HK1187074A - Skin permeating and cell entering (space) peptides and methods of use thereof - Google Patents

Description

Skin penetrating and cell entering (SPACE) peptides and methods of use thereof

Cross Reference to Related Applications

This application claims priority to the following U.S. provisional patent application nos.: 61/411,884 entitled "Peptides to facility Drug Delivery" (Peptides that Facilitate Drug Delivery) filed on 9.11.2010; 61/527,574 entitled "Skin penetrating and Cell organizing (SPACE) Peptides and Methods of Use Thereof" (Skin penetrating and Cell Entering (SPACE) Peptides and Methods of Use Thereof) filed on 25.8.2011; and 61/528,036 entitled "Skin penetrating and Cell organizing (SPACE) Peptides and Methods of Use Thereof," filed on 26.8.2011, which are incorporated herein by reference in their entirety and for all purposes.

Government rights

The present invention was made with government support under federal grant 1UO1 HL080718 awarded by the National Institutes of Health and federal grant 1S10RR017753-01 awarded by the National center for Research Resources. The government has certain rights in the invention.

Background

The skin, the largest organ of the human body, is the host of many skin diseases, which collectively represent a large group of human health disorders. Thus, successful delivery of therapeutic agents, such as macromolecules (e.g., siRNA), into the skin has been the subject of active development. However, achieving this goal of local siRNA delivery is extremely challenging, and with some exceptions has been very difficult to achieve. The main challenge is the poor skin penetration of macromolecules. Among the various physico-chemical approaches proposed to enhance macromolecular penetration, peptide carriers have emerged as potential candidates due to their ease of use, diversity, and potential to target cell subsets within the skin. Penetration tests across the Stratum Corneum (SC) have been performed on a variety of peptides originally identified as delivering drugs into the cytoplasm, including TAT, polyarginine, magainin, and drosophila membrane penetrating peptide (peptide), and a few have shown some efficacy in delivering small molecules into the epidermis. In contrast, only one peptide TD-1 was specifically shown to penetrate SC and have the ability to enhance systemic absorption with topical application. While a variety of peptides are known to penetrate cell membranes and a few to penetrate SC, there is a need for peptides that can simultaneously enhance macromolecular penetration and other activities across SC and/or across the cell membranes of viable epidermal and dermal cells.

Summary of The Invention

The present disclosure provides peptides and peptide compositions that facilitate the delivery of an active agent or active agent carrier, wherein the compositions are capable of penetrating the cell membrane of an SC and/or a living cell.

In a first aspect, the present disclosure provides a composition comprising a peptide comprising the amino acid sequence 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), or STHFIDT (SEQ ID NO:6), wherein the peptide is coupled to an active agent or an active agent carrier comprising the active agent, and wherein the composition is capable of penetrating the stratum corneum upon contact with the Stratum Corneum (SC) or penetrating cells upon contact with the cells.

In one embodiment of the first aspect, the composition is capable of penetrating the Stratum Corneum (SC) as well as penetrating cells.

In one embodiment of the first aspect, the amino acid sequence comprises CTGSTQHQC (SEQ ID NO:7), CHSALTKHC (SEQ ID NO:8), CKTGSHNQC (SEQ ID NO:9), CMGPSSMLC (SEQ ID NO:10), CTDPNQLQC (SEQ ID NO:11) or CSTHFIDTC (SEQ ID NO: 12).

In one embodiment of the first aspect, the amino acid sequence comprises ACTGSTQHQCG (SEQ ID NO:13), ACHSALTKHCG (SEQ ID NO:14), ACKTGSHNQCG (SEQ ID NO:15), ACMGPSSMLCG (SEQ ID NO:16), ACTDPNQLQCG (SEQ ID NO:17) or ACSTHFIDTCG (SEQ ID NO: 18). In one embodiment, wherein the amino acid sequence comprises ACTGSTQHQCG (SEQ ID NO:13), ACHSALTKHCG (SEQ ID NO:14), ACKTGSHNQCG (SEQ ID NO:15), ACMGPSSMLCG (SEQ ID NO:16), ACTDPNQLQCG (SEQ ID NO:17), or ACSTHFIDTCG (SEQ ID NO:18), the peptide is a cyclic peptide comprising a Cys-Cys disulfide bond.

In one embodiment of the first aspect, the composition is capable of penetrating the cell membrane of a living non-human animal cell.

In one embodiment of the first aspect, the composition is capable of penetrating the cell membrane of a living human cell.

In one embodiment of the first aspect, the composition is capable of penetrating the cell membrane of a living epidermal or dermal cell.

In one embodiment of the first aspect, the composition is capable of penetrating the cell membrane of a living immune cell.

In one embodiment of the first aspect, the active agent comprises a macromolecule. In one embodiment, the macromolecule comprises a protein. In one embodiment, the protein comprises an antibody or fragment thereof comprising at least one paratope.

In one embodiment of the first aspect, wherein the active agent comprises a macromolecule, the macromolecule comprises a nucleic acid. In one embodiment, the nucleic acid is DNA. In one embodiment, the nucleic acid is RNA. In one embodiment, wherein the nucleic acid is RNA, the RNA is an interfering RNA. In one embodiment, wherein the RNA is an interfering RNA, the interfering RNA is an shRNA. In one embodiment, wherein the RNA is an interfering RNA, the interfering RNA is a miRNA. In one embodiment, wherein the RNA is an interfering RNA, the interfering RNA is siRNA. In one embodiment, wherein the interfering RNA is siRNA, the siRNA is IL-10 siRNA. In one embodiment, wherein the interfering RNA is siRNA, the siRNA is CD86 siRNA. In one embodiment, wherein the interfering RNA is an siRNA, the siRNA is KRT6a siRNA. In one embodiment, wherein the interfering RNA is siRNA, the siRNA is TNFR1 siRNA. In one embodiment, wherein the interfering RNA is siRNA, the siRNA is TACesiRNA. In one embodiment, wherein the interfering RNA is siRNA, the siRNA is mutation-specific siRNA.

In one embodiment of the first aspect, the active agent is a pharmaceutical compound.

In one embodiment of the first aspect, the active agent comprises a detectable agent. In one embodiment, wherein the active agent comprises a detectable agent, the detectable agent comprises a fluorescent label. In one embodiment, wherein the active agent comprises a detectable agent, the detectable agent comprises a radioactive label.

In one embodiment of the first aspect, the active agent is a nanoparticle.

In one embodiment of the first aspect, the active agent is a low molecular weight compound.

In one embodiment of the first aspect, the active agent is an inhibitor of IL-10 biological activity. In one embodiment, wherein the active agent is an inhibitor of IL-10 biological activity, the active agent is selected from the group consisting of IL-10siRNA and an antibody or fragment thereof that binds IL-10.

In one embodiment of the first aspect, the peptide is coupled to the active agent.

In one embodiment of the first aspect, the peptide is coupled to an active agent carrier comprising the active agent. In one embodiment, wherein the peptide is coupled to an active agent carrier comprising the active agent, the active agent carrier is a liposome. In one embodiment, wherein the peptide is coupled to an active agent carrier comprising the active agent, the active agent carrier is a nanoparticle. In one embodiment, wherein the peptide is coupled to an active agent carrier comprising the active agent, the active agent carrier is a polymeric micelle.

In a second aspect, the present disclosure provides a composition comprising a peptide comprising the amino acid sequence 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), or STHFIDT (SEQ ID NO:6), wherein the peptide is associated with an active agent or an active agent carrier comprising the active agent, wherein the association results from hydrophobic, electrostatic, or van der waals interactions, and wherein the composition is capable of penetrating the stratum corneum upon contact with the Stratum Corneum (SC) or penetrating cells upon contact with the cells.

In one embodiment of the second aspect, the composition is capable of penetrating the Stratum Corneum (SC) as well as penetrating cells.

In one embodiment of the second aspect, the amino acid sequence comprises CTGSTQHQC (SEQ ID NO:7), CHSALTKHC (SEQ ID NO:8), CKTGSHNQC (SEQ ID NO:9), CMGPSSMLC (SEQ ID NO:10), CTDPNQLQC (SEQ ID NO:11) or CSTHFIDTC (SEQ ID NO: 12).

In one embodiment of the second aspect, the amino acid sequence comprises ACTGSTQHQCG (SEQ ID NO:13), ACHSALTKHCG (SEQ ID NO:14), ACKTGSHNQCG (SEQ ID NO:15), ACMGPSSMLCG (SEQ ID NO:16), ACTDPNQLQCG (SEQ ID NO:17) or ACSTHFIDTCG (SEQ ID NO: 18). In one embodiment, wherein the amino acid sequence comprises ACTGSTQHQCG (SEQ ID NO:13), ACHSALTKHCG (SEQ ID NO:14), ACKTGSHNQCG (SEQ ID NO:15), ACMGPSSMLCG (SEQ ID NO:16), ACTDPNQLQCG (SEQ ID NO:17), or ACSTHFIDTCG (SEQ ID NO:18), the peptide is a cyclic peptide comprising a Cys-Cys disulfide bond.

In a third aspect, the present disclosure provides a composition comprising a peptide comprising an amino acid sequence comprising a fragment of three consecutive amino acids selected from one of the following amino acid sequences: TGSTQHQ (SEQ ID NO:1), HSALTHH (SEQ ID NO:2), KTGSHNQ (SEQ ID NO:3), MGPSSML (SEQ ID NO:4), TDPNQLQ (SEQ ID NO:5) or STHFIDT (SEQ ID NO:6), wherein the peptide is coupled to an active agent or an active agent carrier comprising the active agent, and wherein the composition is capable of penetrating the stratum corneum upon contact with the Stratum Corneum (SC) or penetrating cells upon contact with the cells.

In one embodiment of the third aspect, the composition is capable of penetrating the Stratum Corneum (SC) as well as penetrating cells.

In one embodiment of the third aspect, the amino acid sequence comprises a fragment of four consecutive amino acids selected from one of the following amino acid sequences: TGSTQHQ (SEQ ID NO:1), HSALTPH (SEQ ID NO:2), KTGSHNQ (SEQ ID NO:3), MGPSSML (SEQ ID NO:4), TDPNQLQ (SEQ ID NO:5) or STHFIDT (SEQ ID NO: 6).

In one embodiment of the third aspect, the amino acid sequence comprises a fragment of five consecutive amino acids selected from one of the following amino acid sequences: TGSTQHQ (SEQ ID NO:1), HSALTPH (SEQ ID NO:2), KTGSHNQ (SEQ ID NO:3), MGPSSML (SEQ ID NO:4), TDPNQLQ (SEQ ID NO:5) or STHFIDT (SEQ ID NO: 6).

In one embodiment of the third aspect, the amino acid sequence comprises a fragment of six consecutive amino acids selected from one of the following amino acid sequences: TGSTQHQ (SEQ ID NO:1), HSALTPH (SEQ ID NO:2), KTGSHNQ (SEQ ID NO:3), MGPSSML (SEQ ID NO:4), TDPNQLQ (SEQ ID NO:5) or STHFIDT (SEQ ID NO: 6).

In a fourth aspect, the present disclosure provides a composition comprising a peptide comprising an amino acid sequence comprising a fragment of three consecutive amino acids selected from one of the following amino acid sequences: TGSTQHQ (SEQ ID NO:1), HSALTPKH (SEQ ID NO:2), KTGSHNQ (SEQ ID NO:3), MGPSSML (SEQ ID NO:4), TDPNQLQ (SEQ ID NO:5) or STHFIDT (SEQ ID NO:6), wherein the peptide is associated with an active agent or an active agent carrier comprising the active agent, wherein the association results from hydrophobic, electrostatic or van der Waals interactions, and wherein the composition is capable of penetrating the stratum corneum upon contact with the Stratum Corneum (SC) or penetrating cells upon contact with cells.

In one embodiment of the fourth aspect, the composition is capable of penetrating the Stratum Corneum (SC) as well as penetrating cells.

In one embodiment of the fourth aspect, the amino acid sequence comprises a fragment of four consecutive amino acids selected from one of the following amino acid sequences: TGSTQHQ (SEQ ID NO:1), HSALTPH (SEQ ID NO:2), KTGSHNQ (SEQ ID NO:3), MGPSSML (SEQ ID NO:4), TDPNQLQ (SEQ ID NO:5) or STHFIDT (SEQ ID NO: 6).

In one embodiment of the fourth aspect, the amino acid sequence comprises a fragment of five consecutive amino acids selected from one of the following amino acid sequences: TGSTQHQ (SEQ ID NO:1), HSALTPH (SEQ ID NO:2), KTGSHNQ (SEQ ID NO:3), MGPSSML (SEQ ID NO:4), TDPNQLQ (SEQ ID NO:5) or STHFIDT (SEQ ID NO: 6).

In one embodiment of the fourth aspect, the amino acid sequence comprises a fragment of six consecutive amino acids selected from one of the following amino acid sequences: TGSTQHQ (SEQ ID NO:1), HSALTPH (SEQ ID NO:2), KTGSHNQ (SEQ ID NO:3), MGPSSML (SEQ ID NO:4), TDPNQLQ (SEQ ID NO:5) or STHFIDT (SEQ ID NO: 6).

In a fifth aspect, the present disclosure provides an isolated peptide comprising an amino acid sequence selected from one of the following sequences: ACTGSTQHQCG (SEQ ID NO:13), ACHSALTKHCG (SEQ ID NO:14), ACKTGSHNQCG (SEQ ID NO:15), ACMGPSSMLCG (SEQ ID NO:16), ACTDPNQLQCG (SEQ ID NO:17) and ACSTHFIDTCG (SEQ ID NO: 18).

In one embodiment of the fifth aspect, the peptide comprises repeat units of one or more of ACTGSTQHQCG (SEQ ID NO:13), ACHSALTKHCG (SEQ ID NO:14), ACKTGSHNQCG (SEQ ID NO:15), ACMGPSSMLCG (SEQ ID NO:16), ACTDPNQLQCG (SEQ ID NO:17) and ACSTHFIDTCG (SEQ ID NO: 18). In one embodiment, wherein the peptide comprises a repeating unit of one or more of ACTGSTQHQCG (SEQ ID NO:13), ACHSALTKHCG (SEQ ID NO:14), ACKTGSHNQCG (SEQ ID NO:15), ACMGPSSMLCG (SEQ ID NO:16), ACTDPNQLQCG (SEQ ID NO:17) and ACSTHFIDTCG (SEQ ID NO:18), the unit is repeated 2 to 50 times. In one embodiment, wherein the peptide comprises repeating units of one or more of ACTGSTQHQCG (SEQ ID NO:13), ACHSALTKHCG (SEQ ID NO:14), ACKTGSHNQCG (SEQ ID NO:15), ACMGPSSMLCG (SEQ ID NO:16), ACTDPNQLQCG (SEQ ID NO:17) and ACSTHFIDTCG (SEQ ID NO:18), each unit separated by an intervening peptide sequence.

In one embodiment of the fifth aspect, the peptide is a cyclic peptide comprising a Cys-Cys disulfide bond.

In a sixth aspect, the present disclosure provides an isolated polypeptide comprising repeat units of one or more of: TGSTQHQ (SEQ ID NO:1), HSALTPH (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).

In one embodiment of the sixth aspect, the unit is repeated 2 to 50 times.

In one embodiment of this sixth aspect, each unit is separated by an intervening peptide sequence.

In a seventh aspect, the present disclosure provides an isolated polypeptide consisting essentially of repeat units of one or more of: TGSTQHQ (SEQ ID NO:1), HSALTPH (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). In one embodiment, wherein the isolated polypeptide consists essentially of repeat units of one or more of: TGSTQHQ (SEQ ID NO:1), HSALTPH (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).

In an eighth aspect, the present disclosure provides a method of delivering an active agent to a subject, comprising: administering to the subject a composition comprising a peptide comprising the amino acid sequence TGSTQHQ (SEQ ID NO:1), HSALTPKH (SEQ ID NO:2), KTGSHNQ (SEQ ID NO:3), MGPSSML (SEQ ID NO:4), TDPNQLQ (SEQ ID NO:5), or STHFIDT (SEQ ID NO:6), wherein the peptide is coupled to an active agent or an active agent carrier comprising the active agent, and wherein the composition is capable of penetrating the Stratum Corneum (SC) of the subject or penetrating cells of the subject.

In one embodiment of the eighth aspect, the composition is capable of penetrating the Stratum Corneum (SC) of the subject and penetrating cells of the subject.

In one embodiment of the eighth aspect, the administering is topical administering.

In one embodiment of the eighth aspect, the amino acid sequence comprises CTGSTQHQC (SEQ ID NO:7), CHSALTKHC (SEQ ID NO:8), CKTGSHNQC (SEQ ID NO:9), CMGPSSMLC (SEQ ID NO:10), CTDPNQLQC (SEQ ID NO:11) or CSTHFIDTC (SEQ ID NO: 12).

In one embodiment of the eighth aspect, the amino acid sequence comprises ACTGSTQHQCG (SEQ ID NO:13), ACHSALTKHCG (SEQ ID NO:14), ACKTGSHNQCG (SEQ ID NO:15), ACMGPSSMLCG (SEQ ID NO:16), ACTDPNQLQCG (SEQ ID NO:17) or ACSTHFIDTCG (SEQ ID NO: 18). In one embodiment, wherein the amino acid sequence comprises ACTGSTQHQCG (SEQ ID NO:13), ACHSALTKHCG (SEQ ID NO:14), ACKTGSHNQCG (SEQ ID NO:15), ACMGPSSMLCG (SEQ ID NO:16), ACTDPNQLQCG (SEQ ID NO:17), or ACSTHFIDTCG (SEQ ID NO:18), the peptide is a cyclic peptide comprising a Cys-Cys disulfide bond.

In one embodiment of the eighth aspect, the composition is capable of penetrating the cell membrane of a living non-human animal cell.

In one embodiment of the eighth aspect, the composition is capable of penetrating the cell membrane of a living human cell.

In one embodiment of the eighth aspect, the composition is capable of penetrating the cell membrane of a living epidermal or dermal cell.

In one embodiment of the eighth aspect, the composition is capable of penetrating the cell membrane of a living immune cell.

In one embodiment of the eighth aspect, the active agent comprises a macromolecule. In one embodiment, the macromolecule comprises a protein. In one embodiment, the protein comprises an antibody or fragment thereof comprising at least one paratope.

In one embodiment of the eighth aspect, wherein the active agent comprises a macromolecule, the macromolecule comprises a nucleic acid. In one embodiment, the nucleic acid is DNA. In one embodiment, the nucleic acid is RNA. In one embodiment, wherein the nucleic acid is RNA, the RNA is an interfering RNA. In one embodiment, wherein the RNA is an interfering RNA, the interfering RNA is an shRNA. In one embodiment, wherein the RNA is an interfering RNA, the interfering RNA is a miRNA. In one embodiment, wherein the RNA is an interfering RNA, the interfering RNA is siRNA. In one embodiment, wherein the interfering RNA is siRNA, the siRNA is IL-10 siRNA. In one embodiment, wherein the interfering RNA is siRNA, the siRNA is CD86 siRNA. In one embodiment, wherein the interfering RNA is an siRNA, the siRNA is KRT6a siRNA. In one embodiment, wherein the interfering RNA is siRNA, the siRNA is TNFR1 siRNA. In one embodiment, wherein the interfering RNA is siRNA, the siRNA is TACesiRNA. In one embodiment, wherein the interfering RNA is siRNA, the siRNA is mutation-specific siRNA.

