • transdermal delivery compositions can be evaluated for their ability to transport low and high molecular weight delivered agents across the skin.
  • the next example describes several different formulations of transdermal delivery composition that were made to comprise various delivered agents, demonstrating the wide-range of utility of aspects of the invention.
  • transdermal delivery composition containing various delivered agents are provided.
  • the formulations described include: compositions for removing age spots and restoring skin brightness, compositions for advanced pain relief, muscle relaxers, hormone replacement products, wound healing formulations, products for reducing fine lines and wrinkles, stretch mark reducing products, growth factor products, and anti-psoriasis products.
  • Aloe Vera (whole leaf) concentrate 100 ml
  • Aloe Vera (whole leaf) concentrate 5 ml
  • Bioserum which is obtainable from Atrium Biosciences, Ontario Canada, may contain one or more of the following: placental protein, amniotic fluid, calf skin extract, and serum protein.
  • phenochem may contain one or more of the following: Methyl Paraben, Ethyl Paraben, Propyl Paraben, Butyl Paraben, and Isobutyl Paraben, and sodium methylparaban imidizolidinyl urea.
  • Additional components that may be included in some formulations of products that reduce the appearance of fine lines and wrinkles include: igepal cephene distilled, synasol, ethoxylated glycerides, trisodium EDTA, potassium sorbate, citric acid, ascorbic acid, and distilled water.
  • one formulation contains: Collagen (Marine), Distilled Water, Igepal Cephene Distilled, Methyl Paraben, Ethyl Paraben, Propyl Paraben, Butyl Paraben, Isobutyl Paraben, Synasol, Serum Protein, Purified Water, Amniotic Fluid. Placental Protein.
  • Calfskin Extract Hydrolyzed Collagen Sodium Methylparaben Imidazolidinyl Urea. Ethoxylated Glycerides, Trisodium EDTA, Potassium Sorbate, Citric Acid, and Ascorbic Acid.
  • the following example describes experiments that employed two different skin cell model systems to evaluate the ability of a transdermal delivery composition containing collagen to transport collagen to skin cells.
  • a transdermal delivery composition of the invention comprising marine type 1 collagen or native collagen efficiently transported the delivered agent to skin cells.
  • Two different in vitro skin cell model systems were used, human cadaver skin and a cellulose acetate skin cell model system. Based on the physiology of the skin, three possible pathways exist for passive transport of molecules through the skin to the vascular network: (1) intercellular diffusion through the lipid lamellae; (2) transcellular diffusion through both the keratinocytes and lipid lamellae; and (3) diffusion through appendages (hair follicles and sweat ducts).
  • the cellulose acetate skin model evaluates the ability of the delivered agent to transport using the first two pathways and the human cadaver skin evaluates the ability to use all three pathways.
  • the transdermal delivery composition comprising collagen was applied to the cellulose acetate and the human cadaver skin in a diffusion chamber and the results were recorded after 10 minutes, 30 minutes and one hour.
  • the diffused material was analyzed by a spectrophotometer (Hitachi U2000 multiscan spectrophotometer). A portion of the diffused material was also separated on a gel by electrophoresis and the collagen was stained using a collagen-specific dye. A portion of the diffused material was also immunoprecipitated using polyclonal antibodies specific for collagens types 1 -7 and the immunoprecipitates were analyzed by immunodiffusion.
  • the table below provides the collagen concentration in the various samples of transdermal delivery compositions tested.
  • the protein concentration was determined using a micro-protein assay (Bio-Rad).
  • the transdermal delivery composition containing either marine collagen or native collagen was applied to the human cadaver skin and the cellulose acetate skin model systems.
  • the penetration studies were performed in a diffusion chamber and the results were recorded at 10 minutes, 30 minutes and an hour later. Sections of skin or cellulose acetate were stained with a collagen specific dye and a light microscope was used to visualize the transported collagen.
  • TABLE 21 provides the results of these experiments. Note, that the native collagen appeared to penetrate the skin in less time than the marine collagen. This may be due to the differing concentrations of collagen used in the transdermal delivery compositions (i.e., the concentration of the native collagen was 0.40 mg/ml and the concentration of the marine collagen was 1.14 mg/ml). Nevertheless, by one hour, almost all of both types of collagen had penetrated the skin in the model systems employed.
  • a portion of the diffused material was then separated by electrophoresis and visualized by staining with a collagen-specific dye.
  • the penetrated marine collagen remained intact during and after the analysis because the labeled marine collagen detected in the diffused material was observed to have the same molecular weight as marine collagen that had not undergone the analysis (control sample).
  • the results showed that the marine collagen prior to the penetration study and after the penetration study maintained its molecular structure around 500 kilodaltons (KD).
  • KD kilodaltons
  • the native collagen also maintained a molecular weight around 500KD before and after penetration of the epidermis, demonstrating that the native collagen that was delivered by the transdermal delivery composition, like the marine collagen, remained intact into the epidermis.
  • a clinical study was performed to evaluate the ability of a transdermal delivery composition containing collagen, prepared as described herein, to reduce wrinkles and fine lines and otherwise restore skin tone to subjects in need thereof.
  • the medial half of the facial region including the neck and the upper chest areas were assigned as the regions under investigation.
  • digital pictures were taken at days 0, 3, 7, 14 and 21 of the regions under investigation of the face including the symmetrical region of the face where the product was not applied.
  • Micrometer measurements of the wrinkles were then made from the digital pictures and also from the facial areas under investigation.
  • Subjects invited to participate in the study had facial wrinkles and were 25 years or older. Non-facial wrinkle individuals were also invited and served as the control group. The source of subjects for the study was randomly selected from the ethnically diverse population group ages ranging from 25 years to 88 years old. TABLE 25 Description of the subjects participating in the study
  • transdermal delivery composition comprising marine collagen as a wrinkle reducer is 10.29% when applied twice daily for 21 days.
  • percent reduction of the wrinkles varies with the various areas of the face where it is applied, with 17.4% reduction around the eye regions and 15.20% at the temporal cheeks at the high end and around 9% at the chin and mouth regions.
  • the next example sets forth experiments that demonstrate that transdermal delivery compositions containing ethoxylated oils of less than 20 ethoxylations/molecule transfer a delivered agent to the skin more effectively than transdermal delivery compositions containing ethoxylated oils of 20 or more ethoxylations/molecule.
  • transdermal delivery composition formulations containing collagen (1.2mg/ml) and an ethoxylated oil having different amounts of ethoxylations/molecule are prepared.
  • Formulations containing ethoxylated oil of either 12, 16, 18, 20, 24, and 30 ethoxylations/molecule, water, and marine collagen (1.2mg/ml) are made.
  • Approximately 0.5ml of each of these formulations are applied to human cadaver skin in a diffusion chamber and the penetration of collagen is monitored over time (e.g., 10 minutes, 30 minutes, 45 minutes and one hour). Sections of the skin are taken, stained with a collagen specific dye, and the stained sections are analyzed under a light microscope.
  • the collagen that has penetrated the skin at the various time points above is collected from the diffusion chamber and analyzed in a spectrophotometer. As above, a greater amount of collagen will be detected in samples collected at the various time points with formulations containing less than 20 ethoxylations/molecule than formulations containing 20 or more ethoxylations/molecule. Formulations containing less than 20 ethoxylations/molecule will also be observed to penetrate the skin faster than formulations containing 20 or more ethoxylations/molecule.
  • transdermal delivery compositions containing ethoxylated fatty acids having 10-19 ethoxylations/molecule transfer a delivered agent to the skin as effectively as transdermal delivery compositions containing ethoxylated oils having 10-19 ethoxylations/molecule.
  • a transdermal delivery composition containing collagen (1.2mg/ml) and an ethoxylated fatty moiety having 16 ethoxylations/molecule, water and marine collagen is made.
  • Several transdermal delivery compositions containing ethoxylated fatty moieties and having 16 ethoxylations/molecule, water, and marine collagen are made.
  • Approximately 0.5ml of each of these formulations are applied to human cadaver skin in a diffusion chamber and the penetration of collagen is monitored over time (e.g., 10 minutes, 30 minutes, 45 minutes and one hour). Sections of the skin are taken, stained with a collagen specific dye, and the stained sections are analyzed under a light microscope.
  • the collagen that has penetrated the skin at the various time points above is collected from the diffusion chamber and analyzed in a spectrophotometer. As above, approximately the same amount of collagen will be detected in samples collected at the various time points with formulations containing ethoxylated fatty moieties as compared to formulations containing ethoxylated oils. Formulations containing ethoxylated fatty moieties will also be observed to penetrate the skin at approximately the same rate as formulations containing ethoxylated oils.
  • a transdermal delivery composition containing collagen (1.2mg/ml) and an ethoxylated oil having 16 ethoxylations/molecule, water and marine collagen is made. A portion of the composition is transferred to a cartridge adapted for the exemplary transdermal delivery device described herein.
  • the transdermal delivery device is preset to load approximately 0.5ml of the formulation. Approximately 0.5ml of the formulation is applied to human cadaver skin, either manually, or using the transdermal delivery device, in a diffusion chamber and the penetration of collagen is monitored over time (e.g., 10 minutes, 30 minutes, 45 minutes and one hour). Sections of the skin are taken, stained with a collagen specific dye, and the stained sections are analyzed under a light microscope. Several samples are prepared and treated at the same time.
  • the amount of collagen-specific staining seen in stained skin sections collected is substantially more consistent in the samples in which the formulation is administered via the transdermal delivery device than in the samples in which the formulation is delivered manually.
  • the collagen that has penetrated the skin at the various time points above is collected from the diffusion chamber and analyzed in a spectrophotometer.
  • the amount of collagen detected in samples collected from the various samples in which the formulation is delivered via the transdermal delivery device shows considerably less variation than the amounts of collagen calculated from samples in which the formulation was applied manually.
  • Transdermal delivery compositions comprising the delivered agents disclosed below are prepared as described herein.
  • Preferred transdermal delivery compositions comprise formulations such as those described in Example 4 above, wherein the immunogenic compositions disclosed below are the delivered agents, rather than collagen, testosterone, and the like, present in transdermal delivery compositions described in Example 4.
  • a novel nucleic acid and protein corresponding to the NS3/4A domain of HCV was cloned from a patient infected with HCV (SEQ. ID. NO.: 163).
  • a Genebank search revealed that the cloned sequence had the greatest homology to HCV sequences but was only 93% homologous to the closest HCV relative (accession no AJ 278830).
  • This novel peptide SEQ. ID. NO.: 164 and fragments thereof (e.g., SEQ. ID.
  • nucleic acids encoding these molecules can be incorporated in one or more of the transdermal delivery compositions described herein. It was also discovered that both the NS3/4A gene (SEQ. ID. NO.: 163) and corresponding peptide (SEQ. ID. NO.: 164) were immunogenic in vivo.
  • mutants of the novel NS3/4A peptide were created. It was discovered that truncated mutants (e.g., SEQ. ID. NOs.: 174 and 175) and mutants that lack a proteolytic cleavage site (SEQ. ID. NOs.: 165-173), were also immunogenic in vivo. These novel peptides (SEQ. ID. NOs.: 165-173) and fragments thereof (e.g., SEQ. ID.
  • nucleic acids that are any number of consecutive amino acids between at least 3-600 (e.g., 3, 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, or 600 amino acids in length), nucleic acids encoding these molecules, vectors having said nucleic acids, and cells having said vectors, nucleic acids, or peptides can be incorporated in one or more of the transdermal delivery compositions described herein..
  • 3-600 e.g., 3, 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, or 600 amino acids in length
  • nucleic acids encoding these molecules e.g., 3, 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70,
  • a codon-optimized nucleic acid encoding NS3/4A was also created and was found to be immunogenic.
