WO2021058463A1 - Système de liposomes comprenant un lipide, un composé actif et un peptide vecteur - Google Patents

Système de liposomes comprenant un lipide, un composé actif et un peptide vecteur Download PDF

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WO2021058463A1
WO2021058463A1 PCT/EP2020/076395 EP2020076395W WO2021058463A1 WO 2021058463 A1 WO2021058463 A1 WO 2021058463A1 EP 2020076395 W EP2020076395 W EP 2020076395W WO 2021058463 A1 WO2021058463 A1 WO 2021058463A1
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seq
liposome
carrier system
based carrier
vector
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PCT/EP2020/076395
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Vinzenz OJI
Frederic Valentin
Margitta DATHE
Heike NIKOLENKO
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Westfälische Wilhelms-Universität Münster
Forschungsverbund Berlin E.V.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1275—Lipoproteins; Chylomicrons; Artificial HDL, LDL, VLDL, protein-free species thereof; Precursors thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22

Definitions

  • the present invention is related to a liposome-based carrier system wherein the liposomes comprise at least one lipid (L), at least one active compound (AC) and at least one vector peptide (VP).
  • the at least one vector peptide (VP) comprises at least one vector sequence (VS): (GKKKK) n (SEQ ID NO: 1), wherein n is an integer from 1 to 8 and at least one acyl group, wherein the acyl group is a group with the formula: R-C(O)-, wherein R is (C -C 2 o) alkyl or a (C 4 - C 20 ) alkylene group.
  • the liposomes have an average diameter of 65 to 1000 nm.
  • the present invention is directed to the use of the liposome-based carrier system for use in medicine, in particular for use in treating or preventing of peeling skin syndromes (PSS), the Netherton Syndrome, SAM Syndrome psoriasis vulgaris, atopic dermatitis, impetigo, microbial eczema, microbial eczema associated with dermatomycosis, NISCH Syndrome, androgenetic alopecia, alopecia areata, bullous im m unodermatoses, and epidermolysis bullosa, skin tightening, and dry skin conditions.
  • the invention is directed to pharmaceutical compositions comprising the liposome-based carrier system.
  • PSS1 OMIM:270300
  • CDSN corneodesmosin
  • PSS3 OMIM: 610190
  • PSS5 OMIM: 617115
  • PSS6 OMIM 618084
  • PSS2 OMIM: 609796
  • PSS4 OMIM:607936
  • PSS2 in some instances exhibit clinic aspects of the generalized form 9,10 .
  • Clinical, ultrastructural, and pathophysiologic aspects of PSS1 demonstrate a beautiful relationship between LEKTI deficiency (Netherton syndrome, OMIM: 256500) and desmoglein-1 deficiency (SAM syndrome, OMIM: 615508) 11 ,12 .
  • the disorder presents at birth or few days later and affected persons develop cutaneous pronounced erythroderma with skin abnormalities and suffer from lifelong patchy peeling of the skin.
  • CDSN is a basic phosphoprotein exclusively expressed in cornified epithelia and hair follicles 20 . In the epidermis it is expressed in the stratum granulosum and discharged by lamellar granules.
  • the stratum corneum In the stratum corneum it is localized extracellularly in the corneodesmosomes where it is covalently cross-linked to the cornified cell envelope (CCE) and mediates cell-cell cohesion 21 .
  • CCE cell envelope
  • the adhesive properties are attributable to glycine-rich domains, and undergo sequential proteolysis resulting in desquamation of corneocytes 22,23 .
  • the absence of CDSN is characterized by subcorneal splitting and enhanced detachment of corneocytes similar to the situation in Netherton Syndrome.
  • PSS1 is an ultra rare form of a congenital ichthyosis which causes severe psychological stress and a tremendous reduction in the quality of life in those affected. The treatment is entirely symptom relieving and totally inadequate.
  • balneotherapies and topical therapy with ointments 24 It is mainly limited to balneotherapies and topical therapy with ointments 24 . Until now no specific or efficient therapy is available. A potential new therapy could be based for example on strengthening of the cell-cell cohesion in the transition area of the stratum granulosum to stratum corneum by topical delivery of suitable active substances such as recombinantly expressed human CDSN (rhCDSN).
  • rhCDSN recombinantly expressed human CDSN
  • the present invention is directed to a liposome-based carrier system, wherein the liposomes comprise: a) at least one lipid (L), b) at least one active compound (AC) and c) at least one vector peptide (VP) and wherein the at least one vector peptide (VP) comprises a) at least one vector sequence (VS): (GKKKK) n (SEQ ID NO: 1), wherein n is an integer from 1 to 8, preferably 2 to 6, more preferably 3 to 5, most preferably 3; b) at least one acyl group, preferably at least two acyl groups, wherein the acyl group is a group with the formula: R-C(O)-, wherein R is (C 4 -C 2 o) alkyl or a (C 4 -C 2 o) alkylene group; and wherein the liposomes have an average diameter of 65 to 1000 nm.
  • the liposomes comprise: a) at least one lipid (L), b) at least
  • the present invention is directed to a liposome-based carrier systems for use in medicine, in particular for use in treating or prevention of peeling skin syndromes (PSS), the Netherton Syndrome, SAM Syndrome, psoriasis vulgaris, atopic dermatitis, impetigo, microbial eczema, microbial eczema associated with dermatomycosis, NISCH Syndrome, androgenetic alopecia, alopecia areata, bullous immunodermatoses, and epidermolysis bullosa, skin tightening, and dry skin conditions.