In one embodiment of the eighth aspect, the active agent is a pharmaceutical compound.

In one embodiment of the eighth aspect, the active agent comprises a detectable agent. In one embodiment, wherein the active agent comprises a detectable agent, the detectable agent comprises a fluorescent label. In one embodiment, wherein the active agent comprises a detectable agent, the detectable agent comprises a radioactive label.

In one embodiment of the eighth aspect, the active agent is a nanoparticle.

In one embodiment of the eighth aspect, the active agent is a low molecular weight compound.

In one embodiment of the eighth aspect, the active agent is an inhibitor of IL-10 biological activity. In one embodiment, wherein the active agent is an inhibitor of IL-10 biological activity, the active agent is selected from the group consisting of IL-10siRNA and an antibody or fragment thereof that binds IL-10.

In one embodiment of the eighth aspect, the peptide is coupled to the active agent.

In one embodiment of the eighth aspect, the peptide is coupled to an active agent carrier comprising the active agent. In one embodiment, wherein the peptide is coupled to an active agent carrier comprising the active agent, the active agent carrier is a liposome. In one embodiment, wherein the peptide is coupled to an active agent carrier comprising the active agent, the active agent carrier is a nanoparticle. In one embodiment, wherein the peptide is coupled to an active agent carrier comprising the active agent, the active agent carrier is a polymeric micelle.

In a ninth aspect, the present disclosure provides a method of delivering an active agent to a subject, comprising: administering to the subject a composition comprising a peptide comprising the amino acid sequence TGSTQHQ (SEQ ID NO:1), HSALTPKH (SEQ ID NO:2), KTGSHNQ (SEQ ID NO:3), MGPSSML (SEQ ID NO:4), TDPNQLQ (SEQ ID NO:5), or STHFIDT (SEQ ID NO:6), wherein the peptide is associated with an active agent or an active agent carrier comprising the active agent, wherein the association results from hydrophobic, electrostatic, or van der Waals interactions, and wherein the composition is capable of penetrating the Stratum Corneum (SC) of the subject or penetrating cells of the subject.

In one embodiment of the ninth aspect, the composition is capable of penetrating the Stratum Corneum (SC) of the subject and penetrating cells of the subject.

In one embodiment of the ninth aspect, the administering is topical administering.

In one embodiment of the ninth aspect, the amino acid sequence comprises CTGSTQHQC (SEQ ID NO:7), CHSALTKHC (SEQ ID NO:8), CKTGSHNQC (SEQ ID NO:9), CMGPSSMLC (SEQ ID NO:10), CTDPNQLQC (SEQ ID NO:11) or CSTHFIDTC (SEQ ID NO: 12).

In one embodiment of the ninth aspect, the amino acid sequence comprises ACTGSTQHQCG (SEQ ID NO:13), ACHSALTKHCG (SEQ ID NO:14), ACKTGSHNQCG (SEQ ID NO:15), ACMGPSSMLCG (SEQ ID NO:16), ACTDPNQLQCG (SEQ ID NO:17) or ACSTHFIDTCG (SEQ ID NO: 18). In one embodiment, wherein the amino acid sequence comprises ACTGSTQHQCG (SEQ ID NO:13), ACHSALTKHCG (SEQ ID NO:14), ACKTGSHNQCG (SEQ ID NO:15), ACMGPSSMLCG (SEQ ID NO:16), ACTDPNQLQCG (SEQ ID NO:17), or ACSTHFIDTCG (SEQ ID NO:18), the peptide is a cyclic peptide comprising a Cys-Cys disulfide bond.

In a tenth aspect, the present disclosure provides a method of treating a subject having a skin disease, comprising: administering to the subject a composition comprising a peptide comprising the amino acid sequence TGSTQHQ (SEQ ID NO:1), HSALTPKH (SEQ ID NO:2), KTGSHNQ (SEQ ID NO:3), MGPSSML (SEQ ID NO:4), TDPNQLQ (SEQ ID NO:5), or STHFIDT (SEQ ID NO:6), wherein the peptide is coupled to a dermatologically active agent or a dermatologically active agent carrier comprising the active agent, and wherein the composition is capable of penetrating the Stratum Corneum (SC) of the subject or penetrating cells of the subject.

In one embodiment of the tenth aspect, the composition is capable of penetrating the Stratum Corneum (SC) of the subject and penetrating cells of the subject.

In one embodiment of the tenth aspect, the administration is topical administration.

In one embodiment of the tenth aspect, the amino acid sequence comprises ACTGSTQHQCG (SEQ ID NO:13), ACHSALTKHCG (SEQ ID NO:14), ACKTGSHNQCG (SEQ ID NO:15), ACMGPSSMLCG (SEQ ID NO:16), ACTDPNQLQCG (SEQ ID NO:17) or ACSTHFIDTCG (SEQ ID NO: 18). In one embodiment, wherein the amino acid sequence comprises ACTGSTQHQCG (SEQ ID NO:13), ACHSALTKHCG (SEQ ID NO:14), ACKTGSHNQCG (SEQ ID NO:15), ACMGPSSMLCG (SEQ ID NO:16), ACTDPNQLQCG (SEQ ID NO:17), or ACSTHFIDTCG (SEQ ID NO:18), the peptide is a cyclic peptide comprising a Cys-Cys disulfide bond.

In an eleventh aspect, the present disclosure provides a method of treating a subject having a skin disease, comprising: administering to the subject a composition comprising a peptide comprising the amino acid sequence TGSTQHQ (SEQ ID NO:1), HSALTPKH (SEQ ID NO:2), KTGSHNQ (SEQ ID NO:3), MGPSSML (SEQ ID NO:4), TDPNQLQ (SEQ ID NO:5), or STHFIDT (SEQ ID NO:6), wherein the peptide is associated with a dermatologically active agent or a dermatologically active agent carrier comprising the active agent, wherein the association results from hydrophobic, electrostatic, or van der Waals interactions, and wherein the composition is capable of penetrating the Stratum Corneum (SC) of the subject or penetrating cells of the subject.

In one embodiment of the eleventh aspect, the composition is capable of penetrating the Stratum Corneum (SC) of the subject and penetrating cells of the subject.

In one embodiment of the eleventh aspect, the administering is topical administration.

In one embodiment of the eleventh aspect, the amino acid sequence comprises ACTGSTQHQCG (SEQ ID NO:13), ACHSALTKHCG (SEQ ID NO:14), ACKTGSHNQCG (SEQ ID NO:15), ACMGPSSMLCG (SEQ ID NO:16), ACTDPNQLQCG (SEQ ID NO:17) or ACSTHFIDTCG (SEQ ID NO: 18). In one embodiment, wherein the amino acid sequence comprises ACTGSTQHQCG (SEQ ID NO:13), ACHSALTKHCG (SEQ ID NO:14), ACKTGSHNQCG (SEQ ID NO:15), ACMGPSSMLCG (SEQ ID NO:16), ACTDPNQLQCG (SEQ ID NO:17), or ACSTHFIDTCG (SEQ ID NO:18), the peptide is a cyclic peptide comprising a Cys-Cys disulfide bond.

In a twelfth aspect, the present disclosure provides a method of treating a subject having, suspected of having, or susceptible to a disease caused at least in part by expression of an mRNA, comprising administering to the subject a composition comprising a peptide comprising the amino acid sequence 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), or STHFIDT (SEQ ID NO:6), wherein the peptide is coupled to an interfering RNA targeting the mRNA or a vector comprising the interfering RNA, wherein the composition is capable of penetrating the Stratum Corneum (SC) of the subject or cells of the subject, and wherein expression of the mRNA is thereby attenuated.

In one embodiment of the twelfth aspect, the composition is capable of penetrating the Stratum Corneum (SC) of the subject and penetrating cells of the subject.

In one embodiment of the twelfth aspect, the administration is topical administration.

In one embodiment of the twelfth aspect, the amino acid sequence comprises ACTGSTQHQCG (SEQ ID NO:13), ACHSALTKHCG (SEQ ID NO:14), ACKTGSHNQCG (SEQ ID NO:15), ACMGPSSMLCG (SEQ ID NO:16), ACTDPNQLQCG (SEQ ID NO:17) or ACSTHFIDTCG (SEQ ID NO: 18). In one embodiment, wherein the amino acid sequence comprises ACTGSTQHQCG (SEQ ID NO:13), ACHSALTKHCG (SEQ ID NO:14), ACKTGSHNQCG (SEQ ID NO:15), ACMGPSSMLCG (SEQ ID NO:16), ACTDPNQLQCG (SEQ ID NO:17), or ACSTHFIDTCG (SEQ ID NO:18), the peptide is a cyclic peptide comprising a Cys-Cys disulfide bond.

In a thirteenth aspect, the present disclosure provides a method of treating a subject having, suspected of having, or susceptible to a disease caused at least in part by expression of an mRNA, comprising administering to the subject a composition comprising a peptide comprising the amino acid sequence 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), or STHFIDT (SEQ ID NO:6), wherein the peptide is associated with an interfering RNA that targets the mRNA or a vector comprising the interfering RNA, wherein the association results from hydrophobic, electrostatic, or van der waals interactions, wherein the composition is capable of penetrating the Stratum Corneum (SC) of the subject or cells of the subject, and wherein expression of the mRNA is thereby attenuated.

In one embodiment of the thirteenth aspect, the composition is capable of penetrating the Stratum Corneum (SC) of the subject and penetrating cells of the subject.

In one embodiment of the thirteenth aspect, the administering is topical administering.

In one embodiment of the thirteenth aspect, the amino acid sequence comprises ACTGSTQHQCG (SEQ ID NO:13), ACHSALTKHCG (SEQ ID NO:14), ACKTGSHNQCG (SEQ ID NO:15), ACMGPSSMLCG (SEQ ID NO:16), ACTDPNQLQCG (SEQ ID NO:17) or ACSTHFIDTCG (SEQ ID NO: 18). In one embodiment, wherein the amino acid sequence comprises ACTGSTQHQCG (SEQ ID NO:13), ACHSALTKHCG (SEQ ID NO:14), ACKTGSHNQCG (SEQ ID NO:15), ACMGPSSMLCG (SEQ ID NO:16), ACTDPNQLQCG (SEQ ID NO:17), or ACSTHFIDTCG (SEQ ID NO:18), the peptide is a cyclic peptide comprising a Cys-Cys disulfide bond.

In a fourteenth aspect, the present disclosure provides a method of attenuating mRNA expression in a subject in need thereof, comprising administering to the subject a composition comprising a peptide comprising the amino acid sequence 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), or STHFIDT (SEQ ID NO:6), wherein the peptide is coupled to an siRNA targeting the mRNA or a vector comprising an siRNA targeting the mRNA, wherein the composition is capable of penetrating the Stratum Corneum (SC) of the subject or cells of the subject, and wherein expression of the mRNA is thereby attenuated.

In one embodiment of the fourteenth aspect, the mRNA is IL-10mRNA and the siRNA is IL-10 siRNA.

In one embodiment of the fourteenth aspect, the mRNA is a CD86mRNA and the siRNA is a CD86 siRNA.

In one embodiment of the fourteenth aspect, the mRNA is KRT6a mRNA and the siRNA is KRT6a siRNA.

In one embodiment of the fourteenth aspect, the mRNA is a TNFR1mRNA and the siRNA is a TNFR1 siRNA.

In one embodiment of the fourteenth aspect, the mRNA is a TACE mRNA and the siRNA is a TACE siRNA.

In one embodiment of the fourteenth aspect, the composition is capable of penetrating the Stratum Corneum (SC) of the subject and penetrating cells of the subject.

In one embodiment of the fourteenth aspect, the administering is topical administering.

In one embodiment of the fourteenth aspect, the amino acid sequence comprises ACTGSTQHQCG (SEQ ID NO:13), ACHSALTKHCG (SEQ ID NO:14), ACKTGSHNQCG (SEQ ID NO:15), ACMGPSSMLCG (SEQ ID NO:16), ACTDPNQLQCG (SEQ ID NO:17) or ACSTHFIDTCG (SEQ ID NO: 18). In one embodiment, wherein the amino acid sequence comprises ACTGSTQHQCG (SEQ ID NO:13), ACHSALTKHCG (SEQ ID NO:14), ACKTGSHNQCG (SEQ ID NO:15), ACMGPSSMLCG (SEQ ID NO:16), ACTDPNQLQCG (SEQ ID NO:17), or ACSTHFIDTCG (SEQ ID NO:18), the peptide is a cyclic peptide comprising a Cys-Cys disulfide bond.

In a fifteenth aspect, the present disclosure provides a method of attenuating mRNA expression in a subject in need thereof, comprising administering to the subject a composition comprising a peptide comprising the amino acid sequence 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), or STHFIDT (SEQ ID NO:6), wherein the peptide is associated with a siRNA targeting the mRNA or a vector comprising a siRNA targeting the mRNA, wherein the association results from hydrophobic, electrostatic, or van der waals interactions, wherein the composition is capable of penetrating the Stratum Corneum (SC) of the subject or cells of the subject, and wherein expression of the mRNA is thereby attenuated.

In one embodiment of the fifteenth aspect, the mRNA is IL-10mRNA and the siRNA is IL-10 siRNA.

In one embodiment of the fifteenth aspect, the mRNA is a CD86mRNA and the siRNA is a CD86 siRNA.

In one embodiment of the fifteenth aspect, the mRNA is KRT6a mRNA and the siRNA is KRT6a siRNA.

In one embodiment of the fifteenth aspect, the mRNA is a TNFR1mRNA and the siRNA is a TNFR1 siRNA.

In one embodiment of the fifteenth aspect, the mRNA is a TACE mRNA and the siRNA is a TACE siRNA.

In one embodiment of the fifteenth aspect, the composition is capable of penetrating the Stratum Corneum (SC) of the subject and penetrating cells of the subject.

In one embodiment of the fifteenth aspect, the administering is topical administering.

In one embodiment of the fifteenth aspect, the amino acid sequence comprises ACTGSTQHQCG (SEQ ID NO:13), ACHSALTKHCG (SEQ ID NO:14), ACKTGSHNQCG (SEQ ID NO:15), ACMGPSSMLCG (SEQ ID NO:16), ACTDPNQLQCG (SEQ ID NO:17) or ACSTHFIDTCG (SEQ ID NO: 18). In one embodiment, wherein the amino acid sequence comprises ACTGSTQHQCG (SEQ ID NO:13), ACHSALTKHCG (SEQ ID NO:14), ACKTGSHNQCG (SEQ ID NO:15), ACMGPSSMLCG (SEQ ID NO:16), ACTDPNQLQCG (SEQ ID NO:17), or ACSTHFIDTCG (SEQ ID NO:18), the peptide is a cyclic peptide comprising a Cys-Cys disulfide bond.

In a sixteenth aspect, the present disclosure provides a composition comprising a peptide consisting essentially of the amino acid sequence 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), or sttdt hfq (SEQ ID NO:6), wherein the peptide is coupled to an active agent or an active agent carrier comprising the active agent, and wherein the composition is capable of penetrating the stratum corneum upon contact with the Stratum Corneum (SC) or penetrating cells upon contact with the cells.

Other aspects and embodiments will be apparent to one of ordinary skill upon reading this specification.

Brief Description of Drawings

Figure 1 shows the identification of skin penetrating peptides by in vitro phage display in pig skin. (a) The phage library was applied to the donor compartment of the FDC. Phages that were found to penetrate the skin into the recipient chamber were collected, amplified, and used for subsequent rounds of screening. The skin penetration ability of each clone was confirmed by diffusion experiments and confocal microscopy. To confirm the ability of the peptide to penetrate the skin, the peptide was separated from the phage, and penetration into the skin was visually confirmed by confocal microscopy; (b) the percentage of appearance of each high-frequency peptide sequence in rounds 3 to 5 of phage display screening; (c, d) confocal microscopy images of skin penetration of SPACE and control peptide into pig skin, respectively; (e, f) skin penetration of Alexa Fluor 488-labeled streptavidin coupled to biotinylated SPACE peptide and Alexa Fluor488 streptavidin alone. The inset shows an enlarged view of the highlighted portion in the main image. Scale bar 200 μm.

Fig. 2 shows confocal microscopy images showing passage of fluorescently labeled molecules through the skin. (a, b) penetration of the fluorescently labeled phage-displayed peptide into the pig skin. (c, d) the biotinylated peptide-streptavidin coated quantum dots penetrate into the pig skin. (e, f) the fluorescently labeled peptide penetrates into the human skin. (g, h) top view of fluorescently labeled peptides in human skin (top view SC). (a, c, e, g) represents an image of the SPACE peptide, and (b, d, f, h) represents an image of the control peptide. Scale bar 200 μm.

FIG. 3 shows an image of a fluorescently labeled peptide 30 minutes after penetrating into the skin of a live mouse. Three representative images are provided for each case. (a-c) penetration of fluorescently labeled control peptide, and (d-f) penetration of fluorescently labeled SPACE peptide. Scale bar 50 μm.

FIG. 4 shows images of fluorescently labeled peptides 2 hours after penetrating into the skin of a live mouse. Three representative images are provided for each case. (a-c) penetration of fluorescently labeled control peptide, and (d-f) penetration of fluorescently labeled SPACE peptide. Scale bar 50 μm.

FIG. 5 shows the results of images of fluorescent-labeled peptides bound to defatted stratum corneum (a, b) and FTIR spectra of stratum corneum before and after peptide treatment (c-f). Degreased SC images under uv (a) and visible light (different samples) (b). (i, ii, iii) represent SPACE peptide, control peptide and no peptide, respectively. Scale bar 1 cm. Note that the stratum corneum samples shown above were degreased to ensure comparison of the binding capacity of the peptides, not the transport capacity. Thus, the differences between the control and SPACE peptides as seen above may not translate directly into their effect on skin penetration. (c, d) 1400-1700cm of spectra for SPACE peptide and control peptide, respectively^-1And (4) a zone. (e, f) 2800-3000cm spectra for SPACE peptide and control peptide, respectively^-1And (4) a zone. The initial and final spectra are indicated by arrows.

Fig. 6 provides FTIR spectra of amide I and amide II regions of the stratum corneum before and after peptide-free treatment.

FIG. 7 shows graphical results of inulin penetration and conductivity enhancement of pigskin after co-incubation with Control (CP) and SPACE peptide.

Figure 8 shows penetration of the SPACE peptide into various cell lines. (a, c, e) confocal images of cells without peptide treatment, (b, d, f) confocal images of cells incubated with fluorescently labeled SPACE peptide for 24 hours. (a, b) human keratinocytes, (c, d) human fibroblasts, and (e, f) Human Umbilical Vein Endothelial Cells (HUVECs). (g) Magnified images of SPACE peptide internalization in human keratinocytes. (h) Mean fluorescence intensity after 24 hours internalization of control peptide (open bars) and SPACE peptide (solid bars) in HUVEC, fibroblasts and keratinocytes. Error bars indicate standard deviation (N > or ═ 30). Scale bar 100 μm (a-f) and 20 μm (g).