  • the nucleic acid of SEQ. ID. NO.: 163 was analyzed for codon usage and the sequence was compared to the codons that are most commonly used in human cells. Because HCV is a human pathogen, it was unexpected to discover that the virus had not yet evolved to use codons that are most frequently found to encode human proteins (e.g., optimal human codons).
  • a total of 435 nucleotides were replaced to generate the codon-optimized synthetic NS3/4A nucleic acid (SEQ. ID. NO. 197).
  • the NS3/4A peptide encoded by the codon-optimized nucleic acid sequence (SEQ. ID. NO.: 198) was 98% homologous to HCV-I and contained a total of 15 different amino acids.
  • the codon optimized nucleic acid (MSLFl or coNS3/4A) (SEQ. ID. NO.: 197) was found to be more efficiently translated in vitro than the native NS3/4A and that mice immunized with the MSLFl containing construct generated significantly more NS3/4A specific antibodies than mice immunized with a wild-type NS3/4A containing construct. Further, mice immunized with the MSLFl containing construct were found to prime NS3 -specific CTLs more effectively and exhibit better in vivo tumor inhibiting immune responses than mice immunized with wild-type NS3/4A containing constructs.
  • compositions described above are useful as immunogens, which can be administered alone or in conjunction with an adjuvant in one or more of the transdermal delivery compositions described herein.
  • Preferred embodiments include compositions that comprise one or more of the nucleic acids and/or peptides described above with or without an adjuvant. That is, some of the compositions described herein are prepared with or without an adjuvant and comprise, consist, or consist essentially of a NS3/4A peptide (SEQ. ID. NO.: 164 or SEQ. ID.
  • nucleic acids encoding these molecules can be incorporated in one or more of the transdermal delivery compositions described herein.
  • SEQ. ID. NOs.: 176 and 177) or a nucleic acid encoding one or more of these molecules e.g., SEQ. ID.
  • nucleotides can be formulated into one or more of the transdermal delivery systems described herein. Additional compositions are prepared with or without an adjuvant and comprise, consist, or consist essentially of one or more of the NS3/4A mutant peptides (SEQ. ID.
  • nucleic acids encoding these molecules can be incorporated in one or more of the transdermal delivery compositions described herein.
  • compositions comprising ribavirin and an antigen (e.g., one or more of the previously described HCV peptides or nucleic acids) enhance and/or facilitate an animal's immune response to the antigen. That is, it was discovered that ribavirin is a very effective "adjuvant," which for the purposes of this disclosure, refers to a material that has the ability to enhance or facilitate an immune response to a particular antigen.
  • the adjuvant activity of ribavirin was manifested by a significant increase in immune-mediated protection against the antigen, an increase in the titer of antibody raised to the antigen, and an increase in proliferative T cell responses.
  • compositions that comprise ribavirin and one or more of the peptides or nucleic acids described herein, which are formulated into one or more of the transdermal delivery compositions described herein are embodiments of the invention.
  • These compositions can vary according to the amount of ribavirin, the form of ribavirin, as well as the sequence of the HCV nucleic acid or peptide.
  • Embodiments of the invention also include methods of making and using the compositions above. Some methods involve the making of nucleic acids encoding NS3/4A, codon-optimized NS3/4A, mutant NS34A, fragments thereof that are any number of consecutive nucleotides between at least, less than or equal to, or more than 50, 60, 75, 80, 90, 100, 110, 120, 130, 140, 150, 160 , 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 consecutive nucleotides in length or the complement thereof, peptides corresponding to said nucleic acids, constructs comprising said nucleic acids, and cells containing said compositions.
  • Preferred methods concern the making of vaccine compositions or immunogenic preparations that comprise, consist, or consist essentially of the newly discovered NS3/4A fragment, codon-optimized NS3/4A, or an NS3/4A mutant (e.g., a truncated mutant or a mutant lacking a proteolytic cleavage site), or a fragment thereof or a nucleic acid encoding one or more of these molecules, as described above.
  • Preferred nucleic acids for use with the methods described herein include SEQ. ID. NOs.: 163, 197, nucleic acids encoding SEQ. ID.
  • compositions described above can be made by providing an adjuvant (e.g., ribavirin), providing an HCV antigen (e.g., an antigen encoded by SEQ. ID. NOs.: 163, 197, nucleic acids encoding SEQ.
  • an adjuvant e.g., ribavirin
  • an HCV antigen e.g., an antigen encoded by SEQ. ID. NOs.: 163, 197, nucleic acids encoding SEQ.
  • Methods of enhancing or promoting an immune response in an animal, including humans, to an antigen are also provided. Such methods can be practiced, for example, by identifying an animal in need of an immune response to HCV and providing said animal a composition comprising one or more of the nucleic acids or peptides above and an amount of adjuvant that is effective to enhance or facilitate an immune response to the antigen/epitope.
  • the antigen and the adjuvant are administered separately, instead of in a single mixture.
  • the adjuvant is administered a short time before or a short time after administering the antigen.
  • Preferred methods involve providing the animal in need with ribavirin and NS3/4A (e.g., SEQ.
  • NS3/4A codon-optimized NS3/4A
  • a mutant NS3/4A e.g., SEQ. ID. NOs.: 165-175
  • a fragment thereof e.g., SEQ. ID. NOs.: 176-188
  • any number of consecutive amino acids between at least 3-50 (e.g., 3, 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acids in length) or a nucleic acid encoding any one or more of said molecules.
  • some embodiments include a transdermal delivery system described herein (see e.g., Example 4) comprising, optionally, ribavirin and an NS3/4A nucleic acid provided by SEQ. ID. NOs.: 163, 197, nucleic acids encoding SEQ. ID.
  • nucleic acids at least, less than or equal to, or more than 50, 60, 75, 80, 90, 100, 110, 120, 130, 140, 150, 160 , 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 consecutive nucleotides in length or the complement thereof .
  • an immunogen comprising one or more of the HCV nucleic acids or peptides described herein are used to prepare a medicament for the treatment and/or prevention of HCV infection.
  • an individual in need of a medicament that prevents and/or treats HCV infection is identified and said individual is provided a transdermal delivery system comprising ribavirin and an HCV antigen such as NS3/4A (e.g., SEQ. ID. NO.: 164), codon-optimized NS3/4A (e.g., SEQ. ID. NO.: 198), or a mutant NS3/4A (e.g., SEQ. ID.
  • HCV infection a transdermal delivery system described herein (see e.g., Example 4) comprising, optionally, ribavirin and an NS3/4A nucleic acid provided by SEQ. ID.
  • a novel nucleic acid and protein corresponding to the NS3/4A domain of HCV was cloned from a patient infected with HCV (SEQ. ID. NOs.: 163 and 164).
  • a Genebank search revealed that the cloned sequence had the greatest homology to HCV sequences but was only 93% homologous to the closest HCV relative (accession no AJ 278830).
  • a truncated mutant of the novel NS3/4A peptide and NS3/4A mutants, which lack a proteolytic cleavage site, (as well as corresponding nucleic acids) were also created. Further, a human codon-optimized NS3/4A nucleic acid and peptide were created.
  • novel peptides and nucleic acids encoding said peptides were potent immunogens that can be mixed with adjuvants so as to make a composition that induces a recipient to provide an immune response to HCV.
  • the cloning of the novel NS3/4A gene and the creation of the various NS3/4A mutants and codon optimized NS3/4A gene are described in the following example.
  • NS3/4A sequence was amplified from the serum of an HCV- infected patient (HCV genotype Ia) using the Polymerase Chain Reaction (PCR). Total RNA was extracted from serum, and cDNA synthesis and PCR were performed according to standard protocols (Chen M et al., J. Med. Virol. 43:223-226 (1995)). The cDNA synthesis was initiated using the antisense primer "NS4KR" (5'-CCG TCT AGA TCA GCA CTC TTC CAT TTC ATC-3' (SEQ. ID. NO.: 190)).
  • a 2079 base pair DNA fragment of HCV corresponding to amino acids 1007 to 1711, which encompasses the NS3 and NS4A genes, was amplified.
  • a high fidelity polymerase (Expand High Fidelity PCR, Boehringer-Mannheim, Mannheim, Germany) was used with the "NS3KF" primer (5'-CCT GAA TTC ATG GCG CCT ATC ACG GCC TAT-3' (SEQ. ID. NO.: 191) and the NS4KR primer.
  • the NS3KF primer contained a EcoRI restriction enzyme cleavage site and a start codon and the primer NS4KR contained a Xbal restriction enzyme cleavage site and a stop codon.
  • the amplified fragment was then sequenced (SEQ. ID. NO.: 163). Sequence comparison analysis revealed that the gene fragment was amplified from a viral strain of genotype Ia. A computerized BLAST search against the Genbank database using the NCBI website revealed that the closest HCV homologue was 93% identical in nucleotide sequence.
  • the amplified DNA fragment was then digested with EcoRI and Xbal, and was inserted into a pcDNA3.1/His plasmid (Invitrogen) digested with the same enzymes.
  • the NS3/4A-pcDNA3.1 plasmid was then digested with EcoRI and Xba I and the insert was purified using the QiaQuick kit (Qiagen, Hamburg, Germany) and was ligated to a EcoRI/Xba I digested pVAX vector (Invitrogen) so as to generate the NS3/4A-pVAX plasmid.
  • the rNS3 truncated mutant was obtained by deleting NS4A sequence from the NS3/4A DNA.
  • the NS3 gene sequence of NS3/4A-pVAX was PCR amplified using the primers NS3KF and 3'NotI (5'-CCA CGC GGC CGC GAC GAC CTA CAG-3' (SEQ. ID. NO.: 192)) containing EcoRI and Not I restriction sites, respectively.
  • the NS3 fragment (1850 bp) was then ligated to a EcoRI and Not I digested p VAX plasmid to generate the NS3-pVAX vector. Plasmids were grown in BL21 E.coli cells. The plasmids were sequenced and were verified by restriction cleavage and the results were as to be expected based on the original sequence.
  • Table 29 describes the sequence of the proteolytic cleavage site of NS3/4A, referred to as the breakpoint between NS3 and NS4A. This wild-type breakpoint sequence was mutated in many different ways so as to generate several different NS3/4A breakpoint mutants. TABLE 29 also identifies these mutant breakpoint sequences.
  • the fragments listed in TABLE 29 are preferred immunogens that can be incorporated with or without an adjuvant (e.g., ribavirin) into a transdermal delivery composition for administration to an animal so as to induce an immune response in said animal to HCV.
  • an adjuvant e.g., ribavirin
  • NS3/4A-TPT-pVAX TKYMTCMSADLEWIPTWVLVGGVL SEQ. ID. NO.: 180
  • NS3/4A-SSSSCST-pVAX TKYMTCMSADSSSSCSTWVLVGGVL (SEQ. ID. NO.: 186)
  • NS3A/4A-VVWTST-PYAX TKYMTCMSADWVVTSTWVLVGGVL (SEQ. H). NO.: 187) NS5-pVAX ASEDWCCSMSYTWTG (SEQ. ID. NO.: 189)
  • NS5A/B-pVAX SSEDWCCSMWVLVGGVL (SEQ. ID. NO.: 188)
  • the wild type sequence for the NS3/4A fragment is NS3/4A-pVAX.
  • the NS3/4A breakpoint is identified by underline, wherein the Pl position corresponds to the first Thr (T) and the Pl' position corresponds to the next following amino acid the NS3/4A-pVAX sequence.
  • the NS3 protease cleaves between the Pl and Pl' positions.
  • the NS3/4A-pVAX plasmid was mutagenized using the QUICKCHANGETM mutagenesis kit (Stratagene), following the manufacturer's recommendations.
  • the plasmid was amplified using the primers 5'- CTGGAGGTCGTCACGCCTACCTGGGTGCTCGTT-3' (SEQ. ID. NO.: 193) and 5'- ACCGAGCACCCAGGTAGGCGTGACGACCTCCAG-3' (SEQ. ID. NO.: 194) resulting in NS3/4A-TPT-pVAX.
