WO2014123543A2 - Peptides entrant dans les cellules et traversant la peau (space) et leurs procédés d'utilisation - Google Patents

Peptides entrant dans les cellules et traversant la peau (space) et leurs procédés d'utilisation Download PDF

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Publication number
WO2014123543A2
WO2014123543A2 PCT/US2013/025543 US2013025543W WO2014123543A2 WO 2014123543 A2 WO2014123543 A2 WO 2014123543A2 US 2013025543 W US2013025543 W US 2013025543W WO 2014123543 A2 WO2014123543 A2 WO 2014123543A2
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WIPO (PCT)
Prior art keywords
peptide
composition
carrier
active agent
seq
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PCT/US2013/025543
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English (en)
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WO2014123543A3 (fr
Inventor
John A. MURASKI Jr.
Samir Mitragotri
Ming Chen
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Convoy Therapeutics
The Regents Of The University Of California
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Priority to PCT/US2013/025543 priority Critical patent/WO2014123543A2/fr
Publication of WO2014123543A2 publication Critical patent/WO2014123543A2/fr
Publication of WO2014123543A3 publication Critical patent/WO2014123543A3/fr

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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
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/728—Hyaluronic acid
    • 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
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A61K38/13—Cyclosporins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links
    • C07K7/645—Cyclosporins; Related peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • compositions comprising the presently disclosed peptides and/or conjugates, wherein the compositions are capable of penetrating a stratum corneum (SC) layer when contacted therewith or penetrating a cell when contacted therewith, as well as methods for employing the claimed peptides, conjugates, and/or compositions to deliver active agents to subjects.
  • SC stratum corneum
  • Skin the largest organ of the human body, is a host to numerous dermatological diseases which collectively represent a large category of human health conditions. Accordingly, successful delivery of therapeutics, e.g., macromolecules such as siRNA, into skin has become a topic of active research and development. The goal of topical siRNA delivery, however, is extremely challenging and with some exceptions, has been very difficult to accomplish. The primary challenge is poor skin penetration of macromolecules. Among various physico- chemical methods proposed to enhance penetration of macromolecules, peptide carriers have emerged as potential candidates owing to their simplicity of use, diversity and potential ability to target cellular sub-types within the skin.
  • macromolecules such as siRNA
  • peptides including TAT, polyarginine, meganin, and penetratin which were initially identified for delivering drugs into the cytoplasm of cells, have been tested for penetration across the stratum corneum (SC) and a few have shown some efficacy in delivering small molecules into the epidermis.
  • SC stratum corneum
  • TD-1 only one peptide, has been specifically shown to penetrate the SC and possess the ability to enhance systemic uptake of topically applied drugs.
  • peptides are known to penetrate cellular membranes and a few to penetrate the SC, peptides that simultaneously enhance the penetration of macromolecules and other actives across the SC and/or across the cellular membranes of viable epidermal and dermal cells are needed.
  • the presently disclosed subject matter provides compositions comprising a peptide, an active agent, and a carrier comprising the active agent.
  • the peptide comprises an amino acid sequence set forth in any of SEQ ID NOs: 1-18; the peptide is associated with and/or conjugated to the active agent, the carrier, or both; the carrier is selected from the group consisting of a micelle, a liposome, an ethosome, and combinations thereof; and/or the composition is capable of penetrating a stratum corneum (SC) layer when contacted therewith or penetrating a cell when contacted therewith, and optionally wherein the composition further comprises one or more free peptides comprising an amino acid sequence set forth in any of SEQ ID NOs: 1-18.
  • SC stratum corneum
  • the composition is capable of penetrating the SC layer and penetrating the cell.
  • the peptide is a cyclic peptide comprising an amino acid sequence as set forth in any of SEQ ID NOs: 7-18 and a Cys-Cys disulfide bond.
  • the composition is capable of penetrating the cellular membrane of viable non-human animal cells; viable human cells; viable epidermal or dermal cells; and/or viable immunological cells.
  • the active agent comprises a macromolecule, optionally a protein, a nucleic acid, a pharmaceutical compound, a detectable moiety, a small molecule, and/or a nanoparticle.
  • the protein comprises an antibody or a fragment thereof comprising at least one paratope.
  • the macromolecule comprises a nucleic acid, optionally DNA or RNA, and further optionally wherein the nucleic acid is an interfering RNA, an shRNA, an miRNA, or an siRNA.
  • the siRNA is designed to interfere with expression of a gene product selected from the group consisting of an IL-10 gene product, an IL-4 gene product, an CD86 gene product, a KRT6a gene product, a TNFR1 gene product, and a TACE gene product.
  • the siRNA is a mutation-specific siRNA.
  • the pharmaceutical compound is cyclosporin A (CsA) or hyaluronic acid (HA).
  • the pharmaceutical compound is CsA
  • the CsA is encapsulated by the carrier
  • the peptide is conjugated to the carrier.
  • the carrier is an ethosome and the composition further comprises one or more free peptides comprising an amino acid sequence set forth in any of SEQ ID NOs: 1-18.
  • the active agent comprises a detectable agent, optionally a fluorescent label or a radioactive label.
  • the presently disclosed subject matter also provides compositions comprising a peptide, an active agent, and a carrier comprising the active agent, wherein the peptide comprises an amino acid sequence set forth in any of SEQ ID NOs: 1-18; the peptide is associated with an active agent and/or a carrier comprising the active agent, wherein the association results from hydrophobic, electrostatic or van der Walls interactions; the carrier is selected from the group consisting of a micelle, a liposome, an ethosome, and combinations thereof; and the composition is capable of penetrating a stratum corneum (SC) layer when contacted therewith or penetrating a cell when contacted therewith, and further wherein the composition optionally comprises one or more free peptides comprising an amino acid sequence set forth in any of SEQ ID NOs: 1-18.
  • the peptide is a cyclic peptide comprising (i) an amino acid sequence as set forth in any of SEQ ID NOs: 7-18, and (i
  • the presently disclosed subject matter also provides in some embodiments methods for delivering an active agent to a subject.
  • the methods comprise administering to the subject a composition comprising a peptide comprising an amino acid sequence set forth in any of SEQ ID NOs: 1- 8, wherein the peptide is conjugated to an active agent or an active agent carrier comprising the active agent and/or is associated with an active agent and/or a carrier comprising the active agent, wherein the association results from hydrophobic, electrostatic or van der Walls interactions; the carrier is selected from the group consisting of a micelle, a liposome, an ethosome, and combinations thereof; and the composition is capable of penetrating the stratum corneum (SC) of the subject or penetrating a cell of the subject, and optionally wherein the composition further comprises one or more free peptides comprising an amino acid sequence set forth in any of SEQ ID NOs: 1-18.
  • SC stratum corneum
  • the composition is formulated for topical administration.
  • the peptide is a cyclic peptide comprising an amino acid sequence as set forth in any of SEQ ID NOs: 7-18 and a Cys-Cys disulfide bond.
  • the composition is capable of penetrating the cellular membrane of viable non-human animal cells, viable human cells, viable epidermal cells, viable dermal cells, and/or viable immunological cells.
  • the active agent comprises a macromolecule, optionally a protein, a nucleic acid, a pharmaceutical compound, a detectable moiety, a small molecule, and/or a nanoparticle.
  • the protein comprises an antibody or a fragment thereof comprising at least one paratope.
  • the macromolecule comprises a nucleic acid, optionally a DNA molecule.
  • the nucleic acid is NA, optionally an interfering RNA, further optionally an shRNA, an miRNA, or an siRNA.
  • the siRNA is designed to interfere with expression of a gene product selected from the group consisting of an IL-10 gene product, an IL-14 gene product, an CD86 gene product, a KRT6a gene product, a TNFR1 gene product, and a TACE gene product.
  • the siRNA is a mutation-specific siRNA.
  • the pharmaceutical compound is cyclosporin A (CsA) or hyaluronic acid (HA).
  • the pharmaceutical compound is CsA
  • the CsA is encapsulated by the carrier
  • the peptide is conjugated to the carrier.
  • the carrier is an ethosome and the composition further comprises one or more free peptides comprising an amino acid sequence set forth in any of SEQ ID NOs: 1-18.
  • the methods comprise administering to the subject a composition comprising a peptide comprising an amino acid sequence set forth in any of SEQ ID NOs: 1-18, wherein the peptide is conjugated to an active agent or an active agent carrier comprising the active agent and/or is associated with an active agent and/or a carrier comprising the active agent, wherein the association results from hydrophobic, electrostatic or van der Walls interactions; the carrier is selected from the group consisting of a micelle, a liposome, an ethosome, and combinations thereof; and the composition is capable of penetrating the stratum corneum (SC) of the subject or penetrating a cell of the subject, and optionally wherein the composition further comprises one or more free peptides comprising an amino acid sequence set forth in any of SEQ ID NOs: 1-18.
  • SC stratum corneum
  • the composition is formulated for topical administration.
  • the peptide is a cyclic peptide comprising (i) an amino acid sequence as set forth in any of SEQ ID NOs: 7-18; and (ii) a Cys-Cys disulfide bond.
  • the composition is capable of penetrating the cellular membrane of viable non-human animal cells, viable human cells, viable epidermal cells, viable dermal cells, and/or viable immunological cells.
  • the active agent comprises a macromolecule, optionally a protein, a nucleic acid, a pharmaceutical compound, a detectable moiety, a small molecule, and/or a nanoparticle.
  • the protein comprises an antibody or a fragment thereof comprising at least one paratope.
  • the macromolecule comprises a nucleic acid, optionally a DNA molecule.
  • the nucleic acid is RNA, optionally an interfering RNA, further optionally an shRNA, an miRNA, or an siRNA.
  • the siRNA is designed to interfere with expression of a gene product selected from the group consisting of an IL-10 gene product, an IL-14 gene product, an CD86 gene product, a KRT6a gene product, a TNFR1 gene product, and a TACE gene product.
  • the siRNA is a mutation-specific siRNA.
  • the pharmaceutical compound is cyclosporin A (CsA) or hyaluronic acid (HA).
  • the pharmaceutical compound is CsA
  • the CsA is encapsulated by the carrier
  • the peptide is conjugated to the carrier.
  • the carrier is an ethosome and the composition further comprises one or more free peptides comprising an amino acid sequence set forth in any of SEQ ID NOs: 1-18.
  • the presently disclosed subject matter also provides methods for treating a subject having, suspected of having, and/or susceptible to a disorder resulting at least in part from expression of an mRNA.
