WO2005120546A2 - Compositions and methods for topical diagnostic and therapeutic transport - Google Patents

Compositions and methods for topical diagnostic and therapeutic transport Download PDF

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Publication number
WO2005120546A2
WO2005120546A2 PCT/US2005/006931 US2005006931W WO2005120546A2 WO 2005120546 A2 WO2005120546 A2 WO 2005120546A2 US 2005006931 W US2005006931 W US 2005006931W WO 2005120546 A2 WO2005120546 A2 WO 2005120546A2
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WO
WIPO (PCT)
Prior art keywords
composition according
positively charged
carrier
composition
biologically active
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2005/006931
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English (en)
French (fr)
Other versions
WO2005120546A3 (en
Inventor
Michael D. Dake
Jacob M. Waugh
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Revance Therapeuticals Inc
Original Assignee
Essentia Biosystems Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to CA2558676A priority Critical patent/CA2558676C/en
Priority to MXPA06009897A priority patent/MXPA06009897A/es
Priority to US10/591,486 priority patent/US8974774B2/en
Priority to BRPI0508440-7A priority patent/BRPI0508440A/pt
Priority to EP05759520A priority patent/EP1734984A4/en
Priority to CN2005800117508A priority patent/CN101083907B/zh
Priority to AU2005251676A priority patent/AU2005251676B2/en
Priority to KR1020067020211A priority patent/KR101284710B1/ko
Application filed by Essentia Biosystems Inc filed Critical Essentia Biosystems Inc
Priority to JP2007501983A priority patent/JP2007527431A/ja
Publication of WO2005120546A2 publication Critical patent/WO2005120546A2/en
Priority to IL177815A priority patent/IL177815A/en
Anticipated expiration legal-status Critical
Priority to NO20064414A priority patent/NO20064414L/no
Publication of WO2005120546A3 publication Critical patent/WO2005120546A3/en
Priority to US14/642,580 priority patent/US10172877B2/en
Priority to US16/241,885 priority patent/US20200101097A1/en
Priority to US17/100,806 priority patent/US20210069224A1/en
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/28—Dragees; Coated pills or tablets, e.g. with film or compression coating
    • A—HUMAN NECESSITIES
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    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
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    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
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    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4418—Non condensed pyridines; Hydrogenated derivatives thereof having a carbocyclic group directly attached to the heterocyclic ring, e.g. cyproheptadine
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    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
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    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/4886—Metalloendopeptidases (3.4.24), e.g. collagenase
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    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
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Definitions

  • Skin protects the body's organs from external environmental threats and acts as a thermostat to maintain body temperature. It consists of several different layers, each with specialized functions. The major layers include the epidermis, the dermis and the hypodermis.
  • the epidermis is a stratifying layer of epithelial cells that overlies the dermis, which consists of connective tissue. Both the epidermis and the dermis are further supported by the hypodermis, an internal layer of adipose tissue.
  • the epidermis the topmost layer of skin, is only 0.1 to 1.5 millimeters thick
  • the epidermis consists of keratinocytes and is divided into several layers based on their state of differentiation.
  • the epidermis can be further classified into the stratum corneum and the viable epidermis, which consists of the granular melphigian and basal cells.
  • the stratum corneum is hygroscopic and requires at least 10% moisture by weight to maintain its flexibility and softness. The hygroscopicity is attributable in part to the water-holding capacity of keratin. When the horny layer loses its softness and flexibility it%ecomes rough and brittle, resulting in dry skin.
  • the dermis which lies just beneath the epidermis, is 1.5 to 4 millimeters thick.
  • the hypodermis is the deepest layer of the skin. It acts both as an insulator for body heat conservation and as a shock absorber for organ protection (Inlander, Skin, New York, NY: People's Medical Society, 1-7 (1998)). In addition, the hypodermis also stores fat for energy reserves.
  • the pH of skin is normally between 5 and 6. This acidity is due to the presence of amphoteric amino acids, lactic acid, and fatty acids from the secretions of the sebaceous glands.
  • the term "acid mantle” refers to the presence of the water-soluble substances on most regions of the skin.
  • the buffering capacity of the skin is due in part to these secretions stored in the skin's horny layer.
  • Wrinkles one of the telltale signs of aging, can be caused by biochemical, histological, and physiologic changes that accumulate from environmental damage (Benedetto, International Journal of Dermatology, 38:641-655 (1999)). In addition, there are other secondary factors that can cause characteristic folds, furrows, and creases of facial wrinkles (Stegman et al., The Skin of the Aging Face Cosmetic Dermatological Surgery, 2 nd ed., St. Louis, MO: Mosby Year Book: 5-15 (1990)).
  • One of the principal functions of skin is to provide a barrier to the transportation of water and substances potentially harmful to normal homeostasis.
  • the body would rapidly dehydrate without a tough, semi-permeable skin.
  • the skin helps to prevent the entry of harmful substances into the body.
  • a number of strategies have been developed to selectively increase the permeability of skin with variable success.
  • non-protein non-nucleotide therapeutic agent such as antifungals cannot penetrate the skin efficiently, in order to provide the therapeutic effects antifungal agents, it must currently be injected into the skin or administered systemically.
  • the Federal Food and Drug Administration has approved such a procedure, for treatment of fungal infection.
  • the antifungal agent is administered by monitored injection or dosage.
  • Topical application of antifungal agents provides a local delivery for a safer and more desirable treatment alternative due to painless nature of application, reduced training to apply the antifungal therapeutic, smaller doses necessary to affect and to reach a therapeutic clinical result and limiting side effects typically associated with systemic delivery.
  • antigenic agents suitable for immunization cannot penetrate the skin efficiently, in order to provide the therapeutic effects of antigenic agents suitable for immunization the toxin must currently be injected into the skin.
  • the Federal Food and Drug Administration has approved such a procedure, for treatment of for example, malaria, rabies, anthrax, tuberculosis, or related to childhood immunizations such as hepatitis B, diptheria, pertussis, tetanus, Haemophilus influenza type b, inactivated poliovirus, measles, mumps, rubella, varicella, pneumococcus, hepatitis A, and influenza.
  • the antigenic agent for immunization is administered by monitored injection.
  • Topical application of antigenic agent for immunization provides for a safer and more desirable treatment alternative due to painless nature of application, the larger treatment surface area that can be covered, reduced training to apply the therapeutic, smaller doses necessary to affect and to reach a therapeutic clinical result.
  • Transdermal administration of other therapeutics is also an area of great interest due, for instance, to the potential for decreased patient discomfort, direct administration of therapeutic agents into the bloodstream, and the opportunities for monitored delivery via the use of specially constructed devices and/or of controlled release formulations and techniques.
  • the present invention provides new methods and compositions that are broadly applicable to compositions of diverse therapeutic or cosmeceutical agents that can be targeted or imaged to maximize delivery to a particular site.
  • This invention further relates to formulations for transdermal delivery of proteins other than insulin, botulinum toxin, antibody fragments, and VEGF - preferably those having a molecular weight of less than 20,000 kD.
  • protein-based agents can include for example an antigen suitable for immunization.
  • the present invention relates to formulations for transdermal delivery of a non-protein non-nucleotide therapeutic agent such as for example certain antifungals.
  • the invention specifically excludes insulin, botulinum toxins, VEGF and antibody fragments when the term "therapeutic" or "biologically active protein" is employed. Since antigens suitable for immunization have other biological activities such as mounting an immune response, these remain included in the appropriate aspects of this invention, however.
  • This invention further relates to formulations for transdermal delivery of a non-protein non-nucleotide therapeutic agent such as antifungals.
  • Suitable antifungal agents include, for example, amphotericin B, fluconazole, flucytosine, itraconazole, ketoconazole, clotrimazole, econozole, griseofulvin, miconazole, nystatin or ciclopirox and the like.
  • This invention further relates to formulations for transdermal delivery of antigenic agents suitable for immunization can be protein-based antigens, non-protein non- nucleotide agents or hybrids thereof. Suitable antigens include, for example, those for environmental agents, pathogens or biohazards.
  • Suitable agents preferably include, for example, malaria, rabies, anthrax, tuberculosis, or related to childhood immunizations such as hepatitis B, diptheria, pertussis, tetanus, Haemophilus influenza type b, inactivated poliovirus, measles, mumps, rubella, varicella, pneumococcus, hepatitis A, and influenza.
  • antigens suitable for immunization have other biological activities such as mounting an immune response, these remain included in the appropriate aspects of this invention, however.
  • Agents which do not readily cross skin but are substantially smaller than for example insulin - most preferably agents less than 20,000 kD - or have different physiochemical properties can be delivered through still another aspect of this invention.
  • antigens desirable for immunizations can be transported across skin without injection through the present invention. The result affords an r injection-free alternative to childhood immunizations or potentially important biohazards or environmental hazards.
  • non-protein, non-nucleotide therapeutic such as certain of the antifungal agents, for example, have been characterized by poor topical penetration particularly for fungal infections such as oncomychosis or infection of the fingernails and nail plates.
  • This invention accordingly further relates to topical formulations for transdermal delivery of therapeutic and diagnostic substances, including proteins particularly those having a molecular weight of less than 20,000 kD or other biologically active agent such as, for example, a non-protein non-nucleotide therapeutic agent such as certain antifungals or alternately an agent for immunization. Since antigens suitable for immunization have other biological activities such as mounting an immune response, these remain included in the appropriate aspects of this invention, however.
  • Suitable antifungal agents include, for example, amphotericin B, fluconazole, flucytosine, itraconazole, ketoconazole, clotrimazole, econozole, griseofulvin, miconazole, nystatin or ciclopirox and the like.
  • Suitable agents preferably include, for example, malaria, rabies, anthrax, tuberculosis, or related to childhood immunizations such as hepatitis B, diptheria, pertussis, tetanus, Haemophilus influenza type b, inactivated poliovirus, measles, mumps, rubella, varicella, pneumococcus, hepatitis A, and influenza.
  • This invention provides a composition having a biologically active protein and a carrier.
  • the carrier includes a polymeric backbone having attached positively charged branching groups and is present in an effective amount for transdermal delivery.
  • the association between the carrier and the biologically active protein is non-covalent.
  • Another object of this invention is to provide a composition containing a non- protein, non-nucleotide biologically active agent and a carrier.
  • the carrier includes a polymeric backbone having attached positively charged branching groups and is present in an effective amount for transdermal delivery.
  • the association between the carrier and the non- protein, non-nucleotide biologically active agent is non-covalent.
  • kits for administration of a biologically active protein to a subject includes a device for delivering the biologically active protein to the skin or epithelium of the subject and a composition having a polymeric carrier with attached positively charged branching groups.
  • the positively charged branching groups may be selected from -(gly) nl -(arg) n , HIV-TAT and fragments thereof, and Antennapedia PTD and fragments thereof, where the subscript nl is an integer of from 0 to about 20, and the subscript n2 is independently an odd integer of from about 5 to about 25.
  • the association between the carrier and the biologically active protein is non-covalent.
  • This invention also provides a method of administering a biologically active protein to a subject.
  • the method includes topically applying to the skin or epithelium of the subject the protein in conjunction with an effective amount of a carrier.
  • the carrier includes a polymeric backbone having attached positively charged branching groups.
  • the association between the carrier and the biologically active protein is non-covalent.
  • the invention provides a method of administering a non-protein, non-nucleotide biologically active agent to a subject.
  • the method includes topically applying to the skin or epithelium of the subject the biologically active agent in conjunction with an effective amount of a carrier.
  • the carrier may include a polymeric backbone having attached positively charged branching groups.
  • the association between the carrier and the biologically active agent is non-covalent.
  • One object of this invention is to provide a composition containing an antigen suitable for immunization and a carrier.
  • the carrier includes a polymeric backbone having attached positively charged branching groups and is present in an effective amount for transdermal delivery. The association between the carrier and the antigen is non-covalent.
  • Another object of this invention is to provide a kit for administration of an antigen suitable for immunization to a subject.
  • the kit includes a device for delivering the antigen suitable for immunization to the skin or epithelium and a composition with a carrier.