  • the plasmid was amplified using the primers 5'-
  • the sequence of the previously isolated and sequenced unique NS3/4A gene (SEQ. ID. NO.: 163) was analyzed for codon usage with respect to the most commonly used codons in human cells. A total of 435 nucleotides were replaced to optimize codon usage for human cells. The sequence was sent to Retrogen Inc. (6645 Nancy Ridge Drive, San Diego, CA 92121) and they were provided with instructions to generate a full-length synthetic codon optimized NS3/4A gene. The codon optimized NS3/4A gene had a sequence homology of 79% within the region between nucleotide positions 3417-5475 of the HCV-I reference strain. A total of 433 nucleotides differed. On an amino acid level, the homology with the HCV-I strain was 98% and a total of 15 amino acids differed.
  • the plasmid DNA used for in vivo injection was purified using Qiagen DNA purification columns, according to the manufacturers instructions (Qiagen GmbH, Hilden, FRG). The concentration of the resulting plasmid DNA was determined spectrophotometrically (Dynaquant, Pharmacia Biotech, Uppsala, Sweden) and the purified DNA was dissolved in sterile phosphate buffer saline (PBS) at concentrations of 1 mg/ml.
  • PBS sterile phosphate buffer saline
  • HepG2 cells were transiently transfected with the wtNS3/4A and the coNS3/4A plasmids. These experiments revealed that the coNS3/4A plasmid generated 11 -fold higher expression levels of the NS3 protein when compared to the wtNS3/4A plasmid, as determined by densitometry and a standard curve of recombinant NS3. Since the wtNS3/4A and the coNS3/4A plasmids are identical in size it is unlikely that there would be any major differences in transfections efficiencies between the plasmids. Staining of coNS3/4A plasmid transfected, and SFV infected, BHK cells revealed a similar perinuclear and cytoplasmic distribution of the NS3 as previously observed, confirming an unchanged subcellular localization.
  • transdermal delivery system include the nucleic acid embodiments described herein and, in particular, nucleic acids that include nucleotides encoding the HCV peptides described herein (SEQ. ID. NOs.: 163-173 or SEQ. ID. NO.: 198) or a fragment thereof (e.g., SEQ. ID. NOs.: 176 and 177) containing any number of consecutive amino acids between at least 3-50 (e.g., 3, 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length).
  • Some embodiments for example, include genomic DNA, RNA, and cDNA encoding these HCV peptides.
  • the HCV nucleotide embodiments not only include the DNA sequences shown in the sequence listing (e.g., SEQ. ID. NO.: 163 or SEQ. ID. NO.: 197) but also include nucleotide sequences encoding the amino acid sequences shown in the sequence listing (e.g., SEQ. ID. NOs.: 164-173 or SEQ. ID.
  • nucleotide sequence that hybridizes to the DNA sequences shown in the sequence listing under stringent conditions (e.g., hybridization to filter-bound DNA in 0.5 M NaHPO 4 , 7.0% sodium dodecyl sulfate (SDS), 1 mM EDTA at 50 0 C) and washing in 0.2 X SSC/0.2% SDS at 50°C and any nucleotide sequence that hybridizes to the DNA sequences that encode an amino acid sequence provided in the sequence listing (SEQ. ID. NOs.: 164-173 or SEQ. ID.
  • transdermal delivery systems of the invention also include fragments, modifications, derivatives, and variants of the sequences described above. Desired embodiments, for example, include nucleic acids having at least 25 consecutive bases of one of the novel HCV sequences or a sequence complementary thereto and preferred nucleic acids include SEQ. ID. NOs.: 163, 197, nucleic acids encoding SEQ. ID.
  • nucleic acids at least, less than or equal to, or more than 50, 60, 75, 80, 90, 100, 110, 120, 130, 140, 150, 160 , 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 consecutive nucleotides in length or the complement thereof .
  • aspects of the invention include transdermal delivery systems comprising SEQ. ID. NOs.: 163, 197, nucleic acids encoding SEQ. ID.
  • nucleic acids at least, less than or equal to, or more than 50, 60, 75, 80, 90, 100, 110, 120, 130, 140, 150, 160 , 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 consecutive nucleotides in length or the complement thereof .
  • nucleic acid described herein that can be formulated into one or more of the transdermal delivery systems described herein can have any number of consecutive nucleotides between about 12 to approximately 2112 consecutive nucleotides of SEQ. ID. NO.: 163 or SEQ. ID. NO.: 197.
  • Some nucleic acid fragments for incorporation into a transdermal delivery system described herein include nucleic acids comprising, consisting of, or consisting essentially of at least 12-15, 15-20, 20-30, 30-50, 50-100, 100-200, 200-500, 500-1000, 1000-1500, 1500-2079, or 1500-2112 consecutive nucleotides of SEQ. ID.
  • nucleic acid embodiments can also be altered by substitution, addition, or deletion so long as the alteration does not significantly affect the structure or function (e.g., ability to serve as an immunogen) of the HCV nucleic acid. Due to the degeneracy of nucleotide coding sequences, for example, other sequences that encode substantially the same HCV amino acid sequence as depicted in SEQ. ID. NOs.: 164-175 or SEQ. ID. NO.: 198 can be used in some embodiments. These include, but are not limited to, nucleic acid sequences encoding all or portions of HCV peptides (SEQ. ID.
  • nucleic acids that can be used in embodiments of the invention are said to be comprising, consisting of, or consisting essentially of nucleic acids encoding any one of SEQ. ID. NOs.: 164-189 or SEQ. ID. NO.: 196 in light of the modifications above.
  • probes that complement these molecules can be designed and manufactured by oligonucleotide synthesis.
  • Desirable probes comprise a nucleic acid sequence of (SEQ. ID. NO.: 163) that is unique to this HCV isolate.
  • These probes can be used to screen cDNA from patients so as to isolate natural sources of HCV, some of which may be novel HCV sequences in themselves. Screening can be by filter hybridization or by PCR, for example.
  • the labeled probe preferably contains at least 15-30 base pairs of the nucleic acid sequence of (SEQ. ID. NO.: 163) that is unique to this NS3/4A peptide.
  • the hybridization washing conditions used are preferably of a medium to high stringency.
  • the hybridization can be performed in 0.5M NaHPO 4 , 7.0% sodium dodecyl sulfate (SDS), 1 mM EDTA at 42°C overnight and washing can be performed in 0.2X SSC/0.2% SDS at 42°C.
  • SDS sodium dodecyl sulfate
  • washing can be performed in 0.2X SSC/0.2% SDS at 42°C.
  • HCV nucleic acids can also be isolated from patients infected with HCV using the nucleic acids described herein. Accordingly, RNA obtained from a patient infected with HCV is reverse transcribed and the resultant cDNA is amplified using PCR or another amplification technique.
  • the primers are preferably obtained from the NS3/4A sequence (SEQ. ID. NO.: 163).
  • RT-PCR Reverse Transcriptase Asymmetric Gap Ligase Chain Reaction
  • RNA is isolated, following standard procedures. A reverse transcription reaction is performed on the RNA using an oligonucleotide primer specific for the most 5' end of the amplified fragment as a primer of first strand synthesis. The resulting RNA/DNA hybrid is then "tailed" with guanines using a standard terminal transferase reaction. The hybrid is then digested with RNAse H, and second strand synthesis is primed with a poly-C primer. Thus, cDNA sequences upstream of the amplified fragment are easily isolated. For a review of cloning strategies which can be used, see e.g., Sambrook et al., 1989, supra.
  • primers on either side of the sequence to be amplified are added to a suitably prepared nucleic acid sample along with dNTPs and a thermostable polymerase, such as Taq polymerase, PfU polymerase, or Vent polymerase.
  • a thermostable polymerase such as Taq polymerase, PfU polymerase, or Vent polymerase.
  • the nucleic acid in the sample is denatured and the primers are specifically hybridized to complementary nucleic acid sequences in the sample.
  • the hybridized primers are then extended. Thereafter, another cycle of denaturation, hybridization, and extension is initiated. The cycles are repeated multiple times to produce an amplified fragment containing the nucleic acid sequence between the primer sites.
  • PCR has further been described in several patents including US Patents 4,683,195, 4,683,202 and 4,965,188.
  • the primers are selected to be substantially complementary to a portion of the nucleic acid sequence of (SEQ. ID. NO.: 163) that is unique to this NS3/4A molecule, thereby allowing the sequences between the primers to be amplified.
  • primers can be any number between at least 16-20, 20-25, or 25-30 nucleotides in length.
  • Tm melting temperature
  • the formation of stable hybrids depends on the melting temperature (Tm) of the DNA.
  • Tm depends on the length of the primer, the ionic strength of the solution and the G+C content. The higher the G+C content of the primer, the higher is the melting temperature because G: C pairs are held by three H bonds whereas A:T pairs have only two.
  • the G+C content of the amplification primers described herein preferably range between 10% and 75 %, more preferably between 35% and 60%, and most preferably between 40% and 55 %.
  • the appropriate length for primers under a particular set of assay conditions can be empirically determined by one of skill in the art.
  • the spacing of the primers relates to the length of the segment to be amplified.
  • amplified segments carrying nucleic acid sequence encoding HCV peptides can range in size from at least about 25 bp to the entire length of the HCV genome. Amplification fragments from 25- 1000 bp are typical, fragments from 50-1000 bp are preferred and fragments from 100- 600 bp are highly preferred. It will be appreciated that amplification primers can be of any sequence that allows for specific amplification of the NS3/4A region and can, for example, include modifications such as restriction sites to facilitate cloning.
  • the PCR product can be subcloned and sequenced to ensure that the amplified sequences represent the sequences of an HCV peptide.
  • the PCR fragment can then be used to isolate a full length cDNA clone by a variety of methods.
  • the amplified fragment can be labeled and used to screen a cDNA library, such as a bacteriophage cDNA library.
  • the labeled fragment can be used to isolate genomic clones via the screening of a genomic library.
  • an expression library can be constructed utilizing cDNA synthesized from, for example, RNA isolated from an infected patient, hi this manner, HCV geneproducts can be isolated using standard antibody screening techniques in conjunction with antibodies raised against the HCV gene product. (For screening techniques, see, for example, Harlow, E. and Lane, eds., 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor).
  • Embodiments of the invention also include (a) DNA vectors that contain any of the foregoing nucleic acid sequence and/or their complements (i.e., antisense); (b) DNA expression vectors that contain any of the foregoing nucleic acid sequences operatively associated with a regulatory element that directs the expression of the nucleic acid; and (c) genetically engineered host cells that contain any of the foregoing nucleic acid sequences operatively associated with a regulatory element that directs the expression of the coding sequences in the host cell.
  • These recombinant constructs are capable of replicating autonomously in a host cell. Alternatively, the recombinant constructs can become integrated into the chromosomal DNA of a host cell.
  • Such recombinant polynucleotides typically comprise an HCV genomic or cDNA polynucleotide of semi-synthetic or synthetic origin by virtue of human manipulation. Therefore, recombinant nucleic acids comprising these sequences and complements thereof that are not naturally occurring are provided.
  • nucleic acids encoding an HCV peptide or nucleic acids having sequences that complement an HCV gene as they appear in nature can be employed, they will often be altered, e.g., by deletion, substitution, or insertion, and can be accompanied by sequence not present in humans.
  • regulatory elements include, but are not limited to, inducible and non-inducible promoters, enhancers, operators and other elements known to those skilled in the art that drive and regulate expression.
  • Such regulatory elements include, but are not limited to, the cytomegalovirus hCMV immediate early gene, the early or late promoters of SV40 adenovirus, the lac system, the trp system, the TAC system, the TRC system, the major operator and promoter regions of phage A, the control regions of fd coat protein, the promoter for 3- phosphoglycerate kinase, the promoters of acid phosphatase, and the promoters of the yeast-mating factors.