  • the methods comprise administering to the subject a composition comprising a peptide comprising an amino acid sequence set forth in any of SEQ ID NOs: 1-18, wherein the peptide is conjugated to an interfering RNA which targets the mRNA or an active agent carrier comprising an interfering RNA which targets the mRNA and/or is associated with an interfering RNA which targets the mRNA and/or a carrier comprising an interfering RNA which targets the mRNA, wherein the association results from hydrophobic, electrostatic or van der Walls interactions; the carrier is selected from the group consisting of a micelle, a liposome, an ethosome, and combinations thereof; and the composition is capable of penetrating the stratum corneum (SC) of the subject or penetrating a cell of the subject, and optionally wherein the composition
  • the composition is formulated for topical administration.
  • the composition is capable of penetrating the cellular membrane of viable non-human animal cells, viable human cells, viable epidermal cells, viable dermal cells, and/or viable immunological cells.
  • the peptide is a cyclic peptide comprising (i) an amino acid sequence as set forth in any of SEQ ID NOs: 7-18; and (ii) a Cys-Cys disulfide bond.
  • the active agent comprises a macromoiecule, optionally a protein, a nucleic acid, a pharmaceutical compound, a detectable moiety, a small molecule, and/or a nanoparticle.
  • the protein comprises an antibody or a fragment thereof comprising at least one paratope.
  • the macromoiecule comprises a nucleic acid, optionally a DNA molecule.
  • the nucleic acid is RNA, optionally an interfering RNA, further optionally an shRNA, an miRNA, or an siRNA.
  • the siRNA is designed to interfere with expression of a gene product selected from the group consisting of an IL-10 gene product, an CD86 gene product, a KRT6a gene product, a TNFR1 gene product, and a TACE gene product.
  • the siRNA is a mutation-specific siRNA.
  • the pharmaceutical compound is cyclosporin A (CsA) or hyaluronic acid (HA).
  • the pharmaceutical compound is CsA
  • the CsA is encapsulated by the carrier
  • the peptide is conjugated to the carrier.
  • the carrier is an ethosome and the composition further comprises one or more free peptides comprising an amino acid sequence set forth in any of SEQ ID NOs: 1-18.
  • the presently disclosed subject matter also provides in some embodiments methods for attenuating expression of an mRNA of a subject in need thereof.
  • the methods comprise administering to the subject a composition comprising a peptide comprising an amino acid sequence set forth in any of SEQ ID NOs: 1-18, wherein the peptide is conjugated to an interfering RNA which targets the mRNA or an active agent carrier comprising an siRNA which targets the mRNA and/or is associated with an siRNA which targets the mRNA and/or a carrier comprising an siRNA which targets the mRNA, wherein the association results from hydrophobic, electrostatic or van der Walls interactions; the carrier is selected from the group consisting of a micelle, a liposome, an ethosome, and combinations thereof; and the composition is capable of penetrating the stratum corneum (SC) of the subject or penetrating a cell of the subject, and optionally wherein the composition further comprises one or more free peptides comprising an amino acid sequence set forth in any of
  • the composition is formulated for topical administration.
  • the composition is capable of penetrating the cellular membrane of viable non-human animal cells, viable human cells, viable epidermal cells, viable dermal cells, and/or viable immunological cells.
  • the active agent comprises a macromolecule, optionally a protein, a nucleic acid, a pharmaceutical compound, a detectable moiety, a small molecule, and/or a nanoparticle.
  • the protein comprises an antibody or a fragment thereof comprising at least one paratope.
  • the macromolecule comprises a nucleic acid, optionally a DNA molecule.
  • the nucleic acid is RNA, optionally an interfering RNA, further optionally an shRNA, an miRNA, or an siRNA.
  • the siRNA is designed to interfere with expression of a gene product selected from the group consisting of an IL-10 gene product, an CD86 gene product, a KRT6a gene product, a TNFR1 gene product, and a TACE gene product.
  • the siRNA is a mutation-specific siRNA.
  • the pharmaceutical compound is cyclosporin A (CsA) or hyaluronic acid (HA).
  • the pharmaceutical compound is CsA
  • the CsA is encapsulated by the carrier
  • the peptide is conjugated to the carrier.
  • the carrier is an ethosome and the composition further comprises one or more free peptides comprising an amino acid sequence set forth in any of SEQ ID NOs: 1-18.
  • compositions comprising a peptide an active agent, and a carrier comprising the active agent.
  • the peptide consists essentially of an amino acid sequence set forth in any of SEQ ID NOs: 1-18; the peptide is conjugated to the active agent, the carrier, or both; the carrier is selected from the group consisting of a micelle, a liposome, an ethosome, and combinations thereof; and/or the composition is capable of penetrating a stratum corneum (SC) layer when contacted therewith or penetrating a cell when contacted therewith.
  • the composition optionally comprises one or more free peptides comprising an amino acid sequence set forth in any of SEQ ID NOs: 1-18.
  • the presently disclosed subject matter also provides in some embodiments the presently disclosed compositions formulated for use in a cosmetic preparation.
  • the formulated composition has a pH of from about 2 to about 10, optionally of from about 4 to about 8.
  • compositions and methods for delivering active agents to subjects are provided.
  • Figure 1 is an exemplary scheme for conjugating a SPACE Peptide of the presently disclosed subject matter to Cyclosporin A (CsA) employing a CsA epoxide intermediate.
  • CsA Cyclosporin A
  • Figures 2A-2F are infrared (IR) spectra ( Figures 2A-2E) and mass spectrometry (Figure 2F) traces of reactants, intermediates, and products from an exemplary synthesis using the scheme of Figure 1.
  • Figure 2A is an IR spectrum trace of the SPACE Peptide powder showing a characteristic absorption band at about 700-800 cm "1 (circled).
  • Figure 2B is an IR spectrum trace of the CsA powder showing a characteristic absorption band at about 2900 cm "1 (circled).
  • Figure 2C is an IR spectrum trace of the conjugation powder showing characteristic SPACE Peptide absorption band at about 700-800 cm “1 (circled) and the characteristic CsA absorption band at 2900 cm “1 (circled).
  • Figure 2D is a comparison of IR spectrum traces comparing the SPACE Peptide and final conjugation products showing that the conjugation product has the same characteristic absorption band as the SPACE Peptide does (circled area on right side of Figure 2D).
  • the circled area on the left side of Figure 2D is the characteristic CyA absorption band present in the CsA- SPACE trace that is absent in the SPACE Peptide trace.
  • Figure 2E is a comparison of IR spectrum traces comparing the CsA and final conjugation products showing that the conjugation product also has the same characteristic absorption band as does CsA (circled area on the left side of Figure 2E).
  • the circled area on the right side of Figure 2E shows that CsA does not have a band characteristic of SPACE peptide.
  • Figure 2F is a mass spectrometry trace of reactants (SPACE Peptide (SPACE) and an epoxide of cyclosporin A (CsA-Epoxide)) and a product (SPACE Peptide-conjugated cyclosporin A (CsA-SP)) of the presently disclosed subject matter.
  • Figures 3A and 3B are schematic diagrams of in vitro skin penetration tests that can be employed for testing the abilities of the compositions of the presently disclosed subject matter to deliver active agents to various layers of the skin.
  • Figure 4 is a series of bar graphs showing the results of employing a SPACE
  • FIG. 5 is a schematic representation of the measurement of partitioning of SPACE peptide.
  • FITC-SPACE peptide was incubated epidermis-SC and dermis for 48 hours at 37°C and the amount of SPACE peptide partitioned into the tissue was measured.
  • Figure 6 is a schematic diagram of an exemplary generalized method for preparing SPACE Peptide/lipid conjugates.
  • Figure 7 is a schematic diagram of an exemplary generalized method for preparing SPACE Peptide/lipid conjugates and SPACE Peptide-conjugated ethosomes. It is noted that in Figure. 7, the specific concentrations listed are exemplary only and are not intended to limit the generalized methods depicted.
  • Figures 8A-8C are a series of bar graphs depicting the results of delivery of different SPACE-associated cargos to the skin and different compartments thereof.
  • Figure 8A is a bar graph depicting delivery of FITC by a composition containing a FITC-conjugated SPACE Peptide (bar A); a composition containing a FITC- conjugated SPACE Peptide in conjunction with free SPACE Peptide (bar B); a composition containing a SPACE Peptide-conjugated liposome encapsulating FITC (bar C); a composition containing a SPACE Peptide-conjugated ethosome encapsulating FITC (bar D); and a composition containing a SPACE Peptide- conjugated ethosome encapsulating FITC in conjunction with free SPACE Peptide (bar E).
  • Figure 8B is a bar graph depicting delivery of FITC by Compositions A-E of Figure 8A to various layers of the skin. In each of the five sets of bars, the delivery to SC-1 , SC-2, SC-3, epidermis, dermis, and the receiver, left to right respectively, is depicted.
  • Figure 8C is a bar graph showing delivery of a fluorescent label to EPIDERMTM (blue; left column of each pair) and a pig skin model (pink; right column of each pair) using different formulations the contain SPACE Peptides.
  • a FITC-labeled SPACE Peptide penetrated into EPIDERMTM, encapsulation of the FITC-labeled SPACE Peptide into a SPACE Peptide- conjugated ethosome further enhanced penetration in to EPIDERMTM, and the addition of a free SPACE Peptide into the formulation further enhanced skin penetration both in EPIDERMTM and in the pig skin model.
  • Figure 9 is a depiction of an exemplary SPACE Peptide-conjugated ethosome that, in some embodiments, can be employed to deliver cyclosporin A to the skin.
  • the general characteristics for the SPACE Peptide-conjugated ethosome listed in the Figure are intended to be exemplary only.
  • Figure 10 is a bar graph depicting delivery of cyclosporin A (CsA) encapsulated in an exemplary SPACE Peptide-conjugated ethosome composition (Etsm/CsA) into various layers of the skin.
  • CsA cyclosporin A
  • Etsm/CsA SPACE Peptide-conjugated ethosome composition
  • Figures 11A-11 D are a series of bar graphs depicting delivery of hyaluronic acid (HA) using the SPACE-Peptide conjugates of the presently disclosed subject matter.
  • Figure 11A is a bar graph depicting delivery of HA encapsulated in a SPACE Peptide-conjugated ethosome (Etsm/HA) to various skin layers using HA encapsulated in a SPACE Peptide-conjugated ethosome (Etsm/HA).
  • Figure 11 B is a bar graph comparing delivery of HA encapsulated in a SPACE Peptide-conjugated ethosome (Etsm/HA) to various skin layers in the presence or absence of Free SPACE Peptides.
  • FIG. 11C is a bar graph comparing delivery of HA encapsulated in a SPACE Peptide-conjugated ethosome (Etsm/HA) to various skin layers in the presence or absence of Free SPACE Peptides.
  • the numbers in parentheses in the x-axis labels show the concentration of SPACE-lipid employed in the formulations.