  • the carrier includes a polymeric backbone having attached positively charged branching groups selected from -(gly)nl-(arg)n2, HIV-TAT and fragments thereof, and Antennapedia PTD, where the subscript nl is an integer of from 0 to about 20, and the subscript n2 is independently an odd integer of from about 5 to about 25.
  • the association between the carrier and the antigen is non-covalent.
  • Yet another object of this invention is to provide a method of administering an antigen suitable for immunization to a subject.
  • the method includes topically applying to the skin or epithelium of the subject the antigen suitable for immunization in conjunction with an effective amount of a carrier.
  • the carrier contains a polymeric backbone having attached positively charged branching groups. The association between the carrier and the antigen is non-covalent. •-; ⁇ »*».*— -
  • This invention also provides a composition containing an imaging moiety and/or a targeting agent and a carrier.
  • the carrier includes a polymeric backbone having attached positively charged branching groups and is present in an effective amount for transdermal delivery.
  • the association between the carrier and the imaging moiety or targeting agent is non-covalent.
  • the present invention provides a composition comprising a non- covalent complex of: a) a positively-charged backbone; and b) at least one member selected from: i) a first negatively-charged backbone having a plurality of attached imaging moieties, or a plurality of negatively-charged imaging moieties; ii) a second negatively-charged backbone having a plurality of attached targeting agents, or a plurality of negatively-charged targeting moieties; iii) a non-protein non-nucleotide biologically active agent iv) a therapeutic protein other than insulin, botulinum toxin, antibody fragments, or VEGF. wherein the complex carries a net positive charge.
  • the biological agents in this aspect of the invention, can be either a therapeutic agent or a cosmeceutical agent.
  • the invention specifically excludes insulin, botulinum toxins, VEGF and antibody fragments when the term "therapeutic” or "biologically active protein" is employed. Since antigens suitable for immunization have other biological activities such as mounting an immune response, these remain included in the appropriate aspects of this invention, however.
  • candidate agents can be used to determine in vivo efficacy in these non-covalent complexes.
  • the present invention provides a composition comprising a non-covalent complex of a positively-charged backbone having at least one attached efficiency group and an agent for molecular imaging, for example an optical imaging agent.
  • the agent will be targeted to a particular agent for diagnostic and/or therapeutic effect.
  • an optical imaging agent can be associated with a positively-charged backbone and a component to target melanoma for targeted topical diagnosis of melanoma.
  • the present invention pro vides-a method for delivery of a biological agent to a cell surface in a subject, said method comprising administering to said subject a composition as described above.
  • the present invention provides a method for preparing a pharmaceutical or cosmeceutical composition, the method comprising combining a positively charged backbone component and at least one member selected from: i) a first negatively-charged backbone having a plurality of attached imaging moieties, or a plurality of negatively-charged imaging moieties; ii) a second negatively-charged backbone having a plurality of attached targeting agents, or a plurality of negatively-charged targeting moieties; iii) a non-protein non-nucleotide biologically active agent iv) a therapeutic protein other than insulin, botulinum toxins, VEGF and antibody fragments with a pharmaceutically or cosmeceutically acceptable carrier to form a non-covalent complex having a net positive charge.
  • the present invention provides a kit for formulating a pharmaceutical or cosmeceutical delivery composition, the kit comprising a positively charged backbone component and at least one component selected from groups i) through iv) above, along with instructions for preparing the delivery composition.
  • this invention relates to a composition comprising a biologically active agent such as protein having a molecular weight of less than 20,000 kD and other biologically active agents such as, for example, a non-protein non-nucleotide therapeutic agent such as certain antifungals or alternately an agent for immunization, and a carrier comprising a positively charged carrier having a backbone with attached positively charged branching or "efficiency" groups, all as described herein.
  • the biologically active agent may be protein-based (e.g., a protein having a molecular weight of less than 20,000 kD), a non-protein, non nucleotide therapeutic agent (e.g., certain antifungal agents), or an antigen for immunization.
  • Suitable antifungal agents include, for example, amphotericin B, fluconazole, flucytosine, itraconazole, ketoconazole, clotrimazole, econozole, griseofulvin, miconazole, nystatin or ciclopirox and the like.
  • the antigenic agents suitable for immunization can be protein-based antigens which do not therapeutically alter blood glucose levels, non-protein non-nucleotide agents or hybrids thereof.
  • the agents included are themselves antigens suitable for immunization.
  • Suitable antigens include, for example, those for environmental agents, pathogens or biohazards.
  • Suitable antigens include those that may be used for immunizations against malaria, rabies, anthrax, tuberculosis, or those related to childhood immunizations such as hepatitis B, diptheria, pertussis, tetanus, Haemophilus influenza type b, inactivated poliovirus, measles, mumps, rubella, varicella, pneumococcus, hepatitis A, and influenza.
  • the positively charged carrier is a long-chain positively charged polypeptide or a positively charged nonpeptidyl polymer, for example, a polyalkyleneimine.
  • Proteins and non-protein, non-nucleotide therapeutics that are not normally capable of crossing the skin or epithelium appreciably [relative to the complex of the same agent and the carriers of the present invention] and that do not have a therapeutic effect on lowering blood glucose have widely differing surface and physiochemical properties that normally would make it uncertain whether a technique that afforded transdermal delivery of, for example, insulin would be applicable to the protein and non- protein therapeutics.
  • carriers of this invention that have a positively charged backbone with positively charged branching groups, as described herein, are quite surprisingly capable of providing transdermal delivery of protein and non-protein therapeutics.
  • Such carriers suited for transdermal delivery of particular proteins can easily be identified using tests such as those described in the Examples.
  • a protein may, for example be a small protein having a molecular weight of less than 20,000 kD.
  • therapeutic in the context of blood glucose refers to a decline in blood glucose levels sufficient to alleviate acute symptoms or signs of hyperglycemia, for example in diabetic patients.
  • This invention also provides a method for preparing a pharmaceutical or cosmeceutical composition that comprises combining a carrier comprising a positively charged polypeptide or a positively charged nonpeptidyl polymer such as a long-chain polyalkyleneimine (where the polypeptide or nonpeptidyl polymer has positively charged branching or "efficiency" groups as defined herein) with a biologically active agent such as, for example, protein having a molecular weight of less than 20,000 kD.
  • the carrier may be combined with other biologically active agents such as, for example, a non- protein, non-nucleotide therapeutic agent (e.g., certain antifungals) or alternatively an agent for immunization.
  • the present invention also provides a kit for preparing or formulating a composition that comprises the carrier and a therapeutic substance, as well as such additional items that are needed to produce a usable formulation, or a premix that may in turn be used to produce such a formulation.
  • a kit may consist of an applicator or other device for applications of the compositions or components thereof according to the methods of the present invention.
  • device can refer, for example, to an instrument or applicator suitable for delivering, mixing or otherwise preparing the compositions according to the methods of the present invention.
  • This invention also provides devices for transdermal transmission of a biologically active agent that is contained within a composition that includes a carrier comprising a positively charged polypeptide of preferably short chain to intermediate chain length or another long-chain nonpeptidyl polymeric carrier that has positively charged branching or "efficiency" groups as defined herein.
  • a biologically active agent that is contained within a composition that includes a carrier comprising a positively charged polypeptide of preferably short chain to intermediate chain length or another long-chain nonpeptidyl polymeric carrier that has positively charged branching or "efficiency" groups as defined herein.
  • Such devices may be as simple in construction as a skin patch, or may be more complicated devices that may include means for dispensing and monitoring the dispensing of the composition, and optionally means for monitoring the condition of the subject in one or more aspects, including monitoring the reaction of the subject to the substances being dispensed.
  • the device may contain only a therapeutic biologically active agent and a carrier that may be applied separately to the skin.
  • the invention also comprises a kit that includes both a device for dispensing via the skin and a material that contains a positively charged carrier or backbone, and that is suitable for applying to the skin or epithelium of a subject.
  • the invention also comprises a method for administering a biologically active agent that includes topically administering an effective amount of the biologically active agent in conjunction with a positively charged polypeptide or non- polypeptidyl polymer such as a polyalkyleneimine having positively charged branching groups, as described herein.
  • a biologically active agent that includes topically administering an effective amount of the biologically active agent in conjunction with a positively charged polypeptide or non- polypeptidyl polymer such as a polyalkyleneimine having positively charged branching groups, as described herein.
  • a composition containing the positively charged carrier may first be applied to the skin of the subject, followed by applying a skin patch or other device containing the biologically active agent
  • the invention also relates to methods of applying a biologically active agent to epithelial cells, including those other than epithelial skin cells, for example, epithelia ophthalmic cells or cells of the gastrointestinal system.
  • a biologically active agent to epithelial cells, including those other than epithelial skin cells, for example, epithelia ophthalmic cells or cells of the gastrointestinal system.
  • Figure 2 provides a schematic representation of several embodiments of the invention.
  • Figures 3-4 represent the results of transdermal delivery of a plasmid containing the transgene for E. coli beta-galactosidase as described in Example 2.
  • Figure 5 represents the results of transdermal delivery of a plasmid containing the transgene for E. coli beta-galactosidase as described in Example 3.
  • Figure 6 represents the results of transdermal delivery of a plasmid containing the transgene for E. coli beta-galactosidase ' as described in Example 4.
  • Figure 7 represents the results of transdermal delivery of a botulinum toxin as described in Example 5.
  • Figure 8 is a photographic depiction of the results of transdermal delivery of a botulinum toxin as described in Example 6.
  • Figure 9 is a photographic depiction that the imaging complexes of Example 9 follow the brightfield distribution (panels a and c) for melanoma pigmented cells with fluorescent optical imaging agents (panels b and d) for two different fields and different magnifications (panels a and b at 10X versus panels c and d at 40X magnifications).
  • Figure 10 is a photograph depiction showing positive NeutrAvidin staining as described in Example 10 at two different magnifications. Parts (a) and (b) of Figure 10a are at 10X magnification and parts (c) and (d) are at 20X magnification. Parts (e) and (f) of
  • Figure 10(b) are at 20X magnification for repeated staining.
  • Figure 11 represents the results for relative toxicity for carrier backbones as described in Example 11.
  • Figure 12 represents the results of transdermal gene delivery efficiency as described in Example 11.
  • Figure 13 is a photographic depiction of selective delivery of optical imaging probe to CEA-positive cells showing a brightfield image of colon carcinoma and fibroblasts co-culture (panel a) and fluorescence image of colon carcinoma (panel b) as described in
  • the present invention provides a component-based system for selective, persistent delivery of imaging agents or other therapeutic agents.
  • Individual features for the compositions can be selected by designating desired components in bedside formulations.
  • imaging and specific targeting moieties are provided to form a non-covalent (preferably ionic) complex with a positive backbone.
  • the invention obviates the need for attaching components in precise locations on a positive backbone, in contrast to other strategies which increase complexity and expense and decrease efficiency to a level that no successful combination has yet been reported due to steric limitations.
  • certain substances can be transdermally delivered by use of certain positively charged carriers alone, without requiring the inclusion of a negative backbone.
  • the substance or a derivative thereof has sufficient functionalities to associate with the carriers of the present invention non-covalently, preferably ionically.
  • the term "sufficient" in this context refers to an association that can be determined, for example, by change in particle sizing or functional spectrophotometry versus the components alone.
  • the components are shown as (1) a solid backbone having attached positively charged groups (also referred to as efficiency groups shown as darkened circles attached to a darkened bar), for example (Gly) nl -(Arg) n2 (wherein the subscript nl is an integer of from 3 to about 5, and the subscript n2 is an odd integer of from about 7 to about 17) or TAT domains; (2) imaging moieties (open triangles attached to a light bar); (3) targeting agents and/or (4) biologically active agents (open circles attached to a light bar) such as non-protein non-nucleotide therapeutic agents or protein-based therapeutics other than insulin, botulinum toxins, VEGF and antibody fragments;
  • Figure 2 illustrates various examples of multicomponent compositions wherein the groups are depicted as set out in Figure 1.