FIG. 9 shows the results of images of fluorescently labeled peptides after 6 hours of penetration into MDA-MBA-231 human breast cancer cells (a-c). Images of cells treated without peptide (a), control peptide (b) and SPACE peptide (c). Fig. 7 also shows confocal images (d-f) of GFP-expressing endothelial cells after treatment with GFP siRNA complexed with Lipofectamine at a scale bar of 50 μm. (d) Treating cells with Lipofectamine alone (no siRNA), (e) treating cells with Lipofectamine complexed with GFP siRNA, and (f) treating cells with Lipofectamine complexed with SPACE-GFP siRNA. Scale bar 200 μm.

FIG. 10 shows the results of cellular mechanisms and toxicity studies. (a) Peptide internalization of control and SPACE peptides in human keratinocytes at 4 ℃ and with the endocytosis inhibitors deoxy-D-glucose, chlorpromazine, nystatin and EIPA (% control). (b) Cell proliferation of human keratinocytes in the presence of 0.1mg/mL and 1.0mg/mL control peptides (open bars) or SPACE peptides (solid bars). Error bars indicate standard deviation (N-4).

Figure 11 shows the results of siRNA delivery using the SPACE peptide. (a) Percentage knockdown of GPF in GFP expressing endothelial cells. Error bars indicate SD (N > or ═ 30), (b) percentage of IL-10 protein levels in mice 24 hours post treatment. Error bars indicate SE (N > or ═ 3), NS indicates no significant difference (p > 0.15), (c) percent knockdown of GAPDH protein levels in mice 72 hours post-treatment. Error bars indicate dose dependence of SE (N > or ═ 3), (d) GAPDH knockdown after topical application of siRNA. Error bars indicate SE (N > or ═ 3).

Figure 12 shows the results of GAPDH knockdown using peptide-coupled GAPDH sirns at different application times. Reduction of GAPDH protein levels 4, 24, and 72 hours after SPACE-GAPDH siRNA application.

Definition of

Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

If a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. If the stated range includes one or both of the stated limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, exemplary methods and materials are now described. All publications and applications mentioned herein are incorporated by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. In the event that any application or publication incorporated by reference conflicts with the present disclosure, the present disclosure controls.

It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a peptide" includes a plurality of such peptides, and reference to "the agent" includes reference to one or more agents and equivalents thereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such publication by virtue of prior invention. In addition, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

It is understood that throughout this disclosure, reference to amino acids is made according to the single or three letter code. For the convenience of the reader, the single and three letter amino acid codes are provided as follows:

g glycine Gly P proline Pro

Alanine Ala V Val

L leucine Leu I isoleucine Ile

M methionine Met C cysteine Cys

F phenylalanine Phe Y tyrosine Tyr

W Trp H His of Tryptophan

K lysine Lys R arginine Arg

Q Glutamine Gln N asparagine Asn

E glutamic acid Glu D aspartic acid Asp

S serine Ser T threonine Thr

As used herein, the term "active agent" refers to an agent, such as a protein, peptide, nucleic acid (including, for example, nucleotides, nucleosides, and analogs thereof), or small molecule drug, that provides a desired pharmacological effect upon administration to a subject, such as a human or non-human animal, alone or in combination with other active or inert components. The above definitions include precursors, derivatives, analogs and prodrugs of the active agents. The term "active agent" is also used herein to broadly refer to any agent, such as a protein, peptide, nucleic acid (including, for example, nucleotides, nucleosides, and analogs thereof), or small molecule drug, coupled to or associated with or contained by an active agent carrier as described herein.

The term "coupled" as used in the context of the penetrating peptide compositions described herein refers to a covalent or ionic interaction between two entities (e.g., molecules, compounds, or combinations thereof).

The term "associate," as used in the context of penetrating peptide compositions described herein, 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 waals interactions.

The terms "polypeptide" and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length (which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids), as well as polypeptides having modified peptide backbones. The term includes fusion proteins, including but not limited to fusion proteins with heterologous amino acid sequences, fusions with heterologous and native leader sequences, with or without an N-terminal methionine residue; an immunolabeling protein; a fusion protein formed with a detectable fusion partner, for example, a fusion protein comprising a fluorescent protein, β -galactosidase, luciferase, or the like as a fusion partner; and so on.

The terms "antibody" and "immunoglobulin" include antibodies or immunoglobulins of any isotype, antibody fragments that retain specific binding to an antigen (including but not limited to Fab, Fv, scFv, and Fd fragments), chimeric antibodies, humanized antibodies, single chain antibodies, and antigen-binding portions and fragments comprising an antibodyA fusion protein of a non-antibody protein. The antibody can be detectably labeled, for example, with a radioisotope, an enzyme that produces a detectable product, a fluorescent protein, or the like. The antibody may further be coupled to other moieties, such as members of a specific binding pair, e.g., biotin (a member of a biotin-avidin specific binding pair), and the like. These terms also encompass Fab ', Fv, F (ab')₂And other antibody fragments that retain specific binding to the antigen.

Antibodies can exist in a variety of other forms, including, for example, Fv, Fab and (Fab')₂And bifunctional (i.e., bispecific) hybrid antibodies (e.g., Lanzavecchia et al, Eur. J. Immunol.17, 105(1987)) as well as in single chain (e.g., Huston et al, Proc. Natl. Acad. Sci. U.S.A., 85, 5879-. (see generally Hood et al Immunology, Benjamin, N.Y., 2 nd edition (1984), and Hunkapiller and Hood, Nature, 323, 15-16 (1986)).

The terms "nucleic acid," "nucleic acid molecule," and "polynucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length (deoxyribonucleotides or ribonucleotides or analogs thereof). These terms encompass, for example, DNA, RNA, and modified forms thereof. The polynucleotide may have any three-dimensional structure and may perform any known or unknown function. Non-limiting examples of polynucleotides include genes, gene fragments, 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 may be linear or circular.

"RNA interference" (RNAi) is the process by which double-stranded RNA (dsRNA) is used to silence gene expression. Without wishing to be bound by any particular theory, RNAi begins with cleavage of the longer dsRNA into small interfering rnas (sirnas) by RNaseIII-like enzyme butylase (dicer). siRNA are dsRNA, typically about 19 to 28 nucleotides, or 20 to 25 nucleotides, or 21 to 23 nucleotides in length, and typically comprise a 2-3 nucleotide 3 ' overhang and 5 ' phosphate and 3 ' hydroxyl termini. One strand of the siRNA is incorporated into a ribonucleoprotein complex called the RNA-induced silencing complex (RISC). RISC uses this siRNA strand to recognize mRNA molecules that are at least partially complementary to the bound siRNA strand, and then cleaves these target mrnas or inhibits their translation. The siRNA strand incorporated into RISC is called the guide strand or antisense strand. The other siRNA strand, termed the passenger or sense strand, is eliminated from the siRNA and is at least partially homologous to the target mRNA. It will be appreciated by those skilled in the art that in principle either strand of the siRNA can be incorporated into the RISC and used as the guide strand. However, the siRNA can be designed (e.g., by decreasing the stability of the siRNA duplex at the 5' end of the antisense strand) to favor incorporation of the antisense strand into RISC.

RISC-mediated cleavage of mRNA whose sequence is at least partially complementary to the guide strand results in a decrease in the steady state level of that mRNA and the corresponding protein encoded by the mRNA. Alternatively, RISC can also reduce the expression of the corresponding protein by translational inhibition without cleaving the target mRNA. Other RNA molecules can interact with RISC and silence gene expression. Examples of other RNA molecules that can interact with RISC include short hairpin RNA (shrna), single stranded siRNA, microrna (mirna), and dicer substrate 27-mer duplexes, i.e., RNA molecules containing one or more chemically modified nucleotides, one or more deoxyribonucleotides, and/or one or more non-phosphodiester linkages. The term "siRNA" as used herein refers to double-stranded interfering RNA unless otherwise indicated. To facilitate the discussion of the present invention, all RNA molecules that can interact with RISC and participate in RISC-mediated gene expression changes are referred to as "interfering RNAs". Thus, siRNA, shRNA, miRNA and dicer substrate 27-mer duplexes are a subset of "interfering RNA".

A "substitution" results from the substitution of one or more amino acids or nucleotides with a different amino acid or nucleotide, respectively, when compared to the amino acid sequence or nucleotide sequence of a polypeptide. If the substitution is conservative, the amino acid substituted into the polypeptide has similar structural or chemical properties (e.g., charge, polarity, hydrophobicity, etc.) as the amino acid it replaces. Conservative substitutions of naturally occurring amino acids typically result in the replacement of a first amino acid with a second amino acid in the same group as the first amino acid, where an exemplary set of amino acids is shown below: (1) acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; (2) basic (positively charged), such as arginine, histidine and lysine; (3) neutral polar amino acids such as glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; and (4) neutral nonpolar amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. In some embodiments, polypeptide variants may have "non-conservative" changes, in which the substituted amino acids differ in structural and/or chemical properties.

A "deletion" is defined as a change in an amino acid or nucleotide sequence in which one or more amino acid or nucleotide residues, respectively, are absent when compared to the amino acid sequence or nucleotide sequence of a naturally occurring polypeptide. In the context of polypeptide or polynucleotide sequences, a deletion may involve a 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.

An "insertion" or "addition" is an amino acid or nucleotide sequence change that results in the addition of one or more amino acid or nucleotide residues, respectively, when compared to the amino acid sequence or nucleotide sequence of a naturally occurring polypeptide. "insertion" generally refers to the addition of one or more amino acid residues within the amino acid sequence of a polypeptide, while "addition" may be an insertion or refer to the addition of amino acid residues at the N-or C-terminus. In the context of a polypeptide or polynucleotide sequence, an insertion or addition may have 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 that differs in amino acid sequence from that naturally occurring, or in the environment in which it is found in the context of an expression system or viral particle.

"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 may be native or non-native to the host cell, wherein the exogenous native nucleic acid or polypeptide elevates the level of the encoded gene product or polypeptide in the recombinant host cell relative to the level of the encoded gene product or polypeptide present in the host cell prior to introduction of the exogenous molecule.

As used herein, the terms "determining," "measuring," "evaluating," and "analyzing" are used interchangeably and include both quantitative and qualitative determinations.

As used herein, the term "isolated" when used in the context of isolating a compound refers to a compound of interest that is in an environment that is different from the environment in which the compound naturally occurs. "isolated" is intended to include compounds that are within a sample that is substantially enriched in a compound of interest and/or in which the compound of interest is partially or substantially purified.

As used herein, the term "substantially pure" refers to a compound that is removed from its natural environment and is at least 60% free, 75% free, or 90% free of other components with which it is naturally associated.

A "coding sequence" or a sequence "encoding" a selected polypeptide is a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo, for example, when placed under the control of appropriate regulatory sequences (or "control elements"). The boundaries of the coding sequence are generally determined by a start codon at the 5 '(amino) terminus and a translation stop codon at the 3' (carboxyl) 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. The transcription termination sequence may be located 3' to the coding sequence. Other "control elements" may also be associated with a coding sequence. The DNA sequence encoding the polypeptide may be optimized for expression in a selected cell by using codons preferred by the selected cell to represent a DNA copy of the desired polypeptide coding sequence.

"encoded by" refers to a nucleic acid sequence that encodes a gene product (e.g., a polypeptide). If the gene product is a polypeptide, the polypeptide sequence or portion thereof comprises 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 the polypeptide encoded by the nucleic acid sequence.

"operably linked" refers to an arrangement of elements wherein the components are configured to perform their usual function. In the case of a promoter, a promoter operably linked to a coding sequence will have an effect on the expression of the coding sequence. Promoters or other control elements need not be contiguous with the coding sequence, so long as they function to direct its expression. For example, an untranslated yet transcribed sequence can be inserted between a promoter sequence and a coding sequence, while the promoter sequence can still be considered "operably linked" to the coding sequence.

By "nucleic acid construct" is meant a nucleic acid sequence that is configured to contain one or more functional units that together are not found in nature. Examples include circular, linear, double-stranded, extrachromosomal DNA molecules (plasmids), cosmids (plasmids containing COS sequences from lambda phage), viral genomes containing non-native nucleic acid sequences, and the like.

A "vector" is capable of transferring a gene sequence to a target cell. In general, "vector construct", "expression vector" and "gene transfer vector" refer to any nucleic acid construct capable of directing the expression of a gene of interest and capable of transferring a gene sequence to a cell of interest, either by genomic integration of all or a portion of the vector or transient or heritable maintenance of the vector as an extrachromosomal element. Thus, 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 operably linked to an expression cassette promoter. Such expression cassettes can be constructed into "vectors", "vector constructs", "expression vectors" or "gene transfer vectors" in order to transfer the expression cassette into a cell of interest. Thus, the term includes cloning and expression vehicles as well as viral vectors.

Techniques for determining the "sequence identity" of nucleic acids and amino acids are known in the art. Typically, such techniques involve determining the nucleotide sequence of the gene mRNA and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Generally, "identity" refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotide or polypeptide sequences, respectively. Two or more sequences (polynucleotides or amino acids) 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 sequence multiplied by 100. Approximate alignments of nucleic acid sequences are provided by local homology algorithms in the following literature: smith and Waterman, Advances in applied Mathemetics, 2: 482-489(1981). The algorithm can be implemented by using the algorithm compiled by Dayhoff, Atlas of protein Sequences and structures, m.o. Dayhoff, supplement 5, 3: 353-: 6745 application 6763(1986) normalized scoring matrix to amino acid sequences.

An exemplary implementation of this algorithm to determine percent sequence identity is provided by genetics computer Group (Madison, Wis.) in the "BestFit" application. Default parameters for this method are described in Wisconsin Sequence Analysis Package Program Manual, 8 th edition (1995) (available from Genetics Computer Group, Madison, WI). Another way to establish percent identity in the context of the present invention is to use the MPSRCH package of the program owned by the copyright of university of Edinburgh, developed by John f.collins and shanes.sturrok, and distributed by IntelliGenetics, Inc. With this set of software, the Smith-Waterman algorithm can be employed, with default parameters being used for the scoring table (e.g., gap open penalty of 12, gap extension penalty of 1, gap of 6). With the data generated, the "match" value reflects "sequence identity". Other suitable programs for calculating percent identity or similarity between sequences are well known in the art, for example, another alignment program is BLAST, using default parameters. For example, BLASTN and BLASTP may be used with the following default parameters: the genetic code is standard; no filter; two chains; cutoff is 60; the expected value is 10; BLOSUM 62; stated as 50 sequences; the sorting mode is high score; database-not redundant-GenBank + EMBL + DDBJ + PDB + GenBank CDS translation library + Swiss protein database + stupdate + PIR. Details of these programs can be found on the internet, with http: v/in front of blast, ncbi, nlm, nih, gov/blast.

Alternatively, in the context of polynucleotides, homology can be determined by: polynucleotides are hybridized under conditions that form a stable duplex between the homologous regions, then digested with a single strand specific nuclease, and the size of the digested fragments is determined.

Two DNA or two polypeptide sequences are "substantially homologous" to each other if 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 molecule, as determined using the methods described above. As used herein, substantially homologous also refers to sequences that exhibit complete identity to a specified DNA or polypeptide sequence. Substantially homologous DNA sequences can be identified in DNA hybridization experiments, for example, under stringent conditions (as defined for that particular system). Defining suitable hybridization conditions is within the skill of the art. See, e.g., Sambrook and Russel, molecular cloning: ALaborory Manual third edition, (2001) Cold Spring Harbor laboratory Press, Cold Spring Harbor, NY.

A first polynucleotide is "derived" from a second polynucleotide when it has a nucleotide sequence that is the same or substantially the same as a region of the second polynucleotide, its cDNA, its complement, or when it exhibits sequence identity as described above. The term is not meant to require or imply that the polynucleotide must be obtained from the source in question (although this is also contemplated), but may be made by any suitable method.

A first polypeptide (or peptide) is "derived from" a second polypeptide (or peptide) when (i) encoded by a first polynucleotide derived from the second polynucleotide, or (ii) exhibits sequence identity to the second polypeptide as described above. The term is not meant to require or imply that the polypeptide must be obtained from the source in question (although this is also contemplated), but may be made by any suitable method.

The term "in combination" as used herein refers to a use wherein the first treatment is administered, e.g., throughout the administration of the second treatment; wherein the period of administration of the first treatment overlaps with the administration of the second treatment, e.g., administration of the first treatment begins before administration of the second treatment and administration of the first treatment ends before administration of the second treatment ends; wherein administration of the second treatment begins before administration of the first treatment and administration of the second treatment ends before administration of the first treatment ends; wherein administration of the first treatment begins before administration of the second treatment begins and administration of the second treatment ends before administration of the first treatment ends; wherein administration of the second treatment begins before administration of the first treatment begins and administration of the first treatment ends before administration of the second treatment ends. Thus, "combination" may also refer to a regimen involving administration of two or more treatments. As used herein, "in combination" also refers to the administration of two or more treatments, which may be administered in the same or different formulations, by the same or different routes, and in the same or different dosage forms.

The terms "treat," "treating," and the like refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or a symptom thereof, and/or may be therapeutic in terms of a partial or complete cure for the disease and/or adverse effects caused by the disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing disease development in a subject who may be predisposed to having the disease but has not yet been diagnosed as having the disease; (b) inhibiting a disease, i.e., arresting its development or progression; and (c) ameliorating the disease, i.e., reversing the disease and/or alleviating one or more symptoms of the disease. "treatment" is also intended to encompass delivery of an agent so as to provide a pharmacological effect, even in the absence of a disease or disorder. For example, "treatment" encompasses delivery of a penetrating peptide composition that can elicit an immune response or generate immunity in the absence of a disease condition, e.g., in the case of a vaccine.

"subject," "host," and "patient" are used interchangeably herein to refer to an animal, human, or non-human that may be treated according to the methods of the present disclosure or to which a peptide composition according to the present disclosure may be administered to achieve a desired effect. Generally, the subject is a mammalian subject.

The term "dermatitis" as used herein refers to skin inflammation and includes, for example, allergic contact dermatitis, urticaria, atonic dermatitis (dry skin of the lower leg), atopic dermatitis, contact dermatitis (including irritant contact dermatitis and contact dermatitis due to urushiol), eczema, gravitational dermatitis, nummular dermatitis, otitis externa, perioral dermatitis, and seborrheic dermatitis.

The term "stratum corneum" refers to the outer stratum corneum layer of the epidermis, consisting of flattened, keratinized, anucleated, dead, or exfoliated cells.

As used in the claims, the term "comprising" synonymous with "including", "containing", and "characterized by" is inclusive or open-ended and does not exclude additional, unrecited elements and/or method steps. "comprising" is a term of art that indicates the presence of the stated elements and/or steps, but that other elements and/or steps may be added and still fall within the scope of the associated subject matter.

The phrase "consisting of" as used herein excludes any elements, steps and/or ingredients not specifically recited. For example, when the phrase "consisting of" appears in the clause of the subject matter of the claims and does not immediately follow the preamble, it only limits the elements described in that clause; other elements as a whole are not excluded from the claims.