  • PSS peeling skin syndromes
  • the present invention is directed to a method comprising delivering at least one active compound (AC) by a liposome-based carrier system ex vivo, to the outer cell membrane of keratinocytes and/or human epidermal equivalents (HEEs), wherein the liposomes comprise: a) at least one lipid (L), b) at least one active compound (AC) and c) at least one vector peptide (VP), wherein the at least one vector peptide (VP) comprises a) at least one vector sequence (VS): (GKKKK) n (SEQ ID NO: 1)., wherein n is an integer from 1 to 8, preferably 2 to 6, more preferably 3 to 5, most preferably 3 and b) at least one, preferably at least two acyl groups, wherein the acyl group is a group with the formula: R-C(O)-, wherein R is (C 4 -C 2 o) alkyl or a (C 4 -C 2 o
  • the invention relates to a liposome-based carrier system, wherein the liposomes comprise a) at least one lipid (L), b) at least one active compound (AC) which is optionally human corneodesmosin (rhCDSN) and c) at least one vector peptide (VP) according to Formula (I): (VS)-(Z) wherein the vector sequence (VS) is (GKKKK) n (SEQ ID NO: 1)., wherein n is an integer from 1 to 8, preferably 2 to 6, more preferably 3 to 5, most preferably 3 and (Z) is PE-PEG-S-Spacer wherein PE-PEG is 1,2-steaoryl-sn-glycero-3-phosphoethanolamine-N-
  • the spacer is preferably -CH 2 ) 2 -CO-WG-, more preferably the vector peptide (VP) is a peptid with the sequence palmitoyl- WK(pal m itoy l)G KKKKG KKKKG KKKK (SEQ ID NO: 20).
  • the invention relates to a method comprising delivering at least one active compound (AC) by a liposome-based carrier system ex vivo, to the outer cell membrane of keratinocytes and/or human epidermal equivalents (HEEs), wherein the liposomes comprise a) at least one lipid (L), b) at least one active compound (AC) which is optionally human corneodesmosin (rhCDSN) and c) at least one vector peptide (VP) according to Formula (I): (VS)-(Z) wherein the vector sequence (VS) is (GKKKK) n (SEQ ID NO: 1), wherein n is an integer from 1 to 8, preferably 2 to 6, more preferably 3 to 5, most preferably 3 and (Z) is PE-PEG-S-Spacer wherein PE-PEG is 1 ,2-steaoryl-sn-glycero-3-phosphoethanolamine-N-
  • a pharmaceutical composition comprising the liposome-based carrier system and at least one pharmaceutically acceptable carrier.
  • the inventive liposome-based carrier sytem allows the transport of proteins or other suitable substances for example corneodesmosin CDSN or hyaluronic acid, to the keratinocytes, protected by the liposomes from proteolysis and degradation.
  • the inventive liposome-based carrier system accumulates in and/or at the outer cell membrane of keratinocytes and/or human epidermal equivalents (HEEs) without accumulation in the inner cell beyond the outer cell membrane of these cells (e.g. example 4, Figures 10 to 14).
  • HOEs human epidermal equivalents
  • the inventive liposome-based carrier system exhibts low toxicity and sufficient stability (e.g. example 3, Figures 1A to 1D).
  • the inventive liposome-based carrier system allows the use of liposomes, with a size with good skin penetration, through the stratum corneum.
  • liposome-based carrier systems known in the prior art using liposomes of similar size promote uptake in the inner part of the cell of comparable cells, such as keratinocytes.
  • PSS peeling skin syndromes
  • the Netherton Syndrom or similar diseases as mentioned above.
  • Fig. 1 Cell viability assay and Liposome stability.
  • Liposome stability of empty (C) and CDSN loaded (D) POPC-DPPEr LUVs extruded through a 100nm filter) over a period of 28 days at different temperatures.
  • Fig. 2 Liposomal localization on primary keratinocytes.
  • the peptide concentration was 1mM and (C
  • Green carboxyfluorescein (FAM)-labelled P2K12 lipopeptide
  • red DPPEr (Rhodamine B)-labelled lipid of POPC LUVs.
  • Purple CellMaskTM Deep Red plasma membrane stain, indicating the membrane and confirms the viability of the cells. Scalebar represents 20 pm.
  • Fig. 4 Liposome cargo localization on normal and CDSN deficient primary keratinocytes.
  • the peptide concentration was 1mM and (C
  • Green GFP fluorescence of the fusion protein
  • red DPPEr (Rhodamine B)- labelled lipid of POPC LUVs.
  • Purple CellMaskTM Deep Red plasma membrane stain, indicating the membrane and confirms the viability of the cells. Scalebar represents 20 pm.
  • Fig. 5 Liposomal penetration. Penetration capability of P2K12 associated POPC LUVs on three-dimensional epidermal equivalents. Epidermal equivalents were treated epicutanously two times (day 3 and day 5 for 3 h) with P2K12 associated POPC LUVs (A) as well as P2K12-associated POPC LUVs loaded with a rhCDSN:GFP fusion protein (B). For all preparations the peptide concentration was 2pM and (C
  • Fig. 6 Effect of P2K12/POPC-CDSN LUVs on CDSN expression of epidermal equivalents.
  • CDSN deficient epidermal equivalents were treated epicutanously two times (day 3 and day 5 for 3 h) with P2K12 associated POPC LUVs loaded with rhCDSN (C).
  • Untreated CDSN deficient epidermal equivalents (B) as well as untreated epidermal equivalents generated with normal keratinocytes (A) serve as control.
  • the peptide concentration of the preparations was 2mM and (C
  • Epidermal equivalents were harvested on day 7 and CDSN immunofluorescence staining was performed on cryosections.
  • Fig. 7 Effect of P2K12/POPC-CDSN LUVs on the structure of epidermal equivalents. CDSN deficient epidermal equivalents were treated epicutanously two times (day 3 and day 5 for 3 h)
  • Fig. 8 Topical Toluidine blue penetration assay on CDSN deficient human epidermal equivalents of one patient (Patient 1). CDSN deficient epidermal equivalents were treated epicutanously two times (day 4 and day 6 for 3 h) with P2K12 associated POPC LUVs loaded with rhCDSN (C). Untreated epidermal equivalents generated with normal keratinocytes (A) as well as untreated (B) and P2K12/POPC LUV treated (D) CDSN deficient epidermal equivalents serve as control.