  • a first multi-component composition is illustrated in which a positively charged backbone has associated an imaging component, a targeting component, a therapeutic.
  • a second multi-component composition is illustrated which is designed for diagnostic/prognostic imaging.
  • the positively charged backbone is complexed with both optical imaging components and targeting components for example recognizing melanoma to create a topical melanoma detection platform .
  • the present invention described more fully below, provides a number of additional compositions useful in therapeutic and diagnostic programs.
  • biologically active agent refers to a therapeutic agent that cures a disease or alleviates a health-related problem (including health-related problems that are subjectively assessed and/or cosmetic).
  • the biologically active agent may be a therapeutic protein, and in certain embodiments, is preferably a protein with a molecular weight of less than 20,000 kD.
  • the invention specifically excludes insulin, botulinum toxins, VEGF and antibody fragments when the term "therapeutic" or “biologically active protein” is employed. Since antigens suitable for immunization have other biological activities such as mounting an immune response, these remain included in the appropriate aspects of this invention, however.
  • the biologically active agent may be a non-protein, non-nucleotide agent, (e.g., certain antifungal agents). Other non-limiting examples of suitable biologically active agents are provided as discussed herein.
  • the association between carriers as described herein and the biologically active agent is by non-covalent interaction, which can include, for example, ionic interactions, hydrogen bonding, van der Waals forces, or combinations thereof.
  • the present invention provides a composition comprising a non-covalent complex of: a) a positively-charged backbone; and b) at least one member selected from: i) a first negatively-charged backbone having a plurality of attached imaging moieties, or a plurality of negatively-charged imaging moieties; ii) a second negatively-charged backbone having a plurality of attached targeting agents, or a plurality of negatively-charged targeting moieties; iii) a non-protein non-nucleotide biologically active agent iv) a therapeutic protein other than insulin, botulinum toxins, VEGF and antibody fragments, wherein the complex carries a net positive charge.
  • the composition comprises at least two members selected from groups i) through iv). In another group of embodiments, the composition comprises at least one member from each of groups i) and ii), and one selected from either iii) or iv).
  • the positively-charged backbone has a length of from about 1 to 4 times the combined lengths of the members from group b).
  • the positively charged backbone has a charge ratio of from about 1 to 4 times the combined charge of the members from group b).
  • the charge density is uniform and the length and charge ratios are approximately the same. Size to size (length) ratios can be determined based on molecular studies of the components or can be determined from the masses of the components
  • positively charged it is meant that the carrier has a positive charge under at least some solution-phase conditions, more preferably at least under some physiologically compatible conditions. More specifically, “positively charged” as used herein, means that the group in question contains functionalities that are charged under all pH conditions, such as a quaternary arnine, or containing a functionality which can acquire positive charge under certain solution-phase conditions, such as pH changes in the case of primary amines. More preferably, “positively charged” as used herein refers to those functionalities that have the behavior of associating with anions over physiologically compatible conditions. Polymers with a multiplicity of positively-charged moieties need not be homopolymers, as will be apparent to one skilled in the art.
  • positively charged moieties are well known in the prior art and can be employed readily, as will be apparent to those skilled in the art.
  • the positively charged carriers described in this invention which themselves do not have a therapeutic activity represent novel compounds which have utility, for example, in compositions and methods as described herein.
  • these novel compounds which include any carrier which comprises a positively charged backbone having attached positively charged branching groups as described herein and which does not itself have a therapeutic biologic activity.
  • the invention specifically excludes antibody fragments when the term "therapeutic” or "biologically active protein" is employed. Since antigens suitable for immunization have other biological activities such as mounting an immune response, these remain included in the appropriate aspects of this invention, however.
  • the present invention provides a composition comprising a biologically active agent and a carrier comprising a positively charged backbone.
  • the biologically active agent may be, for example, a protein (particularly those having a molecular weight of less than 20,000 kD), a non-protein non-nucleotide therapeutic agent (such as certain antifungals) or an agent for immunization.
  • the carrier may be, for instance, a positively charged polypeptide or nonpeptidyl polymer, which may be either a hetero- or homopolymer such as a polyalkyleneimine.
  • the polypeptide or nonpeptidyl polymer may have positively charged branching or "efficiency" groups as defined herein.
  • Each protein-based therapeutic and non-nucleotide non-protein therapeutic has distinct physiochemical properties which alter the characteristics of the total complex.
  • Such positively charged carriers are among the materials described below as positively charged backbones.
  • the invention also provides a method for administering a therapeutically effective amount of a biologically active agent -comprising applying to the skin or epithelium of the subject (which may be a human or other mammal) the biologically active agent and an amount of the positively charged backbone having branching groups that is effective to provide transdermal delivery of the protein to the subject.
  • the protein and the positively charged carrier may be applied as a pre-mixed composition, or may be applied separately to the skin or epithelium.
  • the protein may be in a skin patch or other device and the carrier may be contained in a liquid or other type of composition that is applied to the skin before application of the skin patch.
  • the positively-charged backbone also referred to as a positively charged
  • “carried' is typically a linear chain of atoms, either with groups in the chain carrying a positive charge at physiological pH, or with groups carrying a positive charge attached to side chains extending from the backbone.
  • the positively charged backbone itself will not have a defined enzymatic or therapeutic biologic activity.
  • the linear backbone is a hydrocarbon backbone which is, in some embodiments, interrupted by heteroatoms selected from nitrogen, oxygen, sulfur, silicon and phosphorus. The majority of backbone chain atoms are usually carbon. Additionally, the backbone will often be a polymer of repeating units (e.g., amino acids, poly(ethyleneoxy), poly(propyleneamine), polyal yleneirnine, and the like) but can be a heteropolymer.
  • the positively charged backbone is a polypropyleneamine wherein a number of the amine nitrogen atoms are present as ammonium groups (tetra-substituted) carrying a positive charge.
  • the positively charged backbone is a nonpeptidyl polymer, which may be a hetero- or homo- polymer such as a polyalkyleneimine, for example a polyethyleneimine or polypropyleneimine, having a molecular weight of from about 10,000 to about 2,500,000, preferably from about 100,000 to about 1,800,000, and most preferably from about 500,000 to about 1,400,000.
  • the backbone has attached a plurality of side-chain moieties that include positively charged groups (e.g., ammonium groups, pyridinium groups, phosphonium groups, sulfonium groups, guanidinium groups, or amidinium groups).
  • the sidechain moieties in this group of embodiments can be placed at spacings along the backbone that are consistent in separations or variable. Additionally, the length of the sidechains can be similar or dissimilar.
  • the sidechains can be linear or branched hydrocarbon chains having from one to twenty carbon atoms and terminating at the distal end (away from the backbone) in one of the above-noted positively charged groups.
  • the association between the carrier and the biologically active agent is by non-covalent interaction, non-limiting examples of which include ionic interactions, hydrogen bonding, van der Waals forces, or combinations thereof.
  • the positively charged backbone is a polypeptide having multiple positively charged sidechain groups (e.g., lysine, arginine, omithine, homoarginine, and the like).
  • the polypeptide has a molecular weight of from about 10,000 to about 1,500,000, more preferably from about 25,000 to about 1,200,000, most preferably from about 100,000 to about 1,000,000.
  • the sidechains can have either the D- or L-form (R or S configuration) at the center of attachment.
  • the backbone can be an analog of a polypeptide such as a peptoid.
  • a peptoid is a polyglycine in which the sidechain is attached to the backbone nitrogen atoms rather than the ⁇ -carbon atoms. As above, a portion of the sidechains will typically terminate in a positively charged group to provide a positively charged backbone component. Synthesis of peptoids is described in, for example, U.S. Patent No. 5,877,278. As the term is used herein, positively charged backbones that have a peptoid backbone construction are considered "non-peptide" as they are not composed of amino acids having naturally occurring sidechains at the ⁇ -carbon locations.
  • sidechain groups can be appended that carry a positively charged group.
  • the sulfonamide-linked backbones (-SO 2 NH- and -NHSO 2 -) can have sidechain groups attached to the nitrogen atoms.
  • the hydroxyethylene (-CH(OH)CH 2 -) linkage can bear a sidechain group attached to the hydroxy substituent.
  • linkage chemistries to provide positively charged sidechain groups using standard synthetic methods.
  • the positively charged backbone is a polypeptide having branching groups (also referred to as efficiency groups) comprising -(gly) nl -(arg) n2 , HIV-TAT or fragments thereof, or the protein transduction domain of Antennapedia, or a fragment thereof, in which the subscript nl is an integer of from 0 to 20, more preferably 0 to 8, still more preferably 2 to 5, and the subscript n2 is independently an odd integer of from about 5 to about 25, more preferably about 7 to about 17, most preferably about 7 to about 13.
  • branching groups also referred to as efficiency groups
  • nl is an integer of from 0 to 20, more preferably 0 to 8, still more preferably 2 to 5
  • the subscript n2 is independently an odd integer of from about 5 to about 25, more preferably about 7 to about 17, most preferably about 7 to about 13.
  • HIV-TAT fragment has the formula (gly) p -RGRDDRRQRRR-(g ⁇ y) q , (gly) p -YGRKKRRQRRR-(gly) q or (gly)p"RjKKRRQRRR-(gly)q wherein the subscripts p and q are each independently an integer of from 0 to 20 and the fragment is attached to the backbone via either the C-terminus or the N-terminus of the fragment.
  • Preferred HIV-TAT fragments are those in which the subscripts p and q are each independently integers of from 0 to 8, more preferably 2 to 5.
  • the positively charged side chain or branching group is the Antennapedia (Antp) protein transduction domain (PTD), or a fragment thereof that retains activity.
  • the positively charged carrier includes side-chain positively charged branching groups in an amount of at least about 0.05 %, as a percentage of the total carrier weight, preferably from about 0.05 to about 45 weight %, and most preferably from about 0.1 to about 30 weight %.
  • the most preferred amount is from about 0.1 to about 25 %.
  • the backbone portion is a polylysine and positively charged branching groups are attached to the lysine sidechain amino groups.
  • the polylysine used in this particularly preferred embodiment has a molecular weight of from about 10,000 to about 1,500,000, preferably from about 25,000 to about 1 ,200,000, and most preferably from about 100,000 to about 1 ,000,000. It can be any of the commercially available (Sigma Chemical Company, St. Louis, Missouri, USA) polylysines such as, for example, polylysine having MW > 70,000, polylysine having MW of 70,000 to 150,000, polylysine having MW 150,000 to 300,000 and polylysine having MW > 300,000.
  • the selection of an appropriate polylysine will depend on the remaining components of the composition and will be sufficient to provide an overall net positive charge to the composition and provide a length that is preferably from one to four times the combined length of the negatively charged components.
  • Preferred positively charged branching groups or efficiency groups include, for example, -gly-gly-gly-gly-arg-arg-arg-arg-arg-arg (-Gly 3 Arg 7 ) or HIV-TAT.
  • the positively charged backbone is a long chain polyalkyleneimine such as a polyethyleneimine, for example, one having a molecular weight of about 1,000,000.
  • the positively charged backbones or carrier molecules comprising polypeptides or polyalkyleneimines, having the branching groups described above, are novel compounds and form an aspect of this invention.
  • the positively charged carrier is a polypeptide (e.g., lysine, arginine, omithine, homoarginine, and the like) having multiple positively charged side-chain groups, as described above.
  • the polypeptide has a molecular weight of at least about 10,000.
  • the positively charged carrier is a nonpeptidyl polymer such as a polyalkyleneimine having multiple positively charged side-chain groups having a molecular weight of at least about 100,000.
  • Such polyalkyleneimines include polyethylene- and polypropyleneimines.
  • the positively charged carrier molecule includes positively charged branching or efficiency groups, comprising -(gly) n r(ai"g)n2 s , in which the subscript nl is an integer of from 0 to 20 more preferably 0 to 8, still more preferably 2 to 5, and the subscript n2 is independently an odd integer of from about 5 to about 25, more preferably from about 7 to about 17, and most preferably from about 7 to about 13, HIV-TAT or fragments thereof, or Antennapedia PTD or a.fragment thereof.