As used herein, the phrase "consisting essentially of limits the scope of the relevant disclosure or claims to the specified materials and/or steps, as well as those that do not materially affect the basic and novel characteristics of the disclosed and/or claimed subject matter. For example, in some embodiments a peptide of the presently disclosed subject matter can "consist essentially of a" core amino acid sequence, "meaning that the peptide can comprise 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 substantially affect a desired biological activity of the peptide.

As for the terms "comprising," "consisting essentially of," and "consisting of," if one of these three terms is used herein, the presently disclosed subject matter may include the use of either of the other two terms. For example, the presently disclosed subject matter relates in some embodiments to compositions comprising the amino acid sequence TGSTQHQ (SEQ ID NO: 1). It is to be understood that the presently disclosed subject matter thus also encompasses peptides which in some embodiments consist essentially of the amino acid sequence TGSTQHQ (SEQ ID NO: 1); and in some embodiments a peptide consisting of the amino acid sequence TGSTQHQ (SEQ ID NO: 1). Similarly, it should also be understood that the methods of the presently disclosed subject matter include, in some embodiments, the steps disclosed herein and/or recited in the claims, that they consist essentially of, in some embodiments, and that they consist of, in some embodiments, the steps disclosed herein and/or recited in the claims.

Detailed description of illustrative embodiments of the invention

The present disclosure relates to peptides capable of penetrating the cell membrane of SC and/or living cells (e.g., epidermal and dermal cells), both alone and when coupled to or associated with an active agent or active agent carrier. Related compositions and methods are also described herein.

Penetrating peptides

The present disclosure provides peptides capable of penetrating SC and/or penetrating living cells after administration. These peptides are referred to herein as "penetrating peptides". In some embodiments, these penetrating peptides are capable of penetrating the cell membrane of living 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 1 below.

TABLE 1

In some embodiments, a penetrating peptide according to the present disclosure comprises an amino acid sequence comprising a fragment of 3, 4, 5, 6, or 7 consecutive amino acids selected from one of the following amino acid sequences: TGSTQHQ (SEQ ID NO:1), HSALTPH (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).

In some embodiments, a penetrating peptide according to the present disclosure has an amino acid sequence of 8 to 11, 12 to 15, or 16 to 19 amino acids in length, comprising an amino acid sequence selected from one of the following amino acid sequences: TGSTQHQ (SEQ ID NO:1), HSALTPH (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). In some embodiments, a penetrating peptide according to the present disclosure has 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. The penetrating peptides according to the present disclosure may be cyclized by any of a variety of known crosslinking methods. In some embodiments, a penetrating peptide according to the present disclosure may be provided in a cyclized conformation (i.e., as a cyclic peptide) in which a Cys-Cys disulfide bond is present. In some embodiments, a penetrating peptide according to the present disclosure has an amino acid sequence 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) comprising an internal amino acid sequence selected from one of the following amino acid sequences, wherein the amino acid sequence of the peptide comprises at least a first Cys located outside the internal sequence in the N-terminal direction and at least a second Cys located outside the internal sequence in the C-terminal direction.

In some embodiments, a penetrating peptide according to the present disclosure comprises an amino acid sequence comprising an internal fragment of 3, 4, 5, or 6 contiguous amino acids selected from one of the following amino acid sequences: TGSTQHQ (SEQ ID NO:1), HSALTPKH (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 comprises at least a first Cys located outside the internal sequence in the N-terminal direction and at least a second Cys located outside the internal sequence in the C-terminal direction.

Penetrating peptides disclosed herein include those having the provided amino acid sequences, as well as peptides having one or more amino acid substitutions (e.g., one or more conservative amino acid substitutions) relative to the provided sequences, wherein the peptides retain the ability to penetrate SC or penetrate cells.

Active agent

The ability of the above peptides to penetrate SC and/or penetrate cell membranes of living cells (e.g., epidermal and dermal cells) upon topical administration while coupled or associated with a molecular cargo (e.g., a low molecular weight compound or macromolecule) makes them suitable for facilitating the delivery of a variety of active agents known in the art.

General classes of activities that can be delivered include, for example, proteins, peptides, nucleic acids, nucleotides, nucleosides, and analogs thereof; and pharmaceutical compounds, such as low molecular weight compounds.

Active agents that can be delivered using the penetrating peptides disclosed herein include agents that act in: peripheral nerves, adrenergic receptors, cholinergic receptors, skeletal muscles, the cardiovascular system, smooth muscles, the blood circulation system, synaptic points, neuroeffector junctions, endocrine and hormonal systems, the immune system, the reproductive system, the skeletal system, the autologous system, the digestive and excretory systems, the histamine system and the central nervous system.

Suitable active agents may for example be selected from: dermatological agents, antitumor agents, cardiovascular agents, renal agents, gastrointestinal agents, rheumatic agents, immunological agents, nerve agents, and the like.

Suitable dermatological agents may include, for example: local anesthetics, anti-inflammatory agents, anti-infective agents, acne treatments, antiviral agents, antifungal agents, psoriasis agents (e.g., topical corticosteroids), and the like.

In some embodiments, suitable dermatological agents are 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), methyl p-methoxycinnamate (CAS registry No. 832-01-9), 4-estrene-17 β -ol-3-one (CAS registry No. 62-90-8), ethyl p-anisoyl acetate (CAS registry No. 2881-83-6), dihydrouracil (CAS registry No. 1904-98-9), lopinavir (registry No. 192725-17-0), ritanserin (CAS registry No. 87051-43-2), Nilotinib (CAS registry No. 641571-10-0), rocuronium (CAS registry No. 119302-91-9), 6- (1-hydroxyethyl) -1-azabicyclo (3.2.0) heptane-3, 7-dione-2-carboxylic acid p-nitrobenzyl ester (CAS registry No. 74288-40-7), abamectin (CAS registry No. 71751-41-2), paliperidone (CAS registry No. 144598-75-4), gemifloxacin (CAS registry No. 175463-14-6), valrubicin (CAS registry No. 56124-62-0), mizoribine (CAS registry No. 50924-49-7), solifenacin succinate (CAS registry No. 242478-38-2), lapatinib (CAS registry No. 231277-92-2), Dydrogesterone (CAS registry number: 152-62-5), 2-dichloro-N- [ (1R, 2S) -3-fluoro-1-hydroxy-1- (4-methylsulfonylphenyl) propan-2-yl ] acetamide (CAS registry number: 73231-34-2), tilmicosin (CAS registry number: 108050-54-0), efavirenz (CAS registry number: 154598-52-4), pirarubicin (CAS registry number: 72496-41-4), nateglinide (CAS registry number: 105816-04-4), epirubicin (CAS registry number: 56420-45-2), entecavir (CAS registry number: 142217-69-4), etoricoxib (CAS registry number: 202409-33-4), Cilnidipine (CAS registry number: 132203-70-4), doxorubicin hydrochloride (CAS registry number: 25316-40-9), escitalopram (CAS registry number: 128196-01-0), sitagliptin phosphate monohydrate (CAS registry number: 654671-77-9), acitretin (CAS registry number: 55079-83-9), rizatriptan benzoate (CAS registry number: 145202-66-0), doripenem (CAS registry number: 148016-81-3), atracurium besylate (CAS registry number: 64228-81-5), nilutamide (CAS registry number: 63612-50-0), 3, 4-dihydroxybenzene ethanol (CAS registry number: 10597-60-1), ketanserin tartrate (CAS registry number: 83846-83-7), ozagrel (CAS registry number: 82571-53-7), Eprosartan mesylate (CAS registry No. 144143-96-4), ranitidine hydrochloride (CAS registry No. 66357-35-5), 6, 7-dihydro-6-mercapto-5H-pyrazolo [1, 2-a ] [1, 2, 4] triazolium chloride (CAS registry No. 153851-71-9), sulfapyridine (CAS registry No. 144-83-2), teicoplanin (CAS registry No. 61036-62-2), tacrolimus (CAS registry No. 104987-11-3), lumiracoxib (CAS registry No. 220991-20-8), allyl alcohol (CAS registry No. 107-18-6), protected meropenem (CAS registry No. 96036-02-1), neramexane (CAS registry No. 121032-29-9), Pimecrolimus (CAS registry number: 137071-32-0), 4- [ 6-methoxy-7- (3-piperidin-1-ylpropoxy) quinazolin-4-yl ] -N- (4-prop-2-yloxyphenyl) piperazine-1-carboxamide (CAS registry number: 387867-13-2), ritonavir (CAS registry number: 155213-67-5), adapalene (CAS registry number: 106685-40-9), aprepitant (CAS registry number: 170729-80-3), eplerenone (CAS registry number: 107724-20-9), rasagiline mesylate (CAS registry number: 735-16179-1), miltefosine (CAS registry number: 58066-85-6), raltavir potassium (CAS registry number: 871038-72-1), Dasatinib monohydrate (CAS registry number: 863127-77-9), oxomazine (CAS registry number: 3689-50-7), pramipexole (CAS registry number: 104632-26-0), parecoxib sodium (CAS registry number: 198470-85-8), tigecycline (CAS registry number: 220620-09-7), toltrazuril (CAS registry number: 69004-03-1), vinflunine (CAS registry number: 162652-95-1), drospirenone (CAS registry number: 67392-87-4), daptomycin (CAS registry number: 103060-53-3), montelukast sodium (registry number: 151767-02-1), brinzolamide (CAS registry number: 138890-62-7), malavir (CAS registry number: 376348-65-1), Doxercalciferol (CAS registry number: 54573-75-0), oxolinic acid (CAS registry number: 14698-29-4), daunorubicin hydrochloride (CAS registry number: 23541-50-6), nizatidine (CAS registry number: 76963-41-2), idarubicin (CAS registry number: 58957-92-9), fluoxetine hydrochloride (CAS registry number: 59333-67-4), ascomycin (CAS registry number: 11011-38-4), beta-Methylvinylphosphate (MAP) (CAS registry number: 90776-59-3), amorolfine (CAS registry number: 67467-83-8), fexofenadine hydrochloride (CAS registry number: 83799-24-0), ketoconazole (CAS registry number: 65277-42-1), 9, 10-difluoro-2, 3-dihydro-3-methyl-7-oxo-7H-pyrido-1 (CAS registry No. 82419-35-0), ketoconazole (CAS registry No. 65277-42-1), terbinafine hydrochloride (CAS registry No. 78628-80-5), amorolfine (CAS registry No. 78613-35-1), methoxsalen (CAS registry No. 298-81-7), olopatadine hydrochloride (CAS registry No. 113806-05-6), zinc pyrithione (CAS registry No. 13463-41-7), olopatadine hydrochloride (CAS registry No. 140462-76-6), cyclosporine (registry No. 59865-13-3), and botulinum toxin and its analogs and vaccine components.

Proteins, polypeptides and peptides as active agents

Proteins that can be used in the depot (depot) formulations disclosed herein can include, for example: molecules such as cytokines and their receptors, and chimeric proteins comprising cytokines or their receptors, including, for example, tumor necrosis factors alpha and beta, their receptors and derivatives thereof; renin; growth hormones including human growth hormone, bovine growth hormone, methionyl 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 pancreatic hormone releasing factor; a lipoprotein; colchicine; prolactin; corticotropin; thyroid stimulating hormone; oxytocin; a vasopressin; somatostatin; lysine vasopressin; a pancreatin; leuprorelin; alpha-1-antitrypsin; an insulin a chain; insulin B chain; proinsulin; follicle stimulating hormone; a calcitonin; luteinizing hormone; luteinizing Hormone Releasing Hormone (LHRH); LHRH agonists and antagonists; glucagon; coagulation factors, such as factor VIIIC, factor IX, tissue factor and von willebrand factor (von Willebrands factor); anti-coagulation factors, such as protein C; atrial natriuretic peptides; a pulmonary surfactant; plasminogen activators other than tissue-type plasminogen activator (t-PA), such as urokinase; bombesin; thrombin; a hematopoietic growth factor; enkephalinase; RANTES (activated regulatory normal T cell expression and secretion factors); human macrophage inflammatory protein (MIP-1-alpha); serum albumin, such as human serum albumin; mullerian-inhibiting substances (mullerian-inhibiting substance); a relaxin a chain; a relaxin B chain; (ii) prorelaxin; mouse gonadotropin-related peptides; chorionic gonadotropin; gonadotropin releasing hormone; bovine growth hormone; porcine growth hormone; microbial proteins, such as beta-lactamases; a DNA enzyme; a statin; an activin; vascular Endothelial Growth Factor (VEGF); hormone or growth factor receptors; an integrin; protein A or D; rheumatoid factor; a neurotrophic factor, such as bone-derived neurotrophic factor (BDNF), neurotrophic factor-3, 4, -5, or-6 (NT-3, NT-4, NT-5, or NT-6) or a nerve growth factor, such as NGF-beta; platelet Derived Growth Factor (PDGF); fibroblast growth factors, such as acidic FGF and basic FGF; epidermal Growth Factor (EGF); transforming Growth Factors (TGF), such as TGF-alpha and TGF-beta, including TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, or TGF-beta 5; insulin-like growth factors I and II (IGF-I and IGF-II); des (1-3) -IGF-I (brain IGF-I), insulin-like growth factor binding protein; CD proteins such as CD-3, CD-4, CD-8 and CD-19; erythropoietin; an osteogenesis inducing factor; an immunotoxin; bone Morphogenetic Protein (BMP); interferons, such as interferon- α (e.g., interferon α 2A), - β, - γ, - λ, and consensus interferon; colony Stimulating Factors (CSF), such as M-CSF, GM-CSF, and G-CSF; interleukins (IL), such as IL-1 through IL-10; superoxide dismutase; a T cell receptor; surface membrane proteins; a decay accelerating factor; viral antigens, such as a portion of the HIV-1 envelope glycoprotein, gp120, gp160, or fragments thereof; a transporter protein; a homing receptor; an address element; fertility inhibitors, such as prostaglandins; a fertility promoting agent; a regulatory protein; antibodies (including fragments thereof) and chimeric proteins, such as immunoadhesins; precursors, derivatives, prodrugs and analogs of these compounds, and pharmaceutically acceptable salts of these compounds, or their precursors, derivatives, prodrugs and analogs.

Suitable proteins or peptides may be natural or recombinant and include, for example, fusion proteins.

In some embodiments, the protein is a growth hormone, such as human growth hormone (hGH), recombinant human growth hormone (rhGH), bovine growth hormone, methionyl human growth hormone, des-phenylalanine human growth hormone, and porcine growth hormone; insulin, insulin a chain, insulin B chain and proinsulin; or growth factors 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 factors I and II (IGF-I and IGF-II).

Peptides suitable for use as active agents in the injectable biodegradable delivery reservoirs disclosed herein include, but are not limited to, glucagon-like peptide 1(GLP-1) and precursors, derivatives, prodrugs and analogs thereof.

Nucleic acids as active agents

Nucleic acid active agents include nucleic acids and precursors, derivatives, prodrugs and analogs thereof, such as therapeutic nucleotides, nucleosides and analogs thereof; a therapeutic oligonucleotide; and therapeutic polynucleotides. The active agents selected from this group can be used, inter alia, as anticancer and antiviral agents. Suitable nucleic acid active agents can include, for example, ribozymes, antisense oligodeoxynucleotides, aptamers, and siRNA. Examples of suitable nucleoside analogs include, but are not limited to, cytarabine (araCTP), gemcitabine (dFdCTP), and floxuridine (FdUTP). In some embodiments, a suitable nucleic acid active agent is an interfering RNA, such as an shRNA, miRNA, or siRNA. Suitable siRNAs include, for example, IL-7 (interleukin 7) siRNA, IL-10 (interleukin 10) siRNA, IL-22 (interleukin 22) siRNA, IL-23 (interleukin 23) siRNA, CD86siRNA, KRT6A (keratin 6A) siRNA, K6A N171K (keratin 6A N171K) siRNA, TNF α (tumor necrosis factor α) siRNA, TNFR1 (tumor necrosis factor receptor 1) siRNA, TACE (tumor necrosis factor (TNF) - α convertase) siRNA, RRM2 (ribonucleotide reductase subunit 2) siRNA, and VEGF (vascular endothelial growth factor) siRNA. The mRNA sequences of the human gene targets of these sirnas are known in the art. For 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; for 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.: AF 301620.1; 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.: NM-001206925.1; for KRT6a, see, e.g., GenBank accession No.: NM-005554.3; for TNF α, see, e.g., GenBank accession No.: NM-000594.2; for TNFR1, see, e.g., GenBank accession No.: NM-001065.3; for TACE, see, e.g., GenBank accession No.: NM-003183.4; for RRM2, see, e.g., GenBank accession No.: NM _001165931.1 and GenBank accession No.: NM-001034.3; for VEGF, see, e.g., GenBank accession No.: NM _001025366.2, GenBank accession No.: NM _001025367.2, GenBank accession No.: NM _001025368.2, GenBank accession No.: NM _001025369.2, GenBank accession No.: NM-001025370.2, NM-001033756.2, GenBank accession number: NM _001171622.1 and GenBank accession No.: NM _ 003376.5.

In addition, various methods and techniques for selecting a particular mRNA target sequence during siRNA design are known in the art. See, e.g., the common siRNA design tool provided by Whitehead institute of biological Research at MIT. The tool can be found on a website, and the website address is http:// directly precondingjura. wi.

Additional active agent compounds

A variety of additional active agent compounds can be used in the injectable depot compositions disclosed herein. Suitable compounds may include compounds that are directed to one or more of the following drug targets: tricyclic domains, carboxypeptidases, 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, metalloproteinases, transporter ATPases, carboxylic ester hydrolases, peroxidases, lipoxygenases, dopa decarboxylases, A/G cyclases, methyltransferases, sulfonylurea receptors, other transporters (e.g. dopamine transporters, GABA transporters 1, noradrenaline transporters, potassium-transporting ATPase alpha chain 1, sodium- (potassium) -chloride co-transporters 2, serotonin transporters, synaptovesicamine transporters and thiazine-sensitive sodium-chloride co-transporters), Electrochemical nucleoside transporters, voltage-gated ion channels, GABA receptors (Cys-loop), acetylcholine receptors (Cys-loop), NMDA receptors, 5-HT3 receptors (Cys-loop), ligand-gated ion channels Glu: kainite, AMPA Glu receptor, acid sensitive ion channel aldosterone, Ryanodine receptor (Ryanodine receptor), vitamin K epoxide reductase, MetGluR-like GABA_BReceptor, inward rectification K⁺Channels, NPC1L1, MetGluR-like calcium-sensitive receptors, aldehyde dehydrogenases, tyrosine 3-hydroxylase, aldose reductase, xanthine dehydrogenase, ribonucleoside reductase, dihydrofolate reductase, IMP dehydrogenase, thioredoxin reductase, dioxygenase, myoinositol monophosphatase, phosphodiesterase, adenosine deaminase, peptidyl-prolyl isomerase, thymidylate synthase, aminotransferase, farnesyl pyrophosphate synthase, protein kinase, carbonic anhydrase, tyrosine dehydrogenase, farnesyl pyrophosphate synthase, tyrosine dehydrogenase, farnesyl-pyruvate synthase, farnesyl-pyruvate, farnesyl,Tubulin, troponin, I κ B kinase- β inhibitors, amine oxidases, cyclooxygenases, cytochrome P450, thyroxine 5-deiodinase, steroid dehydrogenases, HMG-CoA reductase, steroid reductase, dihydroorotate oxidase, epoxide hydrolase, transporter atpase, translocator, glycosyltransferase, nuclear receptor: NR3 receptor, nuclear receptor: NR1 receptor and topoisomerase.