  • Figure 11 D is a bar graph comparing delivery of HA encapsulated in a SPACE Peptide-conjugated ethosome (Etsm/HA) to various skin layers in the presence or absence of Free SPACE Peptides at pH 4 or at pH 8.
  • Figures 12A and 12B are bar graphs depicting delivery of an exemplary siRNA encapsulated in a SPACE Peptide-conjugated ethosome.
  • the y-axis presents a percentage of the 100 ⁇ applied dose that was found in the indicated layers 24 hours after application to a 2 cm 2 sample after 24 hours.
  • SEQ ID NOs: 1-18 are the amino acid sequences of eighteen (18) exemplary SPACE Peptides.
  • the SPACE Peptides are cyclic peptides that include an intrapeptide Cys-Cys disulfide bond.
  • SEQ ID NO. 19 is a nucleotide sequence of an siRNA that is targeted to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). In some embodiments, SEQ ID NO. 19 is modified at the 5'-terminus, the 3'-terminus, or both.
  • active agent refers to an agent, e.g., a protein, peptide, nucleic acid (including, e.g., nucleotides, nucleosides and analogues thereof) or small molecule drug, that provides a desired pharmacological effect upon administration to a subject, e.g., a human or a non-human animal, either alone or in combination with other active or inert components. Included in the above definition are precursors, derivatives, analogues and prodrugs of active agents.
  • active agent can also be used herein to refer generally to any agent, e.g., a protein, peptide, nucleic acid (including, e.g., nucleotides, nucleosides and analogues thereof) or small molecule drug, conjugated or associated with a penetrating peptide as described herein or attached to or encompassed by an active agent carrier as described herein.
  • agent e.g., a protein, peptide, nucleic acid (including, e.g., nucleotides, nucleosides and analogues thereof) or small molecule drug, conjugated or associated with a penetrating peptide as described herein or attached to or encompassed by an active agent carrier as described herein.
  • conjugated refers to a covalent or ionic interaction between two entities, e.g., molecules, compounds or combinations thereof.
  • association refers to a non-covalent interaction between two entities, e.g., molecules, compounds or combinations thereof mediated by one or more of hydrophobic, electrostatic, and van der Walls interactions.
  • polypeptide and protein refer to a polymeric form of amino acids of any length, which can include coded and non- coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
  • the term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and native leader sequences, with or without N- terminal methionine residues; immunologically tagged proteins; fusion proteins with detectable fusion partners, e.g., fusion proteins including as a fusion partner a fluorescent protein, ⁇ -galactosidase, luciferase, etc.; and the like.
  • antibody and “immunoglobulin” include antibodies or immunoglobulins of any isotype, fragments of antibodies which retain specific binding to antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins including an antigen-binding portion of an antibody and a non-antibody protein.
  • the antibodies can be detectably labeled, e.g., with a radioisotope, an enzyme which generates a detectable product, a fluorescent protein, and the like.
  • the antibodies can be further conjugated to other moieties, such as members of specific binding pairs, e.g., biotin (member of biotin-avidin specific binding pair), and the like. Also encompassed by the terms are Fab', Fv, F(ab') 2 , and other antibody fragments that retain specific binding to antigen.
  • Antibodies can exist in a variety of other forms including, for example, Fv, Fab, and (Fab') 2 , as well as bi-functional (i.e., bi-specific) hybrid antibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and in single chains (e.g., Huston ei al., Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883 (1988) and Bird et al., Science, 242, 423-426 (1988), which are incorporated herein by reference). See generally, Hood et al., Immunology. Benjamin, N.Y., 2nd ed. (1984), and Hunkapiller and Hood, Nature, 323, 15-16 (1986).
  • nucleic acid refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • the terms encompass, e.g., DNA, RNA and modified forms thereof.
  • Polynucleotides can have any three-dimensional structure, and can perform any function, known or unknown.
  • Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers.
  • the nucleic acid molecule can be linear or circular.
  • RNA interference is a process by which double-stranded RNA
  • RNAi is used to silence gene expression.
  • siRNAs small interfering RNAs
  • RISC RNA-induced silencing complex
  • siRNA strand uses this siRNA strand to identify mRNA molecules that are at least partially complementary to the incorporated siRNA strand, and then cleaves these target mRNAs or inhibits their translation.
  • the siRNA strand that is incorporated into RISC is known as the guide strand or the antisense strand.
  • the other siRNA strand known as the passenger strand or the sense strand, is eliminated from the siRNA and is at least partially homologous to the target mRNA.
  • siRNA can be designed (e.g., via decreased siRNA duplex stability at the 5' end of the antisense strand) to favor incorporation of the antisense strand into RISC.
  • RISC-mediated cleavage of mRNAs having a sequence at least partially complementary to the guide strand leads to a decrease in the steady state level of that mRNA and of the corresponding protein encoded by the mRNA.
  • RISC can also decrease expression of the corresponding protein via translational repression without cleavage of the target mRNA.
  • Other RNA molecules can interact with RISC and silence gene expression.
  • RNA molecules that can interact with RISC include short hairpin RNAs (shRNAs), single-stranded siRNAs, microRNAs (miRNAs), and dicer-substrate 27-mer duplexes, RNA molecules containing one or more chemically modified nucleotides, one or more deoxyribonucleotides, and/or one or more non-phosphodiester linkages.
  • shRNAs short hairpin RNAs
  • siRNAs single-stranded siRNAs
  • miRNAs microRNAs
  • dicer-substrate 27-mer duplexes RNA molecules containing one or more chemically modified nucleotides, one or more deoxyribonucleotides, and/or one or more non-phosphodiester linkages.
  • siRNA refers to a double-stranded interfering RNA unless otherwise noted.
  • interfering RNAs RNA molecules that can interact with RISC and participate in RISC-mediated changes in gene expression.
  • siRNAs, shRNAs, miRNAs, and dicer-substrate 27- mer duplexes are, therefore, subsets of "interfering RNAs.
  • substitution results from the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively as compared to an amino acid sequence or nucleotide sequence of a polypeptide. If a substitution is conservative, the amino acid that is substituted into a polypeptide has similar structural or chemical properties (which can include, but are not limited to charge, polarity, hydrophobicity, and the like) to the amino acid that it is substituting.
  • amino acid groups are as follows: (1 ) acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; (2) basic (positively charged) amino acids such as arginine, histidine, and lysine; (3) neutral polar amino acids such as glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; and (4) neutral non-polar amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine.
  • polypeptide variants can have "non-conservative" changes, where the substituted amino acid differs in structural and/or chemical properties.
  • a “deletion” is defined as a change in either amino acid or nucleotide sequence in which one or more amino acid or nucleotide residues, respectively, are absent as compared to an amino acid sequence or nucleotide sequence of a naturally occurring polypeptide.
  • a deletion can involve deletion of 2, 5, 10, up to 20, up to 30, or up to 50 or more amino acids, taking into account the length of the polypeptide or polynucleotide sequence being modified, if desired.
  • an “insertion” or “addition” is that change in an amino acid or nucleotide sequence which has resulted in the addition of one or more amino acid or nucleotide residues, respectively, as compared to an amino acid sequence or nucleotide sequence of a naturally occurring polypeptide.
  • “Insertion” generally refers to addition to one or more amino acid residues within an amino acid sequence of a polypeptide, while “addition” can be an insertion or refer to amino acid residues added at the N- or C-termini.
  • an insertion or addition can be of up to 10, up to 20, up to 30, or up to 50 or more amino acids.
  • Non-native “non-endogenous”, and “heterologous”, in the context of a polypeptide, are used interchangeably herein to refer to a polypeptide having an amino acid sequence or, in the context of an expression system or a viral particle, present in an environment different to that found in nature.
  • Exogenous in the context of a nucleic acid or polypeptide is used to refer to a nucleic acid or polypeptide that has been introduced into a host cell.
  • Exogenous nucleic acids and polypeptides can be native or non-native to the host cell, where an exogenous, native nucleic acid or polypeptide provides for elevated levels of the encoded gene product or polypeptide in the recombinant host cell relative to that found in the host cell prior to introduction of the exogenous molecule.
  • the terms “determining”, “measuring”, “assessing”, and “assaying” are used interchangeably and include both quantitative and qualitative determinations.
  • isolated when used in the context of an isolated compound, refers to a compound of interest that is in an environment different from that in which the compound naturally occurs. "Isolated” is meant to include compounds that are within samples that are substantially enriched for the compound of interest and/or in which the compound of interest is partially or substantially purified.
  • substantially pure refers to a compound that is removed from its natural environment and is at least 60% free, 75% free, or 90% free from other components with which it is naturally associated.
  • a “coding sequence” or a sequence that "encodes” a selected polypeptide is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide, for example, in vivo when placed under the control of appropriate regulatory sequences (or “control elements”).
  • the boundaries of the coding sequence are typically determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
  • a coding sequence can include, but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral or prokaryotic DNA, and synthetic DNA sequences.
  • a transcription termination sequence can be located 3' to the coding sequence.
  • Other "control elements" can also be associated with a coding sequence.
  • a DNA sequence encoding a polypeptide can be optimized for expression in a selected cell by using the codons preferred by the selected cell to represent the DNA copy of the desired polypeptide coding sequence.
  • Encoded by refers to a nucleic acid sequence which codes for a gene product, such as a polypeptide.
  • the gene product is a polypeptide
  • the polypeptide sequence or a portion thereof contains an amino acid sequence of at least 3 to 5 amino acids, 8 to 10 amino acids, or at least 15 to 20 amino acids from a polypeptide encoded by the nucleic acid sequence.
  • “Operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function.
  • a promoter that is operably linked to a coding sequence will have an effect on the expression of a coding sequence.
  • the promoter or other control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof.
  • intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked" to the coding sequence.
  • nucleic acid construct it is meant a nucleic acid sequence that has been constructed to comprise one or more functional units not found together in nature. Examples include circular, linear, double-stranded, extrachromosomal DNA molecules (plasmids), cosmids (plasmids containing COS sequences from lambda phage), viral genomes including non-native nucleic acid sequences, and the like.
  • plasmids extrachromosomal DNA molecules
  • cosmids plasmids containing COS sequences from lambda phage
  • viral genomes including non-native nucleic acid sequences, and the like.
  • a “vector” is capable of transferring gene sequences to target cells.
  • vector construct means any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target cells, which can be accomplished by genomic integration of all or a portion of the vector, or transient or inheritable maintenance of the vector as an extrachromosomal element.
  • vector transfer vector means any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target cells, which can be accomplished by genomic integration of all or a portion of the vector, or transient or inheritable maintenance of the vector as an extrachromosomal element.