  • the side-chain or branching groups have the general formula -(gly) n i-(arg) n2 as described above.
  • the branching or efficiency groups are HIV-TAT fragments that have the formula (gly) p -RGRDDRRQRRR-(gly) q , (gly) p -YGRK RRQRRR-(gly) q , or (gly) p -RK RRQRRR-(gly) q , wherein the subscripts p and q are each independently an integer of from 0 to 20 and the fragment is attached to the carrier molecule via either the C- terminus or the N-terminus of the fragment.
  • the side branching groups can have either the D- or L-form (R or S configuration) at the center of attachment.
  • Preferred HIV-TAT fragments are those in which the subscripts p and q are each independently integers of from 0 to 8, more preferably 2 to 5.
  • Other preferred embodiments are those in which the branching groups are Antennapedia PTD groups or fragments thereof that retain the group's activity. These are known in the art, for instance, from Console et al., J. Biol. Chem. 278:35109 (2003).
  • the carrier is a polylysine with positively charged branching groups attached to the lysine side-chain amino groups.
  • the polylysine used in this particularly preferred embodiment can be any of the commercially available (Sigma Chemical Company, St. Louis, Missouri, USA, e.g.) polylysines such as, for example, polylysine having MW > 70,000, polylysine having MW of 70,000 to 150,000, polylysine having MW 150,000 to 300,000 and polylysine having MW > 300,000. However, preferably the polylysine has MW of at least about 10,000.
  • Preferred positively charged branching groups or efficiency groups include, for example, -gly-gly-gly-gly-arg-arg-arg-arg-arg-arg-arg-arg (-Gly 3 Arg 7 ), HIV-TAT or fragments of it, and Antennapedia PTD or fragments thereof.
  • the multicomponent compositions of the present invention comprise at least one component from the following: i) a first negatively-charged backbone having a plurality of attached imaging moieties, or a plurality of negatively-charged imaging moieties; ii) a second negatively-charged backbone having a plurality of attached targeting agents, or a plurality of negatively-charged targeting moieties; iii) a non-protein non-nucleotide biologically active agent iv) a therapeutic protein other than insulin, botulinum toxins, VEGF and antibody fragments.
  • the positively charged backbone or carrier may be used alone to provide transdermal delivery of certain types of substances.
  • Combinations of biologically active agents as described herein such as, for example, combinations of non-nucleotide non-protein therapeutics such as antifungal agents or proteins other than insulin, botulinum toxins, VEGF and antibody fragments (particularly those having a molecular weight of less than 20,000 kD), and antigenic agents suitable for immunization can also be employed in these compositions.
  • some embodiments or compositions will include an imaging agent such as an agent suitable for magnetic resonance imaging or optical imaging. These embodiments or compositions may, for example, afford topical delivery of an optical imaging agent for melanoma.
  • the negatively-charged backbones when used to carry the imaging moieties, targeting moieties and therapeutic agents, can be a variety of backbones (similar to those described above) having multiple groups carrying a negative charge at physiological pH.
  • the imaging moieties, targeting moieties and therapeutic agents with sufficient surface negatively charged moieties will not require attachment of an additional backbone for ionic complexation with the positively-charged backbones as will be readily apparent to one skilled in the art.
  • "Sufficient" in this context implies that a suitable density of negatively- charged groups is present on the surface of the imaging moieties, targeting moieties or therapeutic agents to afford an ionic attraction with the positively-charged backbones described above.
  • the substance or a derivative thereof has sufficient negative charge to associate with the positively charged carriers of the present invention non- covalently.
  • other uncharged moieties can be employed to at sufficient density to afford non-ionic, non-covalent association with the carrier backbones of the present invention, as will be apparent to one skilled in the art.
  • the term "sufficient" in the context of ionic or non-ionic non-covalent interactions can be determined for example by a change in particle sizing or functional spectrophotometry versus the components alone.
  • Suitable negatively-charged groups are carboxylic acids, phosphinic, phosphonic or phosphoric acids, sulfinic or sulfonic acids, and the like.
  • the negatively-charged backbone is an oligosaccharide (e.g., dextran).
  • the negatively- charged backbone is a polypeptide (e.g., poly glutamic acid, poly aspartic acid, or a polypeptide in which glutamic acid or aspartic acid residues are interrupted by uncharged a ino acids).
  • the moieties described in more detail below imaging moieties, targeting agents, and therapeutic agents can be attached to a backbone having these pendent groups, typically via ester linkages.
  • amino acids which interrupt negatively-charged amino acids or are appended to the terminus of the negatively-charged backbone can be used to attach imaging moieties and targeting moieties via, for example, disulfide linkages (through a cysteine residue), amide linkages, ether linkages (through serine or threonine hydroxyl groups) and the like.
  • the imaging moieties and targeting moieties can themselves be small anions in the absence of a negatively charged polymer.
  • the imaging moieties, targeting moieties and therapeutic agents can also be themselves covalently modified to afford sufficient surface negatively charged moieties for -ionic complexation with the positively-charged backbones as will be readily apparent to one skilled in the art.
  • the substance or a derivative thereof has sufficient negative charge to associate with the positively charged carriers of the present invention non-covalently.
  • the term "sufficient" in this context refers to an association that can be determined for example by change in particle sizing or functional spectrophotometry versus the components a
  • a variety of diagnostic or imaging moieties are useful in the present invention and are present in an effective amount that will depend on the condition being diagnosed or imaged, the route of administration, the sensitivity of the agent, device used for detection of the agent, and the like.
  • radiopaque contrast agents for X-ray imaging will include inorganic and organic iodine compounds (e.g., diatrizoate), radiopaque metals and their salts (e.g., silver, gold, platinum and the like) and other radiopaque compounds (e.g., calcium salts, barium salts such as barium sulfate, tantalum and tantalum oxide).
  • inorganic and organic iodine compounds e.g., diatrizoate
  • radiopaque metals and their salts e.g., silver, gold, platinum and the like
  • other radiopaque compounds e.g., calcium salts, barium salts such as barium sulfate, tantalum and tantalum oxide.
  • Suitable paramagnetic contrast agents include gadolinium diethylene triaminepentaacetic acid (Gd-DTPA) and its derivatives, and other gadolinium, manganese, iron, dysprosium, copper, europium, erbium, chromium, nickel and cobalt complexes, including complexes with 1, 4,7,10-tetraazacyclododecane- N,N',N",N'"-tetraacetic acid (DOTA), ethylenediaminetetraacetic acid (EDTA), 1,4,7,10- tetraazacyclododecane-N,N',N"-triacetic acid (DO3A), l,4,7-triazacyclononane-N,N',N"- triacetic acid (NOTA), l,4,8,ll-tetraazacyclotetradecane-N,N',N",N'"-tetraacetic acid (TETA), hydroxybenzylethylene
  • Suitable superparamagnetic contrast agents include magnetites, superparamagnetic iron oxides, monocrystalline iron oxides, particularly complexed forms of each of these agents that can be attached to a negatively charged backbone.
  • CT contrast agents including iodinated and noniodinated and ionic and nonionic CT contrast agents, as well as contrast agents such as spin-labels or other , diagnostically effective agents.
  • Suitable optical imaging agents include, for example, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Cy7.5, Oregon green 488, Oregon green 500, Oregon, green 514, Green fluorescent prote ⁇ - AM, Texas Red, Hex, TET, and HAMRA.
  • Other examples of diagnostic agents include markers.
  • markers or labels such as radionuclides, fluors, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (particularly haptens), and the like. Still other useful substances are those labeled with radioactive species or components, such as "mTc glucoheptonate.
  • imaging agents are neutral at physiological pH and will preferably be attached to a negatively-charged backbone or covalently modified to include sufficient negatively-charged moieties to form a complex with the positively- charged carrier.
  • Other imaging agents carry sufficient negative charge to form a complex with the positively-charged carrier, even in the absence of a negatively-charged backbone.
  • the substance or a derivative thereof has sufficient negative charge to associate with the positively charged carriers of the present invention non-covalently.
  • the term "sufficient" in this context refers to an association that can be determined, for example, by change in particle sizing or functional spectrophotometry versus the components alone.
  • An example of a negatively-charged imaging moiety is phosphate ion, which is useful for magnetic resonance imaging.
  • a variety of targeting agents are useful in the compositions described herein.
  • the targeting agents are attached to a negatively-charged backbone as described for the imaging moieties above.
  • the targeting agents can be any element that makes it possible to direct a therapeutic agent or another component of the composition to a particular site or to alter the tropism of the complex relative to that of the complex without the targeting agent.
  • the targeting agent can be an extracellular targeting agent.
  • Such an agent can also be an intracellular targeting agent, allowing a therapeutic agent to be directed towards particular cell compartments (e.g, mitochondria, nucleus, and the like).
  • the agent most simply can be a small anion which, by virtue of changing the net charge distribution, alters the tropism of the complex from more highly negative cell surfaces and extracellular matrix components to a wider variety of cells or even specifically away from the most highly negative surfaces.
  • the targeting agent or agents are preferably linked, covalently or non- covalently, to a negatively-charged backbone according to the invention.
  • the ta gSihg agent is covalently attached to polyaspartate, sulfated or phosphorylated dextran, and the like that serves as a negatively-charged backbone component, preferably via a linking group.
  • the targeting agent is a fusogenic peptide for promoting cellular transfection (i.e., for favoring the passage of the composition or its various elements across membranes, or for helping in the egress from endosomes or for crossing the nuclear membrane).
  • the targeting agent can also be a cell receptor ligand for a receptor that is present at the surface of the cell type, such as, for example, a sugar, transferrin, or asialo-orosomucoid protein.
  • a cell receptor ligand for a receptor that is present at the surface of the cell type, such as, for example, a sugar, transferrin, or asialo-orosomucoid protein.
  • Other useful targeting agents include sugars, peptides, hormones, vitamins, cytokines, small anions, lipids or sequences or fractions derived from these elements and which allow specific binding with their corresponding receptors.
  • the targeting agents are sugars and/or peptides cell receptor ligands or fragments thereof, receptors or receptor fragments, and the like.
  • the targeting agents are ligands of growth factor receptors, of cytokine receptors, or of cell lectin receptors or of adhesion protein receptors.
  • the targeting agent can also be a sugar which makes it possible to target lectins such as the asialoglycoprotein receptors.
  • a targeting agent is used in the absence of a negatively-charged backbone.
  • the targeting agent carries sufficient negatively charged moieties to form an ionic complex with the positively-charged carrier described above.
  • the substance or a derivative thereof has sufficient negative charge to associate with the positively charged carriers of the present invention non- covalently.
  • the term "sufficient" in this context refers to an association that can be determined for example by change in particle sizing or functional spectrophotometry versus the components alone.
  • Suitable negatively-charged targeting agents for this group of embodiments are protein-based targeting agents having a net negative charge at physiological pH, as well as targeting agents that can facilitate adhesion to a particular cell surface, such as small polyanions (e.g., phosphate, aspartate and citrate) which may change targeting based upon net surface charge of the cell to be targeted.
  • small polyanions e.g., phosphate, aspartate and citrate
  • a variety of biologically active agents including both therapeutic and cosmeceutical agents, are useful in the present invention and are present in an effective amount that will depend on the condition being treated, prophylactically or otherwise, the route of administration, the efficacy of the agent and patient's size and susceptibility to the treatment regimen.
  • the invention specifically excludes botulinum toxins
  • VEGF and antibody fragments when the term "therapeutic” or "biologically active protein” is employed.
  • the invention specifically excludes therapeutic proteins capable of achieving therapeutic alterations of blood glucose levels (e.g, to treat hyperglycemia), such as insulin. Since antigens suitable for immunization have other biological activities (such as mounting an immune response), these remain included in the appropriate aspects of this invention, however.