In some embodiments, the active agent is a compound that targets one of: rhodopsin-like GPCRs, nuclear receptors, ligand-gated ion channels, voltage-gated ion channels, penicillin binding proteins, myeloperoxidase-like, sodium: the neurotransmitter co-transporter family, DNA topoisomerase II, fibronectin III and cytochrome P450.

In some embodiments, the active agent is an anti-cancer agent. Suitable anti-cancer agents include, but are not limited to: actinomycin D, alemtuzumab, allopurinol sodium, amifostine, amsacrine, anastrozole, cytarabine monophosphate (Ara-CMP), asparaginase, azacytidine (Azacytadine), bendamustine, bevacizumab, bicalutamide, bleomycin (such as bleomycin A)₂And B₂) Bortezomib, busulfan, camptothecin sodium salt, capecitabine, carboplatin, carmustine, cetuximab, chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, dacarbazine, daunorubicin liposomes, dacarbazine, decitabine, docetaxel, doxorubicin liposomes, epirubicin, estramustine, etoposide phosphate, exemestane, floxuridine, fludarabine phosphate, 5-fluorouracil, fotemustine, fulvestrant, gemcitabine, goserelin, altretamine, hydroxyurea, idarubicin, ifosfamide, imatinib, irinotecan, ixabepilone, lapatinib, letrozole, leuprolide, lomustine, mechlorethamine, melphalan, 6-mercaptopurine, capecitabine, cisplatin, dacarbazine, dactylosine, dactylosin, and doxepin, Methotrexate, mithramycin, mitomycin C, mitotane, mitoxantrone, nimustine, Ofatumumab, oxaliplatin,Paclitaxel, panitumumab, pemetrexed, pentostatin, pertuzumab, picoplatin, pipobroman, plerixafor, procarbazine, ranitidine, rituximab, streptozotocin, temozolomide, teniposide, 6-thioguanine, thiotepa, topotecan, trastuzumab, trooshusuofan, trittamine, trimetrexate, uracil mustard, valrubicin, vinblastine, vincristine, vindesine, vinorelbine and analogs, precursors, derivatives and prodrugs thereof. It should be noted that two or more of the above compounds can be used in combination in the penetrating peptide compositions of the present disclosure.

Active agents of interest for use in the disclosed penetrating peptide compositions may also include opioids and derivatives thereof as well as opioid receptor agonists and antagonists, such as naltrexone, naloxone, nalbuphine, fentanyl, sufentanil, oxycodone, and pharmaceutically acceptable salts and derivatives thereof.

In some embodiments, the active agent is a small molecule or low molecular weight compound, such as 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).

In some embodiments, the active agent is a label. Suitable labels include, for example, radioisotopes, fluorescers, chemiluminescent agents, 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 the compositions disclosed herein at any suitable concentration. Suitable concentrations may vary depending on the potency of the active agent, the half-life of the active agent, and the like. Furthermore, the penetrating peptide compositions according to the present disclosure may comprise one or more active agents, e.g., a combination of two or more of the above active agents.

Active agent carrier

As described herein before, one or more active agents can be coupled to or associated with a penetrating peptide to provide a penetrating peptide composition according to the present disclosure. Alternatively, a penetrating peptide composition according to the present disclosure may comprise a penetrating peptide as disclosed herein coupled or associated with an active agent carrier, which in turn comprises an active agent attached thereto and/or disposed therein.

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. For example, liposomes or lipid particles can be prepared according to U.S. patent No. 5,077,057(Szoka, Jr.). Liposomes formed from a non-phospholipidic (nonphosphal lipid) component with lipid bilayer formation potential were purified in biochim, biophysis, acta, 19: 227, 232 (1982). For preparation, purification, modification and loading of Liposomes, see generally New, r.c.c., liposomees: APracical Approach, (1990) Oxford University Press Inc., N.Y.

A general discussion of techniques for preparing liposomes and drug-encapsulated liposomes can be found in U.S. Pat. No. 4,224,179 (Schneider). See also Mayer et al, Chemistry and Physics of lipids, 40: 333-345(1986). For encapsulation of active agent dry powder compositions, see also U.S. Pat. No. 6,083,539. For incorporation of active agents into nanoparticles see, for example, m.m. devilliers et al (ed.), Nanotechnology in Drug Delivery, (2009) american associates of Pharmaceutical Scientists. For incorporation of active agents into micelles, see, e.g., d.r.lu and s.oie, Cellular Drug Delivery: principles and Practice, (2004) Humana Press inc.

Linking peptides to active agents and active agent carriers

The penetrating peptide as described herein may be coupled to or associated with an active agent. Alternatively, a penetration peptide as disclosed herein can be coupled to or associated with an active agent carrier, which in turn comprises an active agent (examples of which are discussed above) attached thereto and/or disposed therein. Coupling techniques generally result in the formation of one or more covalent bonds between the penetrating peptide and the active agent or active agent carrier, while association techniques typically utilize one or more of hydrophobic, electrostatic, or van der waals interactions.

A variety of techniques can be used to couple or associate the peptide to the active agent. Similarly, a variety of techniques can be used to couple or associate the peptide to an active agent carrier, e.g., a liposome, nanoparticle, or micelle as described herein.

For example, if the active agent is a peptide or polypeptide, the entire composition comprising 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 comprising the penetrating peptide. Additional methods of coupling peptides to other peptides or polypeptides include: such as Rostovtsev et al (2002) Angew. chem. int. Ed.41: 2596, 2599 and Tornoe et al (2002) J. org. chem.67: 3057-3064 copper catalyzed azide/alkyne [3+2] cycloaddition "click chemistry"; such as Baskin et al (2007) PNAS volume 104, stage 43: 167393-16797 said azide/DIFO (difluorocyclooctyne) without copper click chemistry; such as Lin et al (2005) j.am.chem.soc.127: 2686 Azide/phosphine "Staudinger reaction" as described in 2695; such as Saxon and Bertozzi (2000), Science287(5460) on month 3, 17: the azido/triarylphosphine "modified Staudinger reaction" described in 2007-10; and as Casey (2006) j.of chem.edu. volume 83, stage 2: 192-195, Lynn et al (2000) J.Am.chem.Soc.122: 6601-: 401 — 408 said catalyzed olefin cross-metathesis.

If the active agent is a low molecular weight compound or small molecule, the low molecular weight compound or small molecule can be coupled to a penetrating peptide as described herein using a variety of techniques, such as Loh et al, chemcommun (camb), 11/28/2010; 46(44): 8407-9 (electronic publication No. 10/7/2010). See also Thomson s, Methods molmed, (2004); 94: 255-65, which describe amine and sulfhydryl residues coupling small molecule carboxyl, hydroxyl and amine residues to proteins.

Methods of coupling peptides to active agent carriers (e.g., liposomes) are also known in the art. See, e.g., G.Gregoriadis (eds.), Liposome Technology third edition, volume II, Entransaction of Drugs and Other materials into Liposomes, (2007), Informata Healthcare, New York, NY, which describes techniques for coupling peptides to Liposome surfaces. For covalent attachment of proteins to Liposomes, see New, r.c.c., Liposomes: APracial Approach, (1990) Oxford University Press, N.Y., page 163-182.

Administration of penetrating peptide compositions as pharmaceutical formulations

One skilled in the art will appreciate that there are a variety of suitable methods by which the penetrating peptide composition can be administered to a subject or host, e.g., a patient, in need thereof, and that while more than one route can be used to administer a particular composition, a particular route can provide a more immediate and more effective response than another route. Pharmaceutically acceptable excipients are also well known to those skilled in the art and are readily available. The choice of excipient will be determined in part by the particular compound and by the particular method used to administer the composition. Thus, there are a variety of suitable formulations of the penetrating peptide composition. The following methods and excipients are exemplary only and are in no way limiting.

Formulations suitable for oral administration may include: (a) liquid solutions, such as an effective amount of the compound dissolved in a diluent (e.g., water, saline, or orange juice); (b) a capsule, sachet or tablet, each containing a predetermined amount of active agent as a solid or as a granulate; (c) suspensions in suitable liquids; (d) suitable emulsions and (e) hydrogels. The tablet may comprise one or more of: lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, gum arabic, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, wetting agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenges may comprise the active ingredient in a flavoring agent, typically sucrose and acacia or tragacanth, while confectionery lozenges comprise the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, an emulsion, a gel, etc., in addition to the active ingredient, with excipients known in the art.

The penetrating peptide formulation may be formulated as an aerosol formulation for administration by inhalation. These aerosol formulations may be placed in a pressurized acceptable propellant, such as dichlorodifluoromethane, propane, nitrogen, and the like. They may also be formulated as medicaments in non-pressurized formulations, for example for use in nebulizers or nebulizers.

Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may contain suspending agents, solubilising agents, thickening agents, stabilising agents and preservatives. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may 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 may be prepared from sterile powders, granules and tablets of the kind previously described.

Formulations suitable for topical administration may be presented as creams, gels, pastes, patches, sprays or foams.

Suppositories are also provided by mixing with various bases such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams.

Unit dosage forms for oral or rectal administration, such as syrups, elixirs and suspensions, may be provided wherein each dosage unit, such as a teaspoon, tablespoon, tablet or suppository, contains a predetermined amount of the composition. Similarly, unit dosage forms for injection or intravenous administration may comprise the penetrating peptide in the formulation as a solution in sterile water, physiological saline, or another pharmaceutically acceptable carrier.

The term "unit dosage form" as used herein refers to physically discrete units intended as unit doses for use by human and animal subjects, each unit containing a predetermined amount of the penetrating peptide composition calculated to be sufficient to produce the desired effect, in association with a pharmaceutically acceptable diluent, carrier or vehicle. The technical specifications for the new unit dosage forms of the penetrating peptide compositions depend on the particular active agent employed and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the host.

One skilled in the art will readily appreciate that dosage levels may vary depending on the particular compound, the nature of the delivery vehicle, and the like. Suitable dosages for a given compound can be readily determined by one skilled in the art by a variety of methods.

Optionally, the pharmaceutical composition may comprise other pharmaceutically acceptable components, such as buffers, surfactants, antioxidants, viscosity modifiers, preservatives, and the like. Each of these components is well known in the art. See, for example, U.S. patent No. 5,985,310, the disclosure of which is incorporated herein by reference.

Other components suitable for use in penetrating peptide formulations can be found in Remington's pharmaceutical sciences, Mack pub. co., 18 th edition (6 months 1995). In one embodiment, the aqueous cyclodextrin solution further comprises dextrose, e.g., about 5% dextrose.

Administration of penetrating peptide compositions as medical device components

In some embodiments, one or more of the penetrating peptide compositions of the present disclosure can be incorporated into medical devices known in the art, such as drug eluting stents, catheters, fabrics, cements, bandages (liquid or solid), biodegradable polymer reservoirs, and the like. In some embodiments, the medical device is an implantable or partially implantable medical device.

Method of treatment

The term "effective amount" (or, in the therapeutic context, "pharmaceutically effective amount") of a penetrating peptide composition generally refers to an amount of the penetrating peptide composition effective to achieve a desired therapeutic effect, e.g., in the case of a penetrating peptide-siRNA composition, i.e., an amount effective to reduce expression of a targeted mRNA to produce a desired therapeutic effect.

The effective amount of penetrating peptide composition, suitable delivery vehicle, and protocol can be determined by conventional methods. For example, in a therapeutic setting, a physician can begin treatment with a low dose of one or more penetrating peptide compositions in a subject or patient in need thereof, then increase the dose or systematically change the dosage regimen, monitor its effect on the patient or subject, and adjust the dose or treatment regimen to maximize the desired therapeutic effect. Further discussion of optimized dosages and treatment regimens can be found in Benet et al, Goodman & Gilman's The Pharmacological Basis of therapeutics, ninth edition, edited by Hardman et al, McGraw-Hill, New York, (1996), Chapter 1, pages 3-27, and L.A. Bauer, Pharmacotherapy, Apathophysiologic Approach, fourth edition, ediro et al, Appleton & Lange, Stamford, Connecticut, (1999), Chapter 3, pages 21-43, and references cited therein for The reader's reference.

The dosage level and mode of administration will depend on a variety of factors such as the penetrating peptide used, the active agent, the context of use (e.g., the patient to be treated), and the like. Optimizing the mode of administration, dosage levels, and regimen, including monitoring the system to assess efficacy, are routine matters well known to those of ordinary skill in the art.

In one embodiment, the present disclosure provides a method of treating a subject having a skin disease, comprising: administering to a subject a pharmaceutically effective amount of a composition comprising a penetrating peptide disclosed herein, wherein the peptide is coupled or associated with a dermatologically active agent (e.g., a dermatologically active agent disclosed herein) or a dermatologically active agent carrier comprising an active agent.

In one embodiment, the disclosure provides a method of treating a subject having, suspected of having, or susceptible to a disease caused at least in part by expression of mRNA, comprising administering to the subject a pharmaceutically effective amount of a composition comprising a penetrating peptide described herein, wherein the penetrating peptide is coupled to or associated with an interfering RNA or an active agent carrier comprising the interfering RNA, e.g., an mRNA-targeting shRNA, miRNA, or siRNA, or a carrier comprising the interfering RNA.

In one embodiment, the interfering RNA is an siRNA, e.g., an siRNA selected from one of: IL-10siRNA, IL-17siRNA, IL-22siRNA, IL-23siRNA, CD86siRNA, KRT6a siRNA, TNFR1siRNA, TNF α siRNA and TACE siRNA.

In vitro use

In addition to therapeutic methods and other in vivo uses, the penetrating peptide compositions disclosed herein may also be used in the context of in vitro experiments. For example, the penetrating peptides disclosed herein can be used to deliver any of the various active agents discussed herein, as well as potential active agents, into living cells in vitro to determine the potential therapeutic efficacy, toxicity, etc. of the active or potential agent. To this end, the penetrating peptides and penetrating peptide compositions of the present disclosure may be used in the context of drug testing and/or screening.

In some embodiments, a penetrating peptide composition as described herein can be used in an in vitro gene silencing assay, for example, by introducing a penetrating peptide-interfering RNA conjugate that is directed to a gene target, and monitoring the effect on gene expression.

Additional in vitro uses may include the use of penetrating peptides as disclosed herein coupled to or associated with one or more labeling agents (e.g., fluorescers or radiolabels) or one or more labeling agent carriers to label living cells in vitro.

Examples

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Attempts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weights are weight average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric.

Example 1

SC-penetrating peptides were identified using in vitro phage display as follows and as shown generally in panel a in figure 1.

Phage display

A Ph.D. -C7C phage display peptide library (New England Biolabs) was used. The pig skin was studied in Franz diffusion cells (FDC, Permagear). 2x10 to¹¹The pfu (10. mu.L) phage library was placed in a donor compartment of FDC along with 1mL phosphate buffered saline (PBS, pH7.4). After 24 hours, the liquid in the receptor chamber was removed and the titer was determined by adding an aliquot of the receptor solution to 200 μ L of e.coli (e.coli) strain ER2738(New England Biolabs) and plating onto IPTG/Xgal plates. The number of blue plaques formed after 18 hours incubation at 37 ℃ was counted and 20 plaques were randomly selected for sequencing. For subsequent rounds of screening, 1mL of receptor solution was added to 20mL of a 1: 100 diluted overnight culture of ER2738 and grown for 4.5 hours to amplify the phage. Phages were purified by PEG/NaCl precipitation and resuspended in PBS. The amplified phage were then used for the next round of screening. The number of phage placed in the donor chamber was 2X10¹¹pfu remained constant for all 5 rounds of screening.

Five rounds of screening resulted in a reduction of the display library as shown in panel B of figure 1. A sequence AC-TGSTQHQ-CG (SEQ ID NO:13) appears at high frequency in higher rounds and is designated as a skin penetrating and cell entering (SPACE) peptide. The second sequence (AC-HSALTPKH-CG) (SEQ ID NO:14) also appears with high frequency. The third sequence (AC-STHFIDT-CG) (SEQ ID NO:18) appeared at a relatively high frequency in rounds 4 and 5. Note that "AC-" and "-CG" as used herein in the context of peptide sequences refers to the AC and CG portions of sequences derived from phage display systems.

Phage penetration

Following the same procedure as described above (without amplification and subsequent screening steps), penetration of a variety of phage samples, including phage without peptide library, whole phage library, SPACE peptide display phage, and heptaglycine phage, was determined using the number of phage colonies detected in the recipient sample and standard equations to determine penetration.

Phage cloning

To confirm the ability of the phage to penetrate the skin, a ph.d. peptide display cloning system (M13KE vector, New England Biolabs) was used to construct phage that displayed a specific peptide sequence of interest. The peptide sequences of interest were cloned into a ph.d. peptide display cloning system (M13KE vector, New England Biolabs). The peptide sequence was inserted between the KpnI and EagI restriction sites. To distinguish the original M13KE vector from the modified M13KE vector containing the inserted peptide, the reverse primer was engineered to change the EagI restriction site (5 '-CGGCCG-3') (SEQ ID NO: 19) to the SacII restriction site (5 '-CCGCGG-3') (SEQ ID NO: 20) by a two-site mutation. Both forward and reverse primers were used to replicate the entire vector. The forward primer was 5'-GTTCCGCGGAAACTGTTGAAAGTTGTTTAGCAAAATCCC-3' (SEQ ID NO: 21). The reverse primers for TGSTQHQ (SEQ ID NO:1) and THGQTQS (SEQ ID NO: 22) were 5'-TTTCCGCGGAACCTCCACCGCACTGATGCTGCTCGAACCAGTACAAGCAGAGTGAGAATAGAAAGGTACTACTAAAGGAATTGCGAATAATAATTTTTTCAC-3' (SEQ ID NO: 23) and 5 '-TTTCCGCGGAACCTCCACCGCA(AGACTGAGTCTGCCCATGAGT) ACAAGCAGAGTGAGAATAGAAAGGTACTACTAAAGGAATTGCGAATAATAATTTTTTCAC-3' (SEQ ID NO: 24), respectively.

The replication product was purified and then digested with SacII to generate the blunt ends required for ligation of the vector. The modified vector was electroporated into electrocompetent ER2738 cells and immediately placed in 1mL SOC medium (New England Biolabs) and grown for 45 minutes at 37 ℃. The resulting culture was then placed in 50mL of 1: 100 diluted overnight culture and grown for 4.5 hours. The amplified phage were purified using the protocol described above and the titer was determined. Plaques were picked 18 hours later and sequenced to confirm that the peptide was displayed on the phage surface.