  • HCV peptide-encoding nucleic acid sequences and their complementary sequences can be engineered so as to modify their processing or expression.
  • the HCV nucleic acids described herein can be combined with a promoter sequence and/or ribosome binding site, or a signal sequence can be inserted upstream of HCV peptide-encoding sequences so as to permit secretion of the peptide and thereby facilitate harvesting or bioavailability.
  • a given HCV nucleic acid can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction sites or destroy preexisting ones, or to facilitate further in vitro modification.
  • Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site-directed mutagenesis. (Hutchinson et al., J. Biol. Chem., 253:6551 (1978)).
  • the nucleic acids encoding the HCV peptides, described above, can be manipulated using conventional techniques in molecular biology so as to create recombinant constructs that express the HCV peptides.
  • nucleic acids encoding other proteins or domains of other proteins can be joined to nucleic acids encoding an HCV peptide so as to create a fusion protein.
  • Nucleotides encoding fusion proteins can include, but are not limited to, a full length NS3/4A sequence (SEQ. ID. NO.: 164 or SEQ. ID. NO.: 198), mutant NS3/4A sequences (e.g., SEQ. ID. NOs.: 165-173) or a peptide fragment of an NS3/4A sequence fused to an unrelated protein or peptide, such as for example, polyhistidine, hemagglutinin, an enzyme, fluorescent protein, or luminescent protein, as discussed below.
  • the expression constructs described in Example 1 were purified using the Qiagen DNA purification system, according to the manufacturer's instructions and the purified DNA vectors were used to immunize groups of four to ten Balb/c mice.
  • the plasmids were injected directly into regenerating tibialis anterior (TA) muscles as previously described (Davis et al., Human Gene Therapy 4(6):733 (1993)).
  • mice were injected intramuscularly with 50 ⁇ l/TA of 0.0ImM cardiotoxin (Latoxan, Rosans, France) in 0.9% sterile NaCl.
  • 0.0ImM cardiotoxin Latoxan, Rosans, France
  • each TA muscle was injected with 50 ⁇ l PBS containing either rNS3 or DNA.
  • mice were female and were used at 4- 8 weeks of age. For monitoring of humoral responses, all mice received a booster injection of 50 ⁇ l /TA of plasmid DNA every fourth week. In addition, some mice were given recombinant NS3 (rNS3) protein, which was purified, as described herein. The mice receiving rNS3 were immunized no more than twice. All mice were bled twice a month.
  • Enzyme immunosorbent assays were used to detect the presence of murine NS3-specific antibodies. These assays were performed essentially as described (Chen et al., Hepatology 28(1): 219 (1998)). Briefly, rNS3 was passively adsorbed overnight at 4°C to 96-well microtiter plates (Nunc, Copenhagen, Denmark) at 1 ⁇ g/ml in 50 mM sodium carbonate buffer (pH 9.6). The plates were then blocked by incubation with dilution buffer containing PBS, 2% goat serum, and 1% bovine serum albumin for one hour at 37°C. Serial dilutions of mouse sera starting at 1:60 were then incubated on the plates for one hour.
  • Bound murine serum antibodies were detected by an alkaline phosphatase conjugated goat anti-mouse IgG (Sigma Cell Products, Saint Louis, MO) followed by addition of the substrate pNPP (1 tablet/5ml of IM Diethanol amine buffer with 0.5 mM MgC12). The reaction was stopped by addition of IM NaOH and absorbency was read at 405 nm.
  • mice immunized with NS3/4A- pVAX had developed NS3 antibodies, whereas one out of five immunized with NS3- p VAX had developed antibodies ( Figure 1).
  • mice immunized with NS3/4A-pVAX had developed high levels (>104) of NS3 antibodies (mean levels 10800 ⁇ 4830) and one had a titer of 2160.
  • NS3-pVAX developed NS3 antibodies, none of them developed levels as high as that produced by the NS3/4A- ⁇ VAX construct (mean levels 1800 ⁇ 805).
  • the antibody levels elicited by the NS3/4A fusion construct were significantly higher than those induced by NS3-pVAX at six weeks (mean ranks 7.6 v.s 3.4, p ⁇ 0.05, Mann- Whitney rank sum test, and p ⁇ 0.01, Students t-test).
  • immunization with either NS3-pVAX or NS3/4A-pVAX resulted in the production of NS3-specific antibodies, but the NS3/4A containing construct was a more potent immunogen.
  • MSLFl gene was found to be more immunogenic than the native NS3/4A gene since NS3-speciflc antibodies were significantly higher in mice immunized with the MSLFl-pVAX construct at two weeks after the second and third immunization (Table 30). These results confirmed that the codon-optimized MSLFl-pVAX was a more potent B cell immunogen than NS3/4A-pVAX.
  • mice were immunized with the following vectors: wtNS3/4A (wild type NS3/4a), coNS3/4A (codon-optimized NS3/4a or MSLF-I), or wtNS3/4A-SFV (wild-type NS3/4A obtained from SFV expression).
  • Doses of 4 ⁇ g DNA was administered using the gene gun and doses of 107 SFV particles were injected subcutaneously (s.c). The mice were boosted after four weeks.
  • the mice immunized with the wtNS3/4A-SFV developed antibodies already after the first injection suggesting a potent immunogenicity (Figure 10).
  • mice immunized with the coNS3/4A or wtNS3/4A-SFV vectors had developed mean antibody levels over 103 ( Figure 10).
  • none of the mice immunized with the wtNS3/4A plasmid had developed detectable NS3-specific antibodies at six weeks ( Figure 10).
  • both codon optimization and mRNA amplification by SFV results in an increased B cell immunogenicity of the NS3/4A gene.
  • the mutant constructs for example, the NS3/4A-TGT-pVAX vector was comparable to the NS3- pVAX vector (4/10 vs. 0/10; NS, Fisher's exact test).
  • the NS3/4A- ⁇ VAX vector was more potent than the mutant constructs.
  • HCV hepatocellular carcinoma
  • EXAMPLE 1OD To study whether the constructs described above were capable of eliciting a cell-mediated response against NS3, an in vivo tumor growth assay was performed. To this end, an SP2/0 tumor cell line (SP2/0-Agl4 myeloma cell line (H-2d)) stably transfected with the NS 3 /4 A gene was made. The SP2/0 cells were maintained in DMEM medium supplemented with 10% fetal calf serum (FCS; Sigma Chemicals, St.
  • FCS fetal calf serum
  • the pcDNA3.1 plasmid containing the NS3/4A gene was linearized by BgIII digestion. A total of 5 ⁇ g linearized plasmid DNA was mixed with 60 ⁇ g transfection reagent (Superfect, Qiagen, Germany) and the mixture was added to a 50% confluent layer of SP2/0 cells in a 35 mm dish. The transfection procedure was performed according to manufacturer's protocol.
  • Transfected cells were cloned by limiting dilution and selected by addition of 800 ⁇ g geneticin (G418) /ml complete DMEM medium after 14 days.
  • a stable NS3/4A-ex ⁇ ressing SP2/0 clone was identified using PCR and RTPCR and/or a capture EIA using a monoclonal antibody to NS3. All EIAs for the detection of murine NS3 antibodies were essentially performed as follows. In brief, rNS3 (recombinant NS3 protein produced in E.
  • CoIi dialyzed overnight against PBS, and sterile filtered
  • the plates were then blocked by incubation with dilution buffer containing PBS, 2% goat serum, and 1% bovine serum albumin for one hour at +37°C.
  • Serial dilutions of mouse sera starting at 1 :60 were then incubated on the plates for one hour.
  • Bound murine serum antibodies were detected by an alkaline phosphatase conjugated goat anti-mouse IgG (Sigma cellproducts, Saint Louis, Missouri USA) followed by addition of the substrate pNPP (1 tablet/5ml of IM Diethanolamine buffer with 0.5 niM MgCl2). The reaction was stopped by addition of IM NaOH. Absorbance was then read at 405 ran.
  • mice Inhibition of tumor growth in this model is dependent on the priming of cytotoxic T lymphocytes (CTLs).
  • CTLs cytotoxic T lymphocytes
  • groups often mice were immunized i.rn. five times with one month intervals with either lOO ⁇ g NS3- ⁇ VAX or 100 ⁇ g NS3/4A-pVAX.
  • Two weeks after the last immunization 2 x 10 6 SP2/0 or NS3/4A-SP2/0 cells were injected into the right flank of each mouse. Two weeks later the mice were sacrificed and the maximum tumor sizes were measured. There was no difference between the mean SP2/0 and NS3/4A-SP2/0 tumor sizes in the NS3- ⁇ VAX immunized mice. (See TABLE 33).
  • NS3-s ⁇ 2 4 9.750 24.917 4.992 2.496 NS3-spNS3 3 8.000 1.000 1.000 0.57
  • mice were immunized with PBS, rNS3, a control DNA, or the NS3/4A construct, and tumor sizes were determined, as described above.
  • the NS3/4A construct was able to elicit a T-cell response sufficient to cause a statistically significant reduction in tumor size ⁇ See TABLE 34).
  • mice immunized with the NS3/4A-pVAX plasmid the growth of NS3/4A-SP2/0 tumor cells was significantly inhibited as compared to growth of the non-transfected SP2/0 cells. ⁇ See TABLE 35).
  • NS3/4A- ⁇ VAX immunization elicits CTLs that inhibit growth of cells expressing NS3/4A in vivo.
  • mice [0318] In another set of experiments, the inhibition of NS3/4A-expressing SP2/0 tumor growth was evaluated in MSLFl-pVAX immunized Balb/c mice.
  • groups of mice were immunized with different immunogens (4 ⁇ g of plasmid) using a gene gun at weeks zero, four, eight, twelve, and sixteen. Two weeks after the last immunization approximately 2 x 106 NS3/4A-expressing' SP2/0 cells were injected s.c into the right flank of the mouse. The kinetics of the tumor growth was then monitored by measuring the tumor size through the skin at days seven, 11, and 13. The mean tumor sizes were calculated and groups were compared using the Mann- Whitney non-parametric test. At day 14 all mice were sacrificed.
  • NS3/4A-SP2/0 or SP2/0 cells were used as targets. Percent specific lysis was calculated as the ratio between lysis of NS3/4A-SP2/0 cells and lysis of SP2/0 cells. Mice immunized with NS3/4A-pVAX displayed specific lysis over 10% in four out of five tested mice, using an effector to target ratio of 20:1 (See Figures 5A and 5B).
  • in vitro stimulation was carried out for five days in 25-ml flasks at a final volume of 12 ml, containing 5U/ml recombinant murine IL-2 (mIL-2; R&D Systems, Minneapolis, MN).
  • the restimulation culture contained a total of 4O x 10 6 immune spleen cells and 2 x 10 6 irradiated (10,000 rad) syngenic SP2/0 cells expressing the NS3/4A protein.
  • a standard 51 Cr-release assay was performed. Effector cells were harvested and a four- hour 51Cr assay was performed in 96-well U-bottom plates in a total volume of 200 ⁇ l.
  • a total of 1 x 10 6 target cells was labeled for one hour with 20 ⁇ l of 51 Cr (5 mCi/ml) and then washed three times in PBS. Cytotoxic activity was determined at effecto ⁇ target (E:T) ratios of 40:1, 20:1, and 10:1, using 5 x 103 51Cr-labeled target cells/well.
  • spleenocytes were harvested from C57BL/6 mice 12 days after peptide immunization and were resuspended in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-Glutamine, 1OmM HEPES, 100 U/ml Penicillin and 100 ⁇ g/ml Streptomycin, ImM non-essential amino acids, 50 ⁇ M ⁇ -mercaptoethanol, ImM sodium pyruvate.