  • the antigenic agents suitable for immunization can be protein-based antigens which do not therapeutically alter blood glucose levels, non-protein non-nucleotide agents or hybrids thereof. Nucleotides encoding antigens are specifically not suitable for the compositions of the present invention, however. Thus, the agents included are themselves antigens suitable for immunization.
  • Suitable antigens include, for example, those for environmental agents, pathogens or biohazards.
  • Suitable antigenic agents preferably include, for example, antigens related treatments for malaria, rabies, anthrax, tuberculosis, or related to childhood immunizations such as hepatitis B, diptheria, pertussis, tetanus, Haemophilus influenza type b, inactivated poliovirus, measles, mumps, rubella, varicella, pneumococcus, hepatitis A, and influenza.
  • Suitable therapeutic agents that can be attached to a negatively charged backbone can be found in essentially any class of agents, including, for example, analgesic — agents, anti-asthmatic agents, antibiotics, antidepressant agents, anti-diabetic agents, antifungal agents, antiemetics, antihypertensives, anti-impotence agents, anti-inflammatory agents, antineoplastic agents, anti-HIV agents, antiviral agents, anxiolytic agents, contraception agents, fertility agents, antithrombotic agents, prothrombotic agents, hormones, vaccines, immunosuppressive agents, vitamins and the like.
  • sufficient negatively charged groups can be introduced into the therapeutic agent to afford ionic complexation with the positively charged backbones described above.
  • Suitable cosmeceutic agents include, for example, epidermal growth factor
  • therapeutic agents useful in the present invention include such analgesics as lidocaine, novocaine, bupivacaine, procaine, tetracaine, benzocaine, cocaine, mepivacaine, etidocaine, proparacaine ropivacaine, prilocaine and the like; anti- asthmatic agents such as azelastine, ketotifen, traxanox, corticosteroids, cromolyn, nedocromil, albuterol, bitolterol mesylate, pirbuterol, salmeterol, terbutyline, theophylline and the like; antibiotic agents such as neomycin, streptomycin, chloramphenicol, norfloxacin, ciprofloxacin, trimethoprim, sulfamethyloxazole, the ⁇ -lactam antibiotics, tetracycline, and the like; antidepressant agents such as nefo
  • the biological agent is selected from protein having a molecular weight of less than 20,000 kD and other biologically active agents such as, for example, a non-protein non-nucleotide therapeutic agent such as certain antifungals, and antigenic agents for immunization.
  • suitable examples specifically excludes insulin, botulinum toxins, VEGF and antibody fragments.
  • biological or cosmeceutical agents can be used in the absence of a negatively-charged backbone.
  • biological or cosmeceutical agents are those that generally carry a net negative charge at physiological pH to form a complex with the positively-charged carrier.
  • antigens for immunization typically include proteins or glycoproteins, and many antifungal agents, as well as agents for targeted imaging of melanoma with or without an inherent therapeutic potential.
  • the substance or a derivative thereof has sufficient negative charge to associate with the positively charged carriers of the present invention non-covalently.
  • the term "sufficient" in this context refers to an association that can be determined for example by change in particle sizing or functional spectrophotometry versus the components alone.
  • Negatively-charged backbones having attached imaging moieties, targeting agents or therapeutic agents having attached imaging moieties, targeting agents or therapeutic agents
  • the individual compounds are attached to a negatively charged backbone, covalently modified to ⁇ titroduce negatively-charged moieties, or employed directly if the compound contains sufficient negatively-charged moieties to confer ionic complexation to the positively charged backbone described above.
  • the attachment is via a linking group used to covalently attach the particular agent to the backbone through functional groups present on the agent as well as the backbone.
  • linking groups are useful in this aspect of the invention. See, for example, Hermanson, Bioconjugate Techniques, Academic Press, San Diego, CA (1996);
  • the therapeutic, diagnostic or targeting agents will not have an available functional group for attaching to a linking group, and can be first modified to incorporate, for example, a hydroxy, amino, or thiol substituent.
  • the substituent is provided in a non-interfering portion of the agent, and can be used to attach a linking group, and will not adversely affect the function of the agent.
  • the present invention provides compositions comprising a non-covalent complex of a positively-charged backbone having at least one attached efficiency group and
  • the positively-charged backbone can be essentially any of the positively-charged backbones described above, and will also comprise (as with selected backbones above) at least one attached efficiency group.
  • Suitable efficiency groups include, for example, (Gly) nl -(Arg) n2 wherein the subscript nl is an integer of from 3 to about 5, and the subscript n2 is independently an odd integer of from about 7 to about 17; or TAT domains.
  • the TAT domains may have the formula (gly) p -RGRDDRRQRRR-(gly) q , (gly) p -YGRKKRRQRRR-(gly) q , or ( ly) -RKKRRQRRR-( ly)q 5 wherein the subscripts p and q are each independently an integer of from 0 to 20 and the fragment is attached to the carrier molecule via either the C- terminus or the N-terminus of the fragment.
  • the side branching groups can have either the D- or L-form (R or S configuration) at the center of attachment.
  • Preferred HIV-TAT fragments are those in which the subscripts p and q are each independently integers of from 0 to 8, more preferably 2 to 5.
  • the positively above can be used for transdermal delivery of proteins and other biologically active agents (e.g., proteins having a molecular weight of less than 20,000 kD, non-protein non-nucleotide therapeutic agents such as certain antifungal agents, or antigenic agents for immunization).
  • proteins and other biologically active agents e.g., proteins having a molecular weight of less than 20,000 kD, non-protein non-nucleotide therapeutic agents such as certain antifungal agents, or antigenic agents for immunization.
  • the use of the positively charged carrier enables transmission of the protein or marker gene both into and out of skin cells, and delivery of it in an effective amount and active form to an underlying tissue.
  • This embodiment may include a quantity of a small preferably polyvalent anions, (e.g, phosphate, aspartate, or citrate), or may be carried out in the substantial absence of such a polyanion.
  • a small preferably polyvalent anions e.g, phosphate, aspartate, or citrate
  • protein includes protein extracted from natural sources, as well as protein that may be obtained synthetically, via chemical or recombinant means.
  • the protein also may be in a modified form or in the form of, e.g. a recombinant peptide, a fusion protein, or a hybrid molecule.
  • the protein in in some cases may be a portion of a larger protein molecule that possesses the necessary activity.
  • Preferable proteins are those having a molecular weight of less than 20,000 kD [e.g., those that maybe used in transdermal compositions and methods, such as antigens for immunization], which can vary widely in physiochemical properties.
  • compositions of this invention are preferably in the form of products to be applied to the skin or epithelium of subjects or patients (i.e. humans or other mammals in need of the particular treatment).
  • the term "in need” is meant to include both pharmaceutical and health-related needs as well as needs that tend to be more cosmetic, aesthetic, or subjective.
  • the compositions may also be used, for example, for altering or improving the appearance of facial tissue.
  • the compositions are prepared by mixing proteins particularly those having a molecular weight of less than 20,000 kD or other biologically active agent such as for example, a non-protein non-nucleotide therapeutic agent or alternately an agent for immunization to be administered with the positively charged carrier, and usually with one or more additional pharmaceutically acceptable carriers or excipients;
  • proteins particularly those having a molecular weight of less than 20,000 kD or other biologically active agent such as for example, a non-protein non-nucleotide therapeutic agent or alternately an agent for immunization to be administered with the positively charged carrier, and usually with one or more additional pharmaceutically acceptable carriers or excipients;
  • they may contain a simple aqueous pharmaceutically acceptable carrier or diluent, such as saline, which may be buffered.
  • the compositions may contain other ingredients typical in topical pharmaceutical or cosmeceutical compositions, including a dermatologically or pharmaceutically acceptable carrier, vehicle or medium (i.e.
  • compositions or components thereof are suitable for use in contact with these tissues or for use in patients in general without undue toxicity, incompatibility, instability, allergic response, and the like.
  • compositions of the invention may comprise any ingredient conventionally used in the fields under consideration, and particularly in cosmetics and dermatology.
  • the association between the carrier and the biologically active agent is by non-covalent interaction, which can include, for example, ionic interactions, hydrogen bonding, van der Waals forces, or combinations thereof.
  • compositions may be pre-formulated or may be prepared at the time of administration, for example, by providing a kit for assembly at or prior to the time of administration.
  • the therapeutic proteins and the positively charged backbone may be administered in separate form to the patient, for example by providing a kit that contains a skin patch or other dispensing device containing the therapeutic protein and a liquid, gel, cream or the like that contains the positively charged carrier (and optionally other ingredients).
  • the combination is administered by applying the liquid or other composition containing the carrier to the skin, followed by application of the skin patch or other device.
  • compositions of the invention are applied so as to administer an effective amount of a therapeutic proteins or other beneficial substance, such as an imaging or targeting agent.
  • a therapeutic proteins or other beneficial substance such as an imaging or targeting agent.
  • effective amount refers to any composition or method that provides greater transdermal delivery of the biologically active agent relative to the agent in the absence of the carrier.
  • effective amount refers to an amount sufficient to allow a subject to mount an immune response to the antigen after application or a series of applications of the antigen.
  • antifungal agents “effective amount” refers to an amount sufficient to reduce symptoms or signs of fungal infection.
  • compositions may contain an appropriate effective amount of a therapeutic protein or other biologically active agent for application as a single-dose treatment, or may be more concentrated, either for dilution at the place of administration or for use in multiple applications.
  • compositions containing proteins (particularly those having a molecular weight of less than 20,000 kD) or other biologically active agents will contain from about 1 x 10 "20 to about 25 weight % of the biologically active agent and from about 1 x 10 "19 to about 30 weight % of the positively charged carrier.
  • compositions containing a non-protein non-nucleotide therapeutic agent or alternately an agent for immunization will contain from about 1 x 10 "10 to about 49.9 weight % of the antigen and from about 1 x 10 "9 to about 50 weight % of the positively charged carrier.
  • the amount of carrier molecule or the ratio of it to the biologically active agent will depend on which carrier is chosen for use in the composition in question.
  • compositions of this invention may include solutions, emulsions (including microemulsions), suspensions, creams, lotions, gels, powders, or other typical solid or liquid compositions used for application to skin and other tissues where the compositions maybe used.
  • compositions may contain, in addition to biologically active agents and the carrier molecule, other ingredients typically used in such products, such as antimicrobials, moisturizers and hydration agents, penetration agents, preservatives, emulsifiers, natural or synthetic oils, solvents, surfactants, detergents, gelling agents, emollients, antioxidants, fragrances, fillers, thickeners, waxes, odor absorbers, dyestuffs, coloring agents, powders, viscosity-controlling agents and water, and optionally including anesthetics, anti-itch additives, botanical extracts, conditioning agents, darkening or lightening agents, glitter, humectants, mica, minerals, polyphenols, silicones or derivatives thereof, sunblocks, vitamins, and phytomedicinals.
  • other ingredients typically used in such products such as antimicrobials, moisturizers and hydration agents, penetration agents, preservatives, emulsifiers, natural or synthetic oils, solvents, surfactants, detergents, gelling
  • compositions according to this invention may be in the form of controlled- release or sustained-release compositions, wherein the proteins substance to be delivered and the carrier are encapsulated or otherwise contained within a material such that they are released onto the skin in a controlled manner over time.
  • the substance to be delivered and the carrier may be contained within matrixes, liposomes, vesicles, microcapsules, microspheres and the like, or within a solid particulate material, all of which are selected and/or constructed to provide release of the substance or substances over time.
  • the therapeutic substance and the carrier may be encapsulated together (e.g., in the same capsule) or separately (in separate capsules).
  • compositions of this invention are, of course, another aspect of the invention.
  • compositions of this invention are administered by or under the direction of a physician or other health professional. They may be administered in a single treatment or in a series of periodic treatments over time.
  • a composition as described above is applied topically to the skin or to a nail plate and surrounding skin.
  • compositions are administered under the direction of a physician or other health professional. They may be administered in a single treatment or in a series of periodic treatments over time.
  • a composition as described above is applied topically to the skin.
  • Kits for administering the compositions of the inventions may also include a custom applicator suitable for that purpose.