Peptide synthesis

The peptide sequence of SPACE peptide was ACTGSTQHQCG (SEQ ID NO:13), the peptide sequence of Control Peptide (CP) was ACTHGQTQSCG (SEQ ID NO: 25), and a disulfide bond was formed between cysteines to produce a cyclic peptide. 5-carboxyfluorescein (5-FAM) or Fluorescein Isothiocyanate (FITC) conjugated peptides were synthesized by ChinaTech Peptide Co. and RS Synthesis. The dye was placed on the N-terminus of the peptide. Biotinylated forms of both peptides were synthesized by ChinaTech Peptide co.

Example 2

Mathematical model

Penetration of phage SC was unexpected in view of its size (Potts RO and Guy RH (1992) differentiating skin permeability. phase Res9 (5): 663-669; Magnus B, Pugh W and Roberts M (2004) Simple threads defining the pore of complex for transduction. phase Res21 (6): 1047-1054; Migortri S (2003) Modeling skin permeability and hydrophilic tissue base skin permeability Control. J.rolRelease 86 (1): 69-92.). Large solutes (typically MW > 500Da) exhibit poor skin penetration, which transdermal penetration is typically limited by their detection sensitivity. The M13 phage used in this study was a long filamentous particle, approximately 8nm wide and 900nm long. High donor concentration (about 2X 10)¹¹pfu/ml), low detection limit (about 1pfu) and amplification potential facilitate the assessment of phage dermal penetration. The measured penetration of the phage across the pigskin is very low; control phage (phage without peptide library) was 10^-9cm/h, 10 for SPACE phage^-7-10^-6cm/h. Penetration of the phage, although much less than low molecular weight solutes, is still significant and unexpected. Of utmost importanceThe penetration of the phage is sequence specific.

Diffusion through intercellular lipids represents the classical mechanism of transdermal penetration of molecules. However, this mechanism is generally limited to small lipophilic molecules. Penetration of large hydrophilic molecules is relatively less studied. Transdermal transport of such solutes is attributed to two pathways: (i) polar or porous pathways, and (ii) appendages (hair follicles). Mathematical models have been described in the literature to illustrate the effect of two pathways on percutaneous penetration (Tang H, Mitgotris S, Blankschtein D and Langer R (2001) Theoretical description of transdermal transport of hydrophyllic reagents: application to low-frequency sonophoresis. J Pharm Sci90 (5): 545. sub.568; Peck KD, Ghanem AH and Higuchi WI (1994) Hindereddifferentiation of drugs from drugs and active holes of inter and enhanced transdermal peptides. Res. 11 (9. 1306. 1314)). The application of these models to phage transport and their comparison to experimental observations is given below.

The basic principles of these models have been disclosed (Tang H, Mitrapotris S, BlankschteinD and Langer R (2001) the Theoretical description of translational transport of hydrolytic reagents: application to low-frequency speech (translated by English) J Pharm Sci90 (5): 545. sub.568 (English); Peck, Ghanem, and Higuchi WI (1994) articulated description of polar molecules and radioactive site of interactive and iterative transformed human nuclear membrane. the summary of these models is provided below in 11 (1306. sub.1314). These models have been applied to describe the transport of macromolecules, such as dextran with a hydrodynamic radius of 2.6nm, which, although smaller than the radius of the phage (about 4nm), are of the same order of magnitude. The following analysis is based on extrapolation of these models and provides a background of information to interpret phage penetration through the skin.

Polar path: polar (or porous) pathways have been used to describe a variety of hydrophilic solutes (packets)Including macromolecules) transdermal diffusion (Tang H, migrotri S, Blankschtein D and langer r (2001) the ecological description of transdermal transport of hydrophicomentants: application to low-frequency nonphotorepresentation (translated by English) Jpharm Sci90 (5): 545-568 (English); mitrace model S et al (translated by English) Int J Pharm (English), Tezel A, Sens A and Mitrace S (2003) Description of translation of hydrolytic solutions low-frequency and modified pore model A modified pore model, J Pharm Sci92(2) translation of J Pharm Sci 393 (English), Tezel A, Sens S (2002) translation of expression analysis of low-frequency and modified pore model, and English translation of modification J Pharm # 18432 and English translation of J Pharm # 3 (English translation of J Pharm. and S (2002) translation of expression analysis of low-frequency and modification J Pharm. translation of J Pharm. and R2. translation of expression of (as translated in English) J Pharm Sci (English); seto JE, Polat BE, Lopez RF, Blankspchtein D and Langer R (Effects of both excessive and basal surfaces on the transform of hydrophilic polymers: synthetic in view of both fluids and threads-and molecular-tissue and human skin. (translated in English) J Control Release145 (1): 26-32 (English); TangH, Blankschtein D and Langer R (2002) Prediction of both phases and tissues, and J Control extended skin tissue on the transform of both phases, English J7 (7): 898, 9H, B, D, E, D, Es of low-frequency ultrasound on the transdermal validation of mannitol: compatible students with in vivo and in vitro skin (translated by English) J Pharm Sci91 (8): 1776-1794) (english).

In order to cross the Stratum Corneum (SC), hydrophilic solutes need to cross multiple lipid bilayers. However, given the low permeability of hydrophilic solutes across lipid bilayers, it seems unlikely that hydrophilic molecules would diffuse across SCs through the classical partitioning-diffusion process that plays an important role for hydrophobic solutes. It has been proposed that percutaneous penetration of such solutes occurs primarily through defects in the stratum corneum, which exist in a variety of physical forms, including grain boundaries, dislocations, nanoscale pores, or other abnormalities in the skin structure. Hydration of the stratum corneum may further increase the occurrence of such defects. The precise size of these defects depends on the type of defect and may span a length scale of 1-100 nm.

Osmotic coefficient of diffusion of skin hydrophilic osmotic agentThe general formula of (a) is given as follows:

[1]

where ε, τ and L are the porosity, tortuosity and thickness of the membrane, respectively, and D^∞The solute diffusion coefficient for infinite dilution. H (λ) is the steric hindrance factor, where λ is the hydrodynamic radius r of the osmotic agent_hThe ratio to the effective aperture r of the skin (i.e., λ r ═ r)_hR). The relative relationship between H (λ) and λ is given by the theory of hindered transport and is described in literature (11). γ (r) is the pore size distribution in the skin, which has been described for pig skin by the following function (4).

γ(r)＝0.024exp(-0.00045r²) [2]

Equation [2] considers the energetics of pore (or void) formation in a medium such as skin. The probability of pore formation can be related to the free energy of pore formation according to the following general equation.

[3]

Where E is the pore formation free energy per unit area per unit pore. A comparison of equations [2] and [3] shows that the E value of the 4nm radius pores in the pigskin is < 1kT, which is a relatively small value.

The epsilon value of the pigskin was determined to be about 2X10^-5(4). Similarly, it has been shown that the tortuosity of diffusion tau for large hydrophilic solutes in porcine SC is about 1 (4). D_p ^∞Is the solute diffusion coefficient in water, has been calculated using correlations such as the Wilke-Chang or Stoke-Einstein equations as follows.

[4]

Wherein r is_hHas the unit ofD_p ^∞In units of cm²And s. By combining the equations [1 ]]、[2]And [ 4]]The radius r can be estimated as follows_hIs the porous pathway of the solute.

[5]

Wherein the content of the first and second substances,the unit of (2) is cm/h. By substituting(corresponding to the radius of the phage) can be obtainedAbout 10^-8cm/h。

Contribution of the appurtenances: large solutes can also diffuse across the skin through the appendage. Although the density of hair follicles varies greatly with anatomical location, it is estimated that the average density of pig skin follicles is about 10/cm². However, most of the follicles are occupied by hairs and cannot be used for transport. The contribution of the skin penetrating shunt is given by the following equation.

[6]

Wherein phi is_sFraction of skin area occupied by hair follicles available for transport. D_sIs the diffusion coefficient of solute in the hair follicle contents, L_shuntIs the length of the diffusion path through the hair follicle. The fractional area of skin in the hair follicle available for transport is about 10^-4cm²/cm²。D_sThe Wilke-Change equation or Stoke-Einstein relationship may be used for estimation. Assuming that the hair follicle is filled with viscous liquid and considering the large size of the phage, D_sCan be approximately about 10^-8cm²/s。L_shuntIs about 500 μm. By substituting phi_sAnd L_shuntThe above values of (A) can be as followsAbout 10^-7Expression in cm/h.

Thus, the above equation estimates the penetration of the phage across the pigskin to be 10^-7-10^-8In the range of cm/h.

In order to make thisThese estimates were compared to experimental data and phage penetration across the pigskin was measured as described previously herein. Control phage (phage not displaying any peptide) showed about 10^-9Penetration in cm/h. Heptaglycine-displaying phage also displayed approximately 10^-10Low penetration of cm/h. The entire phage display library showed about 10^-8-10^-7cm/h penetration, 10 for the phage displaying the SPACE sequence^-7-10^-6cm/h. These values roughly correspond to theoretical predictions. Note that the measured penetration values may not represent steady state values and may not meet the classical definition of penetration, although these values still provide reasonable values for comparison with theoretical predicted values.

Given that all phage particles used in the study were of the same size, a possible explanation for the higher penetration of the SPACE phage relative to the control phage (not displaying peptide) was due to the peptide displayed on the phage surface. The porous pathway model assumes that the distribution of solutes in the skin is global, i.e., the solutes do not exhibit an affinity to the skin. If SPACE phages would exhibit a higher affinity to the skin, a higher proportion of phages would be caused to cross the skin. Experimental observations do indicate that SPACE increases the affinity of cargo to skin components, especially keratin. Without wishing to be bound by any particular theory, we hypothesize that this increased affinity is the primary reason behind the enhanced penetration of the SPACE phage relative to the control phage.

To further confirm this hypothesis, experiments were conducted in which the effect of excess SPACE peptide on the penetration of SPACE phage across the skin was evaluated. Specifically, SPACE peptide display phages were used in the presence of about 100,000 fold excess of SPACE peptide (about 10 for each SPACE peptide on the phage)⁵Individual free SPACE peptides) were placed on the skin. An excess of SPACE peptide significantly reduced the penetration of SPACE phage, in this case the penetration of SPACE phage was close to that of heptaglycine phage (about 10)^-10cm/h)。

Example 3

Dermal screening

In a separate experiment, phage selection was performed to isolate phages located in the dermis. For dermal screening, the phage display library was also placed in the donor compartment of FDCs. After 24 hours, the fluid in the donor chamber was removed and the skin was left at 60 ℃ for 90 seconds. The epidermis is then removed from the dermis. To extract the phages from the dermis, the dermis is cut into small pieces and then homogenized (IKA disperser). The homogenate was centrifuged at 5,000rpm for 5 minutes, then resuspended in PBS and incubated at room temperature for 5 minutes. The sample was then centrifuged again and washed twice more. After the final centrifugation, the homogenate was resuspended in 1% NP40(Sigma) to elute the remaining phage from the dermis. The eluates were plated and amplified according to the method outlined above. This was repeated for a total of 5 rounds. At the end of the fifth round of screening, approximately 1.9x10 was recovered from the dermis⁵And (4) phage. The phage were sequenced and the three leaders in the fifth round were AC-KTGSHNQ-CG (SEQ ID NO:15) (30%), AC-MGPSSML-CG (SEQ ID NO:16) (30%) and AC-TDPNQLQ-CG (SEQ ID NO:17) (20%). SPACE peptides were selected for further study in view of the similarity between the AC-KTGSHNQ-CG (SEQ ID NO:15) peptide and the SPACE peptides identified above.

Example 4

Penetration of fluorescently labeled phage

Phage clones displaying SPACE peptide were fluorescently labeled and tested for their ability to penetrate into the skin. The phage particles were labeled with the Alexa Fluor488 protein labeling kit (Invitrogen). Alexa Fluor488 contains TFP ester that reacts with the primary amine group on the phage coat protein. 2x10 to¹²pfu in DI water or PBS solution was added to DI water to give a total volume of 500. mu.L. The phage solution was then added to 50 μ L of 1M sodium bicarbonate solution, and the resulting solution was placed in a vial containing a fluorescent dye and left at room temperature for 1 hour. The phage was then purified using PEG/NaCl to remove excess unreacted dye and titer was determined.

Full thickness pigskin was obtained from the ventral region of yorkshire pigs. The skin was stored at-80 ℃ and thawed immediately prior to use. The electrical conductivity of the skin was measured to ensure the integrity of the skin barrier. Skin samples with resistances above 50k Ω were used for the experiments. A fluorescently labeled phage clone displaying SPACE peptide or a scrambled peptide sequence (AC-THGQTQS-CG) (SEQ ID NO: 25) was placed in the donor compartment of FDC. To simulate the phage selection experiment, 2X10 was used¹¹pfu was added to the donor chamber and the skin was harvested 24 hours later. Skin samples were prepared for imaging by confocal microscopy.

Preparation of skin samples for confocal microscopy imaging

Immediately after harvesting, skin samples were placed in 4% paraformaldehyde (Electron Microcopy Sciences) overnight at 4 ℃ and rinsed with DI water. The skin samples were then frozen in OCT cryosection embedding medium and sectioned at a thickness of 20 μm on a cryomicrotome (Leica). The tissue was fixed on positively charged slides in order to attach the tissue to the slide (Fisher Scientific). Slides were washed in DI water for 5 minutes and stained with 5. mu.g/mLHoechest 33342(Invitrogen) for 5 minutes. The slides were then washed in DI water for an additional 5 minutes and then allowed to dry completely at room temperature in the dark. A10. mu. LPermount coverslipper (Fisher Scientific) was placed on top of the skin section along with a coverslip and the slide was sealed. All samples were imaged on a confocal microscope (Leica and Olympus Fluoview 500).

The imaging results of the above experiment are shown in (a) and (b) of fig. 2. The fluorescently labeled phage clones displaying the SPACE peptide showed small but detectable skin penetration into (a). In contrast, the scrambled peptide sequence (AC-THGQTQS-CG) (SEQ ID NO: 25) showed only superficial penetration (b).

Example 5

Penetration of peptides into pig skin

The ability of the SPACE peptide to penetrate the pigskin when removed from the phage was tested as follows. Full thickness pigskin was obtained and prepared as described above. Fluorescently labeled peptide (200. mu.L of a 1mg/mL solution) was placed in the donor compartment of FDC. After 24 hours, the remaining solution in the donor compartment was removed and the FDC was disassembled. The skin sample was retrieved and rinsed with DI water to remove excess peptide or peptide complex on the skin surface. Skin samples were then prepared as described above for confocal microscopy imaging.

The imaging results of the above experiment are shown in (c) and (d) of fig. 1. The SPACE peptide penetrated into the skin when removed from the phage (c). Consistent with observations obtained with intact phage, it was found that the SPACE peptide was concentrated primarily in the dermis. No significant penetration of the control peptide was observed (d).

Example 6

Penetration of peptides and macromolecules in pig skin

The ability of the SPACE peptides to carry macromolecular cargo across SCs was tested as follows. Full thickness pigskin was obtained and prepared as described above. The peptide is first coupled to the macromolecule as described in more detail below, and the peptide-macromolecule complex is then placed in the donor compartment of the FDC. After 24 hours, the remaining solution in the donor compartment was removed and the FDC was disassembled. The skin sample was retrieved and rinsed with DI water to remove excess peptide complexes on the skin surface. Skin samples were then prepared as described above for confocal microscopy imaging.

To couple the peptide to the macromolecular streptavidin, 80 μ L of a 1mg/mL solution of biotinylated peptide was incubated with 20 μ L of a 2mg/mL solution of streptavidin-Alexa Fluor488 conjugate (Invitrogen) for 30 minutes at room temperature. The resulting solution was then placed in the donor compartment of the FDC. Skin samples were harvested after 24 hours and imaged as described above.

Streptavidin passed far through the SC when coupled to biotinylated SPACE peptide, some streptavidin was present in the epidermis and dermis (fig. 1 (e) panel). Streptavidin not coupled to the SPACE peptide showed minimal epidermal penetration (fig. 1 panel (f)).

The ability of the SPACE peptides to carry quantum dots across the SC was also tested. For quantum dot delivery into the skin, 198 μ L of 100ng/mL biotinylated peptide solution was incubated with 2 μ L of QDot525 streptavidin conjugate (Invitrogen) for 1 hour at room temperature. Then 200. mu.L of the suspension was placed in the donor compartment of FDC. Skin samples were harvested after 24 hours and imaged by confocal microscopy as described above.

The SPACE peptide when coupled to streptavidin coated quantum dots resulted in detectable but smaller transport amounts. No significant penetration of quantum dots coupled to the control peptide was observed. See diagrams (c) and (d) of fig. 2, respectively. Without wishing to be bound by any particular theory, the reduced transport may be due to the relatively large size of the quantum dots.

Example 7

Penetration of peptides in human skin

The ability of the SPACE peptide to penetrate human skin when removed from the phage and coupled to a fluorescently labeled form of an exemplary small molecule was tested as follows. Full thickness human skin was obtained from National Disease research exchange. The skin was stored at-80 ℃ and thawed immediately prior to use. The electrical conductivity of the skin was measured to ensure the integrity of the skin barrier. Skin samples with resistances above 50k Ω were used for the experiments. Fluorescently labeled peptide (200. mu.L of a 1mg/mL solution) was placed in the donor compartment of FDC. After 24 hours, the remaining solution in the donor compartment was removed and the FDC was disassembled. The skin sample was retrieved and rinsed with DI water to remove excess peptide or peptide complex on the skin surface. Skin samples were then prepared for confocal microscopy imaging as discussed above.

SPACE peptides were shown to successfully cross human skin and showed similar penetration as present in pig skin. See fig. 2 (e) panel (SPACE) and (f) panel (control). When viewed from above, a high localization of the SPACE peptide within keratinocytes was found, whereas no significant penetration of the control peptide was observed. See panels (g) and (h) of FIG. 2, respectively.

Example 8

Penetration of peptides into the skin of living mice

The ability of the SPACE peptide to penetrate the skin of live mice was tested as follows. Mu.l of fluorescently labeled peptide (1mg/ml) was applied to the skin of the mice. Penetration was assessed at various time points by harvesting the skin, sectioning and viewing under a microscope.

The SPACE peptide penetrated into the skin of the live mice at significantly higher levels than the control peptide (see FIGS. 3 (a) - (f) and 4 (a) - (f), respectively). The application of the SPACE peptide to the skin of mice for 30 minutes (FIG. 3) resulted in penetration, and the application for two hours (FIG. 4) resulted in significant skin penetration and was located deep in the dermis, consistent with that seen in pig skin and human skin.

Example 9

Study of the stratum corneum

To further characterize the ability of the SPACE peptide to penetrate SC, experiments were performed using isolated SC as follows. To separate the SC from the full thickness skin, the skin was placed in a water bath at 60 ℃ for 90 seconds. After removal from the water bath, the epidermis was separated from the dermis. The SC were then placed in a petri dish containing 0.25% trypsin, with the epidermis side down, to remove the epidermis from the SC. The SC was washed in DI water and then allowed to dry completely at room temperature. To degrease the SC, the SC were placed in the following chloroform: methanol solvent mixtures for 15 minutes each: 2: 1(v/v), 1: 1(v/v) and 1: 2 (v/v). To confirm lipid removal, FTIR was performed on SC samples before and after exposure to the solvent mixture.