  • In vitro stimulation was carried out for five days in 25ml flasks in a total volume of 12ml, containing 25 x 10 6 spleen cells and 25 x 10 6 irradiated (2,000 rad) syngeneic splenocytes.
  • the restimulation was performed in the presence of 0.05 ⁇ M NS3/4A H-2Db binding peptide (sequence GAVQNEVTL SEQ. ID. NO.: 199) or a control peptide H-2Db peptide (sequence KAVYNFATM SEQ. ID. NO.: 200).
  • a 51 Cr-release assay was performed.
  • RMA-S target cells were pulsed with 50 ⁇ M peptide for 1.5 hrs at +37 0 C prior to 51 Cr-labelling, and then washed three times in PBS. Effector cells were harvested and the four hour 51 Cr assay was performed as described.
  • Cytotoxic activity was determined at the E:T ratios 60:1, 20:1, and 7:1 with 5 x 10 3 51 Cr- labeled target cells/well. By these assays, it was determined that the MSLFl gene primed higher levels of in vitro lytic activity compared to the NS3/4A-pVAX vector. (See Figure 6A-6L). Similar results were obtained with both the peptide coated H-2b expressing RMA-S cells and NS3/4A-expressing EL-4 cells.
  • the cells were then washed twice in FACS buffer and resuspended in 100 ⁇ l FACS buffer containing 10 ⁇ l/lOO ⁇ l PE conjugated Rat- ⁇ Mouse IgGl secondary antibody and incubated on ice for 30 minutes. The cells were then washed twice in FACS buffer and incubated with 1 ⁇ g/10 6 cells of FITC conjugated ⁇ -mouse CD8 antibody for 30 minutes. The cells were then washed twice in FACS buffer and resuspended in 0.5 ml FACS buffer containing 0.5 ⁇ g/ml of PI. Approximately 200,000 events from each sample were acquired on a FACS Calibur (BDB) and dead cells (PI positive cells) were excluded from the analysis.
  • BDB FACS Calibur
  • the lytic activity of the in vivo primed CTLs were assayed on both NS3- peptide loaded H-2D b expressing RMA-S cells and EL-4 cells stably expressing NS3/4A.
  • the coNS3/4A plasmid and the wtNS3/4A-SFV vector was clearly more efficient than the wtNS3/4A plasmid in priming CTLs that lysed NS3-peptide coated target cells (FIGURE 12).
  • the CTL priming event was enhanced by codon optimization or mRNA amplification of the NS3/4A gene. The difference was less clear when using the NS3/4A-expressing EL-4 cells presumably since this assay is less sensitive (FIGURE 12).
  • the tumor challenge model was then used to evaluate how effective the different immunogens were in priming a protective immunity against growth of NS3/4A-EL-4 tumor cells in vivo. To ensure that the effectiveness of the priming event was studied, all mice were immunized only once. Fully consistent with the in vitro CTL data did we find that only vectors containing NS3/4A were able to rapidly prime protective immune responses as compared to the immunized with the empty pVAX plasmid ( ⁇ 0.05, ANOVA; FIGURE 14). However, this was dependent on NS4A but independent of either codon optimization or mRNA amplification, suggesting that C57BL/6 mice are quite easily protected against tumor growth using genetic immunization.
  • the NS3/4A gene can be used as a vaccine. Although it had been determined that NS3/4A quickly primed in vivo functional CTLs, the effect of therapeutic immunization after the injection of tumor cells was analyzed next. Groups of ten C57BL/6 mice were challenged with 10 6 NS3/4A-EL-4 tumor cells. One group was immunized transdermally with of 4 ⁇ g coNS3/4A at six days, and another group at 12 days, after tumor challenge. After the therapeutic vaccination both groups had significantly smaller tumors as compared to the nonimmunized control group (p ⁇ 0.01, respectively, ANOVA; FIGURE 16).
  • gene gun immunization with the coNS3/4A plasmid also works as a therapeutic vaccine. That is, gene gun immunization using the coNS3/4A gene six to 12 days after inoculation of NS3/4A-expressing tumor cells significantly inhibited tumor growth.
  • a rapid priming of HCV NS3-specific immune responses that are functional in vivo are generated by either DNA based immunization with a codon optimized gene or by mRNA amplification by the SFV replicon.
  • Inbred BALB/c (H-2 d ) and C57BL/6 (H-2 b ) mice were obtained from commercial vendors (M ⁇ llegard, Denmark).
  • B cell ( ⁇ MT) deficient mice were kindly provided by Dr Karin Sandstedt, Karolinska Institutet, Sweden.
  • CD4 deficient C57BL/6 mice were obtained from the breeding facility at the Microbiology and Tumorbiology Centre, Karolinska Institutet. All mice were female and were used at 4-8 weeks of age at the start of the experiments. //.
  • rNS3 The recombinant NS3 (rNS3) protein was kindly provided by Darrell L. Peterson, Department of Biochemistry, Commonwealth University, VA. The production of recombinant NS3 protein (not including NS4A) in E. CoIi has been described in the field. Prior to use the rNS3 protein was dialyzed over night against PBS and sterile filtered.
  • Plasmid DNA used for in vivo injection was purified by using Qiagen DNA purification columns according to the manufacturers instructions (Qiagen GmbH, Hilden, FRG). The concentration of the resulting plasmid DNA was determined spectrophotometrically (Dynaquant, Pharmacia Biotech, Uppsala, Sweden). Purified DNA was dissolved in sterile phosphate buffer saline (PBS) at concentrations of 1 mg/ml. ///. In vitro translation assay
  • HepG2 cells were transiently transfected by standard protocols. In brief, HepG2 cells were plated into 2.5cm 2 wells (0,5 x 10 6 ) in DMEM medium the day before transfection. Two ⁇ g of each plasmid DNA construct (wtNS3/4A and coNS3/4A) was transfected into HepG2 cells using Fugene 6 Transfection Reagent (Roche). After transfection, the HepG2 cells were incubated for 24-48hrs.
  • Cell lysates were analysed by immunoprecipitation followed by SDS- PAGE.
  • transient transfected HepG2 cells were lysed in RIPA buffer (0,15M NaCl, 5OmM Tris, 1% Triton-X 100, 1% Na-deoxycholate and 1% SDS).
  • the cell lysates were immunoprecipitated with protein A sepharose and anti-NS3 polyclonal antibody overnight at 4°C.
  • the washed pellets were re-suspended in SDS sample buffer, heated at 100°C for 5 minutes prior to SDS-PAGE analysis on 4-12% Bis-Tris gel (Invitrogen) and electrotransferred onto Nitrocellulose membranes.
  • Detection of NS3 protein was done according to manufacturer's protocol by using a chemiluminiscence-linked Western blot kit (WesternBreeze; Invitrogen). NS3 protein expression was detected and quantified as a chemiluminiscent signal by using an NS3 -specific polyclonal antibody. Chemiluminiscent signals were detected by using the GeneGnome (Syngene, Cambridge, UK). Quantification of chemiluminiscence Western blots was performed on GeneGnome and units of intensity from each protein band was calculated and compared to a standard curve of rNS3.
  • Baby Hamster Kidney (BHK)-21 cells were maintained in complete BHK medium supplemented with 5% FCS, 10% tryptose phosphate broth, 2mM glutamine, 2OmM Hepes and antibiotics (streptomycin lO ⁇ g/ml and penicillin 100 IU/ml).
  • the wtNS3/4A gene was isolated by PCR as Spel -BStBl fragment and inserted into the Spel-BstBl site of pSFVlOEnh containing a 34 amino acid long translational enhancer sequence of capsid followed by the FMDV 2a cleavage peptide.
  • Packaging of recombinant RNA into rSFV particles was done using a two-helper RNA system. Indirect immunofluorescence of infected BHK cells was performed to determine the titre of the recombinant virus stocks.
  • BHK cells were transient transfected with coNS3/4A- ⁇ VAXl according to standard techniques using Lipofectamine plus reagent (Invitrogen) or infected by rSFV.
  • NS3 protein was detected by indirect immunofluorescence .
  • mice/group mice 5-10 mice/group of female BALB/c (H-2 d ) or C57BL/6 (H- 2 b ) mice, 4-8 weeks old, were immunized by needle injections of lOO ⁇ g of plasmid DNA encoding individual or multiple HCV proteins. Plasmid DNA in PBS was given intramuscularly (Lm.) in the tibialis anterior (TA) muscle. Where indicated in the text, the mice were injected i.m. with 50 ⁇ L/TA of 0,0ImM cardiotoxin (Latoxan, Rosans, France) in 0,9% sterile saline NaCl, five days prior to DNA immunization. The mice were boosted at four-week intervals.
  • plasmid DNA was linked to gold particles (l ⁇ m) according to protocols supplied by the manufacturer (Bio-Rad Laboratories, Hercules, CA). Prior to immunization the abdominal injection area was shaved and the immunization was performed according to the manufacturer's protocol at a helium discharge pressure of 500 psi. Each injection dose contained 4 ⁇ g of plasmid DNA. The mice were boosted with the same dose at monthly intervals.
  • mice were immunized subcutaneously, in the base of the tail, with 1 x 10 7 virus particles diluted in PBS (wtNS3/4A-SFV), in a final volume of 100 ⁇ l.
  • PBS wtNS3/4A-SFV
  • Peptide immunization was performed by subcutaneous immunization in the base of the tail with 100 ⁇ g peptide mixed 1:1 in complete Freunds adjuvant.
  • Serum for antibody detection and isotyping was collected every second or fourth week after the first immunization by retroorbital bleeding of isofluorane- anesthetized mice.
  • the enzyme immuno assays were performed as previously described.
  • the SP2/0-Agl4 myeloma cell line (H-2 d ) was maintained in DMEM medium supplemented with 10% fetal calf serum (FCS; Sigma Chemicals, St. Louis, MO), 2 mM L-Glutamin, 1OmM HEPES, 100 U/ml Penicillin and 100 ⁇ g/ml Streptomycin, ImM non-essential amino acids, 50 ⁇ M ⁇ -mercaptoethanol, ImM sodium pyruvate (GIBCO-BRL, Gaithesburgh, MD).
  • SP2/0-Agl4 cells with stable expression of NS3/4A were maintained in 800 ⁇ g geneticin (G418) /ml complete DMEM medium.
  • the EL-4 lymphoma (H-2 b ) cells were maintained in RPMI 1640 medium supplemented with 10% FCS, 1OmM HEPES, ImM sodium pyruvate, ImM nonessential amino acids, 50 ⁇ M ⁇ -mercaptoethanol, 100U/ml Penicillin and lOO ⁇ g/ml Streptomycin (GIBCO-BRL).
  • EL-4 cells with stable expression of NS3/4A were generated by transfection of EL-4 cells with the linearized NS3/4A-pcDNA3.1 plasmid using the SuperFect (Qiagen GmbH, Hilden, FRG) transfection reagent. The transfection procedure was performed according to manufacturer's protocol. Transfected cells were cloned by limiting dilution and selected by addition of 800 ⁇ g geneticin (G418) /ml complete RPMI 1640 medium.
  • RMA-S cells (a kind gift from Professor Klas Karre, Karolinska Institutet, Sweden) were maintained in RPMI 1640 medium supplemented with 5% FCS, 2 mM L-Glutamin, 100 U/ml Penicillin and 100 ⁇ g/ml Streptomycin. All cells were grown in a humidified 37 0 C, 5% CO 2 incubator.
  • CD4 and CD8 T cell subpopulations were depleted in vivo by intraperitoneal injection of purified hybridoma supernatant.
  • a total of 0.4 mg per mouse per injection of anti-CD4 (clone GKl .5) or anti-CD8 (clone 53-6.7) was injected on days -3, -2, and -1 before tumor challenge, and on days 3, 6, 10, and 13 after challenge.