  • the term includes cloning, and expression vehicles, as well as integrating vectors.
  • An “expression cassette” includes any nucleic acid construct capable of directing the expression of a gene/coding sequence of interest, which is operably linked to a promoter of the expression cassette. Such cassettes can be constructed into a “vector”, “vector construct”, “expression vector”, or “gene transfer vector”, in order to transfer the expression cassette into target cells.
  • the term includes cloning and expression vehicles, as well as viral vectors.
  • sequence identity refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively.
  • Two or more sequences can be compared by determining their "percent identity.”
  • the percent identity of two sequences, whether nucleic acid or amino acid sequences is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100.
  • An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, /Advances in Applied Mathematics, 2: 482-489 (1981). This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M.O. Dayhoff ed., 5 suppl. 3: 353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6): 6745-6763 (1986).
  • the Smith-Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the "Match" value reflects "sequence identity.”
  • Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters.
  • homology can be determined by hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments.
  • Two DNA, or two polypeptide sequences are "substantially homologous" to each other when the sequences exhibit at least about 80%-85%, at least about 85%- 90%, at least about 90%-95%, or at least about 95%-98% sequence identity over a defined length of the molecules, as determined using the methods above.
  • substantially homologous also refers to sequences showing complete identity to the specified DNA or polypeptide sequence.
  • DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See e.g., Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Third Edition, (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  • a first polynucleotide is "derived from" a second polynucleotide if it has the same or substantially the same nucleotide sequence as a region of the second polynucleotide, its cDNA, complements thereof, or if it displays sequence identity as described above. This term is not meant to require or imply the polynucleotide must be obtained from the origin cited (although such is encompassed), but rather can be made by any suitable method.
  • a first polypeptide (or peptide) is "derived from" a second polypeptide (or peptide) if it is (i) encoded by a first polynucleotide derived from a second polynucleotide, or (ii) displays sequence identity to the second polypeptides as described above. This term is not meant to require or imply the polypeptide must be obtained from the origin cited (although such is encompassed), but rather can be made by any suitable method.
  • a first therapy is administered during the entire course of administration of a second therapy; where the first therapy is administered for a period of time that is overlapping with the administration of the second therapy, e.g., where administration of the first therapy begins before the administration of the second therapy and the administration of the first therapy ends before the administration of the second therapy ends; where the administration of the second therapy begins before the administration of the first therapy and the administration of the second therapy ends before the administration of the first therapy ends; where the administration of the first therapy begins before administration of the second therapy begins and the administration of the second therapy ends before the administration of the first therapy ends; where the administration of the second therapy begins before administration of the first therapy begins and the administration of the first therapy ends before the administration of the second therapy ends.
  • in combination can also refer to regimen involving administration of two or more therapies.
  • “In combination with” as used herein also refers to administration of two or more therapies which can be administered in the same or different formulations, by the same or different routes, and in the same or different dosage form type.
  • treatment refers to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or can be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which can be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development or progression; and (c) relieving the disease, i.e., causing regression of the disease and/or relieving one or more disease symptoms.
  • Treatment is also meant to encompass delivery of an agent in order to provide for a pharmacologic effect, even in the absence of a disease or condition.
  • treatment encompasses delivery of a penetrating peptide composition that can elicit an immune response or confer immunity in the absence of a disease condition, e.g., in the case of a vaccine.
  • Subject refers to an animal, human or non-human, amenable to therapy according to the methods of the disclosure or to which a peptide composition according to the present disclosure can be administered to achieve a desired effect.
  • the subject is a mammalian subject.
  • dermatitis refers to inflammation of the skin and includes, for example, allergic contact dermatitis, urticaria, histotic dermatitis (dry skin on the lower legs), atopic dermatitis, contact dermatitis including irritant contact dermatitis and urushiol-induced contact dermatitis, eczema, gravitational dermatitis, nummular dermatitis, otitis externa, perioral dermatitis, and seborrhoeic dermatitis.
  • stratum corneum refers to the horny outer layer of the epidermis, consisting of several layers of flat, keratinized, non-nucleated, dead, or peeling cells.
  • the peptides of the presently disclosed subject matter in some embodiments can "consist essentially of a core amino acid sequence, which indicates that the peptide can include one or more (e.g., 1 , 2, 3, 4, 5, 6, or more) N-terminal and/or C-terminal amino acids the presence of which does not materially affect the desired biological activity of the peptide.
  • compositions comprising the amino acid sequence TGSTQHQ (SEQ ID NO: 1). It is understood that the presently disclosed subject matter thus also encompasses peptides that in some embodiments consist essentially of the amino acid sequence TGSTQHQ (SEQ ID NO: 1); as well as peptides that in some embodiments consist of the amino acid sequence TGSTQHQ (SEQ ID NO: 1).
  • the methods of the presently disclosed subject matter in some embodiments comprise the steps that are disclosed herein and/or that are recited in the claims, that they in some embodiments consist essentially of the steps that are disclosed herein and/or that are recited in the claims, and that they in some embodiments consist of the steps that are disclosed herein and/or that are recited in the claim.
  • the present disclosure provides peptides that are capable of penetrating the SC and/or penetrating viable cells following administration. These peptides are referred to herein as “penetrating peptides" or “SPACE Peptides”. In some embodiments, these penetrating peptides are capable of penetrating the cellular membranes of viable epidermal and dermal cells. Penetrating peptides according to the present disclosure can include, for example, one or more of the amino acid sequences provided in Table 2 below.
  • penetrating peptides include an amino acid sequence including a stretch of three, four, five, six, or seven consecutive amino acids selected from one of the following amino acid sequences TGSTQHQ (SEQ ID NO: 1), HSALTKH (SEQ ID NO: 2), KTGSHNQ (SEQ ID NO: 3), MGPSSML (SEQ ID NO: 4), TDPNQLQ (SEQ ID NO: 5) and STHFIDT (SEQ ID NO: 6).
  • penetrating peptides according to the present disclosure have an amino acid sequence from 8 to 11, 12 to 15, or 16 to 19 amino acids in length, including an amino acid sequence selected from one of the following amino acid sequences TGSTQHQ (SEQ ID NO: 1), HSALTKH (SEQ ID NO: 2), KTGSHNQ (SEQ ID NO: 3), MGPSSML (SEQ ID NO: 4), TDPNQLQ (SEQ ID NO: 5) and STHFIDT (SEQ ID NO: 6).
  • TGSTQHQ SEQ ID NO: 1
  • HSALTKH SEQ ID NO: 2
  • KTGSHNQ SEQ ID NO: 3
  • MGPSSML SEQ ID NO: 4
  • TDPNQLQ SEQ ID NO: 5
  • STHFIDT SEQ ID NO: 6
  • penetrating peptides according to the present disclosure have an amino acid sequence of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acids.
  • penetrating peptides according to the present disclosure can be circularized by any of a variety of known cross-linking methods.
  • a penetrating peptide according to the present disclosure can be provided in a circularized conformation (i.e., as a cyclic peptide) in which a Cys- Cys disulfide bond is present.
  • penetrating peptides have an amino acid sequence including an internal amino acid sequence selected from one of the following amino acid sequences TGSTQHQ (SEQ ID NO: 1), HSALTKH (SEQ ID NO: 2), KTGSHNQ (SEQ ID NO: 3), MGPSSML (SEQ ID NO: 4), TDPNQLQ (SEQ ID NO: 5) and STHFIDT (SEQ ID NO: 6), wherein the amino acid sequence of the peptide includes at least a first Cys positioned external to the internal sequence in the N-terminal direction and at least a second Cys positioned external to the internal sequence in the C-terminal direction.
  • TGSTQHQ SEQ ID NO: 1
  • HSALTKH SEQ ID NO: 2
  • KTGSHNQ SEQ ID NO: 3
  • MGPSSML SEQ ID NO: 4
  • TDPNQLQ SEQ ID NO: 5
  • STHFIDT SEQ ID NO: 6
  • Exemplary penetrating peptides according to the present disclosure that have an amino acid sequence including an internal amino acid sequence of one of SEQ ID NOs: 1-6 include, but are not limited to peptides comprising any of SEQ ID NOs: 7- 18.
  • the two Cys residues present in any of SEQ ID NOs: 7- 18 are employed to form a Cys-Cys disulfide bond.
  • SEQ ID NO: 7 is the amino acid sequence CTGSTQHQC, which includes the internal sequence TGSTQHQ (SEQ ID NO: 1).
  • a cyclic penetrating peptide according to the present disclosure comprises a Cys-Cys disulfide bond between amino acid 1 and amino acid 9 of SEQ ID NO: 7.
  • SEQ ID NO: 8 is the amino acid sequence CHSALTKHC, which includes the internal sequence HSALTKH (SEQ ID NO: 2).
  • a cyclic penetrating peptide according to the present disclosure comprises a Cys-Cys disulfide bond between amino acid 1 and amino acid 9 of SEQ ID NO: 8.
  • SEQ ID NO: 13 is the amino acid sequence ACTGSTQHQCG, which also includes the internal sequence TGSTQHQ (SEQ ID NO: 1).
  • a cyclic penetrating peptide according to the present disclosure comprises a Cys-Cys disulfide bond between amino acid 2 and amino acid 10 of SEQ ID NO: 13.
  • SEQ ID NO: 14 is the amino acid sequence ACHSALTKHCG, which also includes the internal sequence HSALTKH (SEQ ID NO: 2).
  • a cyclic penetrating peptide according to the present disclosure comprises a Cys-Cys disulfide bond between amino acid 2 and amino acid 10 of SEQ ID NO: 14.
  • penetrating peptides include an amino acid sequence including an internal stretch of three, four, five, or six consecutive amino acids selected from one of the following amino acid sequences TGSTQHQ (SEQ ID NO: 1), HSALTKH (SEQ ID NO: 2), KTGSHNQ (SEQ ID NO: 3), MGPSSML (SEQ ID NO: 4), TDPNQLQ (SEQ ID NO: 5) and STHFIDT (SEQ ID NO: 6); and further including at least a first Cys positioned external to the internal sequence in the N-terminal direction and at least a second Cys positioned external to the internal sequence in the C-terminal direction.
  • TGSTQHQ SEQ ID NO: 1
  • HSALTKH SEQ ID NO: 2
  • KTGSHNQ SEQ ID NO: 3
  • MGPSSML SEQ ID NO: 4
  • TDPNQLQ SEQ ID NO: 5
  • STHFIDT SEQ ID NO: 6
  • penetrating peptides disclosed herein include those having the amino acid sequences provided, as well as peptides having one or more amino acid substitutions, e.g., one or more conservative amino acid substitutions, relative to the sequences provided, wherein the peptides retains the capability of penetrating the SC or penetrating a cell.