Fourier Transform Infrared (FTIR) Spectroscopy of SC

FTIR was performed on SC samples to observe the effect of different peptide solutions on SC structure. The SCs were cut into 1.5X 1.5cm pieces and a control spectrum was obtained for each piece prior to exposure to the peptide. Then 2mL of peptide solution was incubated with SC for 24 hours. The SC samples were then rinsed with DI water and allowed to dry completely at room temperature. The spectra of each SC sample were again read and the previous and subsequent spectra were compared to determine the effect of each peptide on SC structure. Using Nicolet Magna850 Photometer at 2cm^-1The spectra were obtained and averaged over 400 scans.

Experiments with isolated human SC showed that the SPACE peptide binds to keratinocyte proteins, most likely to keratin (fig. 5 panels (a) and (b)). Fourier transform infrared spectroscopy (FTIR) studies also confirmed the effect of SPACE peptides on keratin. In particular, SC exposed to the SPACE peptide showed a change in FTIR spectrum, indicating a structural change in keratin (fig. 5 (c)). FTIR showed no significant effect of the control peptide on protein structure compared to that seen in the absence of any peptide (fig. 5 (d) and fig. 6). FTIR also indicated that the SPACE peptide had no detectable effect on SC lipids (fig. 5 (e) and (f) plots). Symmetric CH is not found₂No shift in center frequency was found with changes in the peak area of the tensile peak, indicating that the SPACE peptide did not cause extraction or fluidization of SC lipids. Consistent with FTIR data, exposure to the SPACE peptide did not cause significant changes in skin conductivity (fig. 7). In particular, the skin conductivity increased by about 1.7(+/-0.6) fold after 24 hours incubation with SPACE peptide. The improvement, although higher than observed for the control peptide, was still relatively mild. Similarly, co-incubation of SPACE peptide with large hydrophilic molecule inulin resulted in only a mild increase in its penetration (FIG. 7), indicating that SPACE peptide is primarily effective in enhancing penetration of coupled cargo but not co-administered cargo.

Without wishing to be bound by any particular theory, the primary role of the SPACE peptide may be to enhance partitioning of SCs primarily in keratinocytes, which in turn enhances the ability of the SPACE peptide and conjugate to cross the skin barrier. An additional effect of the peptide on penetration can also be expected as it can affect the keratin structure.

Example 10

Penetration of SPACE peptides into living cells

The ability of the SPACE peptide to penetrate SC has been confirmed and also tested for its ability to penetrate into living cells in cell culture, including keratinocytes, fibroblasts and endothelial cells (HUVEC).

For cell penetration studies, 1.2X 10⁴Individual cells were seeded on poly-d-lysine coated glass bottom plates (MatTek). For HUVEC cells, the culture dishes were coated with 1% gelatin prior to cell seeding. After 4 hours of incubation at 37 ℃, the medium was removed and 20 μ L of 1mg/mL fluorescent peptide solution was added to 180 μ L of medium and subsequently to the cell culture dish. For the control, an equal amount of PBS was added instead of the peptide solution. After addition of the peptide, the cell cultures were incubated under appropriate conditions to study cell penetration (4 ℃ or 37 ℃) and incubated for 6 or 24 hours. Cells were prepared for confocal microscopy imaging.

Cell culture conditions

Adult epidermal keratinocytes (Invitrogen) were cultured in EpiLife medium (Invitrogen) supplemented with human keratinocyte growth additive (Invitrogen), human skin fibroblasts (ATCC) were cultured in DMEM medium (ATCC) supplemented with 10% fetal bovine serum, mixed human umbilical vein endothelial cells (HUVEC, Lonza) were cultured in M199 medium supplemented with 15% fetal bovine serum, 15. mu.g/mL endothelial cell growth additive, 100. mu.g/mL heparin and 2mM L-glutamine in 1% gelatin-coated culture flasks, and MDA-MB-231 breast cancer cells were cultured in DMEM medium (ATCC) supplemented with 10% fetal bovine serum. All cell culture media were supplemented with 100U/mL penicillin and 100. mu.g/mL streptomycin and the cultures were allowed to stand under standard cell culture conditions (37 ℃, 5% CO)₂) And (4) growing.

Preparation of cell culture samples for confocal microscopy imaging

After incubation, cells were washed with hank's balanced salt buffer (HBSS, Lonza) and incubated with 1% trypan blue for 5 min to quench any fluorescence on the cell surface. The cells were then fixed with 4% paraformaldehyde for 3 minutes, followed by another wash in HBSS. The cells were then incubated with Hoechest33342 (5. mu.g/mL) for 5 minutes and then washed in HBSS. The cell culture dishes were then filled with HBSS and imaged using confocal microscopy (Olympus Fluoview 500).

The significant penetration of the SPACE peptide was demonstrated for all cell lines (FIGS. 8 (a) - (f)), and FIG. 8 (g) shows a magnified view of SPACE peptide internalization in keratinocytes. In all cases, the degree of internalization of the SPACE peptide was higher than that of the control peptide, indicating that cell penetration occurred in a sequence-specific manner (fig. 8, panel (h)). SPACE peptide also showed internalization in breast cancer cells (MD-MB-23, FIGS. 9 (a) - (c)). The ability to penetrate all types of test cells suggests that the mode of SPACE entry into cells proceeds via a pathway common to all cell lines studied, rather than due to specific membrane proteins characteristic of keratinocytes.

Example 11

Cell penetration mechanism study

To determine the potential mechanism of cellular penetration of the SPACE peptide, the effect of various endocytosis inhibitors, including incubation at 4 ℃, on internalization was tested in human keratinocytes (see FIG. 10, panel (a)).

Endocytosis inhibitor

For cell mechanism studies, cells were incubated with various endocytosis inhibitors or at 4 ℃ for 1 hour, followed by addition of fluorescently labeled peptides. The endocytosis inhibitors used were EIPA (Invitrogen) and chlorpromazine, nystatin and deoxy-D-glucose (Sigma). EIPA was dissolved in DMSO and used at a concentration of 100. mu.M. Chlorpromazine, nystatin and deoxy-D-glucose were dissolved in sterile water and used at concentrations of 10. mu.g/mL, 25. mu.g/mL and 5mM, respectively. Cells were incubated with fluorescently labeled peptide for 3 hours and then harvested for analysis using flow cytometry.

Preparation of samples for flow cytometry

After incubation with the fluorescently labeled peptide, the medium was removed and the cells were washed 3 times in HBSS for 5 minutes each to remove residual fluorescence. 0.25% trypsin (HyClone) was used to remove cells from the cell culture plate. The cells were then centrifuged at 5,000rpm for 5 minutes to pellet the cells. The cell pellet was resuspended in PBS (ph7.4) on ice and the sample was analyzed using FACS Aria flow cytometer.

Incubation at 4 ℃ significantly reduced the internalization of the SPACE peptide (about 5% uptake compared to 37 ℃) as well as the control peptide, indicating that both enter the cells by an active mechanism (figure 10, panel (a)). This was further confirmed by the use of deoxy-D-glucose, which also resulted in a reduction (about 52%) in the internalization of both peptides (fig. 10 panel (a)). To further assess the nature of active uptake, cells were incubated with the clathrin-mediated endocytosis inhibitor chlorpromazine and the caveolae-mediated endocytosis inhibitor nystatin. Neither reduced cellular internalization of the SPACE peptide or control peptide (fig. 10, panel (a)). Finally, the effect of the megalocytosis inhibitor 5- (N-ethyl-N-isopropyl) amiloride (EIPA) was tested. Exposure of cells to EIPA resulted in a decrease in SPACE internalization of about 50% (fig. 10 panel (a)). In contrast, EIPA had no effect on control peptide internalization. Taken together, these results suggest that macroendocytosis plays an important role in the internalization of SPACE peptides, a conclusion that also applies to other Cell penetrating peptides, see in particular the relevant literature (Nakase I et al (2004) Cellular uptake of aromatic-rich peptides: rolls for Cellular uptake and molecular Ther10 (6): 1011;. Patel LN, Zaro JL and Shen WC (2007) Cellular uptake peptides: intracellular pathways and pharmacological activities. phase Res24 (11): 1977;. 1992). Studies have reported that cargo internalized by megalocytosis is not normally co-localized with endosomes/lysosomes, which means that it is possible to avoid their entry into the lysosome-degrading compartment (Tamaru M, Akita H, Fujiwara T, Kajimoto K and Harashima H (2010) Leptin-derivative peptide, a targeting peptide and for use in a human tissue cell. MTT analysis of keratinocyte cell cultures showed that SPACE peptide was not toxic to cells in the concentration ranges studied here (0.1-1.0mg/mL, FIG. 10 (b)).

Example 12

Knockdown of GFP using SPACE peptide-coupled siRNA

The ability of SPACE peptides to penetrate into a variety of cells makes them excellent candidates for siRNA delivery. This possibility was explored in vitro using Green Fluorescent Protein (GFP) -expressing endothelial cells as model cell lines.

Endothelial cells (ATCC) expressing GFP were grown in DMEM medium supplemented with 10% fetal bovine serum. GFP siRNA, 5 '-GAC GUAAAC GGC CAC AAGUUC N6-3' (SEQ ID NO: 26) (Dharmacon), was coupled to a fluorescently labeled peptide (containing free carboxyl groups) by EDC chemistry.

The 10mM peptide solution was incubated in MES buffer (pH5.5) with equal portions of 10mM N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride solution (EDAC, Sigma) and 9.5mM N-hydroxythiosuccinimide sodium salt solution (NHS, Sigma) for 15 minutes. The amine modified siRNA was then added to the mixture to couple the peptide to the siRNA and allowed to mix overnight.

The peptide-siRNA complexes were added to the appropriate cell culture medium to give a final concentration of 1. mu.M siRNA. The medium with the peptide-siRNA was then added to the cells and allowed to incubate for 48 hours. Cells were imaged using a confocal microscope and image analysis was performed using ImageJ to determine the overall fluorescence intensity of each cell. Knockdown was determined as the percentage of cells in the test case with an intensity at least 30% lower than the observed mean intensity for the control (untreated) cell population.

SPACE peptide-conjugated sirnas caused significant GFP knockdown (fig. 11 panel (a)). In contrast, no significant knockdown was observed for siRNA alone, SPACE conjugated to a control siRNA, or control peptide conjugated to siRNA. To determine whether conjugation of siRNA to SPACE peptide adversely affected the potency of siRNA, both unconjugated siRNA and SPACE-siRNA were combined with lipofectamine^TMRecombines and assesses knockdown. In both cases, knockdown was significant compared to control, i.e., no siRNA treatmentFIGS. (d) - (f) of FIG. 9).

Example 13

In vivo dermal penetration Using peptide-coupled IL-10siRNA

The ability of SPACE peptides to enhance dermal penetration of IL-10siRNA was evaluated as follows. This siRNA was chosen for its potential to treat atopic dermatitis, a major skin disorder. Control peptides were not evaluated in the in vivo siRNA studies due to insignificant knockdown seen with the control peptides when compared to the SPACE peptide and insufficient in vivo skin penetration ability.

The sequences of siRNAs used in the in vivo study were as follows: IL-10: 5 '-GAA UGAAUUUGA CAU CUU CUU N6-3' (SEQ ID NO: 27), and luciferase (control): 5 '-UAA GGC UAU GAA GAG AUA CUU N6-3' (SEQ ID NO: 28). For IL-10, 2-O-methyl modifications were made at all bases. All sirnas were purchased from Dharmacon.

siRNA delivery was performed in 6-8 week old female Balb/C mice (Charles river Laboratories) according to protocols approved by the Institutional Animal Care and use Committee. Mice were placed under anesthesia (1-2% isoflurane) and their back was gently shaved. 200 μ L of 10 μ M peptide-siRNA solution or corresponding control was topically applied to 3cm of the animal's back²Over a large area. The solution was then covered with sterile gauze and breathable bandage. After 24 hours, mice were treated with CO₂Sacrificed and skin samples were immediately collected using a 4mm biopsy punch. Two 4mm biopsy samples were taken randomly from the treatment area and immediately frozen in liquid nitrogen. The skin was then placed in a surfactant combination of 0.5% (w/v)3- (decyldimethylammonium) propanesulfonate (DPS) and Brij30 and homogenized on ice (IKA disperser) for 1 minute to extract the proteins from the skin samples. The homogenate was then centrifuged at 10,000rpm for 5 minutes and the supernatant collected. Total protein concentration was determined using the micro BCA protein quantification kit (Pierce) and IL-10 levels were determined using a mouse IL-10ELISA (Raybiotech).

Application of IL-10siRNA alone without peptide did not significantly affect IL-10 levels compared to mice receiving no treatment, SPACE peptide alone, or SPACE conjugated to luciferase siRNA (control siRNA). In contrast, animals treated with SPACE conjugated to IL-10siRNA and SPACE conjugated to 2-O-methyl modified IL-10siRNA showed a significant reduction in IL-10 levels (FIG. 11, panel (b)).

Example 14

In vivo dermal penetration using peptide-coupled GAPDH siRNA

As another example, a SPACE peptide was conjugated to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) siRNA and its effect on skin GAPDH levels was assessed as follows. This target was chosen because GAPDH is a common housekeeping protein and provides an example of a common siRNA target.

The sequences of siRNAs used in the in vivo study were as follows: GAPDH: 5 '-GUG UGAACC ACG AGAAAU AUU N6-3' (SEQ ID NO: 29), and luciferase (control): 5 '-UAA GGC UAU GAA GAG AUA CUU N6-3' (SEQ ID NO: 28). All sirnas were purchased from Dharmacon.

siRNA delivery was performed in 6-8 week old female Balb/C mice (Charles River Laboratories) according to protocols approved by the institutional animal care and use committee. Mice were placed under anesthesia (1-2% isoflurane) and their back was gently shaved. 200 μ L of 10 μ M peptide-siRNA solution or corresponding control was topically applied to 3cm of the animal's back²Over a large area. The solution was then covered with sterile gauze and breathable bandage. After 72 hours, mice were treated with CO₂Sacrificed and skin samples were immediately collected using a 4mm biopsy punch. Two 4mm biopsy samples were taken randomly from the treatment area and immediately frozen in liquid nitrogen. The skin was then placed in a surfactant combination of 0.5% (w/v)3- (decyldimethylammonium) propanesulfonate (DPS) and Brij30 and homogenized on ice (IKA disperser) for 1 minute to extract the proteins from the skin samples. The homogenate was then centrifuged at 10,000rpm for 5 minutes and collectedAnd (5) clear liquid. Total protein concentration was determined using a micro BCA protein quantification kit (Pierce). Using Kdalert^TMGAPDH levels were measured with the GAPDH assay kit (Ambion).

Animals treated with the SPACE-GAPDH siRNA conjugate resulted in a significant reduction in protein levels compared to controls (untreated, siRNA alone, SPACE peptide alone, and SPACE conjugated to control siRNA, panel (c) of FIG. 11). Knockdown of GAPDH in skin is dose-dependent; 43% of knockdown was observed at 10. mu.M, 21% at 5. mu.M, and 10% at 1. mu.M (FIG. 11 (d)). Knockdown also depends on application time, with longer application times leading to higher knockdown (fig. 12).

Claims (138)

1. A composition comprising a peptide comprising the amino acid sequence TGSTQHQ (SEQ ID NO:1), hsaltkhh (SEQ ID NO:2), KTGSHNQ (SEQ ID NO:3), MGPSSML (SEQ ID NO:4), TDPNQLQ (SEQ ID NO:5) or STHFIDT (SEQ ID NO:6), wherein said peptide is coupled to an active agent or an active agent carrier comprising said active agent, and wherein said composition is capable of penetrating the stratum corneum upon contact with Stratum Corneum (SC) or penetrating cells upon contact with said cells.

2. The composition of claim 1, wherein the composition is capable of penetrating the SC layer and penetrating the cell.

3. The composition of claim 1, wherein the amino acid sequence comprises CTGSTQHQC (SEQ ID NO:7), CHSALTKHC (SEQ ID NO:8), CKTGSHNQC (SEQ ID NO:9), CMGPSSMLC (SEQ ID NO:10), CTDPNQLQC (SEQ ID NO:11), or CSTHFIDTC (SEQ ID NO: 12).

4. The composition of claim 1, wherein the amino acid sequence comprises ACTGSTQHQCG (SEQ ID NO:13), ACHSALTKHCG (SEQ ID NO:14), ACKTGSHNQCG (SEQ ID NO:15), ACMGPSSMLCG (SEQ ID NO:16), ACTDPNQLQCG (SEQ ID NO:17), or ACSTHFIDTCG (SEQ ID NO: 18).

5. The composition of claim 4, wherein the peptide is a cyclic peptide comprising a Cys-Cys disulfide bond.

6. The composition of claim 1, wherein the composition is capable of penetrating the cell membrane of the living non-human animal cell.

7. The composition of claim 1, wherein the composition is capable of penetrating the cell membrane of the living human cell.

8. The composition of claim 1, wherein the composition is capable of penetrating the cell membrane of a living epidermal or dermal cell.

9. The composition of claim 1, wherein the composition is capable of penetrating the cell membrane of a living immune cell.

10. The composition of claim 1, wherein the active agent comprises a macromolecule.

11. The composition of claim 10, wherein the macromolecule comprises a protein.

12. The composition of claim 11, wherein the protein comprises an antibody or fragment thereof comprising at least one paratope.

13. The composition of claim 10, wherein the macromolecule comprises a nucleic acid.

14. The composition of claim 13, wherein the nucleic acid is DNA.

15. The composition of claim 13, wherein the nucleic acid is RNA.

16. The composition of claim 15, wherein the RNA is an interfering RNA.

17. The composition of claim 16, wherein the interfering RNA is an shRNA.

18. The composition of claim 16, wherein the interfering RNA is a miRNA.

19. The composition of claim 16, wherein the interfering RNA is siRNA.

20. The composition of claim 19, wherein the siRNA is IL-10 siRNA.

21. The composition of claim 19, wherein the siRNA is CD86 siRNA.

22. The composition of claim 19, wherein the siRNA is KRT6 asiRNA.

23. The composition of claim 19, wherein the siRNA is a TNFR1 siRNA.

24. The composition of claim 19, wherein the siRNA is TACEsiRNA.

25. The composition of claim 19, wherein the siRNA is a mutation-specific siRNA.

26. The composition of claim 1, wherein the active agent is a pharmaceutical compound.

27. The composition of claim 1, wherein the active agent comprises a detectable agent.

28. The composition of claim 27, wherein the detectable agent comprises a fluorescent label.

29. The composition of claim 27, wherein the detectable agent comprises a radioactive label.

30. The composition of claim 1, wherein the active agent is a nanoparticle.

31. The composition of claim 1, wherein the active agent is a low molecular weight compound.

32. The composition of claim 1, wherein the active agent is an inhibitor of IL-10 bioactivity.

33. The composition of claim 32, wherein the active agent is selected from an IL-10siRNA and an antibody or fragment thereof that binds IL-10.