  • Fig. 9 Compilation of topical Toluidine blue penetration assay on HEEs of three PSS1 patients. HEE of three different PSS1 patients were treated epicutanously two times (day
  • Fig. 10 Binding properties of P2K12 associated POPC-liposomes (28nm) at primary kerantinocytes. Small unilamellar liposomes (28 nm) enter kerantinocytes and are localized intracellulary.
  • Fig. 11 Binding properties of P2K12 associated POPC-liposomes (73nm) at primary kerantinocytes. Large unilamellar liposomes (73nm) accumulate at the cell membrane of keratinocytes and do not enter in the inner part of the cell.
  • Fig. 12 Binding properties of P2K12 associated POPC-liposomes (89nm) at primary kerantinocytes. Large unilamellar liposomes (89nm) accumulate at the cell membrane of keratinocytes and do not enter in the inner part of the cell.
  • Fig. 13 Binding properties of P2K12 associated POPC-liposomes (106nm) at primary kerantinocytes. Large unilamellar liposomes (106nm) accumulate at the cell membrane of keratinocytes and do not enter in the inner part of the cell.
  • Fig. 14 Binding properties of P2K12 associated POPC-liposomes (193nm) at primary kerantinocytes. Large unilamellar liposomes (193nm) accumulate at the cell membrane of keratinocytes and do not enter in the inner part of the cell.
  • Fig. 15 Recombinant expression of human CDSN.
  • Recombinant human CDSN without signal peptide CDSNA33
  • CDSNA33 C-terminal polyhistidine tag
  • Crude extract and elution fractions of the purification were separated on a 12.5% SDS-PAGE gel.
  • Samples were transferred to a PVDF membrane and detected with a specific antibody against CDSN (P-20 SantaCruz, sc-49620) by Western blotting.
  • Fig. 16 Intensity scan to record localization of liposomes more in detail. Intensity scan along the red line in the equator plane of undifferentiated keratinocytes.
  • Purple CellMaskTM Deep Red plasma membrane stain, indicating the membrane and confirming the viability of the cells.
  • Fig. 17 Immunohistochemical Detection of Differentiation Marker. Differentiation marker expressed in normal (CDSN +/+) as well as CDSN deficient (CDSN -/-) HEEs. Transglutaminase activity assay (A-B), filaggrin expression (C-D), claudin 1 expression (E-F).
  • the present invention provides a liposome-based carrier system.
  • a liposome is a structure composed of lipid molecules which consist of a lipophilic and a hydrophilic part.
  • lipids may spontaneously arrange in bilayers.
  • the inner part of the bilayer is stabilized by van der Waals interactions between the lipophilic tails of the lipids in the two layers, while the hydrophilic heads arrange at the surface of the bilayer, and are exposed to the water.
  • Spherical arrangement of the layer encompassing an aqueous interior is called a liposome.
  • the liposome comprises at least one lipid (L).
  • the liposome may comprise at least two different lipids (L), at least three different lipids (L), at least four different lipids (L) or more different lipids (L).
  • the liposome may comprise one lipid bilayer (unilammellar) or more than one lipid bilayer (multilammellar), such as two lipid bilayers (bilammellar).
  • Preferably at least 90%, more preferably 95%, most preferably 99%, even more preferred at least 99.5% of the liposomes in the liposome-based carrier system are unilammellar.
  • the at least one lipid (L) is a phospholipid or a zwitterionic phospholipid, more preferably phosphatidylcholins, particular preferred the at least one lipid (L) is selected from the group consisting of L-a-phosphatidylcholine (Egg PC), 1-palmitoyl-2-oleoyl-glycerol-3- phosphocholine (POPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) (14:0 PC), and 1 ,2 Distearyl-sn-glycero-3- phosphatidylcholin (DSPC), particularly preferred L-a- phosphatidylcholine (Egg PC), most particularly preferred 1-palmitoyl-2-oleoyl-glycerol-3- phosphocholine (POPC).
  • Egg PC L-a-phosphatidylcholine
  • POPC 1-palmitoyl-2-oleoyl-glycerol-3
  • the at least one lipid (L) may comprise further additive lipids, such as ceramides, cholesterol and anionic lipids like glycerols, e.g. 1,2-dipalmitoyl-sn-glycero- 3-phospho-(T-rac-glycerol) DPPG (16:0/16:0) are other suitable components.
  • the liposomes comprise at least one active compound (AC).
  • the active compound (AC) may be located in the lipid bilayer and/or in the aqueous inner part of the liposomes.
  • the liposome may comprise at least two different active compounds (AC), at least three different active compounds (AC), at least four different active compounds (AC) or more.
  • the active compound (AC) may be any compound which is suitable to be transported by the liposome-based carrier system.
  • the active compound (AC) is a biologically active compound, which is preferably useful for application in the prevention or treatment of a disease or condition, more preferably in the prevention or treatment of the diseases and conditions mentioned herein.
  • the active compound (AC) has a molecular weight in the range of 1000 to 500000 g/mol, more preferably in the range of 1000 to 300000 g/mol, most preferably 3000 to 250000 g/mol.
  • the active substance (AC) is selected from the group consisting of at least one protein and hyaluronic acid and derivatives thereof, and cross-linked hyaluronic acid and derivatives thereof.
  • the active substance is selected from the group consisting of at least one protein with a sequence identity of at least 70% of one of the following human corneodesmosin (SEQ ID NO. 2), rhCDSN (SEQ ID NO. 3), desmoglein 1 (SEQ ID NO.
  • identity or “sequence identity” is meant a property of sequences that measures their similarity or relationship.
  • identity or “sequence identity” as used in the present invention means the percentage of pair-wise identical residues - following (homology) alignment of a sequence of a polypeptide of the invention with a sequence in question- with respect to the number of residues in the longer of these two sequences. Identity is measured by dividing the number of identical residues by the total number of residues and multiplying the product by 100.
  • the percentage of sequence homology or sequence identity can, for example, be determined herein using the program BLASTP, version blastp 2.2.5 (November 16, 2002; cf. Altschul, S. F. et al. (1997) Nucl. Acids Res. 25, 3389-3402).