  • a custom applicator can include for example a prosthetic nail plate, a lacquer, a nail polish with a color agent, a gel, or a combination of any or all of these.
  • the invention relates to methods for the*t ⁇ pical administration of the combination of the positively charged carrier described above with an effective amount of a biologically active agent (e.g, a proteins with a molecular weight of less than 20,000 kD, antigens suitable for immunization, antifungal agents or a non-protein, non-nucleotide therapeutic agent).
  • a biologically active agent e.g, a proteins with a molecular weight of less than 20,000 kD, antigens suitable for immunization, antifungal agents or a non-protein, non-nucleotide therapeutic agent.
  • the administration can be effected by the use of a composition according to the invention that contains appropriate types and amounts of these two substances specifically carrier and biologically active agent.
  • the invention also includes the administration of these two substances in combination, though not necessarily in the same composition.
  • the therapeutic substance may be incorporated in dry form in a skin patch or other dispensing device and the positively charged carrier may be applied to the skin surface before application of the patch so that the two act together, resulting in the desired transdermal delivery.
  • the two substances carrier and biologically active agent
  • the present invention provides a method for preparing a pharmaceutical composition, the method comprising combining a positively charged backbone component and at least one member selected from: i) a first negatively-charged backbone having a plurality of attached imaging moieties, or a plurality of negatively-charged imaging moieties; ii) a second negatively-charged backbone having a plurality of attached targeting agents, or a plurality of negatively-charged targeting moieties; iii) a non-protein non-nucleotide biologically active agent iv) a therapeutic protein other than insulin, botulinum toxins, VEGF, or antibody fragments with a pharmaceutically acceptable carrier to form a non-covalent complex having a net positive charge.
  • the positively charged backbone or carrier may be used alone to provide transdermal delivery of certain types of substances.
  • compositions and methods comprising about 1 x 10 "20 to about 25 weight % of the biologically active agent and from about 1 x 10 "19 to about 30 weight % of the positively charged carrier.
  • compositions and methods containing a non- nucleotide, non-protein therapeutic such as an antifungal agent, selective imaging agents for diagnosis of skin disorders such as melanoma, or an antigenic agent suitable " for *" immunization, where the compositions and methods contain from 1 x 10 "10 to about 49.9 weight % of the antigen and from about 1 x 10 "9 to about 50 weight % of the positively charged carrier.
  • compositions are prepared by mixing the positively charged backbone component with the desired components of interest (e.g., targeting, imaging or therapeutic components) in ratios and a sequence to obtain compositions having a variable net positive charge.
  • the compositions can be prepared, for example, at bedside using pharmaceutically acceptable carriers and diluents for administration of the composition.
  • the compositions can be prepared by suitable mixing of the components and then lyophilized and stored (typically at room temperature or below) until used or formulated into a suitable delivery vehicle.
  • compositions can be formulated to provide mixtures suitable for various modes of administration, non-limiting examples of which include topical, cutaneous, oral, rectal, vaginal, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, and transdermal.
  • the pharmaceutical compositions of the invention preferably contain a vehicle which is pharmaceutically acceptable for an injectable formulation, in particular for direct injection into the desired organ, or for topical administration (to skin and/or mucous membrane).
  • the pharmaceutical compositions may in particular be sterile, isotonic solutions or dry compositions (e.g, freeze-dried compositions), which may be reconstituted by the addition of sterilized water or physiological saline, to prepare injectable solutions.
  • compositions when the compositions are to be applied topically (e.g., when transdermal delivery is desired) the component or components of interest can be applied in dry form to the skin (e.g., via by using a skin patch), where the skin is separately treated with the positively charged backbone or carrier. In this manner the overall composition is ' essentially formed in situ and administered to the patient or subject.
  • compositions of the present invention can be delivered to a subject, cell or target site, either in vivo or ex vivo using a variety of methods.
  • any of the routes normally used for introducing a composition into ultimate contact with the tissue to be treated can be used.
  • the compositions will be administered with pharmaceutically acceptable carriers. Suitable methods of administering such compounds are available and well known to those of skill in the art. Although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
  • Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, e.g., Remington 's Pharmaceutical Sciences, 17 ed. 1985).
  • Administration can be, for example, intravenous, topical, intraperitoneal, subdermal, subcutaneous, transcutaneous, intramuscular, oral, infra-joint, parenteral, intranasal, or by inhalation.
  • Suitable sites of administration thus include, but are not limited to, the skin, bronchium, gastrointestinal tract, eye and ear.
  • the compositions typically include a conventional pharmaceutical carrier or excipient and can additionally include other medicinal agents, carriers, adjuvants, and the like.
  • the formulation will be about 5% to 75% by weight of a composition of the invention, with the remainder consisting of suitable pharmaceutical excipients.
  • the formulations can take the form of solid, semi-solid, lyophilized powder, or liquid dosage forms, such as, for example, tablets, pills, capsules, powders, solutions, suspensions, emulsions, suppositories, retention enemas, creams, ointments, lotions, aerosols or the like.
  • the formulation can contain, along with the biologically active composition, any of the following: a diluent such as lactose, sucrose, dicalcium phosphate, and the like; a distintegrant such as starch or derivatives thereof; a lubricant such as magnesium stearate and the like; and a binder such as starch, gum acacia, polyvinylpyrrolidone, gelatin, cellulose and derivatives thereof.
  • Compositions can be presented in unit-dose or multi-dose sealed containers, such as ampoules or vials. Doses administered to a patient should be sufficient to achieve a beneficial therapeutic response in the patient over time.
  • a sustained-release formulation can be administered to an organism or to cells in culture and can carry the desired compositions.
  • the sustained- release composition can be administered to the tissue of an organism, for example, by injection.
  • sustained-release it is meant that the composition is made available for uptake by surrounding tissue or cells in culture for a period of time longer than would be achieved by administration of the composition in a less viscous medium, for example, a saline solution.
  • the compositions, alone or in combination with other suitable components can be made into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation.
  • Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
  • propellants such as dichlorodifluoromethane, propane, nitrogen, and the like.
  • the compositions can also be delivered as dry powder (e.g., Nektar).
  • Formulations suitable for parenteral administration include aqueous and non- aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic and compatible with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • Other methods of administration include, but are not limited to, administration using angioplastic balloons, catheters, and gel formations. Methods for angioplastic balloon, catheter and gel formation delivery are well known in the art.
  • compositions of the present invention can by tailored for a variety of imaging uses.
  • virtual colonoscopy can be performed using the component-based system for imaging.
  • virtual colonoscopy involves essentially infusing contrast into a colon and visualizing the images on CT, then reconstructing a 3-D image. Similar techniques could be employed for MR. However, feces, mucous, and air all serve as contrast barriers and can give an artificial surface to the colon wall reconstruction. Addition of a cellular-targeting contrast would help overcome these barriers to provide a true wall reconstruction and help avoid both false- positives and false-negatives.
  • the component-based system could be applied here.
  • the cationic efficiency backbone could be applied with a single contrast agent, for example a CT, MR, or optical contrast agent.
  • a single contrast agent for example a CT, MR, or optical contrast agent.
  • the component based system offers the additional option of adding a specific second agent.
  • This agent could consist of a cationic efficiency backbone, a different imaging moiety, and targeting components, for example targeting two antigens characteristic of colon cancer.
  • the imaging moieties from the simple to the diagnostic could be selected so that one was CT contrast and the other MR contrast, or so that both were MR contrast with one being a T2 agent and the other a Tl agent.
  • any regions specific for a tumor antigen could be visualized and overlaid on the original reconstraction.
  • therapeutic agents could be incorporated into the targeted diagnostic system as well. Similar strategies could be applied to regional enteritis and ulcerative colitis (and again combined with therapy).
  • optical imaging moieties and detection methods could be employed, for example, in the case of melanoma diagnosis or management, preferably in conjunction with a fluorescent imaging moiety. In these embodiments, detection can be visual, image-aided or entirely image-based for example by darkfield image analysis.
  • This example illustrates transdermal delivery of a very large complex, namely a plasmid containing the blue fluorescent protein (BFP) transgene, using a positively charged backbone or carrier of the invention.
  • BFP blue fluorescent protein
  • the positively charged backbone was assembled by covalently attaching
  • -Gly 3 Arg 7 to polylysine MW 150,000 via the carboxyl of the terminal glycine to free amines of the lysine sidechains at a degree of saturation of 18% (i.e., 18 out of each 100 lysine residues is covalently attached to a -Gly 3 Arg 7 ).
  • the modified backbone was designated "KNR2" to denote a second size of the peptidyl carrier.
  • the control polycation was unmodified polylysine (designated "K2", Sigma Chemical Co., St. Louis, MO) of the same size and from the same lot.
  • MD containing the entire transgene for blue fluorescent protein (BFP) and partial flanking sequences driven by a cytomegalovirus (CMV) promoter was employed.
  • BFP serves as an identifiable marker for cells that have been fransfected, then transcribe and translate the gene and can be directly visualized (i.e. without additional staining) under fluorescence microscopy.
  • CMV cytomegalovirus
  • K2 at a 4:1 charge ratio to a 0.5 mg/mL solution of a plasmid expressing blue fluorescent protein driven by a CMV promoter.
  • KNR2 at a ratio of 15:1 to a 0.5 mg/mL solution of a plasmid expressing blue fluorescent protein driven by a CMV promoter.
  • KNR2 at a ratio of 10:1 to a 0.5 mg/mL solution of a plasmid expressing blue fluorescent protein driven by a CMV promoter.
  • KNR2 at a ratio of 4:1 to a 0.5 mg/mL solution of a plasmid expressing blue fluorescent protein driven by a CMV promoter.
  • KNR2 at a ratio of 1.25: 1 to a 0.5 mg/mL solution of a plasmid expressing blue fluorescent protein driven by a CMV promoter.
  • Total positive staining was determined by blinded observer via batch image analysis using Image Pro Plus software (Media Cybernetics, Silver Spring, MD) and was normalized to total cross-sectional area to determine percent positive staining for each. Mean and standard error were subsequently determined for each group with analysis of significance at 95% confidence in one way ANOVA repeated measures using Statview software (Abacus, Berkeley, CA).
  • Runs #4 and #5 exhibit statistically significant (PO.05 by one factor ANOVA repeated measures with Fisher PLSD and TUKEY-A posthoc testing) enhancement of gene delivery efficiency relative to both polylysine alone and Superfect. Toxicities:
  • Mean toxicity data are as follows (reported in AU at OD595; low values, such as present with saline alone correlate with low toxicity, while higher values, such as present in condition 1 indicate a high cellular toxicity): Saline - 0.057 A; 1) 3.460 A; 2) 0.251 A; 3) 0.291 A 4) 0.243 A 5) 0.297 A: 6) 0.337 A.
  • a less toxic, more efficient gene delivery can be accomplished with a ratio of
  • This example illustrates the transport of a large nucleotide across skin by a carrier of the invention after a single administration.
  • -Gly 3 Arg 7 to polylysine (MW 150,000) via the carboxyl of the terminal glycine to free amines of the lysine sidechains at a degree of saturation of 18% (i.e., 18 out of each 100 lysine residues is covalently attached to a -Gly 3 Arg 7 ).
  • the modified backbone was designated "KNR2" as before.
  • the control polycation was unmodified polylysine (designated "K2", Sigma Chemical Co., St. Louis, MO) of the same size and from the same lot.
  • An additional control polycation, Superfect (Qiagen) which is an activated dendrimer-based agent, was selected as a reference for high transfection rates (i.e. simultaneous positive control and reference for state-of-the art efficiency versus toxicity in vitro).
  • Therapeutic agent selection :
  • ⁇ gal E. Coli beta-galactosidase
  • CMV cytomegalovirus
  • ⁇ gal serves as an identifiable marker for cells which have been fransfected, then transcribe and translate the gene and can be directly visualized after specific staining for the foreign enzyme.
  • ⁇ gal serves as an identifiable marker for cells which have been fransfected, then transcribe and translate the gene and can be directly visualized after specific staining for the foreign enzyme.