  • Flow cytometric analysis of peripheral blood mononuclear cell populations at days 0, 3, 6, 10, and 13 demonstrated that more than 85% of the CD4 and CD8 T cells were depleted.
  • mice were inoculated with the tumor cells as described above. After six or 12 days the mice were immunized once. The tumor growth was monitored from day 6 to day 20.
  • Antibodies and MHC Ig fusion protein
  • the restimulation culture contained a total of 25 x 10 6 immune spleen cells and 2,5 x 10 6 irradiated (10,000 rad) syngenic EL-4 cells expressing the NS3/4A protein.
  • a standard 51 Cr-release assay was performed. Effector cells were harvested and a four-hour 51 Cr assay was performed in 96-well U-bottom plates in a total volume of 200 ⁇ l.
  • a total of 1 x 10 6 target cells (NS3/4A expressing EL-4 cells) was labelled for one hour at +37 0 C with 20 ⁇ l Of 51 Cr (5 mCi/ml) and then washed three times in PBS.
  • effectors and 51 Cr-labeled target cells were added to wells at effector:target (E:T) ratios of 60:1, 20:1, and 7:1.
  • E:T effector:target
  • the level of cytolytic activity was determined after incubation of effectors and targets for 4 hour at +37 °C. 100 ⁇ l supernatant was harvested and the radioactivity was measured with a ⁇ -counter.
  • Splenocytes from DNA or rSFV immunised mice were harvested from C57BL/6 mice and were resuspended in complete RPMI 1640 medium as previously described.
  • in vitro stimulation was carried out for five days by mixing 25 x 10 6 spleen cells and 25 x 10 6 irradiated (2,000 rad) syngeneic splenocytes.
  • the restimulation was performed in the presence of 0,05 ⁇ M NS3/4A H-2D b binding peptide (sequence GAVQNEVTL (SEQ ID NO.: 199)).
  • a four hour 51 Cr-release assay was performed using 51 Cr-labelled peptide pulsed RMA-S cells as targets. Cytotoxic activity was determined at the E:T ratios 60:1, 20:1, and 7:1.
  • NS3 -peptide specific CD8+ T cells were analysed by ex-vivo staining of spleen cells from DNA or rSFV immunized mice with recombinant soluble dimeric mouse H-2D b :Ig fusion protein as previously described, hi brief, spleen cells were resuspended in PBS/1% FCS (FACS buffer) and incubated with Fc-blocking antibodies. Cells were then washed and incubated with H-2D b :Ig preloaded with NS3/4A derived peptide.
  • HCV peptides or derivatives thereof which can be used in the transdermal delivery compositions described herein include but are not limited to, those containing as a primary amino acid sequence all of the amino acid sequence substantially as depicted in the Sequence Listing (SEQ. ID. NOs.: 164-173) and SEQ. ID. NO.: 198) and fragments of SEQ. ID. NOs.: 164-173 and SEQ. ID. NO.: 198 that are at least four amino acids in length (e.g., SEQ. ID. NOs.: 176-178) including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change. Preferred fragments of a sequence of SEQ.
  • HCV peptides can be, for example, at least 12-704 amino acids in length (e.g., any number between 12-15, 15-20, 20-25, 25-50, 50-100, 100-150, 150-250, 250-500 or 500-704 amino acids in length).
  • Embodiments also include HCV peptides that are substantially identical to those described above. That is, HCV peptides that have one or more amino acid residues within SEQ. ID. NOs.: 164-173 and SEQ. ID. NO.: 198 and fragments thereof that are substituted by another amino acid of a similar polarity that acts as a functional equivalent, resulting in a silent alteration. Further, the HCV peptides can have one or more amino acid residues fused to SEQ. ID. NOs.: 164-173 and SEQ. ID. NO.: 198 or a fragment thereof so long as the fusion does not significantly alter the structure or function (e.g., immunogenic properties) of the HCV peptide.
  • HCV peptides that are substantially identical to those described above. That is, HCV peptides that have one or more amino acid residues within SEQ. ID. NOs.: 164-173 and SEQ. ID. NO.: 198 and fragments thereof that are substituted by another amino acid
  • Substitutes for an amino acid within the sequence can be selected from other members of the class to which the amino acid belongs.
  • the non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine, and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • the aromatic amino acids include phenylalanine, tryptophan, and tyrosine. Accordingly, the peptide embodiments of the invention are said to be consisting essentially of SEQ. ID. NOs.: 164-189 and SEQ. ID. NO.: 198 in light of the modifications described above.
  • HCV peptides described herein can be prepared by chemical synthesis methods (such as solid phase peptide synthesis) using techniques known in the art such as those set forth by Merrifield et al., J. Am. Chem. Soc. 85:2149 (1964), Houghten et al., Proc. Natl. Acad. Sci. USA, 82:51 :32 (1985), Stewart and Young fSolid phase peptide synthesis. Pierce Chem Co., Rockford, IL (1984), and Creighton, 1983, Proteins: Structures and Molecular Principles, W. H. Freeman & Co., N.Y. Such polypeptides can be synthesized with or without a methionine on the amino terminus. Chemically synthesized HCV peptides can be oxidized using methods set forth in these references to form disulfide bridges.
  • HCV peptides described herein can be chemically synthesized, it can be more effective to produce these polypeptides by recombinant DNA technology.
  • Such methods can be used to construct expression vectors containing the HCV nucleotide sequences described above, for example, and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.
  • RNA capable of encoding an HCV nucleotide sequence can be chemically synthesized using, for example, synthesizers. See, for example, the techniques described in Oligonucleotide Synthesis, 1984, Gait, M. J. ed., IRL Press, Oxford.
  • HCV peptides concern cell lines that have been engineered to express the embodied HCV peptides.
  • some cells are made to express the HCV peptides of SEQ. ID. NOs.: 164-173 and SEQ. ID. NO.: 198 or fragments of these molecules (e.g., SEQ. ID. NOs.: 176-188).
  • a variety of host-expression vector systems can be utilized to express the embodied HCV peptides.
  • Suitable expression systems include, but are not limited to, microorganisms such as bacteria (e.g., E. coli or B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing HCV nucleotide sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing the HCV nucleotide sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the HCV sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing HCV sequences; or mammalian
  • a number of expression vectors can be advantageously selected depending upon the use intended for the HCV gene product being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of HCV peptide or for raising antibodies to the HCV peptide, for example, vectors which direct the expression of high levels of fusion protein products that are readily purified can be desirable.
  • vectors include, but are not limited, to the E.
  • coli expression vector pUR278 (Ruther et al, EMBO J., 2:1791 (1983), in which the HCV coding sequence can be ligated individually into the vector in frame with the lacZ coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res., 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem., 264:5503-5509 (1989)); and the like.
  • the pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione S- transferase (GST).
  • fusion proteins are soluble and can be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • the PGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • AcNPV Autographa californica nuclear polyhedrosis virus
  • the virus grows in Spodoptera frugiperda cells.
  • the HCV coding sequence can be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
  • Successful insertion of an HCV gene coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus, (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene).
  • HCV nucleotide sequence of interest can be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene can then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the HCV gene product in infected hosts.
  • HCV initiation signals can also be required for efficient translation of inserted HCV nucleotide sequences. These signals include the ATG initiation codon and adjacent sequences.
  • exogenous translational control signals including, perhaps, the ATG initiation codon
  • the initiation codon can be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert.
  • exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (See Bittner et al., Methods in Enzymol, 153:516-544 (1987)).
  • a host cell strain can be chosen, which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products are important for the function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used.
  • mammalian host cells include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, and WI38.
  • stable expression For long-term, high-yield production of recombinant proteins, stable expression is preferred.
  • cell lines that stably express the HCV peptides described above can be engineered.
  • host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g., promoter, enhancer sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells are allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn are cloned and expanded into cell lines. This method is advantageously used to engineer cell lines which express the HCV gene product.
  • a number of selection systems can be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et al., Cell 11 :223 (1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sd. USA 48:2026 (1962)), and adenine phosphoribosyltransferase (Lowy, et al., Cell 22:817 (1980)) genes can be employed in tk ⁇ , hgprf or aprf cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler, et al., Proc. Natl. Acad. Sd. USA 77:3567 (1980); O ⁇ are, et al., Proc. Natl. Acad. Sd. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. ScL USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al., J. MoI. Biol. 150:1 (1981)); and hygro, which confers resistance to hygromycin (Santerre, et al., Gene 30:147 (1984)).
  • any fusion protein can be readily purified by utilizing an antibody specific for the fusion protein being expressed.
  • a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines. (Janknecht, et al., Proc. Natl. Acad. Sd. USA 88: 8972- 8976 (1991)).
  • the gene of interest is subcloned into a vaccinia recombination plasmid such that the gene's open reading frame is translationally fused to an amino-terminal tag consisting of six histidine residues.
  • Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni 2+ nitriloacetic acid-agarose columns and histidine-tagged proteins are selectively eluted with imidazole-containing buffers.
  • the example below describes a method that was used to express the HCV peptides encoded by the embodied nucleic acids.
  • the in vitro analysis revealed that all proteins were expressed to high amounts from their respective expression constructs.
  • the rNS3 construct (NS3-pVAX vector) produced a single peptide of approximately 6IkDa
  • the mutant constructs e.g., the TGT construct (NS3/4A-TGT- ⁇ VAX) and the RGT construct (NS3/4A-RGT-pVAX)
  • the mutant constructs e.g., the TGT construct (NS3/4A-TGT- ⁇ VAX) and the RGT construct (NS3/4A-RGT-pVAX)
  • the cleaved product produced from the expressed NS3/4A peptide was approximately 61 kDa, which was identical in size to the rNS3 produced from the NS3- ⁇ VAX vector.
  • sequences, constructs, vectors, clones, and other materials comprising the embodied HCV nucleic acids and peptides can be in enriched or isolated form.
  • enriched means that the concentration of the material is many times its natural concentration, for example, at least about 2, 5, 10, 100, or 1000 times its natural concentration, advantageously 0.01%, by weight, preferably at least about 0.1% by weight.
  • Enriched preparations from about 0.5% or more, for example, 1%, 5%, 10%, and 20% by weight are also contemplated.
  • isolated requires that the material be removed from its original environment (e.g., the natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide present in a living animal is not isolated, but the same polynucleotide, separated from some or all of the coexisting materials in the natural system, is isolated. It is also advantageous that the sequences be in purified form.
  • the term "purified” does not require absolute purity; rather, it is intended as a relative definition. Isolated proteins have been conventionally purified to electrophoretic homogeneity by Coomassie staining, for example. Purification of starting material or natural material to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.
  • HCV gene products described herein can also be expressed in plants, insects, and animals so as to create a transgenic organism.
  • Desirable transgenic plant systems having an HCV peptide include Arahadopsis, maize, and Chlamydomonas.
  • Desirable insect systems having an HCV peptide include, but are not limited to, D. melanogaster and C. elegans.
  • Animals of any species including, but not limited to, amphibians, reptiles, birds, mice, hamsters, rats, rabbits, guinea pigs, pigs, micro-pigs, goats, dogs, cats, and non-human primates, e.g., baboons, monkeys, and chimpanzees can be used to generate transgenic animals having an embodied HCV molecule.
  • These transgenic organisms desirably exhibit germline transfer of HCV peptides described herein.
  • Any technique known in the art is preferably used to introduce the HCV transgene into animals to produce the founder lines of transgenic animals or to knock out or replace existing HCV genes.
  • Such techniques include, but are not limited to pronuclear microinjection (Hoppe, P. C. and Wagner, T. E., 1989, U.S. Pat. No. 4,873,191); retrovirus mediated gene transfer into germ lines (Van der Putten et al., Proc. Natl. Acad. ScL, USA 82:6148-6152 (1985)); gene targeting in embryonic stem cells (Thompson et al., Cell 56:313-321 (1989)); electroporation of embryos (Lo, MoI Cell. Biol.