34. The composition of claim 1, wherein the peptide is coupled to the active agent.

35. The composition of claim 1, wherein the peptide is coupled to an active agent carrier comprising the active agent.

36. The composition of claim 35, wherein the active agent carrier is a liposome.

37. The composition of claim 35, wherein the active agent carrier is a nanoparticle.

38. The composition of claim 35, wherein the active agent carrier is a polymeric micelle.

39. A composition comprising a peptide comprising the amino acid sequence 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), or STHFIDT (SEQ ID NO:6), wherein said peptide is associated with an active agent or an active agent carrier comprising said active agent, wherein said association results from hydrophobic, electrostatic, or van der waals interactions, and wherein said composition is capable of penetrating the Stratum Corneum (SC) upon contact therewith or penetrating cells upon contact therewith.

40. The composition of claim 39, wherein the composition is capable of penetrating the SC layer and penetrating the cell.

41. The composition of claim 39, wherein the amino acid sequence comprises CTGSTQHQC (SEQ ID NO:7), CHSALTKHC (SEQ ID NO:8), CKTGSHNQC (SEQ ID NO:9), CMGPSSMLC (SEQ ID NO:10), CTDPNQLQC (SEQ ID NO:11), or CSTHFIDTC (SEQ ID NO: 12).

42. The composition of claim 39, wherein the amino acid sequence comprises ACTGSTQHQCG (SEQ ID NO:13), ACHSALTKHCG (SEQ ID NO:14), ACKTGSHNQCG (SEQ ID NO:15), ACMGPSSMLCG (SEQ ID NO:16), ACTDPNQLQCG (SEQ ID NO:17), or ACSTHFIDTCG (SEQ ID NO: 18).

43. The composition of claim 42, wherein the peptide is a cyclic peptide comprising a Cys-Cys disulfide bond.

44. An isolated peptide comprising an amino acid sequence selected from one of the following sequences: ACTGSTQHQCG (SEQ ID NO:13), ACHSALTKHCG (SEQ ID NO:14), ACKTGSHNQCG (SEQ ID NO:15), ACMGPSSMLCG (SEQ ID NO:16), ACTDPNQLQCG (SEQ ID NO:17) and ACSTHFIDTCG (SEQ ID NO: 18).

45. The isolated peptide of claim 44, wherein the peptide comprises repeating units of one or more of ACTGSTQHQCG (SEQ ID NO:13), ACHSALTKHCG (SEQ ID NO:14), ACKTGSHNQCG (SEQ ID NO:15), ACMGPSSMLCG (SEQ ID NO:16), ACTDPNQLQCG (SEQ ID NO:17), and ACSTHFIDTCG (SEQ ID NO: 18).

46. The isolated peptide according to claim 45, wherein the unit is repeated 2 to 50 times.

47. The isolated peptide according to claim 45, wherein each unit is separated by an intervening peptide sequence.

48. The isolated peptide of claim 44, wherein the peptide is a cyclic peptide comprising a Cys-Cys disulfide bond.

49. An isolated polypeptide comprising a repeat unit of one or more of TGSTQHQ (SEQ ID NO:1), HSALTPKH (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).

50. The isolated peptide according to claim 49, wherein the unit is repeated 2 to 50 times.

51. The isolated peptide according to claim 49, wherein each unit is separated by an intervening peptide sequence.

52. An isolated polypeptide consisting essentially of repeat units of one or more of TGSTQHQ (SEQ ID NO:1), HSALTPKH (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).

53. A method of delivering an active agent to a subject, comprising: administering to a subject a composition comprising a peptide comprising the amino acid sequence TGSTQHQ (SEQ ID NO:1), HSALTPKH (SEQ ID NO:2), KTGSHNQ (SEQ ID NO:3), MGPSSML (SEQ ID NO:4), TDPNQLQ (SEQ ID NO:5), or STHFIDT (SEQ ID NO:6), wherein the peptide is coupled to an active agent or an active agent carrier comprising the active agent, and wherein the composition is capable of penetrating the Stratum Corneum (SC) of the subject or penetrating cells of the subject.

54. The method of claim 53, wherein the composition is capable of penetrating SCs of the subject and penetrating cells of the subject.

55. The method of claim 53, wherein the administration is topical administration.

56. The method of claim 53, wherein the amino acid sequence comprises CTGSTQHQC (SEQ ID NO:7), CHSALTKHC (SEQ ID NO:8), CKTGSHNQC (SEQ ID NO:9), CMGPSSMLC (SEQ ID NO:10), CTDPNQLQC (SEQ ID NO:11), or CSTHFIDTC (SEQ ID NO: 12).

57. The method of claim 56, wherein the amino acid sequence comprises ACTGSTQHQCG (SEQ ID NO:13), ACHSALTKHCG (SEQ ID NO:14), ACKTGSHNQCG (SEQ ID NO:15), ACMGPSSMLCG (SEQ ID NO:16), ACTDPNQLQCG (SEQ ID NO:17), or ACSTHFIDTCG (SEQ ID NO: 18).

58. The method of claim 57, wherein the peptide is a cyclic peptide comprising a Cys-Cys disulfide bond.

59. The method of claim 53, wherein the composition is capable of penetrating a cell membrane of the living non-human animal cell.

60. The method of claim 53, wherein the composition is capable of penetrating the cell membrane of the living human cell.

61. The method of claim 53, wherein the composition is capable of penetrating the cell membrane of the living epidermal or dermal cell.

62. The method of claim 53, wherein the composition is capable of penetrating the cell membrane of the living immune cell.

63. The method of claim 53, wherein the active agent comprises a macromolecule.

64. The method of claim 63, wherein the macromolecule comprises a protein.

65. The method of claim 64, wherein the protein comprises an antibody or fragment thereof comprising at least one paratope.

66. The method of claim 64, wherein the macromolecule comprises a nucleic acid.

67. The method of claim 66, wherein the nucleic acid is DNA.

68. The method of claim 66, wherein the nucleic acid is RNA.

69. The method of claim 68, wherein the RNA is an interfering RNA.

70. The method of claim 69, wherein the interfering RNA is an shRNA.

71. The method of claim 69, wherein the interfering RNA is miRNA.

72. The method of claim 69, wherein the interfering RNA is siRNA.

73. The method of claim 72, wherein the siRNA is IL-10 siRN.

74. The method of claim 72, wherein the siRNA is a CD86 siRNA.

75. The method of claim 72, wherein the siRNA is KRT6 asiRNA.

76. The method of claim 72, wherein the siRNA is a TNFR1 siRNA.

77. The method of claim 72, wherein the siRNA is TACESsiRNA.

78. The method of claim 72, wherein the siRNA is a mutation-specific siRNA.

79. The method of claim 53, wherein the active agent is a pharmaceutical compound.

80. The method of claim 53, wherein the active agent comprises a detectable agent.

81. The method of claim 53, wherein the detectable agent comprises a fluorescent label.

82. The method of claim 53, wherein the detectable agent comprises a radioactive label.

83. The method of claim 53, wherein the active agent is a nanoparticle.

84. The method of claim 53, wherein the active agent is a low molecular weight compound.

85. The method of claim 1, wherein the active agent is an inhibitor of IL-10 bioactivity.

86. The method of claim 85, wherein the active agent is selected from the group consisting of an IL-10siRNA and an antibody or fragment thereof that binds IL-10.

87. The method of claim 53, wherein the peptide is coupled to the active agent.

88. The method of claim 53, wherein the peptide is coupled to an active agent carrier comprising the active agent.

89. The method of claim 88, wherein the active agent carrier is a liposome.

90. The method of claim 88, wherein the active agent carrier is a nanoparticle.

91. The method according to claim 88, wherein the active agent carrier is a polymeric micelle.

92. A method of delivering an active agent to a subject, comprising: administering to a subject a composition comprising a peptide comprising the amino acid sequence TGSTQHQ (SEQ ID NO:1), HSALTPKH (SEQ ID NO:2), KTGSHNQ (SEQ ID NO:3), MGPSSML (SEQ ID NO:4), TDPNQLQ (SEQ ID NO:5), or STHFIDT (SEQ ID NO:6), wherein the peptide is associated with an active agent or an active agent carrier comprising the active agent, wherein the association results from hydrophobic, electrostatic, or van der Waals interactions, and wherein the composition is capable of penetrating the Stratum Corneum (SC) of the subject or penetrating cells of the subject.

93. The method of claim 92, wherein the composition is capable of penetrating the SC of the subject and penetrating the cell of the subject.

94. The method of claim 92, wherein the administration is topical administration.

95. The method of claim 92, wherein the amino acid sequence comprises CTGSTQHQC (SEQ ID NO:7), CHSALTKHC (SEQ ID NO:8), CKTGSHNQC (SEQ ID NO:9), CMGPSSMLC (SEQ ID NO:10), CTDPNQLQC (SEQ ID NO:11), or CSTHFIDTC (SEQ ID NO: 12).

96. The method of claim 95, wherein the amino acid sequence comprises ACTGSTQHQCG (SEQ ID NO:13), ACHSALTKHCG (SEQ ID NO:14), ACKTGSHNQCG (SEQ ID NO:15), ACMGPSSMLCG (SEQ ID NO:16), ACTDPNQLQCG (SEQ ID NO:17), or ACSTHFIDTCG (SEQ ID NO: 18).

97. The method of claim 96, wherein the peptide is a cyclic peptide comprising a Cys-Cys disulfide bond.

98. A method of treating a subject having a skin disease, comprising: administering to the subject a composition comprising a peptide comprising the amino acid sequence TGSTQHQ (SEQ ID NO:1), HSALTPKH (SEQ ID NO:2), KTGSHNQ (SEQ ID NO:3), MGPSSML (SEQ ID NO:4), TDPNQLQ (SEQ ID NO:5), or STHFIDT (SEQ ID NO:6), wherein the peptide is coupled to a dermatologically active agent or a dermatologically active agent carrier comprising the active agent, and wherein the composition is capable of penetrating the Stratum Corneum (SC) of the subject or penetrating cells of the subject.

99. The method of claim 98, wherein the composition is capable of penetrating the SC of the subject and penetrating the cell of the subject.

100. The method of claim 98, wherein the administration is topical administration.

101. The method of claim 98, wherein the amino acid sequence comprises ACTGSTQHQCG (SEQ ID NO:13), ACHSALTKHCG (SEQ ID NO:14), ACKTGSHNQCG (SEQ ID NO:15), ACMGPSSMLCG (SEQ ID NO:16), ACTDPNQLQCG (SEQ ID NO:17), or ACSTHFIDTCG (SEQ ID NO: 18).

102. The method of claim 101, wherein the peptide is a cyclic peptide comprising a Cys-Cys disulfide bond.

103. A method of treating a subject having a skin disease, comprising: administering to a subject a composition comprising a peptide comprising the amino acid sequence TGSTQHQ (SEQ ID NO:1), HSALTPKH (SEQ ID NO:2), KTGSHNQ (SEQ ID NO:3), MGPSSML (SEQ ID NO:4), TDPNQLQ (SEQ ID NO:5), or STHFIDT (SEQ ID NO:6), wherein the peptide is associated with a dermatologically active agent or a dermatologically active agent carrier comprising the active agent, wherein the association results from hydrophobic, electrostatic, or van der Waals interactions, and wherein the composition is capable of penetrating the Stratum Corneum (SC) of the subject or penetrating cells of the subject.

104. The method of claim 103, wherein the composition is capable of penetrating the SC of the subject and penetrating the cell of the subject.

105. The method of claim 103, wherein the administration is topical administration.

106. The method of claim 103, wherein the amino acid sequence comprises ACTGSTQHQCG (SEQ ID NO:13), ACHSALTKHCG (SEQ ID NO:14), ACKTGSHNQCG (SEQ ID NO:15), ACMGPSSMLCG (SEQ ID NO:16), ACTDPNQLQCG (SEQ ID NO:17), or ACSTHFIDTCG (SEQ ID NO: 18).

107. The method of claim 106, wherein the peptide is a cyclic peptide comprising a Cys-Cys disulfide bond.

108. A method of treating a subject having, suspected of having, or susceptible to a disease caused at least in part by expression of an mRNA, comprising administering to the subject a composition comprising a peptide comprising the amino acid sequence TGSTQHQ (SEQ ID NO:1), HSALTKH H (SEQ ID NO:2), KTGSHNQ (SEQ ID NO:3), MGPSSML (SEQ ID NO:4), TDQLQ (SEQ ID NO:5), or STHFIDT (SEQ ID NO:6), wherein the peptide is coupled to an interfering RNA targeting the mRNA or a vector comprising the interfering RNA, wherein the composition is capable of penetrating the Stratum Corneum (SC) of the subject or cells of the subject, and wherein expression of the mRNA is thereby attenuated.

109. The method of claim 108, wherein the composition is capable of penetrating the SC of the subject and penetrating the cell of the subject.

110. The method of claim 108, wherein the administration is topical administration.

111. The method of claim 108, wherein the amino acid sequence comprises ACTGSTQHQCG (SEQ ID NO:13), ACHSALTKHCG (SEQ ID NO:14), ACKTGSHNQCG (SEQ ID NO:15), ACMGPSSMLCG (SEQ ID NO:16), ACTDPNQLQCG (SEQ ID NO:17), or ACSTHFIDTCG (SEQ ID NO: 18).

112. The method of claim 111, wherein the peptide is a cyclic peptide comprising a Cys-Cys disulfide bond.

113. A method of treating a subject having, suspected of having, or susceptible to a disease caused at least in part by expression of an mRNA, comprising administering to the subject a composition comprising a peptide comprising the amino acid sequence TGSTQHQ (SEQ ID NO:1), HSALTHH (SEQ ID NO:2), KTGSHNQ (SEQ ID NO:3), MGPSSML (SEQ ID NO:4), TDQLQ (SEQ ID NO:5), or STHFIDT (SEQ ID NO:6), wherein the peptide is associated with an interfering RNA targeting the mRNA or a carrier comprising the interfering RNA, wherein the association results from hydrophobic, electrostatic, or van der Waals interactions, wherein the composition is capable of penetrating the Stratum Corneum (SC) of the subject or cells of the subject, and wherein expression of the mRNA is thereby attenuated.

114. The method of claim 113, wherein the composition is capable of penetrating the SC of the subject and penetrating the cell of the subject.

115. The method of claim 113, wherein the administration is topical administration.

116. The method of claim 113, wherein the amino acid sequence comprises ACTGSTQHQCG (SEQ ID NO:13), ACHSALTKHCG (SEQ ID NO:14), ACKTGSHNQCG (SEQ ID NO:15), ACMGPSSMLCG (SEQ ID NO:16), ACTDPNQLQCG (SEQ ID NO:17), or ACSTHFIDTCG (SEQ ID NO: 18).

117. The method of claim 116, wherein the peptide is a cyclic peptide comprising a Cys-Cys disulfide bond.

118. A method of attenuating mRNA expression in a subject in need thereof, comprising administering to the subject a composition comprising a peptide comprising the amino acid sequence TGSTQHQ (SEQ ID NO:1), HSALTHH (SEQ ID NO:2), KTGSHNQ (SEQ ID NO:3), MGPSSML (SEQ ID NO:4), TDPNQLQ (SEQ ID NO:5) or STHFIDT (SEQ ID NO:6), wherein the peptide is coupled to an siRNA targeting the mRNA or a vector comprising an siRNA targeting the mRNA, wherein the composition is capable of penetrating the Stratum Corneum (SC) of the subject or cells of the subject, and wherein expression of the mRNA is thereby attenuated.

119. The method of claim 118, wherein the mRNA is an IL-10mRNA and the siRNA is an IL-10 siRNA.

120. The method of claim 118, wherein the mRNA is a CD86mRNA and the siRNA is a CD86 siRNA.

121. The method of claim 118, wherein the mRNA is KRT6amRNA and the siRNA is KRT6a siRNA.

122. The method of claim 118, wherein the mRNA is a TNFR1mRNA and the siRNA is a TNFR1 siRNA.

123. The method of claim 118, wherein the mRNA is TACE mRNA and the siRNA is TACE siRNA.

124. The method of claim 118, wherein the composition is capable of penetrating the SC of the subject and penetrating the cell of the subject.

125. The method of claim 118, wherein the administration is topical administration.

126. The method of claim 118, wherein the amino acid sequence comprises ACTGSTQHQCG (SEQ ID NO:13), ACHSALTKHCG (SEQ ID NO:14), ACKTGSHNQCG (SEQ ID NO:15), ACMGPSSMLCG (SEQ ID NO:16), ACTDPNQLQCG (SEQ ID NO:17), or ACSTHFIDTCG (SEQ ID NO: 18).

127. The method of claim 126, wherein the peptide is a cyclic peptide comprising a Cys-Cys disulfide bond.

128. A method of attenuating mRNA expression in a subject in need thereof, comprising administering to the subject a composition comprising a peptide comprising the amino acid sequence TGSTQHQ (SEQ ID NO:1), HSALTHH (SEQ ID NO:2), KTGSHNQ (SEQ ID NO:3), MGPSSML (SEQ ID NO:4), TDPNQLQ (SEQ ID NO:5), or STHFIDT (SEQ ID NO:6), wherein the peptide is associated with an siRNA targeting the mRNA or a carrier comprising an siRNA targeting the mRNA, wherein the association results from hydrophobic, electrostatic, or van der Waals interactions, wherein the composition is capable of penetrating the Stratum Corneum (SC) of the subject or cells of the subject, and wherein expression of the mRNA is thereby attenuated.

129. The method of claim 128, wherein the mRNA is an IL-10mRNA and the siRNA is an IL-10 siRNA.

130. The method of claim 128, wherein the mRNA is a CD86mRNA and the siRNA is a CD86 siRNA.

131. The method of claim 128, wherein the mRNA is KRT6amRNA and the siRNA is KRT6a siRNA.

132. The method of claim 128, wherein the mRNA is a TNFR1mRNA and the siRNA is a TNFR1 siRNA.

133. The method of claim 128, wherein the mRNA is TACE mRNA and the siRNA is TACE siRNA.

134. The method of claim 128, wherein the composition is capable of penetrating the SC of the subject and penetrating the cell of the subject.

135. The method of claim 128, wherein the administration is topical administration.

136. The method of claim 128, wherein the amino acid sequence comprises ACTGSTQHQCG (SEQ ID NO:13), ACHSALTKHCG (SEQ ID NO:14), ACKTGSHNQCG (SEQ ID NO:15), ACMGPSSMLCG (SEQ ID NO:16), ACTDPNQLQCG (SEQ ID NO:17), or ACSTHFIDTCG (SEQ ID NO: 18).

137. The method of claim 136, wherein the peptide is a cyclic peptide comprising a Cys-Cys disulfide bond.

138. A composition comprising a peptide consisting essentially of the amino acid sequence TGSTQHQ (SEQ ID NO:1), HSALTHH (SEQ ID NO:2), KTGSHNQ (SEQ ID NO:3), MGPSSML (SEQ ID NO:4), TDPNQLQ (SEQ ID NO:5) or STHFIDT (SEQ ID NO:6), wherein said peptide is coupled to an active agent or an active agent carrier comprising said active agent, and wherein said composition is capable of penetrating the Stratum Corneum (SC) upon contact therewith or penetrating cells upon contact therewith.