  • This particular plasmid has a molecular weight of approximately 2,805,000.
  • Group labeled ⁇ Lf S micrograms of ⁇ gal plasmid (p/CMV-sport- ⁇ gal) per final aliquot (i.e. 80 micrograms total) and K2 at a charge ratio of 4:1 were mixed to homogeneity and diluted to 200 microliters with phosphate buffered saline. The resulting composition was mixed to homogeneity with 1.8 ml of Cetaphil and aliquoted in 200 microliter portions for in vivo experiments.
  • Group labeled AMI 8 ' micrograms of ⁇ gal plasmid (p/CMV-sport- ⁇ gal) per final aliquot (i.e. 80 micrograms total) and Superfect at a charge ratio of 5:1 were mixed to homogeneity and diluted to 200 microliters with phosphate buffered saline. The resulting composition was mixed to homogeneity with 1.8 ml of Cetaphil and aliquoted in 200 microliter portions for in vivo experiments.
  • Treated segments were divided into three equal portions the cranial portion was fixed in 10% neutral buffered formalin for 12-16 hours then stored in 70% ethanol until paraffin embedding.
  • the central portion was snap-frozen and employed directly for beta- galactosidase staining at 37 degrees Celsius on sections as previously described (Waugh, J.M., M. Kattash, J. Li, E. Yuksel, M.D. Kuo, M. Lussier, A.B. Weinfeld, R. Saxena, E.D. Rabinovsky, S. Thung, S.L.C. Woo, and S.M. Shenaq.
  • Toxicity was evaluated by dye exclusion on paired sections to those analyzed for efficiency above. Sections only underwent staining for either efficiency or for toxicity since the methods are not reliably co-employed. For toxicity analyses, the sections were immersed in exclusion dye for 5 minutes, then incubated at 37 degrees Celsius for 30 minutes at 10% CO 2 . Any cells that did not exclude the dye in this period of time were considered non-viable. Data handling and statistical analyses:
  • Sections stained as above were photographed in their entirety on a Nikon E600 microscope with plan-apochromat lenses. Resulting images underwent batch image analysis processing using Image Pro Plus software as before with manual confirmation to determine number positive for beta-galactosidase enzyme activity (blue with the substrate method employed here) or cellular toxicity. These results were normalized to total cross-sectional number of cells by nuclear fast red staining for each and tabulated as percent cross-sectional positive staining. Subsequently, mean and standard error were subsequently determined for each group with analysis of significance at 95% confidence in one way ANOVA repeated measures using Statview software (Abacus, Berkeley, CA).
  • Results are summarized in the table below and illustrated in Figure 3.
  • the positively charged peptidyl transdermal delivery carrier achieved statistically significant increases in delivery efficiency and transgene expression versus both K2 (negative control essentially) and the benchmark standard for efficiency, Superfect. While Superfect did achieve statistically significant improvements over K2, KNR2 had greater than an order of magnitude improvement in delivery efficiency versus Superfect in this model system
  • Table 1 Mean and standard error for beta-galactosidase positive cells as percent of total number by treatment group.
  • the peptidyl transdermal carrier can transport large complexes across skin with high efficiencies, particularly given the constraints of transgene expression and total complex size discussed previously. Positive area here, rather than positive number was employed for analyses since (1) the method is greatly simplified and has greater accuracy in image analysis, (2) point demonstrations of efficiencies had already been afforded in II.B conclusively, (3) area measurements provide a broader scope for understanding in vivo results since noncellular components occupy a substantial portion of the cross section, and (4) comparison to still larger nonpeptidyl carrier complexes was facilitated Example 3
  • This example illustrates the transdermal delivery of a large nucleotide-based therapeutic across skin using a positively charged peptidyl carrier of the invention in seven sequential daily applications.
  • the positively charged peptidyl backbone was assembled by covalently attaching -Gly 3 Arg 7 to polylysine (MW 150,000) via the carboxyl of the terminal glycine to free amines of the lysine sidechains at a degree of saturation of 18% (i.e., 18 out of each 100 lysine residues is covalently attached to a -Gly 3 Arg 7 ).
  • the modified backbone was designated "KNR2".
  • the control polycation was unmodified polylysine (designated "K2", Sigma Chemical Co., St. Louis, MO) of the same size and from the same lot.
  • ⁇ gal ⁇ gal
  • CMV cytomegalovirus
  • Group labeled AL1 8 micrograms of ⁇ gal plasmid (p/CMV-sport- ⁇ gal) per final aliquot (i.e. 240 micrograms total) and K2 at a charge ratio of 4:1 were mixed to homogeneity and diluted to 600 microliters with phosphate buffered saline. The resulting composition was mixed to homogeneity with 5.4 ml of Cetaphil and aliquoted in 200 microliter portions for in vivo experiments.
  • mice were euthanized via inhalation of CO 2 , and treated skin segments were harvested at full thickness by blinded observers. Treated segments were divided into three equal portions the cranial portion was fixed in 10% neutral buffered formalin for 12-16 hours then stored in 70% ethanol until paraffin embedding. The central portion was snap-frozen and employed directly for beta-galactosidase staining at 37 degrees
  • Sections stained as above were photographed in their entirety on a Nikon E600 microscope with plan-apochromat lenses. Resulting images underwent batch image analysis processing using Image Pro Plus software as before with manual confirmation to determine area positive for beta-galactosidase enzyme activity. These results were normalized to total cross-sectional area for each and tabulated as percent cross-sectional positive staining. Subsequently, mean and standard error were subsequently determined for each group with analysis of significance at 95% confidence in one way ANOVA repeated measures using Statview software (Abacus, Berkeley, CA).
  • Results are summarized in the table below and illustrated in Figure 5.
  • the peptidyl transdermal delivery carrier achieved statistically significant increases in delivery efficiency and transgene expression versus K2.
  • Table 2 Mean and standard error for cumulative transgene expression of beta-galactosidase as percent of total area after 7 once-daily applications for each treatment group.
  • This example illustrates the transdermal delivery of a large nucleotide-based therapeutic across skin, using a positively charged non-peptidyl carrier of the invention in seven sequential daily applications.
  • the positively charged backbone was assembled by covalently attaching -
  • Gly 3 Arg 7 to polyethyleneimine (PEI, MW 1,000,000) via the carboxyl of the terminal glycine to free amines of the PEI sidechains at a degree of saturation of 30% (i.e., 30 out of each 100 lysine residues is covalently attached to a -Gly 3 Arg ).
  • the modified backbone was designated "PEIR” to denote the large nonpeptidyl carrier.
  • the control polycation was unmodified PEI (designated "PEI", Sigma Chemical Co., St. Louis, MO) of the same size and from the same lot.
  • Therapeutic agent selection [0150] For the present experiment, an 8.5 kilobase plasmid (pSport-based template,
  • Group labeled AT 8 micrograms of ⁇ gal plasmid (p/CMV-sport- ⁇ gal) per final aliquot (i.e. 240 micrograms total) and composite nonpeptidyl carrier PEJR ("PEIR") at a charge ratio of 5:1 were mixed to homogeneity and diluted to 600 microliters with Tris-EDTA buffer. The resulting composition was mixed to homogeneity with 5.4 ml of Cetaphil and aliquoted in 200 microliter portions for in vivo experiments.
  • PEIR composite nonpeptidyl carrier
  • Group labeled AU 8 micrograms of ⁇ gal plasmid (p/CMV-sport- ⁇ gal) per final aliquot (i.e. 240 micrograms total) and highly purified Essentia nonpeptidyl carrier PEIR ("pure PEIR”) at a charge ratio of 5:1 were mixed to homogeneity and diluted to 600 microliters with Tris-EDTA buffer. The resulting composition was mixed to homogeneity with 5.4 ml of Cetaphil and aliquoted in 200 microliter portions for in vivo experiments.
  • pure PEIR highly purified Essentia nonpeptidyl carrier PEIR
  • Treated segments were divided into three equal portions the cranial portion was fixed in 10% neutral buffered formalin for 12-16 hours then stored in 70% ethanol until paraffin embedding. The central portion was snap-frozen and employed directly for beta-galactosidase staining at 37 degrees Celsius on sections as previously described. The treated caudal segment was snap frozen for solubilization studies.
  • Sections stained as above were photographed in their entirety on a Nikon E600 microscope with plan-apochromat lenses. Resulting images underwent batch image analysis processing using Image Pro Plus software with manual confirmation to determine area positive for beta- galactosidase enzyme activity. These results were normalized to total cross-sectional area for each and tabulated as percent cross-sectional positive staining. Subsequently, mean and standard error were subsequently determined for each group with analysis of significance at 95% confidence in one way ANOVA repeated measures using Statview software (Abacus, Berkeley, CA).
  • Results are summarized in the table below and illustrated in Figure 6.
  • the nonpeptidyl transdermal delivery carrier - in both a composite form and in an ulfrapure form - achieved statistically significant increases in delivery efficiency and transgene expression versus PEI.
  • the ulfrapure form of PEIR exhibited trending toward higher efficiencies than standard PEIR consistent with the higher calculated specific activity of the reagent.
  • the nonpeptidyl transdermal carrier can transport large complexes across skin with high efficiencies, particularly given the constraints of transgene expression and total complex size discussed previously. While the efficiencies are not as great as those obtained with the smaller complexes of the peptidyl carriers), significant gains were accomplished. Of note, the distribution of transgene expression using the large nonpeptidyl complexes was almost exclusively hair follicle-based, while the results for the peptidyl carriers were diffuse throughout the cross-sections. Thus, size and backbone tropism can be employed for a nano- mechanical targeting of delivery.
  • This experiment demonstrates the use of a peptidyl carrier to transport a large complex containing an intact labeled protein botulinum toxin across intact skin after a single time administration relative to controls.
  • Botulinum toxin was chosen here as a model system for large proteins, such as agents for immunleation, for example.
  • -Gly 3 Arg 7 to polylysine (MW 112,000) via the carboxyl of the terminal glycine to free amines of the lysine side chains at a degree of saturation of 18% (i.e., 18 out of each 100 lysine residues is covalently attached to a -Gly 3 Arg 7 ).
  • the modified backbone was designated "KNR".
  • the control polycation was unmodified polylysine (designated "K", Sigma Chemical Co., St. Louis, MO) of the same size and from the same lot.
  • Botox® brand of botulinum toxin A (Allergan) was selected for this experiment. It has a molecular weight of approximately 150,000. Preparation of samples: [0159] The botulinum toxin was reconstituted according to the manufacturer's instructions. An aliquot of the protein was biotinylated with a calculated 12-fold molar excess of sulfo-NHS-LC biotin (Pierce Chemical). The labeled product was designated
  • JMW-8 2.0 units of Btox-b per aliquot (i.e. 20 U total) and K at a charge ratio of 4:1 were mixed to homogeneity and diluted to 200 microliters with phosphate buffered saline. The resulting composition was mixed to homogeneity with 1.8 ml of Cetaphil and aliquoted in 200 microliter portions.
  • Biotin visualization was conducted as follows. Briefly, each section was immersed for 1 hour in NeufrAvidin® buffer solution. To visualize alkaline phosphatase activity, cross sections were washed in saline four times then immersed in NBT/BCIP (Pierce Scientific) for 1 hour. Sections were then rinsed in saline and photographed in entirety on a Nikon E600 microscope with plan-apochromat lenses.
  • Total positive staining was determined by blinded observer via batch image analysis using Image Pro Plus software (Media Cybernetics, Silver Spring, MD) and was normalized to total cross-sectional area to determine percent positive staining for each. Mean and standard error were subsequently determined for each group with analysis of significance at 95% confidence in one way ANOVA repeated measures using Statview software (Abacus, Berkeley, CA).
  • Table 4 Mean and standard error for labeled botulinum toxin area as percent of total cross- section after single time topical adminisfration of Btox-b with KNR (JMW-7) or K (JMW-8) for 30 minutes.