  • a molecular cargo e.g., a low molecular weight compound or macromolecule
  • active agents include, for example, proteins, peptides, nucleic acids, nucleotides, nucleosides and analogues thereof; as well as pharmaceutical compounds, e.g., low molecular weight compounds.
  • Active agents which can be delivered using the penetrating peptides disclosed herein include agents which act on the peripheral nerves, adrenergic receptors, cholinergic receptors, the skeletal muscles, the cardiovascular system, smooth muscles, the blood circulatory system, synaptic sites, neuroeffector junction sites, endocrine and hormone systems, the immunological system, the reproductive system, the skeletal system, autacoid systems, the alimentary and excretory systems, the histamine system and the central nervous system.
  • Suitable active agents can be selected, for example, from dermatological agents, anti-neoplastic agents, cardiovascular agents, renal agents, gastrointestinal agents, rheumatologic agents, immunological agents, and neurological agents among others.
  • Suitable dermatological agents can include, for example, local anesthetics, anti-inflammatory agents, anti-infective agents, agents to treat acne, anti-virals, antifungals, and agents for psoriasis such as topical corticosteroids, among others.
  • a suitable dermatological agent is selected from the following list: 16-17A-Epoxyprogesterone (CAS Registry No. 1097-51-4), P- methoxycinnamic acid/4-Methoxycinnamic acid (CAS Registry No. 830-09-1), Octyl Methoxycinnamate (CAS Registry No. 5466-77-3), Octyl Methoxycinnamate (CAS Registry No. 5466-77-3), Methyl p-methoxy cinnamate (CAS Registry No. 832-01-9), 4-ESTREN-17
  • Proteins useful in the disclosed depot formulations can include, for example, molecules such as cytokines and their receptors, as well as chimeric proteins including cytokines or their receptors, including, for example tumor necrosis factor alpha and beta, their receptors and their derivatives; renin; growth hormones, including human growth hormone, bovine growth hormone, methionine-human growth hormone, des-phenylalanine human growth hormone, and porcine growth hormone; growth hormone releasing factor (GRF); parathyroid and pituitary hormones; thyroid stimulating hormone; human pancreas hormone releasing factor; lipoproteins; colchicine; prolactin; corticotrophin; thyrotropic hormone; oxytocin; vasopressin; somatostatin; lypressin; pancreozymin; leuprolide; alpha-1 -antitrypsin; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; luteinizing
  • Suitable proteins or peptides can be native or recombinant and include, e.g., fusion proteins.
  • the protein is a growth hormone, such as human growth hormone (hGH), recombinant human growth hormone (rhGH), bovine growth hormone, methionine-human growth hormone, des-phenylalanine human growth hormone, and porcine growth hormone; insulin, insulin A-chain, insulin B-chain, and proinsulin; or a growth factor, such as vascular endothelial growth factor (VEGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), transforming growth factor (TGF), and insulin-like growth factor-l and -II (IGF-I and IGF-I).
  • hGH human growth hormone
  • rhGH recombinant human growth hormone
  • bovine growth hormone methionine-human growth hormone, des-phenylalanine human growth hormone, and porcine growth hormone
  • insulin insulin A-chain, insulin B-chain, and proinsulin
  • a growth factor such as vascular endothelial growth factor (
  • Suitable peptides for use as the active agent in the injectable, biodegradable delivery depots disclosed herein include, but are not limited to, Glucagon-like peptide-1 (GLP-1) and precursors, derivatives, prodrugs and analogues thereof.
  • GLP-1 Glucagon-like peptide-1
  • Nucleic acid active agents include nucleic acids as well as precursors, derivatives, prodrugs and analogues thereof, e.g., therapeutic nucleotides, nucleosides and analogues thereof; therapeutic oligonucleotides; and therapeutic polynucleotides. Active agents selected from this group can find particular use as anticancer agents and antivirals. Suitable nucleic acid active agents can include for example ribozymes, antisense oligodeoxynucleotides, aptamers and siRNA. Examples of suitable nucleoside analogues include, but are not limited to, cytarabine (araCTP), gemcitabine (dFdCTP), and floxuridine (FdUTP).
  • araCTP cytarabine
  • dFdCTP gemcitabine
  • FdUTP floxuridine
  • a suitable nucleic acid active agent is an interfering RNA, e.g., shRNA, miRNA or siRNA.
  • Suitable siRNAs include, for example, IL-7 (lnterleukin-7) siRNA, IL-10 (lnterleukin-10) siRNA, IL-22 (lnterleukin-22) siRNA, IL-23 (Interleukin 23) siRNA, CD86 siRNA, KRT6a (keratin 6A) siRNA, K6a N171K (keratin 6a N171 K) siRNA, TNFa (tumor necrosis factor a) siRNA, TNFR1 (tumor necrosis factor receptor-1) siRNA, TACE (tumor necrosis factor (TNF)-a converting enzyme) siRNA, RRM2 (ribonucleotide reductase subunit-2) siRNA, and VEGF (vascular endothelial growth factor) siRNA.
  • IL-7 lanterleukin-7
  • IL-7 see e.g., GENBANK® Accession No. NM_000880.3, GENBANK® Accession No. NM_001199886.1 , GENBANK® Accession No. NM_001199887.1 , and GENBANK® Accession No. NM_001199888.1 ;
  • IL-10 see e.g., GENBANK® Accession No. NM_000572.2; for IL-22 see e.g., GENBANK® Accession No. NM_020525.4; for IL-23, see e.g., GENBANK® Accession No.
  • NM_016584.2 and GENBANK® Accession No. AF301620.1 ; for CD86, see e.g., GENBANK® Accession No. NM_175862.4, GENBANK® Accession No. NM_006889.4, GENBANK® Accession No. NM_176892.1 , GENBANK® Accession No. NM_001206924.1 , and GENBANK® Accession No. N _001206925.1 ; for KRT6a, see e.g., GENBANK® Accession No. NM_005554.3; for TNFa, see e.g., GENBANK® Accession No.
  • NM_001025369.2 GENBANK® Accession No. N _001025370.2, NM_001033756.2, GENBANK® Accession No. NM_001171622.1 , and GENBANK® Accession No. NM_003376.5.
  • siRNA design tool provided by the Whitehead Institute of Biomedical Research at MIT. This tool can be located on the internet on the website located by placing http: // directly preceding jura.wi.mit.edu/bioc/siRNAext/.
  • Suitable compounds can include compounds directed to one or more of the following drug targets: Kringle domain, Carboxypeptidase, Carboxylic ester hydrolases, Glycosylases, Rhodopsin-like dopamine receptors, Rhodopsin-like adrenoceptors, Rhodopsin-like histamine receptors, Rhodopsin-like serotonin receptors, Rhodopsin-like short peptide receptors, Rhodopsin-like acetylcholine receptors, Rhodopsin-like nucleotide-like receptors, Rhodopsin-like lipid-like ligand receptors, Rhodopsin-like melatonin receptors, Metalloprotease, Transporter ATPase, Carboxylic ester hydrolases, Peroxidase, Lipoxygenase, DOPA decarboxylase, A/G cyclase
  • the active agent is a compound targeting one of rhodopsin-like GPCRs, nuclear receptors, ligand-gated ion channels, voltage-gated ion channels, penicillin-binding protein, myeloperoxidase-like, sodium: neurotransmitter symporter family, type II DNA topoisomerase, fibronectin type III, and cytochrome P450.
  • the active agent is an anticancer agent.
  • Suitable anticancer agents include, but are not limited to, Actinomycin D, Alemtuzumab, Allopurinol sodium, Amifostine, Amsacrine, Anastrozole, Ara-CMP, Asparaginase, Azacytadine, Bendamustine, Bevacizumab, Bicalutimide, Bleomycin (e.g., Bleomycin A 2 and B 2 ), Bortezomib, Busulfan, Camptothecin sodium salt, Capecitabine, Carboplatin, Carmustine, Cetuximab, Chlorambucil, Cisplatin, Cladribine, Clofarabine, Cyclophosphamide, Cytarabine, dacarbazine, Dactinomycin, Daunorubicin, Daunorubicin liposomal, dacarbazine, Decitabine, Docetaxel, Doxorubicin, Doxor
  • Active agents of interest for use in the disclosed penetrating peptide compositions can also include opioids and derivatives thereof as well as opioid receptor agonists and antagonists, e.g., naltrexone, naloxone, nalbuphine, fentanyl, sufentanil, oxycodone, and pharmaceutically acceptable salts and derivatives thereof.
  • opioids and derivatives thereof as well as opioid receptor agonists and antagonists, e.g., naltrexone, naloxone, nalbuphine, fentanyl, sufentanil, oxycodone, and pharmaceutically acceptable salts and derivatives thereof.
  • the active agent is a small molecule or low molecular weight compound, e.g., a molecule or compound having a molecular weight of less than or equal to about 1000 Daltons, e.g., less than or equal to about 800 Daltons.
  • the active agent is a label.
  • suitable labels include, e.g., radioactive isotopes, fluorescers, chemiluminescers, chromophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, magnetic particles, nanoparticles and quantum dots.
  • the active agent can be present in any suitable concentration in the compositions disclosed herein. Suitable concentrations can vary depending on the potency of the active agent, active agent half-life, etc.
  • penetrating peptide compositions according to the present disclosure can include one or more active agents, e.g., a combination of two or more of the active agents described above. IV. Active Agent Carriers
  • a penetrating peptide composition according to the present disclosure can include a penetrating peptide as disclosed herein conjugated or associated with an active agent carrier (also referred to herein as a "carrier") which in turn includes the active agent attached thereto and/or disposed therein.
  • an active agent carrier also referred to herein as a "carrier”
  • Suitable active agent carriers include, for example, liposomes, nanoparticles, micelles, microbubbles, and the like. Techniques for incorporating active agents into such carriers are known in the art.
  • liposomes or lipidic particles can be prepared in accordance with U.S. Patent No. 5,077,057 to Szoka, Jr. Liposomes formed from nonphosphal lipid components which have the potential to form lipid bilayers are disclosed in Biochim. Biophys. Acta, 19: 227-232 (1982).
  • purification, modification and loading of liposomes see generally, New, R.C.C., Liposomes: A Practical Approach, (1990) Oxford University Press Inc., N.Y.
  • an active agent carrier of the presently disclosed subject matter is an ethosome.
  • Ethosomes are vesicles formed typically from phospholipids in the presence of water and ethanol or another alcohol, and sometimes further in the presence of glycols and other polyols. Ethosomes can be prepared by techniques that would be known to one of ordinary skill in the art, and are set forth in, for example, U.S. Patent Nos. 5,540,934 and 5,716,638, both to Touitou ; and in Godin and Touitou (2003) Crit Rev Ther Drug Carrier Syst 20:63- 102.