  • the percentage of homology is based on the alignment of the entire polypeptide sequences (matrix: BLOSUM 62; gap costs: 11.1; cutoff value set to 10 3 ) including respective sequences. It is calculated as the percentage of numbers of “positives” (homologous amino acids) indicated as result in the BLAST program output divided by the total number of amino acids selected by the program for the alignment.
  • the active compound (AC) has a sequence identity of at least 70%, 71%, 72%, 73%, 74% 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with the respective sequence of the respective protein.
  • the Desmogleins are a group of proteins, which are incorporated in the cell membrane and which play a role as integrale membrane proteie in cell-cell contacts. In the desmoglein family, there are four different forms 1 , 2, 3 and 4 which are coded by different genes, which are all located on the human chromosome 18. In multilayered squamous epithelia mainly desmoglein-3 is present, desmoglein-4 in highly differentiated keratinocytes of hair follicles and desmoglein-1 in the upper layers of the epidermis.
  • Desmoglein-1 (homo sapiens) SEQ ID NO 4:
  • Desmocollins are calcium dependent glyco-proteins, which belong to the desmocollin sub family of the cadhereins. These are found together with desmogleinen in particular in epithelial cells, where those form the adhesive proteins of the cell-cell conncections of desmosoms and are necessary for cell adhesion and desmosome formation.
  • Desmocollin-1 (homo sapiens, isoform Dsda preproprotein), SEQ ID NO 7: MALASAAPGSIFCKQLLFSLLVLTLLCDACQKVYLRVPSHLQAETLVGKVNLEECLKSASLIRSSD PAFRILEDGSIYTTHDLILSSERKSFSIFLSDGQRREQQEIKVVLSARENKSPKKRHTKDTALKRS KRRWAPIPASLMENSLGPFPQHVQQIQSDAAQNYTIFYSISGPGVDKEPFNLFYIEKDTGDIFCT
  • Desmocollin-3 (homo sapiens, isoform Dsc3a preproprotein, SEQ ID NO 8:
  • LEKTI Lympho-epithelial kazal-type-related inhibitor
  • SPINK5 serin protease inhibitor kazal-type 5
  • LEKTI is a protein, which is coded in humans by the SPINK5-gene.
  • LEKTI is a serin protease inhibitor with several domains, which is expressed in the multilayered epithelial tissues. In the epidermis, LEKTI takes part in the regulation of desquamation by its ability to inhibit selectively KLK5, KLK7 and KLK14. Mutations in the SPINKS5-gene could result in the Netherton-syndrome, a disease which is characterized by ichthyosis, faulty cornification, and atopy.
  • LEKTI precursor homo sapiens
  • Des oso es are intercellular connections, which tightly connect neighbor cells. Desmoplaktin is an obligate ingredient of functional desmosomes, which anchors intermediate filaments at desmosomal plaques.
  • Desmoplaktin (homo sapiens), SEQ ID NO 10:
  • Periplaktin is coded in humans by the PPL- gene. It is an ingredient of desmosomes and of the epidermal cornified envelope of the keratinocytes.
  • the N-terminal domaine interacts with the plasma membraneand its C-terminus interacts with intermediate filaments. It forms complexes with envoplaktin by its rod-domaine.
  • Periplaktin may serve as link between the cornified envelope and desmosomes as well as intermediate filaments.
  • Envoplaktin interacts with periplaktin and is an ingredient of the epidermal cornified envelope.
  • Claudines are a group of proteins, which are the most important components of tight junctions. Thight junctions seal in the epithelia the space between the cells and thus protect the body against dehydration and outer influences.
  • Claudin 1 (homo sapiens) SEQ ID NO 13:
  • Occludin is a protein, which takes part, together with the claudin family, in the formation of the tight junctions which are present in epithelia cells.
  • Kallikreins are serine proteases, which take part in the degradation of desmosomal and corneodesmosal proteins. Thus, these proteins play an important role in desquamation of the skin cell.
  • Kallikrein-5 preprotein, homo sapiens
  • SEQ ID NO 15 SEQ ID NO 15:
  • Kallikrein-7 isoform 1, preprotein, homo sapiens
  • SEQ ID NO 16 SEQ ID NO 16
  • Kallikrein-14 preprotein, homo sapiens
  • Filaggrin is formed during cornification of the skin in the keratinocytes. Filaggrins help to cross link keratin filaments by disulfid bridges and thus have a structure forming function in the epidermis. Later the filaments are released and gradually metabolized into hygroscopic amino acids and derivatives of amino acids, which function as natural moisturizer of the skin.
  • Filaggrin (homo sapiens), SEQ ID NO 18:
  • Filaggrin 2 is expressed in the cornified cell cayers and colocalized with corneodesmosin, which plays a decisive role for the maintanance of cell. cell adhesion in this region of the epidermis. A lack of filaggrin 2 in the skin results in a significantly lowered corneodesmosin expression.
  • Filaggrin 2 (homo sapiens), SEQ ID NO 19:
  • Hyaluronic acid naturally is present in the human body and is an important component of different connective tissues and is able to bind large amounts of water.
  • hyaluronic acid is a polymer of a disaccharid based on D-glucoronic acid and N-acetyl-D-glucoseamine according to formula (II).
  • Hyaluronic acid may be used in form of its salts (for example as sodium and/or potassium salt).
  • derivatives of hyaluronic acid may be used, such as cross-linked hyaluronic acid. The preparation of cross-linked hyaluronic acid is known to the person skilled in the art 27 .
  • n is 1 to 1000, preferably 1 to 500, more preferably 1 to 300, most preferably 1 to 200.
  • the at least one active compound (AC) is a protein. Even more particularly preferred, the at least one active compound (AC) is corneodesmosin CDSN, optionally recombinant human corneodesmosin (rhCDSN).
  • the liposome comprises at least one vector peptide (VP).
  • the liposome comprises at least two different vector peptides, at least three different vector peptides, or at least four different vector peptides.