  • the isolated or purified peptide can be used to generate antibodies.
  • antibodies can encompass polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library.
  • Antibodies that recognize the HCV peptides have many uses including, but not limited to, biotechnological applications, therapeutic/prophylactic applications, and diagnostic applications.
  • various hosts including goats, rabbits, rats, mice, and humans etc. can be immunized by injection with an HCV peptide.
  • various adjuvants can be used to increase immunological response.
  • adjuvants include, but are not limited to, ribavirin, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
  • BCG ⁇ Bacillus Calmette-Guerin) and Corynebacterium parvum are also potentially useful adjuvants.
  • Peptides used to induce specific antibodies can have an amino acid sequence consisting of at least four amino acids, and preferably at least 10 to 15 amino acids.
  • short stretches of amino acids encoding fragments of NS3/4A are fused with those of another protein such as keyhole limpet hemocyanin such that an antibody is produced against the chimeric molecule.
  • a composition comprising ribavirin and an HCV peptide (SEQ. ID. NOs.: 164-173 and SEQ. ID. NO.: 198), a fragment thereof containing any number of consecutive amino acids between at least 3-50 (e.g., 3, 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids ) (e.g., SEQ. ID.
  • nucleic acid encoding one or more of these molecules is administered to an animal, preferably a mammal including a human.
  • antibodies capable of specifically recognizing HCV can be generated by injecting synthetic 3-mer, 10- mer, and 15-mer peptides that correspond to an HCV peptide into mice, a more diverse set of antibodies can be generated by using recombinant HCV peptides, prepared as described above.
  • substantially pure peptide is isolated from a transfected or transformed cell.
  • concentration of the peptide in the final preparation is adjusted, for example, by concentration on an Amicon filter device, to the level of a few micrograms/ml.
  • Monoclonal or polyclonal antibody to the peptide of interest can then be prepared as follows:
  • Monoclonal antibodies to an HCV peptide can be prepared using any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique originally described by Koehler and Milstein (Nature 256:495-497 (1975)), the human B-cell hybridoma technique (Kosbor et al. Immunol Today 4:72 (1983)); Cote et al Proc Natl Acad Sd 80:2026-2030 (1983), and the EBV-hybridoma technique Cole et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss Inc, New York N.Y., pp 77-96 (1985).
  • Antibodies can also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al., Proc Natl Acad Sd 86: 3833-3837 (1989), and Winter G. and Milstein C; Nature 349:293-299 (1991).
  • Antibody fragments that contain specific binding sites for an HCV peptide can also be generated.
  • fragments include, but are not limited to, the F(ab') 2 fragments that can be produced by pepsin digestion of the antibody molecule and the Fab fragments that can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
  • Fab expression libraries can be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (Huse W. D. et al. Science 256:1275-1281 (1989)).
  • monoclonal antibodies to an HCV peptide are made as follows. Briefly, a mouse is repetitively inoculated with a few micrograms of the selected protein or peptides derived therefrom over a period of a few weeks. The mouse is then sacrificed, and the antibody producing cells of the spleen isolated. The spleen cells are fused in the presence of polyethylene glycol with mouse myeloma cells, and the excess unfused cells destroyed by growth of the system on selective media comprising aminopterin (HAT media). The successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture is continued.
  • HAT media aminopterin
  • Antibody-producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as ELISA, as originally described by Engvall, E., Meth. Enzymol. 70:419 (1980), and derivative methods thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Davis, L. et al. Basic Methods in Molecular Biology Elsevier, New York. Section 21-2.
  • Polyclonal antiserum containing antibodies to heterogeneous epitopes of a single protein can be prepared by immunizing suitable animals with the expressed protein or peptides derived therefrom described above, which can be unmodified or modified to enhance immunogenicity.
  • Effective polyclonal antibody production is affected by many factors related both to the antigen and the host species. For example, small molecules tend to be less immunogenic than others and can require the use of carriers and adjuvant.
  • host animals vary in response to site of inoculations and dose, with both inadequate or excessive doses of antigen resulting in low titer antisera. Small doses (ng level) of antigen administered at multiple intradermal sites appears to be most reliable.
  • An effective immunization protocol for rabbits can be found in Vaitukaitis, J. et al. J. Clin. Endocrinol. Metab. 33:988-991 (1971).
  • Booster injections are given at regular intervals, and antiserum harvested when antibody titer thereof, as determined semi-quantitatively, for example, by double immunodiffusion in agar against known concentrations of the antigen, begins to fall. See, for example, Ouchterlony, O. et al., Chap. 19 in: Handbook of Experimental Immunology D. Wier (ed) Blackwell (1973). Plateau concentration of antibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12 ⁇ M). Affinity of the antisera for the antigen is determined by preparing competitive binding curves, as described, for example, by Fisher, D., Chap. 42 in: Manual of Clinical Immunology. 2d Ed.
  • Antibody preparations prepared according to either protocol are useful in quantitative immunoassays that determine concentrations of antigen- bearing substances in biological samples; they are also used semi-quantitatively or qualitatively (e.g., in diagnostic embodiments that identify the presence of HCV in biological samples).
  • the next section describes how some of the novel nucleic acids and peptides described above can be used in diagnostics.
  • the embodied diagnostics are classified according to whether a nucleic acid or protein-based assay is used. Some diagnostic assays detect the presence or absence of an embodied HCV nucleic acid sequence in a sample obtained from a patient, whereas, other assays seek to identify whether an embodied HCV peptide is present in a biological sample obtained from a patient. Additionally, the manufacture of kits that incorporate the reagents and methods described herein that allow for the rapid detection and identification of HCV are also embodied. These diagnostic kits can include, for example, an embodied nucleic acid probe or antibody, which specifically detects HCV. The detection component of these kits will typically be supplied in combination with one or more of the following reagents.
  • a support capable of absorbing or otherwise binding DNA, RNA, or protein will often be supplied.
  • Available supports include membranes of nitrocellulose, nylon or derivatized nylon that can be characterized by bearing an array of positively charged substituents.
  • One or more restriction enzymes, control reagents, buffers, amplification enzymes, and non-human polynucleotides like calf-thymus or salmon-sperm DNA can be supplied in these kits.
  • nucleic acid-based diagnostics include, but are not limited to, direct DNA sequencing, Southern Blot analysis, dot blot analysis, nucleic acid amplification, and combinations of these approaches.
  • the starting point for these analysis is isolated or purified nucleic acid from a biological sample obtained from a patient suspected of contracting HCV or a patient at risk of contracting HCV.
  • the nucleic acid is extracted from the sample and can be amplified by RT-PCR and/or DNA amplification using primers that correspond to regions flanking the embodied HCV nucleic acid sequences (e.g., NS3/4A (SEQ. ID. NO.: 163)).
  • nucleic acid probes that specifically hybridize with HCV sequences are attached to a support in an ordered array, wherein the nucleic acid probes are attached to distinct regions of the support that do not overlap with each other.
  • an ordered array is designed to be "addressable” where the distinct locations of the probe are recorded and can be accessed as part of an assay procedure. These probes are joined to a support in different known locations. The knowledge of the precise location of each nucleic acid probe makes these "addressable" arrays particularly useful in binding assays.
  • the nucleic acids from a preparation of several biological samples are then labeled by conventional approaches (e.g., radioactivity or fluorescence) and the labeled samples are applied to the array under conditions that permit hybridization.
  • Nucleic acids present in biological samples can be disposed on a support so as to create an addressable array.
  • the samples are disposed on the support at known positions that do not overlap.
  • the presence of HCV nucleic acids in each sample is determined by applying labeled nucleic acid probes that complement nucleic acids, which encode HCV peptides, at locations on the array that correspond to the positions at which the biological samples were disposed. Because the identity of the biological sample and its position on the array is known, the identification of a patient that has been infected with HCV can be rapidly determined.
  • Any addressable array technology known in the art can be employed.
  • One particular embodiment of polynucleotide arrays is known as GenechipsTM, and has been generally described in US Patent 5,143,854; PCT publications WO 90/15070 and 92/10092. These arrays are generally produced using mechanical synthesis methods or light directed synthesis methods, which incorporate a combination of photolithographic methods and solid phase oligonucleotide synthesis. (Fodor et al, Science, 251:767-777, (1991)).
  • VLSPISTM Very Large Scale Immobilized Polymer Synthesis
  • a wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid assays.
  • There are several ways to produce labeled nucleic acids for hybridization or PCR including, but not limited to, oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
  • a nucleic acid encoding an HCV peptide can be cloned into a vector for the production of an mRNA probe.
  • RNA polymerase such as T7, T3 or SP6 and labeled nucleotides.
  • RNA polymerase such as T7, T3 or SP6 and labeled nucleotides.
  • Suitable reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as, substrates, cofactors, inhibitors, magnetic particles and the like.
  • the presence of an HCV peptide in a protein sample obtained from a patient can also be detected by using conventional assays and the embodiments described herein.
  • antibodies that are immunoreactive with the disclosed HCV peptides can be used to screen biological samples for the presence of HCV infection.
  • antibodies that are reactive to the embodied HCV peptides are used to immunoprecipitate the disclosed HCV peptides from biological samples or are used to react with proteins obtained from a biological sample on Western or Immunoblots.
  • diagnostic embodiments also include enzyme-linked immunosorbant assays (ELISA), radioimmunoassays (RIA), immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA), including sandwich assays using monoclonal and/or polyclonal antibodies specific for the disclosed HCV peptides.
  • ELISA enzyme-linked immunosorbant assays
  • RIA radioimmunoassays
  • IRMA immunoradiometric assays
  • IEMA immunoenzymatic assays
  • sandwich assays are described by David et al., in U.S. Patent Nos. 4,376,110 and 4,486,530.
  • Other embodiments employ aspects of the immune-strip technology disclosed in U.S. Patent Nos. 5,290,678; 5,604,105; 5,710,008; 5,744,358; and 5,747,274.
  • the antibodies described herein are attached to a support in an ordered array, wherein a plurality of antibodies are attached to distinct regions of the support that do not overlap with each other.
  • the protein-based arrays are ordered arrays that are designed to be "addressable" such that the distinct locations are recorded and can be accessed as part of an assay procedure. These probes are joined to a support in different known locations. The knowledge of the precise location of each probe makes these "addressable" arrays particularly useful in binding assays.
  • an addressable array can comprise a support having several regions to which are joined a plurality of antibody probes that specifically recognize HCV peptides present in a biological sample and differentiate the isotype of HCV identified herein.
  • proteins are obtained from biological samples and are then labeled by conventional approaches (e.g., radioactivity, colorimetrically, or fluorescently).
  • the labeled samples are then applied to the array under conditions that permit binding. If a protein in the sample binds to an antibody probe on the array, then a signal will be detected at a position on the support that corresponds to the location of the antibody-protein complex. Since the identity of each labeled sample is known and the region of the support on which the labeled sample was applied is known, an identification of the presence, concentration, and/or expression level can be rapidly determined.
  • an investigator can accurately determine the protein concentration of the particular peptide in a tested sample and can also assess the expression level of the HCV peptide.
  • Conventional methods in densitometry can also be used to more accurately determine the concentration or expression level of the HCV peptide.
  • Proteins present in biological samples can be disposed on a support so as to create an addressable array.
  • the protein samples are disposed on the support at known positions that do not overlap.
  • the presence of an HCV peptide in each sample is then determined by applying labeled antibody probes that recognize epitopes specific for the HCV peptide. Because the identity of the biological sample and its position on the array is known, an identification of the presence, concentration, and/or expression level of an HCV peptide can be rapidly determined.