  • an SPACE Peptide-containing ethosome of the presently disclosed subject matter is prepared by mixing lipids and/or phospholipids, particularly includes at least one functionalized lipid and/or phospholipid with one or more SPACE Peptides in a volume of water that in some embodiments can contain ethanol and/or sodium phosphate buffer.
  • CsA, HA, or any other bioactive agent can also be added to the mixture to allow SPACE Peptide- containing micelles, liposomes, and/or ethosomes for form, which encapsulate the CsA, HA, or other bioactive agent.
  • a SPACE Peptide (in some embodiments, the same SPACE Peptide as used during the micelle/liposome/ethosome formation step, in some embodiments a different SPACE Peptide as used during the micelle/liposome/ethosome formation step, and in some embodiments a mixture of the same and/or one or more different SPACE Peptides as used during the micelle/liposome/ethosome formation step) is added after micelle/liposome/ethosome formation to produce a composition comprising a SPACE Peptide-containing micelle/liposome/ethosome that also comprises one or more free SPACE Peptides.
  • an active agent carrier of the presently disclosed subject matter is a micelle, liposome, and/or ethosome comprising one or more SPACE Peptides conjugated with an alkyl chain.
  • Methods for preparing alkyl- conjugated peptides include but are not limited to those disclosed in, for example, Peters et al. (2009) PNAS Vol. 106, No. 24: 9815-9819.
  • Penetrating peptides as described herein can be conjugated to or associated with an active agent.
  • a penetrating peptide as disclosed herein can be conjugated or associated with an active agent carrier, which in turn includes the active agent attached thereto and/or disposed therein (examples of which are discussed above).
  • Conjugation techniques generally result in the formation of one or more covalent bonds between the penetrating peptide and either the active agent or an active agent carrier while association techniques generally utilize one or more of hydrophobic, electrostatic or van der Walls interactions.
  • a variety of techniques can be used for conjugating or associating a peptide to an active agent.
  • a variety of techniques can be used for conjugating or associating a peptide to an active agent carrier, e.g., liposomes, nanoparticies, or micelle as described herein.
  • the entire composition, including the penetrating peptide can be synthesized using standard amino acid synthesis techniques. Other methods including standard molecular biology techniques can be used to express and purify the entire polypeptide sequence including the penetrating peptide. Additional methods of conjugating peptides to other peptides or polypeptides include Cu-catalyzed azide/alkyne [3+2] cycloaddition "Click Chemistry" as described by Rostovtsev et al. (2002) Angew. Chem. Int. Ed. 41 : 2596-2599 and Tornoe et al. (2002) J. Org. Chem.
  • the active agent is a low molecular weight compound or small molecule
  • a variety of techniques can be utilized to conjugate the low molecular weight compound or small molecule to a penetrating peptide as described herein, e.g., Click chemistry as described in Loh et al., Chem Commun (Camb), 2010 Nov 28;46(44): 8407-9. Epub 2010 Oct 7. See also, Thomson S., Methods Mol Med. (2004) 94: 255-265, describing conjugation of small molecule carboxyl, hydroxyl, and amine residues to amine and sulfhydryl residues on proteins.
  • a SPACE Peptide can be conjugated to cyclosporin A (CsA) as set forth in the method of Figure 1. Briefly, an epoxide derivative of CsA is prepared, and reacted with a SPACE Peptide under conditions wherein the N-terminal amino group of the SPACE Peptide attacks the epoxide ring to produce a CsA-SPACE Peptide conjugate.
  • the CsA-SPACE Peptide conjugate can then be employed as set forth herein, including as is, associated with an active agent carrier, encapsulated by an active agent carrier, etc.
  • a variety of suitable methods of administering a penetrating peptide composition to a subject or host, e.g., patient, in need thereof, are available, and, although more than one route can be used to administer a particular composition, a particular route can provide a more immediate and more effective reaction than another route.
  • Pharmaceutically acceptable excipients are also well known to those who are skilled in the art, and are readily available. The choice of excipient will be determined in part by the particular compound, as well as by the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of the penetrating peptide compositions. The following methods and excipients are merely exemplary and are in no way limiting.
  • Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as solids or granules; (c) suspensions in an appropriate liquid; (d) suitable emulsions and (e) hydrogels.
  • Tablet forms can include one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients.
  • Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles including the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.
  • a flavor usually sucrose and acacia or tragacanth
  • pastilles including the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.
  • Penetrating peptide formulations can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They can also be formulated as pharmaceuticals for non- pressured preparations such as for use in a nebulizer or an atomizer.
  • pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen, and the like.
  • propellants such as dichlorodifluoromethane, propane, nitrogen, and the like. They can also be formulated as pharmaceuticals for non- pressured preparations such as for use in a nebulizer or an atomizer.
  • Formulations suitable for parenteral administration include aqueous and nonaqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic 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.
  • the formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • Formulations suitable for topical administration can be presented as creams, gels, pastes, patches, sprays or foams.
  • Suppository formulations are also provided by mixing with a variety of bases such as emulsifying bases or water-soluble bases.
  • bases such as emulsifying bases or water-soluble bases.
  • Formulations suitable for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, foams.
  • Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions can be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition.
  • unit dosage forms for injection or intravenous administration can comprise the penetrating peptides in a formulation as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of penetrating peptide composition calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle.
  • the specifications for the novel unit dosage forms of the penetrating peptide compositions depend on the particular active agent employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.
  • dose levels can vary as a function of the specific compound, the nature of the delivery vehicle, and the like. Suitable dosages for a given compound are readily determinable by those of skill in the art by a variety of standard methodologies.
  • the pharmaceutical composition can contain other pharmaceutically acceptable components, such a buffers, surfactants, antioxidants, viscosity modifying agents, preservatives and the like.
  • a buffers such as sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium sulfate
  • the aqueous cyclodextrin solution further comprise dextrose, e.g., about 5% dextrose.
  • one or more of the penetrating peptide compositions of the present disclosure can be incorporated into a medical device known in the art, for example, drug eluting stents, catheters, fabrics, cements, bandages (liquid or solid), biodegradable polymer depots and the like.
  • the medical device is an implantable or partially implantable medical device.
  • an effective amount (or, in the context of a therapy, a
  • “pharmaceutically effective amount”) of a penetrating peptide composition generally refers to an amount of the penetrating peptide composition, effective to accomplish the desired therapeutic effect, e.g., in the case of a penetrating peptide-siRNA composition, an amount effective to reduce expression of the targeted mRNA by an amount effective to produce a desired therapeutic effect.
  • Effective amounts of penetrating peptide compositions, suitable delivery vehicles, and protocols can be determined by conventional methodologies. For example, in the context of therapy a medical practitioner can commence treatment with a low dose of one or more penetrating peptide compositions in a subject or patient in need thereof, and then increase the dosage, or systematically vary the dosage regimen, monitor the effects thereof on the patient or subject, and adjust the dosage or treatment regimen to maximize the desired therapeutic effect. Further discussion of optimization of dosage and treatment regimens can be found in Benet ef a/., in Goodman and Gilman's The Pharmacological Basis of Therapeutics, Ninth Edition, Hardman ef a/., Eds., McGraw-Hill, New York, (1996), Chapter 1 , pp.
  • the present disclosure provides a method of treating a subject having a dermatological disease, including: administering to the subject a pharmaceutically effective amount of a composition including a penetrating peptide as disclosed herein, wherein the peptide is conjugated to or associated with a dermatological active agent, e.g., a dermatological active agent as disclosed herein, or a dermatological active agent carrier including the active agent.
  • a dermatological active agent e.g., a dermatological active agent as disclosed herein, or a dermatological active agent carrier including the active agent.
  • the present disclosure provides a method of treating a subject having, suspected of having or susceptible to a disorder resulting at least in part from expression of an mRNA, including administering to the subject a pharmaceutically effective amount of a composition including a penetrating peptide as described herein, wherein the penetrating peptide is conjugated to or associated with an interfering RNA or an active agent carrier including an interfering RNA, e.g., an shRNA, miRNA or siRNA which targets the mRNA or a carrier including the interfering RNA.
  • an interfering RNA e.g., an shRNA, miRNA or siRNA which targets the mRNA or a carrier including the interfering RNA.
  • the interfering RNA is an siRNA, e.g., an siRNA selected from one of the following: an IL-10 siRNA, an IL-14 siRNA, an IL-17 siRNA, an IL-22 siRNA, an IL-23 siRNA, a CD86 siRNA, a KRT6a siRNA, a TNFR1 siRNA, a TNFa siRNA, and a TACE siRNA.
  • siRNAs can be designed to target mRNAs encoding other gene products with desired bioactivities including, but not limited to mRNAs encoding members of the keratin family, the collagen family, other cytokine families, growth factor families, adhesion protein families, angiogenesis-promoting protein families, etc.
  • compositions of the presently disclosed subject matter can be employed in a cosmetic formulation and/or for cosmetic uses.
  • the compositions of the presently disclosed subject matter can be solubilized in a cosmetic carrier such as liposomes, or adsorbed on powdery organic polymers, mineral supports such as talcs and bentonites, and more generally solubilized in, or fixed on, any physiologically acceptable carrier.
  • composition of the presently disclosed subject matter can be applied by any appropriate route, notably oral, parenteral, or topical, and the formulation of the cosmetic compositions can be adapted by the person skilled in the art, in particular for cosmetic or dermatological compositions.
  • the compositions of the presently disclosed subject matter can be formulated for topical administration. These compositions therefore can contain a physiologically acceptable medium (i.e., a medium compatible with the skin and epithelial appendages) and cover all cosmetic or dermatological forms.
  • compositions of the presently disclosed subject matter can also contain various protective or anti-aging active principles intended to promote and supplement the action of the active agents.
  • the following ingredients can be included, either alone or in combination: cicatrizant, anti- age, anti-wrinkle, smoothing, anti-radical, anti-UV agents, agents stimulating the synthesis of dermal macromolecules or energy metabolism, moisturizing, antibacterial, antifungal, anti-inflammatory, anesthetic agents, agents modulating cutaneous differentiation, pigmentation or depigmentation, agents stimulating nail or hair growth.
  • active agents having an anti-radical or antioxidant action chosen from among vitamin C, vitamin E, coenzyme Q10, polyphenolic plant extracts, and/or retinoids, can also be added.
  • compositions of the presently disclosed subject matter can also include other active agents that stimulate the synthesis of dermal macromolecules (laminin, fibronectin, collagen), for example the collagen peptide sold under the name COLLAXYL® by Vincience SA, Sophia Antipolis, France.