  • the at least one vector peptide (VP) comprises at least one vector sequence (VS): (GKKKK) n (SEQ ID NO: 1). Wherein n is an integer from 1 to 8, preferably 2 to 6, more preferably 3 to 5, most preferably 3.
  • the vector sequence (VS) preferably establishes the contact of the liposomes with the target cells.
  • the at least one vector peptide (VP) comprises at least one, preferably at least two acyl groups.
  • the acyl group is a group with the formula: R-C(O)-, wherein R is (C 4 - C 2 o) alkyl or a (C 4 -C 2 o) alkylene group, preferably a (C 4 -C 2 o) alkyl group.
  • R is (C 4 - C 2 o) alkyl or a (C 4 -C 2 o) alkylene group, preferably a (C 4 -C 2 o) alkyl group.
  • the at least one acyl group preferably helps to anchor the vector peptide in the liposome.
  • alkyl refers to a monoradical of a saturated straight or branched hydrocarbon.
  • the alkyl group comprises from 4 to 20 carbon atoms, i.e., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, or 20, carbon atoms more preferably 14 to 20, most preferably 15 to 18 carbon atoms, particular preferred 16 carbon atoms.
  • alkyl groups include butyl, iso-butyl, tert-butyl, n-pentyl, iso-pentyl, sec-pentyl, neo-pentyl, 1,2-dimethyl-propyl, iso-amyl, n- hexyl, iso-hexyl, sec-hexyl, n-heptyl, iso-heptyl, n-octyl, 2-ethyl-hexyl, n-nonyl, n-decyl, n- undecyl, n-dodecyl, hexadecyl and the like.
  • alkenylene refers to a diradical of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond.
  • the maximal number of carbon-carbon double bonds in the alkenylene group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkenylene group by 2 and, if the number of carbon atoms in the alkenylene group is uneven, rounding the result of the division down to the next integer.
  • the maximum number of carbon-carbon double bonds is 4.
  • the alkenylene group has 1 to 4, i.e. , 1 , 2, 3, or 4, carbon-carbon double bonds.
  • the alkenylene group comprises from 4 to 25 carbon atoms, i.e., 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20, carbon atoms, preferably 14 to 20, more preferably 15 to 17 carbon atoms.
  • the alkenylene group comprises from 14 to 20 carbon atoms and 1 , 2, 3, 4, or 5 carbon-carbon double bonds, more preferably it comprises 15 to 17 carbon atoms and 1, 2, 3, or 4 carbon- carbon double bonds.
  • the carbon-carbon double bond(s) may be in cis (Z) or trans (E) configuration.
  • Exemplary alkenylene groups include 1-buten-1 ,2-diyl, 1-buten-1 ,3-diyl, 1-buten- 1,4-diyl, 1-buten-2,3-diyl, 1-buten-2,4-diyl, 1-buten-3,4-diyl, 2-buten-1 ,2-diyl, 2-buten-1 ,3-diyl, 2- buten-1 ,4-diyl, 2-buten-2,3-diyl, 2-buten-2,4-diyl, 2-buten-3,4-diyl, and the like.
  • the vector peptide (VP) has a structure according to Formula (I): (VS)-(Z).
  • the vector sequence (VS) is defined as outlined above.
  • (Z) is a group which preferably mediates the connection of the vector peptide (VP) with the liposome.
  • (Z) may be basically any group which is suitable to connect the vector peptide (VP) with the liposomes.
  • (Z) is a group which may comprise a backbone structure which may be connected with the vector sequence (VS) and to which at least one lipophilic group is attached which can be incorporated in the lipid bilayer of the liposomes.
  • the lipophilic groups may be directly attached to the vector sequence (VS) without the use of a backbone structure.
  • the lipophilic group can be attached at the N-termial a-amino and /or amino group of side chains of suitable amino acids of the backbone structure.
  • These lipophilic groups comprise a least one acyl group as defined above.
  • (Z) may be selected from (acyl)-WK(acyl)-), or (acyl)-K(acyl)W-
  • Acyl is a group with the formula: R-C(O)-, wherein R is (C 4 -C 2 o) alkyl or a (C 4 -C 2 o) alkylene group, preferably a (C 4 -C 20 ) alkyl group, as also defined above.
  • (Z) may be selected from PE-PEG-S-Spacer-.
  • PE-PEG is 1,2- steaoryl-sn-glycero-3-phosphoethanolamine-N-[maleiimide(poly(ethylene glycol)-2000)].
  • the spacer is preferably -(CH 2 ) 2 -CO-WG-.
  • the vector peptide (VP) has the structure according to formula (III): .PE-PEG-S— CH 2 ) 2 -CO-WG-(VS). The preparation of compounds comprising PE-PEG-S-Spacer- has been disclosed in Sauer et. al, Biochemistry 2005, 44, 2021-2029.
  • (Z) is (acyl)-WK(acyl) (SEQ ID NO: 2).
  • the vector peptide (VP) has the sequence palmitoyl- WK(palmitoyl)GKKKKGKKKKGKKKK (SEQ ID NO: 20).
  • the vector peptide (VP) is a cationic lipopeptide, which integrates into the lipid bilayer of the liposomes and which preferably causes the interaction of the liposomes with the respective target cells, for example keratinocytes or and/or human epidermal equivalents (HEEs).
  • the inventive carrier system may accumulate in and/or at the outer cell membrane of the target cell, such as keratinocytes.
  • Accumulation in and/or at the outer cell membrane of the target cell within this invention means that preferably at least 80%, more preferably at least 90%, most preferably at least 95%, even more preferred at least 99% of the overall amount of the liposome-based carrier system which accumulates in and/or at the outer cell membrane of keratinocytes and in the inner cell of the keratinocytes beyond the outer cell membrane, accumulates in and/or at the outer cell membrane of the keratinocytes (mol/mol).
  • the amount of the carrier system which accumulates in and/or at the outer cell membrane is in contact with the target cell, such as the keratinocytes, at least over the vector peptide.