  • compositions comprising HCV nucleic acids or peptides
  • Embodiments of the invention also include NS3/4A fusion proteins or nucleic acids encoding these molecules. For instance, production and purification of recombinant protein may be facilitated by the addition of auxiliary amino acids to form a "tag".
  • tags include, but are not limited to, His-6, Flag, Myc and GST. The tags may be added to the C-terminus, N-terminus, or within the NS3/4A amino acid sequence.
  • Further embodiments include NS3/4A fusion proteins with amino or carboxy terminal truncations, or internal deletions, or with additional polypeptide sequences added to the amino or carboxy terminal ends, or added internally.
  • NS3/4A fusion proteins or truncated or mutated versions thereof, where the residues of the NS3/4A proteolytic cleavage site have been substituted.
  • substitutions include, but are not limited to, sequences where the Pl ' site is a Ser, GIy, or Pro, or the Pl position is an Arg, or where the P8 to P4' sequence is Ser-Ala-Asp-Leu-Glu-Val-Val- Thr-Ser-Thr-T ⁇ -Val (SEQ. ID. NO.: 177).
  • More embodiments concern an immunogen comprising the NS3/4A fusion protein, or a truncated, mutated, or modified version thereof, capable of eliciting an enhanced immune response against NS3.
  • the immunogen can be provided in a substantially purified form, which means that the immunogen has been rendered substantially free of other proteins, lipids, carbohydrates or other compounds with which it naturally associates.
  • Some embodiments contain at least one of the HCV nucleic acids or HCV peptides (e.g., SEQ. ID. NOs.: 163-189, 197, or 198) joined to a support.
  • these supports are manufactured so as to create a multimeric agent.
  • These multimeric agents provide the HCV peptide or nucleic acid in such a form or in such a way that a sufficient affinity to the molecule is achieved.
  • a multimeric agent having an HCV nucleic acid or peptide can be obtained by joining the desired molecule to a macromolecular support.
  • a “support” can be a termed a carrier, a protein, a resin, a cell membrane, a capsid or portion thereof, or any macromolecular structure used to join or immobilize such molecules.
  • Solid supports include, but are not limited to, the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, nitrocellulose strips, membranes, microparticles such as latex particles, animal cells, Duracyte®, artificial cells, and others.
  • An HCV nucleic acid or peptide can also be joined to inorganic carriers, such as silicon oxide material (e.g., silica gel, zeolite, diatomaceous earth or aminated glass) by, for example, a covalent linkage through a hydroxy, carboxy or amino group and a reactive group on the carrier.
  • silicon oxide material e.g., silica gel, zeolite, diatomaceous earth or aminated glass
  • the macromolecular support has a hydrophobic surface that interacts with a portion of the HCV nucleic acid or peptide by a hydrophobic non-covalent interaction.
  • the hydrophobic surface of the support is a polymer such as plastic or any other polymer in which hydrophobic groups have been linked such as polystyrene, polyethylene or polyvinyl.
  • HCV nucleic acid or peptide can be covalently bound to carriers including proteins and oligo/polysaccarides (e.g. cellulose, starch, glycogen, chitosane or aminated sepharose).
  • a reactive group on the molecule such as a hydroxy or an amino group, is used to join to a reactive group on the carrier so as to create the covalent bond.
  • Additional multimeric agents comprise a support that has other reactive groups that are chemically activated so as to attach the HCV nucleic acid or peptide.
  • cyanogen bromide activated matrices epoxy activated matrices, thio and thiopropyl gels, nitrophenyl chloroformate and N-hydroxy succinimide chlorformate linkages, or oxirane acrylic supports are used. (Sigma).
  • Carriers for use in the body are desirably physiological, non-toxic and preferably, non- immunoresponsive.
  • Suitable carriers for use in the body include poly-L-lysine, poly-D, L-alanine, liposomes, capsids that display the desired HCV peptide or nucleic acid, and Chromosorb (Johns-Manville Products, Denver Co.).
  • Ligand conjugated Chromosorb ® (Synsorb-Pk) has been tested in humans for the prevention of hemolytic-uremic syndrome and was reported as not presenting adverse reactions. (Armstrong et al. J. Infectious Diseases 171:1042-1045 (1995)).
  • a naked carrier i.e., lacking an attached HCV nucleic acid or peptide
  • a "prodrug- type” therapy is envisioned in which the naked carrier is administered separately from the HCV nucleic acid or peptide and, once both are in the body of the organism, the carrier and the HCV nucleic acid or peptide are assembled into a multimeric complex.
  • linkers e.g., " ⁇ linkers” engineered to resemble the flexible regions of ⁇ phage
  • linkers e.g., " ⁇ linkers” engineered to resemble the flexible regions of ⁇ phage
  • the determination of an appropriate length of linker that allows for an optimal cellular response or lack thereof, can be determined by screening the HCV nucleic acid or peptide with varying linkers in the assays detailed in the present disclosure.
  • a composite support comprising more than one type of HCV nucleic acid or peptide is also envisioned.
  • a "composite support” can be a carrier, a resin, or any macromolecular structure used to attach or immobilize two or more different HCV nucleic acids or peptides.
  • linkers such as ⁇ linkers
  • the determination of an appropriate length of linker that allows for an optimal cellular response or lack thereof, can be determined by screening the HCV nucleic acid or peptide with varying linkers in the assays detailed in the present disclosure.
  • the multimeric and composite supports discussed above can have attached multimerized HCV nucleic acids or peptides so as to create a "multimerized-multimeric support" and a “multimerized-composite support", respectively.
  • a multimerized ligand can, for example, be obtained by coupling two or more HCV nucleic acids or peptides in tandem using conventional techniques in molecular biology.
  • the multimerized form of the HCV nucleic acid or peptide can be advantageous for many applications because of the ability to obtain an agent with a higher affinity, for example.
  • linkers or spacers, such as flexible ⁇ linkers, between the individual domains that make-up the multimerized agent can also be advantageous for some embodiments.
  • ⁇ linkers of an appropriate length between protein binding domains can encourage greater flexibility in the molecule and can overcome steric hindrance.
  • the insertion of linkers between the multimerized HCV nucleic acid or peptide and the support can encourage greater flexibility and limit steric hindrance presented by the support.
  • the determination of an appropriate length of linker can be determined by screening the HCV nucleic acids or peptides in the assays detailed in this disclosure.
  • Embodiments also include vaccine compositions and immunogen preparations comprising the NS 3 /4 A fusion protein, or a truncated or mutated version thereof, and, optionally, an adjuvant.
  • vaccine compositions and immunogen preparations comprising the NS 3 /4 A fusion protein, or a truncated or mutated version thereof, and, optionally, an adjuvant.
  • an adjuvant an adjuvant
  • Vaccine compositions and immunogen preparations comprising, consisting of, or consisting essentially of either an embodied HCV nucleic acid or HCV peptide or both (e.g., any one or more of SEQ. ID. NOs.: 163-189, 197, or 198) are contemplated. These compositions typically contain an adjuvant, but do not necessarily require an adjuvant. That is many of the nucleic acids and peptides described herein function as immunogens when administered neat.
  • compositions described herein can be manufactured in accordance with conventional methods of galenic pharmacy to produce medicinal agents for administration to animals, e.g., mammals including humans.
  • nucleic acid-based vaccines are known and it is contemplated that these compositions and approaches to immunotherapy can be augmented by reformulation with ribavirin (See e.g., U.S. Pat. No. 5,589,466 and 6,235,888).
  • ribavirin See e.g., U.S. Pat. No. 5,589,466 and 6,235,888.
  • a gene encoding one of the HCV peptides described herein e.g., SEQ. ID. NO.: 163 or SEQ. ID. NO.: 197) is cloned into an expression vector capable of expressing the polypeptide when introduced into a subject.
  • the expression construct is introduced into the subject in a mixture of adjuvant (e.g., ribavirin) or in conjunction with an adjuvant (e.g., ribavirin).
  • adjuvant e.g., ribavirin
  • the adjuvant e.g., ribavirin
  • RNA encoding the HCV polypeptide antigen of interest is provided to the subject in a mixture with ribavirin or in conjunction with an adjuvant (e.g., ribavirin).
  • suitable promoters include Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (HIV) such as the HIV Long Terminal Repeat (LTR) promoter, Moloney virus, ALV, Cytomegalovirus (CMV) such as the CMV immediate early promoter, Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV) as well as promoters from human genes such as human actin, human myosin, human hemoglobin, human muscle creatine and human metalothionein can be used.
  • Simian Virus 40 SV40
  • MMTV Mouse Mammary Tumor Virus
  • HIV HIV Long Terminal Repeat
  • ALV HIV Long Terminal Repeat
  • CMV Cytomegalovirus
  • EBV Epstein Barr Virus
  • RSV Rous Sarcoma Virus
  • polyadenylation signals useful with some embodiments, especially in the production of a genetic vaccine for humans, include but are not limited to, SV40 polyadenylation signals and LTR polyadenylation signals.
  • the SV40 polyadenylation signal which is in pCEP4 plasmid (Invitrogen, San Diego Calif), referred to as the SV40 polyadenylation signal, is used.
  • enhancers may be selected from the group including but not limited to: human actin, human myosin, human hemoglobin, human muscle creatine and viral enhancers such as those from CMV, RSV and EBV.
  • Gene constructs can be provided with mammalian origin of replication in order to maintain the construct extrachromosomally and produce multiple copies of the construct in the cell. Plasmids pCEP4 and pREP4 from Invitrogen (San Diego, CA) contain the Epstein Barr virus origin of replication and nuclear antigen EBNA-I coding region, which produces high copy episomal replication without integration.
  • the genetic vaccines comprise ribavirin and a nucleic acid encoding NS3/4A, NS3, or a fragment or mutant thereof (SEQ. ID. NOs.: 164-188 and 198).
  • SEQ. ID. NOs.: 164-188 and 198 The following example describes the preparation of a genetic immunogen suitable for use in humans.

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Abstract

Les modes de réalisation selon la présente invention concernent la découverte de plusieurs formulations d'une composition de diffusion transdermique permettant d'administrer des composés de masses moléculaires faibles et élevées, de préférence des antigènes et des immunogènes du virus de l'hépatite C (HCV), à un sujet. L'invention concerne lesdites compositions de diffusion transdermique, des dispositifs de diffusion transdermique permettant d'administrer lesdites compositions à des sujets nécessitant des soins, et des procédés de fabrication et d'utilisation desdites compositions et dispositifs.
PCT/US2006/020101 2005-05-25 2006-05-24 Formulations transdermiques contenant des immunogènes du virus de hépatite c WO2008004992A2 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140220063A1 (en) * 2013-02-05 2014-08-07 Nitto Denko Corporation Vaccine composition

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002013855A2 (fr) * 2000-08-17 2002-02-21 Tripep Ab Vaccins contenant de la ribavirine et procedes d'utilisation associes
WO2004048402A2 (fr) * 2002-11-26 2004-06-10 Tripep Ab Gene de fusion ns3/4a non structurel a codons optimises du virus de l'hepatite c
WO2005039464A1 (fr) * 2003-10-10 2005-05-06 Oryxe Composes transdermiques a poids moleculaires faible et eleve

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002013855A2 (fr) * 2000-08-17 2002-02-21 Tripep Ab Vaccins contenant de la ribavirine et procedes d'utilisation associes
WO2004048402A2 (fr) * 2002-11-26 2004-06-10 Tripep Ab Gene de fusion ns3/4a non structurel a codons optimises du virus de l'hepatite c
WO2005039464A1 (fr) * 2003-10-10 2005-05-06 Oryxe Composes transdermiques a poids moleculaires faible et eleve

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140220063A1 (en) * 2013-02-05 2014-08-07 Nitto Denko Corporation Vaccine composition
US10076491B2 (en) * 2013-02-05 2018-09-18 Nitto Denko Corporation Vaccine composition

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