  • active agents that stimulate the synthesis of dermal macromolecules (laminin, fibronectin, collagen)
  • laminin, fibronectin, collagen for example the collagen peptide sold under the name COLLAXYL® by Vincience SA, Sophia Antipolis, France.
  • cosmetic compositions of the presently disclosed subject matter can be present in the form of an aqueous solution, hydroalcoholic or oily solution; an oil in water emulsion, water in oil emulsion or multiple emulsions; creams, suspensions, powders, etc., that are suitable for application on the skin, mucosa, lips, and/or epithelial appendages.
  • These compositions can be more or less fluid and in some embodiments have the appearance of a cream, a lotion, a milk, a serum, a pomade, a gel, a paste, or a foam.
  • They can also be present in solid form, such as a stick, or can be applied on the skin in aerosol form. They can be used as a care product and/or as a skin makeup product.
  • compositions also comprise any additive commonly used in the contemplated field of application as well as the adjuvants necessary for their formulation, such as solvents, thickeners, diluents, antioxidants, colorants, sunscreens, self-tanning agents, pigments, fillers, preservatives, fragrances, odor absorbers, other cosmetic active principles, essential oils, vitamins, essential fatty acids, surface active agents, film-forming polymers, etc.
  • solvents such as solvents, thickeners, diluents, antioxidants, colorants, sunscreens, self-tanning agents, pigments, fillers, preservatives, fragrances, odor absorbers, other cosmetic active principles, essential oils, vitamins, essential fatty acids, surface active agents, film-forming polymers, etc.
  • these adjuvants can, for example, correspond to a concentration ranging from 0.01 to 20% of the total weight of the composition.
  • the fatty phase can represent in some embodiments from 5 to 80% by weight and in some embodiments from 5 to 50% by weight with relation to the total weight of the composition.
  • the emulsifiers and co-emulsifiers used in the composition can be chosen from among those conventionally used in the field under consideration. For example, they can be used in a proportion going from 0.3 to 30% by weight with relation to the total weight of the composition.
  • the penetrating peptide compositions disclosed herein can also be used in the context of in vitro experimentation.
  • the penetrating peptides disclosed herein can be used to deliver any of a wide variety of active agents as discussed herein, as well as potential active agents, into viable cells in vitro to determine the potential therapeutic effect, toxicity, etc. of the active agent or potential active agent.
  • the penetrating peptides and penetrating peptide compositions of the present disclosure can be useful in the context of drug testing and/or screening.
  • penetrating peptide compositions as described herein can be used in in vitro gene silencing experiments, e.g., by introducing a penetrating peptide-interfering RNA conjugate directed to a gene target and monitoring the effect on gene expression.
  • Additional in vitro uses can include the use of penetrating peptides as disclosed herein conjugated or associated with one or more labeling agents (e.g., fluorescent agents or radioactive labels) or one or more labeling agent carriers in order to label viable cells in vitro.
  • Cyclosporin A was conjugated to SPACE Peptides using the generalized scheme depicted in Figure 1.
  • Step I CsA was epoxidized by mixing CsA (50 mg), m-Chloroperoxybenzoic acid (8 mg), and anhydrous Na 2 C0 3 (10 mg) in 5 mL of methylene chloride. The reaction mixture was stirred overnight at room temperature (RT). The reaction mixture was thereafter washed with 20% sodium bisulfite and 10% sodium carbonate. The organic layer was dried over anhydrous sodium carbonate. The solvent was removed under vacuum producing a crystalline product.
  • Step II a SPACE Peptide having the amino acid sequence set forth in SEQ ID NO: 13 was conjugated at its N-terminus to the epoxide of CsA obtained from Step I above.
  • the epoxide of CsA was dissolved in 5 ml of ethanol at RT overnight. 50 mg of SPACE Peptide was added and the mixture was incubated overnight at RT to form a SPACE Peptide/CsA conjugate.
  • Figures 2A-2C show FTIR spectra of SPACE Peptide powder (Figure 2A), CsA powder ( Figure 2B), and a SPACE Peptide-CsA conjugate ( Figure 2C).
  • the characteristic spectra are used to determine the presence of both components in the conjugate as determined by the presence of characteristic features in the spectra.
  • Comparison of the spectrum of the SPACE Peptide-CsA conjugate with either SPACE alone ( Figure 2D) or CsA alone ( Figure 2E) confirmed the presence of both components. Since the SPACE Peptide was highly water soluble and cyclosporin was highly water insoluble, the simultaneous presence of both components indicated their close association.
  • Full thickness pig skin (Lampire Biological Laboratories, Pipersville, PA) was used.
  • the skin was stored at -80°C and defrosted immediately prior to use. Briefly, the skin was allowed to thaw with the stratum corneum (SC) side up left open to the atmosphere. Skin disks of 36 mm were punched out. The subcutaneous fatty tissue was carefully removed from the dermis, and the hair shaft was clipped to no more than 4 mm. The skin pieces were cleaned with PBS (pH 7.4). Subsequently, the integrity of the skin disks were checked with a skin conductivity measurement to ensure that samples were free from any surface irregularity such as tiny holes or crevices in the portion that was used for skin penetration and deposition studies.
  • SC stratum corneum
  • 100-200 ⁇ _ of the test formulation was applied to skin surface using a pipette.
  • the experiments were carried out under occlusion with light protection.
  • the incubation time of the skin with each test formulation was 24 hours.
  • a sample of 1-3 ml_ was withdrawn from the receptor phase for concentration measurement by fluorescence assay using a micro-plate reader (SAFIRE, XFLUOR4, V4.50, Tecan Group Ltd, NY, United States of America) for fluorescence.
  • a sample of 3 ml was withdrawn from the receptor phase for 3 H-CsA measurement by a liquid scintillation counter (TRI- CARB 2100TR, Packard Instrument Company, Downers Grove, IL).
  • the formulations were removed from the skin by washing five times with PBS (pH 7.4). After cleaning, the skin was transferred onto a device for tape-stripping the SC.
  • the epidermis sheet was separated from the dermis with a sterile surgical scalpel and cut into small pieces and collected into a glass vial. Dermis was thereafter cut into small pieces and transferred into a glass vial.
  • FITC- SPACE and Fluorescein-HA For extraction of drug from the separated skin layers, in the case of FITC- SPACE and Fluorescein-HA, 4 ml of methanol and PBS pH 7.4 (1 :1 , v/v) mixture was added to each glass vial. The vials were shaken at 200 rpm on an orbital shaker overnight at room temperature. The dispersions were centrifuged (10 min, 10000 rpm) to subside skin tissue pieces at the bottom. The supernatant was withdrawn, diluted if the concentration was found outside the range of detection, and analyzed by fluorescence measurement.
  • Figure 4 summarizes the results of the tests of the transport barrier in the skin for a fluorescently-labeled SPACE Peptide (FITC-SPACE; SPACE Peptide employed was SEQ ID NO: 13): Labeling with FITC was done during peptide synthesis.
  • the results of three representative tests are presented; (i) placement of SPACE Peptide on intact porcine skin; (ii) placement of SPACE Peptide on skin after the SC was removed by tape stripping, thus exposing the epidermis of the skin; and (iii) placement of SPACE Peptide on skin after SC and epidermis were removed, thus exposing the dermis of the skin.
  • the first case represented normal skin.
  • the second case represented a disease where the SC was compromised
  • the third case represented an extreme case where the epidermis was missing, for example, open wounds.
  • SPACE Peptide-Conjugated Lipids SPACE Peptide-conjugated lipids were synthesized using the basic procedure described herein below and depicted in Figure 6.
  • FITC-SPACE Peptide (FITC-Ahx-ACTGSTQHQCG (SEQ ID NO: 13, with a disulfide bridge between amino acids 2-10) (Ambiopharm, North Augusta, SC) ⁇ Fluorescein Hyaluronic acid (molecular weight 200-325 kDa, Creative
  • SPACE Peptide-conjugated lipids were used to prepare SPACE-Peptide- displaying ethosomes.
  • a general procedure for preparing SPACE-Peptide-displaying ethosomes is presented in Figure 7.
  • Figure 8A is a bar graph showing total penetration of various SPACE Peptide- containing formulations in SC + Epidermis + Dermis + Receiver.
  • column A presents data for FITC-SPACE at 1 mg/ml
  • column B presents data for FITC-SPACE at 1 mg/ml + free SPACE Peptide at 10 mg/ml
  • column C presents data for SPACE-lipids containing FITC
  • column D presents data for SPACE- ethosomes containing FITC
  • column E presents data for SPACE-ethosomes containing FITC-labeled SPACE Peptide + 25 mg/ml free SPACE Peptide.
  • Figure 8B shows that about 2% of the applied does was deposited in epidermis. Additional experiments performed using EPIDERMTM tissue confirmed penetration of SPACE Peptide across the skin.
  • the SPACE Peptide formulations were prepared using the methods described herein above.
  • EPIDERMTM tissues were obtained from MatTek Corporation (Ashland, MA).
  • the EPIDERMTM tissue possessed cornified stratum corneum that was backed by viable epidermis. Thus, this tissue had a combination of barrier properties and living cells.
  • SPACE Peptide showed excellent penetration from SPACE-ethosome formulations across EPIDERMTM tissue.
  • a 100 microliter formulation applied on EPIDERMTM tissue for 24 hours was determined to deliver 25% of SPACE Peptide across EPIDERMTM tissue.
  • Liposomes enhanced penetration into superficial layers but were less effective for deeper layers
  • Figure 8C is a bar graph showing delivery of a fluorescent label to EPIDERMTM (left column of each pair) and a pig skin model (right column of each pair) using different formulations the contain SPACE Peptides.
  • a 100 ⁇ sample of various formulations was placed on a 2 cm 2 skin sample and penetration was tested 24 hours after application.
  • a FITC-labeled SPACE Peptide penetrated into EPIDERMTM, encapsulation of the FITC-labeled SPACE Peptide into a SPACE Peptide-conjugated ethosome further enhanced penetration in to EPIDERMTM, and the addition of a free SPACE Peptide into the formulation further enhanced skin penetration both in EPIDERMTM and in the pig skin model.
  • SPACE ethosomes were used to encapsulate cyclosporin.
  • SPACE ethosomes were prepared using the procedure outlined in EXAMPLE 3 and its penetration into skin was measured using the procedure outlined in EXAMPLE 2.
  • the resultant ethosomes possessed a diameter of about 150 nm and possessed a zeta potential of -50 mV (see Figure 9).