  • the molar ratio of the at least one lipid (L) to the sum of the least one active compound (AC) and the at least one vector peptide (VP) is preferably 3000:1 to 20:1, more preferably 2500:1 to 1000:1, most preferably 2000:1 to 1000:1. Moreover, the molar ratio of the at least one lipid (L) to the sum of the least one active compound (AC) and the at least one vector peptide (VP) may be 100:1 to 5:1 , preferably 50:1 to 10:1, more preferably 20: 1 to15:1.
  • the molar ratio of the at least one vector peptide to the at least one lipid (L) is preferably 0.05% to 5.5%, more preferably 0.08 to 3%, most preferably 0.09 to 2%, particularly preferred 0.09 to 1%, more particularly preferred 0.09 to 0.5%.
  • the concentration of the at least one lipid (L) in the liposome-based carrier system may be 40 to 5000 pmol/L, more preferably 50 to 2500 pmol/L more preferably 50 to 2000 pmol/L, most preferably 50 to 1000 pmol/L
  • the overall concentration of the at least one vector peptide (VP) and the at least one protein in the liposome-based carrier system may be 0.045 to 10 pmol/L, preferably 1 to 5 pmol/L, more preferably 1 to 2 pmol/L.
  • the liposomes have an average diameter of 65 to 1000 nm, preferably 70 to 1000 nm more preferably 75 to 1000 nm, most preferably 80 to 1000 nm.
  • the liposomes may also have an average diameter of 65 to 550 nm, preferably 70 to 550 nm, more preferably 75 to 550 nm, most preferably 80 to 550 nm.
  • the diameter values are average mean size values based on the intensity weighted distribution (z-average) determined by dynamic light scattering (DLS).
  • the liposome-based carrier system may further comprise a polar solvent.
  • the solvent is water.
  • the liposome-based carrier system basically may be used in any suitable buffer system known to the person skilled in the art.
  • the buffer system is a physiologically acceptable buffer system which is suitable for the respective active compound (AC), in particular if the active compound (AC) is a protein.
  • the pH of the liposome-based carrier system is in the range of 6.5 to 8.5, more preferably in the range of 6.8 to 7.8, most preferably in the range of 7 to 7.5, particular preferred 7.35 to 7.45.
  • the pH may be adjusted with an acid or a base, such as HCI or NaOH.
  • the pH may be kept constant by using a physiologically acceptable buffer system. Examples for suitable buffer systems are NaH 2 P0 4 /Na 2 HP0 4 , and Dulbecco’s phosphate-buffered saline.
  • NaCI is present in a concentration of of 100 to 400, preferably 120 to 300, more preferably 125 to 138, most preferably 137 mmol/L.
  • NaCI is present in a concentration of 0.8 to 1%, preferably in a concentration of 0.85 to 9.5, more preferably in a concentration of 0.9%.
  • a suitable medium for the liposome-based carrier system based on water as solvent may be:
  • KH 2 P0 4 0.5 to 2.5, preferably 1 to 2, more preferably 1.3 to 1.9, most preferably 1.8 mmol/L KH 2 P04.
  • all components NaCI, KCI, Na 2 HP0 4 and KH 2 P0 4 are present.
  • the present invention is directed to a liposome-based carrier system, wherein the liposomes comprise: a) at least one lipid (L), b) at least one active compound (AC) and c) at least one vector peptide (VP) wherein the at least one active compound (AC) is corneodesmosin CDSN and the at least one vector peptide (VP) comprises a) at least one vector sequence (VS): (GKKKK) n (SEQ ID NO: 1)., wherein n is an integer from 1 to 8 preferably 2 to 6, more preferably 3 to 5, most preferably 3 and b) at least one, preferably at least two acyl groups, wherein the acyl group is a group with the formula: R-C(O)-, wherein R is (C 4 -C 20 ) alkyl or a (C 4 -C 20 ) alkylene group, preferably a (C 4 -C 20 ) alkyl group; and wherein the lipo
  • the liposome-based carrier system disclosed in the previous paragraph is for use in medicine.
  • the liposome-based carrier system in this embodiment is for use in treating or preventing of peeling skin syndromes (PSS), the Netherton Syndrome, SAM Syndrome, psoriasis vulgaris, atopic dermatitis, impetigo, microbial eczema, microbial eczema associated with dermatomycosis, NISCH Syndrome, androgenetic alopecia, alopecia areata, bullous immunodermatoses, and epidermolysis bullosa, skin tightening, and dry skin conditions.
  • PSS peeling skin syndromes
  • SAM Syndrome the Netherton Syndrome
  • psoriasis vulgaris psoriasis vulgaris
  • atopic dermatitis impetigo
  • microbial eczema microbial eczema associated with dermatomycosis
  • NISCH Syndrome NISCH Syndrome
  • the invention is directed to a liposome-based carrier system, wherein the liposomes comprise: a) at least one lipid (L), b) at least one active compound (AC) and c) at least one vector peptide (VP), wherein the at least one vector peptide (VP) i comprises a) at least one vector sequence (VS): (GKKKK) n (SEQ ID NO: 1)., wherein n is an integer from 1 to 8, preferably 2 to 5, more preferably 3 to 6, most preferably 3 and b) at least one, preferably at least two acyl groups, wherein the acyl group is a group with the formula: R-C(O)-, wherein R is (C 4 -C 2 o) alkyl or a (C 4 -C 2 o) alkylene group, preferably a (C 4 -C 20 ) alkyl group; and wherein the liposomes have an average diameter of 65 to 1000 nm, for
  • the liposome-based carrier system in this embodiment is for use in treating or preventing of peeling skin syndromes (PSS), the Netherton Syndrome, SAM Syndrome, psoriasis vulgaris, atopic dermatitis, impetigo, microbial eczema, microbial eczema associated with dermatomycosis, NISCH Syndrome, androgenetic alopecia, alopecia areata, bullous immunodermatoses, and epidermolysis bullosa, skin tightening, and dry skin conditions.