  • Example 5 demonstrated that the peptidyl transdermal carrier allowed efficient transfer of botulinum toxin after topical administration in a murine model of intact skin. However, this experiment did not indicate whether the complex protein botulinum toxin was released in a functional form after translocation across skin. The following experiment was thus constructed to evaluate whether botulinum toxin can be therapeutically delivered across intact skin as a topical agent using this peptidyl carrier (again, without covalent modification of the protein).
  • -Gly 3 Arg 7 to polylysine MW 112,000 via the carboxyl of the terminal glycine to free amines of the lysine side chains at a degree of saturation of 18% (i.e., 18 out of each 100 lysine residues is covalently attached to a -Gly 3 Arg 7 ).
  • the modified backbone was designated "KNR”.
  • Control polycation was unmodified polylysine (designated "K", Sigma Chemical Co., St. Louis, MO) of the same size and from the same lot.
  • the same botulinum toxin therapeutic agent was used as in Example 5, and was prepared in the same manner.
  • Samples were prepared as follows: Group labeled "JMW-9": 2.0 units of botulinum toxin per aliquot (i.e. 60 U total) and peptidyl carrier KNR at a calculated MW ratio of 4:1 were mixed to homogeneity and diluted to 600 microliters with phosphate buffered saline. The resulting composition was mixed to homogeneity with 5.4 ml of Cetaphil and aliquoted in 200 microliter portions.
  • JMW-9 2.0 units of botulinum toxin per aliquot (i.e. 60 U total) and peptidyl carrier KNR at a calculated MW ratio of 4:1 were mixed to homogeneity and diluted to 600 microliters with phosphate buffered saline. The resulting composition was mixed to homogeneity with 5.4 ml of Cetaphil and aliquoted in 200 microliter portions.
  • JMW-10 2.0 units of botulinum toxin per aliquot (i.e. 60 U total) and K at a charge ratio of 4:1 were mixed to homogeneity and diluted to 600 microliters with phosphate buffered saline. The resulting composition was mixed to homogeneity with 5.4 ml of Cetaphil and aliquoted in 200 microliter portions.
  • JMW-11 2.0 units of botulinum toxin per aliquot (i.e. 60 U total) without polycation was diluted to 600 microliters with phosphate buffered saline. The resulting composition was mixed to homogeneity with 5.4 ml of Cetaphil and aliquoted in 200 microliter portions. Animal experiments to determine therapeutic efficacy after single time treatment with peptidyl carriers and botulinum toxin:
  • mice consistently ambulated toward a paralyzed limb (which occurred in 100% of treated animals and 0% of controls from either control group).
  • a limb treated with botulinum toxin plus the control polycation polylysine or with botulinum toxin without polycation can mobilize digits (as a defense mechanism when picked up), but the limbs treated with botulinum toxin plus the peptidyl carrier KNR ("Essentia Btox lotion”) could not be moved.
  • Table 5 Digital abduction scores 30 minutes after single-time topical application of botulinum toxin with the peptidyl carrier KNR ("JMW-9”), with a control polycation K (“JMW-10”), or alone (“JMW-11").
  • the positively charged backbone was assembled by covalently attaching
  • PEI polyethyleneimine
  • the modified backbone was designated "PEIR” to denote the large nonpeptidyl carrier.
  • Control polycation was unmodified PEI (designated "PEI", Sigma Chemical Co., St. Louis, MO) of the same size and from the same lot.
  • the same botulinum toxin therapeutic agent was used as in example 5.
  • Botulinum toxin was reconstituted from the BOTOX® product according to the manufacturer's instructions. In each case, an excess of polycation was employed to assemble a final complex that had an excess of positive charge as in delivery of highly negative large nucleotide complexes. A net neutral or positive charge prevents repulsion of the protein complex from highly negative cell surface proteoglycans and extracellular matrix.
  • the botulinum toxin dose was standardized across all groups as was total volume and final pH of the composition to be applied topically. Samples were prepared as follows: Group labeled "AZ": 2.0 units of botulinum toxin per aliquot (i.e.
  • BA 2.0 units of botulinum toxin per aliquot (i.e. 60 U total) and PEI at a charge ratio of 5:1 were mixed to homogeneity and diluted to 600 microliters with phosphate buffered saline. The resulting composition was mixed to homogeneity with 5.4 ml of Cetaphil and aliquoted in 200 microliter portions. .
  • Table 7 Repetition 2. Digital abduction scores 30 minutes after single-time topical administration of Botulinum toxin with ulfrapure PEIR ("AZ1"), or control polycation PEI ("BA1"). Mean and standard error are presented.
  • AZ1 Botulinum toxin with ulfrapure PEIR
  • BA1 control polycation PEI
  • botulinum toxin penetration with either peptidyl or nonpeptidyl carriers versus topical botulinum toxin without the carrier further establishes utility for transdermal penetration of antigens for immunization, particularly for immunization with antigens that cross skin poorly otherwise such as botulinum.
  • Delivery o a functional botulinum toxin ensures that at least four distinct epitopes have been delivered transdermally in an intact state; the fact that functional botulinum toxin was not delivered in the absence of the carrier in either example confirms that the carrier affords significant immunization potential relative to the agent in the absence of the carrier.
  • TAT efficiency factors as well as assembly of these carriers with botulinum toxins.
  • Biosciences Corp., Piscataway, NJ was slu ⁇ ied in sterile lx PBS.
  • the column was standardized by elution of FITC dexfrans (Sigma, St Louis, MO) having 19kD molecular weight.
  • the lyophilized reaction mixture from above was dissolved in a small volume PBS and applied to the column. It was eluted by successive applications of 1 ml PBS. Fractions were collected with the first one consisting of the first 3 ml eluted, including the reaction volume. Subsequent fractions were 1 ml.
  • the modified backbone was designated "KNR2".
  • the control polycation was unmodified polylysine (designated “K2", Sigma Chemical Co., St. Louis, MO) of the same size and from the same lot.
  • K2 Sigma Chemical Co., St. Louis, MO
  • a murine monoclonal antibody to a conserved human melanoma domain, ganglioside 2, (IgG3, US Biologicals, Swampscott, MA) was covalently attached to a short polyaspartate anion chain (MW 3,000) via EDC coupling as above to generate a derivatized antibody designated "Gang2As ⁇ ".
  • an anionic imaging agent was designed using an oligonucleotide as a polyanion wherein the sequence was ATGC-J (designated “ATGC-J” henceforth) with "J” representing a covalently attached Texas Red fluorophore, (Sigma Genosys, Woodlands, TX).
  • ATGC-J designated "ATGC-J” henceforth
  • J representing a covalently attached Texas Red fluorophore
  • the resulting complexes were applied to hydrated CellTek Human Melanoma slides and control CellTek Cytokeratin Slides (SDL, Des Plaines, IL) and incubated for 5 minutes before photographic evaluation of fluorescence distribution versus brightfield distribution of melanoma pigment in the same field. Additional controls without ATGC-J or without Gang2Asp were also employed.
  • Revitix proteins [Organogenesis, Canton, MA] were biotinylated and stored at
  • a 2.0 cm x 2.0 cm dose site on the dorsum of each mouse was carefully shaved with a hair clipper (Oster) two days before the first day of treatment application. Animals did not undergo further depilatory treatment. Animals were anesthetized via inhalation of isoflurance only during the application of treatments in Cetaphil moisturizing cream (Galderma, Fort Worth ,TX) and had metered 200 microliter doses of the appropriate treatment applied to the cranial portion of dorsal back skin (selected because the mouse cannot reach this region with mouth or limbs).
  • mice were euthanized via inhalation of CO 2 , and freated skin segments were harvested at full thickness by blinded observers at 8 hours post application of the last treatment. Treated segments were divided into three equal portions the cranial portion was fixed in 10% neutral buffered formalin for 12-16 hours then stored in 70% ethanol until paraffin embedding. The central portion was employed for NeutrAvidin, Hematoxylin & Eosin, and Chloroesterase-specific staining. The freated caudal segment was snap frozen for solubilization studies.
  • Revitix proteins were labeled with biotin and good labeling on variety of proteins was shown.
  • NeutrAvidin staining was used to determine transdermal delivery of Revitix protein.
  • the photographs of control group (panel a and c and e) vs. treatment group (b and d and f) at two different magnifications are shown in Figure 10, where a and b are at 1 OX magnification and c through f are at 20X magnification.
  • the mean for positive NeutrAvidin staining was used for comparison.
  • the positively charged peptidyl backbone was assembled by covalently attaching ⁇ -Arg to polylysine (MW 150,000) via the carboxyl of the terminal glycine to free amines of the lysine sidechains at a degree of saturation of 18% (i.e., 18 out of each 100 lysine residues is covalently attached to a -Arg ).
  • the modified backbone was designated "KNR".
  • the control polycation was unmodified polylysine (designated "K", Sigma Chemical Co., St. Louis, MO) of the same size and from the same lot.
  • the probe is a multi-component system that self-assembles based on electrostatic interactions. Such a system allows for easy substitution of functional moieties.
  • the central component is a carrier backbone that has an excess of positive charges and multiple CPPs attached. All cargos are negatively charged.
  • the final complex has a net positive charge to maintain transport activity.
  • Targeting moieties a) Monoclonal antibody to carcinoembryonic antigen (CEA; clone CD66e, US Biological) covalently conjugated to anionic pofyaspartate (3K MW) via EDC coupling; b) Confrol — Monoclonal antibody to actin (clone 3G1, US Biological) conjugated to polyaspartate
  • KNR achieved 20X greater efficiency in transdermal delivery and fransgene expression of BFP versus confrol (5% ⁇ 2.12% vs. 0.25% ⁇ 0.06%, PO.01), validating the feasibility of topical delivery of complexes large enough for molecular imaging.
  • fluorescein and TR signals even though each was a distinct component of the complex, co-localized in 40.2% of pixels of the colon carcinoma cells (phi correlation 0.74, PO.001).
  • Control fibroblast cells were minimally labeled with fluorescein or TR while 87.6% ⁇ 8.3% of colon carcinoma cells were positive for fluorescein signal.
  • Relative toxicity for carrier backbones results are shown in Figure 11 and trandermal gene delivery efficiency results are shown in Figure 12.

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JP2011507558A (ja) * 2007-08-31 2011-03-10 アムジエン・インコーポレーテツド 固体タンパク質製剤
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US10201594B2 (en) 2012-10-28 2019-02-12 Revance Therapeutics, Inc. Compositions and methods for safe treatment of rhinitis
US11484580B2 (en) 2014-07-18 2022-11-01 Revance Therapeutics, Inc. Topical ocular preparation of botulinum toxin for use in ocular surface disease
EP3753568A1 (en) 2019-06-21 2020-12-23 Fastox Pharma SA Composition modulating botulinum neurotoxin effect
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KR101284710B1 (ko) 2013-07-23
CN101083907A (zh) 2007-12-05
JP2015108006A (ja) 2015-06-11
WO2005120546A3 (en) 2007-07-19
CA3031270A1 (en) 2005-12-22
AU2005251676B2 (en) 2011-09-29
NZ593986A (en) 2013-07-26
US20050239705A1 (en) 2005-10-27
CN101083907B (zh) 2012-10-10
JP2007527431A (ja) 2007-09-27
BRPI0508440A (pt) 2007-07-24
EP1734984A2 (en) 2006-12-27
US8092788B2 (en) 2012-01-10
US10172877B2 (en) 2019-01-08
AU2005251676A1 (en) 2005-12-22
SG150570A1 (en) 2009-03-30
US8974774B2 (en) 2015-03-10
CR20110355A (es) 2011-09-20
US20150320781A1 (en) 2015-11-12
CA2558676A1 (en) 2005-12-22
US20210069224A1 (en) 2021-03-11
MXPA06009897A (es) 2008-02-07
KR20070024502A (ko) 2007-03-02
IL177815A0 (en) 2006-12-31
NO20064414L (no) 2006-12-04
CA2558676C (en) 2019-04-16
IL177815A (en) 2017-07-31
US20200101097A1 (en) 2020-04-02
US20080038203A1 (en) 2008-02-14

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