  • SPACE-ethosomes delivered substantial amounts of cyclosporin into skin (see Figure 10). Specifically, about 12.1 % of topically applied cyclosporin was determined to penetrate into skin after 24 hours when 100 pL of formulation was applied to 2 cm 2 . A significant quantity (4.2%) penetrated into epidermis. Further, cyclosporin A exhibited substantial localization in the skin. The amount of cyclosporin A in the skin was 1284-fold higher than that in the receiver compartment.
  • the therapeutic concentration of CsA delivered intralesionally is typically in the range of 2-35 pg, which is generally reached only after a 12-injection course over 4 weeks.
  • the data presented in Figure 10 demonstrated that single doses of a 0.5% CsA-containing SPACE ethosome could deliver between 0.3% (dermis) and 4.7% (top SC) of the CsA.
  • 0.5% CsA corresponds to 500 pg/100 ⁇ , meaning that between 1.5 pg and 23.5 pg of CsA were delivered to various skin compartments in 24 hours using CsA-containing SPACE ethosomes.
  • SPACE ethosomes were used to encapsulate HA.
  • SPACE ethosomes were prepared using the procedure outlined in EXAMPLE 3 and its penetration into skin was measured using the procedure outlined in EXAMPLE 2.
  • Figure 11 A SPACE-ethosomes delivered substantial amounts of HA into skin. Specifically, about 17% of topically-applied HA was determined to penetrate into skin when 100 pL of formulation was applied to 2 cm 2 for 24 hours. A significant quantity also penetrated into epidermis: an 8-fold enhancement of epidermal accumulation was found compared to an aqueous solution of HA. Further, cyclosporin A exhibited substantial localization in the skin. The amount of HA in the skin was significantly higher than that in the receiver compartment.
  • Figure 11 B shows the effect of free SPACE concentration on HA delivery from SPACE-ethosome formulations.
  • concentration of SPACE-lipid in all formulations was fixed at 2 mg/ml and all SPACE formulations were prepared using acetate buffer (pH 4) and ethanol.
  • SPACE- ethosome formulations delivered significantly higher amounts of HA.
  • the delivery increased with increasing concentrations of free SPACE Peptide. While SPACE- ethosomes without free SPACE delivered about 2 micrograms of HA per sq. cm, increasing the free SPACE concentration to 50 mg/ml increased the delivered amount to more than 3.5 micrograms per sq. cm.
  • SPACE Peptide concentration in the lipid-conjugated form of HA delivery was also assessed (see Figure 11C).
  • the free SPACE Peptide concentration in the formulations was 0 mg/ml, and all SPACE formulations were prepared using acetate buffer (pH 4) and ethanol. Of the conditions tested, a SPACE-lipid concentration of 5 mg/ml yielded the best delivery, with about 4 micrograms of the applied dose entered the skin per sq. cm.
  • the pH of the formulation was adjusted to 4 by addition of hydrochloric acid (referred to as "HA-202pH” in Figure 11 D).
  • HA-202pH hydrochloric acid
  • Figure 11 D Excellent penetration of HA from this formulation into epidermis was seen (see Figure 11 D). While not wishing to be bound by any particular theory of operation, it appeared that pH played a role in the performance of HA from the formulations since the formulation made at pH 8 delivered less HA than an otherwise identical formulation at pH 4.
  • SPACE ethosomes were also tested for their ability to deliver siRNAs to skin using the following general procedure.
  • DOTAP-Ethosomes containing GAPDH-siRNA or conjugation of GAPDH-siRNA and SPACE For 1 ml of ethosomes, 10 mg of DOTAP and 2 mg of cholesterol was dissolved in 2 ml ethanol and added into SPACE-POPE conjugation solution prepared as described herein above. The solvent (both ethanol and water) system was removed using a rotary evaporator at room temperature.
  • Skin preparation Full thickness pig skin (Lampire Biological Laboratories,
  • the skin was stored at -80°C and defrosted immediately prior to use. Briefly, the skin was allowed to thaw with the stratum corneum side up left open to the atmosphere for at least half hour. Skin disks of 36 mm were punched out. The subcutaneous fatty tissue was carefully removed from the dermis, and the hair shaft was cut off to no more than 4 mm. The skin piece was cleaned with PBS (pH 7.4). The integrity of skin disks was checked with a skin conductivity measurement to ensure that the samples were free from any surface irregularities such as tiny holes or crevices in the portion that was used for skin penetration and deposition studies.
  • the experiments were carried out under occlusion with light protection.
  • the incubation time of the skin with different test formulations was 24 hours.
  • a sample of 1 ml was withdrawn from the receptor phase for concentration measurement by fluorescence assay using a micro- plate reader (SAFIRE, XFLUOR4, V4.50, Tecan Group Ltd, NY, US).
  • the formulations were removed from the skin by being washed five times with PBS (pH 7.4). After cleaning, the skin was transferred onto a device for tape-stripping the SC.
  • the epidermis sheet was separated from the dermis with a surgical sterile scalpel and cut into small pieces and collected into a glass vial. Dermis was also cut into small pieces and transferred into a glass vial.
  • 4 ml of methanol and PBS pH 7.4 (1:1 , v/v) mixture was added to each glass vial.
  • the vials were shaken overnight at 200 rpm on an orbital shaker at room temperature. Afterwards the dispersions were centrifuged (10 min, 10000 rpm) to subside skin tissue pieces at the bottom. The supernatant were withdrawn, diluted if necessary and analyzed by fluorescence measurement.
  • Fluorescent assay of FAM-GAPDH-siRNA and FAM-GAPDH-siRNA-SPACE- Peptide conjugation The concentrations of FAM-GAPDH-siRNA and FAM-GAPDH- siRNA-SPACE-Peptide conjugates were determined by fluorescence spectroscopy. Fluorescence detection was performed at an excitation of 495 nm and an emission of 525 nm for both conjugates.
  • DOTAP-Ethosomes conjugated with SPACE (2 mg/ml) containing FAM-GAPDH- siRNA (25 nmol/ml) or FAM-GAPDH-siRNA-SPACE (25 nmol/ml)
  • ethosomes 10 mg of DOTAP and 2 mg of cholesterol was dissolved in 2 ml ethanol and added into above obtained solution of SPACE-POPE conjugation solution.
  • the solvent (both ethanol and water) system was removed using a rotary evaporator at room temperature.
  • 1 ml of Ethanol/acetic acid buffer (45%, v/v) mixture which contained 25 nmol of GAPDH-siRNA and 50 mg of free SPACE-Peptide, or 1 ml of Ethanol/MES buffer (45%, v/v), which contained 25 nmol of GAPDH-siRNA-SPACE conjugation and 50 mg of free SPACE, was used to hydrate the lipid film.
  • the obtained ethosomal solution was extruded 21 times through a 100 nm polycarbonate membrane using a mini-extruder.
  • PBS buffer salts 0.5 ml, 100 mM, pH 8.0 (water were
  • PBS buffer salts 0.5 ml, 100 mM, pH 8.0 (water were
  • MES buffer for dissolving siRNA
  • Figures 12A and 12B show the efficacy of these formulations in delivering siRNA into skin.
  • SPACE-ethosomal siRNA exhibited high penetration into skin (see Figure 12A).
  • SI-102+ SPACE- ethosomal siRNA
  • FIG. 12B The efficacy of penetration was even higher when SPACE-conjugated siRNA was encapsulated in SPACE-ethosomes (Sl-102c+; see Figure 12B). In this case, 21.7% of the applied dose (100 microliters of 25 nmole/ml) entered the skin through an area of 2 sq. cm in 24 hours.

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Abstract

L'invention concerne des compositions qui facilitent l'administration d'un agent actif ou d'un vecteur d'agent actif, les compositions étant capables de pénétrer dans la couche cornée (SC) et/ou dans les membranes cellulaires de cellules viables. Dans certains modes de réalisation, les compositions comprennent un peptide, un agent actif et un vecteur comprenant l'agent actif, le peptide ayant une séquence d'acides aminés énoncée dans l'un des numéros d'identifications de séquences : 1 à 18 ; le peptide est associé et/ou conjugué à l'agent actif, au vecteur ou aux deux ; le vecteur est choisi dans le groupe constitué d'une micelle, d'un liposome, d'un ethosome et de combinaisons de ceux-ci ; la composition est capable de pénétrer dans la couche cornée (SC) lorsqu'elle est mise en contact avec celle-ci ou de pénétrer dans une cellule lorsqu'elle est mise en contact avec celle-ci, éventuellement la composition comprenant en outre un ou plusieurs peptides libres ayant une séquence d'acides aminés énoncée dans l'un des numéros d'identifications de séquences : 1 à 18. L'invention concerne également des procédés d'administration d'agents actifs à des sujets, des méthodes de traitement de sujets souffrant d'affections dermatologiques et des méthodes d'atténuation de l'expression des ARNm des sujets qui en ont besoin et/ou de traitement de maladies et/ou de troubles.
PCT/US2013/025543 2013-02-11 2013-02-11 Peptides entrant dans les cellules et traversant la peau (space) et leurs procédés d'utilisation WO2014123543A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114107397A (zh) * 2021-11-19 2022-03-01 深圳市大鳄生物科技股份有限公司 递送带负电荷的核酸的递送系统、复合物及药物
WO2022157548A1 (fr) 2021-01-24 2022-07-28 Forrest Michael David Inhibiteurs d'utilisations cosmétiques et thérapeutiques d'atp synthase

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GB0606415D0 (en) * 2006-03-31 2006-05-10 Univ Southampton Topical drug delivery
EP2099496A2 (fr) * 2006-12-08 2009-09-16 Massachusetts Institute of Technology Administration de nanoparticules et/ou d'agents à des cellules
JP2010537632A (ja) * 2007-08-29 2010-12-09 タフツ ユニバーシティー 組織及び細胞への核酸、タンパク質、薬物、及びアデノウイルスの送達を向上させるための細胞透過性ペプチドの製造及び使用方法、組成物並びにキット
WO2010129023A2 (fr) * 2009-04-28 2010-11-11 President And Fellows Of Harvard College Protéines superchargées pour une pénétration cellulaire
CA2811113A1 (fr) * 2010-11-09 2012-05-18 The Regents Of The University Of California Peptides entrant dans les cellules et traversant la peau (space) et leurs procedes d'utilisation
WO2012135805A2 (fr) * 2011-03-31 2012-10-04 modeRNA Therapeutics Administration et formulation d'acides nucléiques génétiquement modifiés

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022157548A1 (fr) 2021-01-24 2022-07-28 Forrest Michael David Inhibiteurs d'utilisations cosmétiques et thérapeutiques d'atp synthase
CN114107397A (zh) * 2021-11-19 2022-03-01 深圳市大鳄生物科技股份有限公司 递送带负电荷的核酸的递送系统、复合物及药物

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