  • PSS peeling skin syndromes
  • Another embodiment of the invention relates to a method comprising delivering at least one active compound (AC) by an liposome-based carrier system ex vivo, wherein the liposomes comprise: a) at least one lipid (L), b) at least one active compound (AC) and c) at least one vector peptide (VP), wherein the at least one vector peptide (VP) comprises a) at least one vector sector sequence (VS): (GKKKK) n (SEQ ID NO: 1)., wherein n is an integer from 1 to 8, preferably 2 to 6, more preferably 3 to 5, most preferably 3 and b) at least one, preferably at least two acyl groups, wherein the acyl group is a group with the formula: R-C(O)-, wherein R is (C 4 -C 20 ) alkyl or a (C 4 -C 20 ) alkylene group, preferably a (C 4 -C 20 ) alkyl group; wherein the liposomes have
  • the invention relates to a method comprising delivering at least one active compound (AC) by an liposome-based carrier system ex vivo, to the outer cell membrane of keratinocytes and/or human epidermal equivalents (HEEs) wherein the liposomes comprise: a) at least one lipid (L), b) at least one active compound (AC) and c) at least one vector peptide (VP), wherein the at least one vector peptide (VP) comprises a) at least one vector sector sequence (VS): (GKKKK) n (SEQ ID NO: 1)., wherein n is an integer from 1 to 8, preferably 2 to 6, more preferably 3 to 5, most preferably 3 and b) at least one, preferably at least two acyl groups, wherein the acyl group is a group with the formula: R-C(O)-, wherein R is (C 4 -C 2 o) alkyl or a (C 4 -C 2 o)
  • the present invention is directed to a pharmaceutical composition
  • a pharmaceutical composition comprising the liposome-based carrier system as described above, and at least one pharmaceutically acceptable carrier.
  • “Pharmaceutical composition” refers to one or more active ingredients, and one or more inert ingredients that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions of the present invention encompass any composition made by admixing the liposome-based carrier system of the present invention and a pharmaceutically acceptable carrier.
  • One preferred embodiment of the pharmaceutical composition is an oil in water formulation and/or the administration in form of a gel.
  • Carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, including but not limited to peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier.
  • Saline and aqueous dextrose are preferred carriers when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions are preferably employed as liquid carriers for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations and the like.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
  • Such compositions will contain a therapeutically effective amount of the items therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration.
  • the invention comprises the following items:
  • a liposome-based carrier system wherein the liposomes comprise: a) at least one lipid (L), b) at least one active compound (AC) and c) at least one vector peptide (VP); wherein the at least one active compound (AC) is selected from the group consisting of at least one protein with a sequence identity of at least 70% of one of the following human corneodesmosin (SEQ ID NO. 2), rhCDSN (SEQ ID NO. 3), desmoglein 1 (SEQ ID NO.
  • the at least one vector peptide (VP) comprises a) at least one vector sequence (VS): (GKKKK) n (SEQ ID NO: 1)
  • a liposome-based carrier system wherein the liposomes comprise: a) at least one lipid (L), b) at least one active compound (AC) and c) at least one vector peptide (VP), wherein the at least one vector peptide (VP) comprises a) at least one vector sequence (VS): (GKKKK) n (SEQ ID NO: 1), wherein n is an integer from 1 to 8, preferably 2 to 6, more preferably 3 to 5, most preferably 3 and b) at least one acyl group, wherein the acyl group is a group with the formula: R-C(O)-, wherein R is (C 4 -C 20 ) alkyl or a (C 4 -C 20 ) alkylene group; and wherein the liposomes have an average diameter of 65 to 1000 nm, for use in a method of treatment or prevention which comprises delivering of at least one active compound (AC) by the liposome-based carrier system, and wherein the liposome-based carrier system accumulate
  • a method comprising delivering at least one active compound (AC) by a liposome-based carrier system ex vivo, wherein the liposomes comprise: a) at least one lipid (L), b) at least one active compound (AC) and c) at least one vector peptide (VP), wherein the at least one vector peptide (VP) comprises a) at least one vector sequence (VS): (GKKKK) n (SEQ ID NO: 1)., wherein n is an integer from 1 to 8, preferably 2 to 6, more preferably 3 to 5, most preferably 3 and b) at least one, preferably at least two acyl groups, wherein the acyl group is a group with the formula: R-C(O)-, wherein R is (C 4 -C 2 o) alkyl or a (C 4 -C 2 o) alkylene group, preferably a (C 4 -C 20 ) alkyl group; wherein the liposomes have an average diameter of 65 to 1000 nm
  • the liposome-based carrier system according to item 1 or item 2 for use in treating or preventing of peeling skin syndromes (PSS), the Netherton Syndrome, SAM Syndrome psoriasis vulgaris, atopic dermatitis, impetigo, microbial eczema, microbial eczema associated with dermatomycosis, NISCH Syndrome, androgenetic alopecia, alopecia areata, bullous immunodermatoses, and epidermolysis bullosa, skin tightening, and dry skin conditions.
  • PSS peeling skin syndromes
  • the liposome-based carrier system according to item 1 to 6 or the method of item 3 and 4, wherein i) the active compound (AC) is recombinant human corneodesmosin (rhCDSN) and/or ii) the vector peptide (VP) is a peptide according to Formula (I): (VS)-(Z), wherein the vector sequence (VS) is (GKKKK) n (SEQ ID NO: 1)., wherein n is an integer from 1 to 8, preferably 2 to 6, more preferably 3 to 5, most preferably 3 and
  • (Z) is a) (acyl)-WK(acyl)- or (acyl)-K(acyl)W- or b) PE-PEG-S-Spacer wherein PE-PEG is 1 ,2-steaoryl-sn-glycero-3-phosphoethanolamine-N-
  • the spacer is preferably -CH 2 )2-CO-WG-, more preferably the vector peptide (VP) is a peptid with the sequence palmitoyl- WK(palmitoyl)GKKKKGKKKKGKKKK (SEQ ID NO: 20).
  • the at least one active substance is selected from the group consisting of at least one protein and hyaluronic acid and derivatives thereof, preferably the active substance is selected from the group consisting of at least one protein with a sequence identity of at least 70% of one of the following: human corneodesmosin (SEQ ID NO. 2), rhCDSN (SEQ ID NO. 3), desmoglein 1 (SEQ ID NO.
  • the at least one lipid (L) is a phospholipid preferably a phosphatidylcholin or a zwitterionic phospholipid more preferably a phosphatidylcholine or eggPC.
  • the liposome-based carrier system according to any one of the items 1 to 9 wherein at least 90% of the liposomes, preferably 95%, more preferably 99% of the liposomes are unilamellar vesicles.
  • the liposomes have an average diameter of i) 70 to 1000 nm, more preferably 75 to 500 nm or ii) 65 to 550 nm, preferably 70 to 550 nm, more preferably 75 to 1000 nm and/or
  • the concentration of the at least one lipid (L) in the liposome-based carrier system may be 40 to 5000 pmol/L, more preferably 50 to 2500 pmol/L, most preferably 50 to 2000 pmol/L and/or
  • the overall concentration of the at least one vector peptide (VP) and the at least one protein in the liposome-based carrier system is 0.045 to 10 pmol/L, preferably 1 to 5 pmol/L, more preferably 1 to 2 pmol/L.
  • the liposome-based carrier system according to any one of the items 1 to 11 , wherein the molar ratio of the at least one lipid (L) to the sum of the at least one active compound (AC) and the at least one vector peptide (VP) is 3000:1 to 20:1, preferably 2500:1 to 1000:1 , most preferably 2000:1 to 1000:1. 13.
  • the liposome-based carrier system according to any one of the items 1 to 11 wherein the molar ratio of the at least one lipid (L) to the sum of the at least one active compound (AC) and the at least one vector peptide (VP) is 100:1 to 5:1, preferably 50:1 to 10:1 , more preferably
  • the liposome-based carrier system according to any one of items 2 and 4 to 13 or the method according to any one of items 3 to 13, wherein at least 80%, preferably at least 90%, more preferably at least 95%, most preferably at least 99% of the overall amount of the liposome-based carrier system which accumulates in and/or at the outer cell membrane of keratinocytes and in the inner cell of the keratinocytes beyond the outer cell membrane, accumulates in and/or at the outer cell membrane of the keratinocytes (mol/mol).
  • a pharmaceutical composition comprising the liposome-based carrier system according to any one of the items 1 to 2, and 10 to 14 and at least one pharmaceutically acceptable carrier.
  • the CDSNcoding sequence was amplified with specific CDSN primer using Phusion® High Fidelity DNA polymerase and was cloned into the expression plasmid pASK-IBA45+ (I BA Lifescience, Germany, Goettingen) between the Xbal and Ncol restriction sites via the In- Fusion® HD Cloning kit from Clontech Laboratories, Inc. (Mountain View, USA) according to the manufacturer’s instructions. Successful cloning was checked by an analytical agarose gel and sequence analysis.
  • the plasmid pASK-IBA45+-CDSNA33 encodes CDSN amino acids 34-529 fused to a C-terminal located histidine-tag enabling purification.
  • a construct for a CDSNA33 fusion protein containing the sequence for CopGFP between the gene and the histidine-tag, was generated in a likewise manner.
  • BL21-Gold(DE3) competent E. coli cells were transformed with the expression plasmids and cultured in LB-medium supplemented with ampicillin at 37 °C under constant agitation. Protein expression was induced at an optical density of 0.5 (OD600) with 100 pg anhydrotetracycline (AHT) for 18 h.
  • rhCDSN was purified by FPLC using an NGC QuestTM Chromatography System (BioRad, Hercules, California) and Bio-Scale Mini Profinity IMAC Cartridges (BioRad,).
  • the cartridge was washed with 50 ml 50 mM NaH2P04, 300 mM NaCI, 20 mM imidazole, 0.5% Triton X- 100, pH 8.0 supplemented with protease inhibitor cocktail p8849.
  • an imidazole gradient with a final concentration of 500 mM were performed. All elution fractions were collected98 with a BioFracTM Fraction Collector (BioRad).
  • a buffer exchange was performed to remove the imidazole.
  • fractions with the highest concentrations were pooled and dialyzed in two steps against a 50 mM phosphate buffer (50 mM phosphate buffer, 150 mM NaCI, pH7.4) via Slide-A-LyzerTM Dialysis Cassettes, 20K MWCO (Thermo Scientific, Waltham, Massachusetts).
  • the purified protein samples were analyzed by a 12.5% SDS-PAGE under reducing conditions, followed by Coomassie staining and immunoblotting.
  • immunoblotting proteins were transferred to a polyvinylidene fluoride (PVDF) membrane via semi-dry blotting method.
  • PVDF polyvinylidene fluoride
  • BSA bovine serum albumin
  • TBS tris-buffered saline
  • immunoblotting was performed with the specific anti-CDSN antibody P-20 (SantaCruz, sc-49620)(1:5000 in TBS supplemented with 0.1% Tween-20 and 1% BSA) and an alkaline phosphatase (AP) conjugated secondary IgG antibody.
  • NBT nitro blue tetrazolium chloride
  • BCIP 5- bromo-4-chloro-3-indolyl-phosphate
  • Trypsin activity was stopped by addition of medium containing fetal calf serum (FCS) and cells were resuspended in keratinocyte serum-free medium (KSFM) supplemented with 10 ng/ml epidermal growth factor (EGF), 50 mg/ml bovine pituitary extract (BPE) (all from Invitrogen, Darmstadt, Germany), 100 U/ml penicillin, and 100 mg/ml streptomycin (PAA, Pasching, Austria). Keratinocytes were incubated at 37 °C, 5% C0 2 and 95% humidity.
  • FCS fetal calf serum
  • BPE bovine pituitary extract
  • HOEs human epidermal equivalents
  • KSFM medium supplemented with 10 ng/ml EGF, 50 mg/ml BPE (all from Invitrogen, Darmstadt, Germany) without antibiotics until they reached a confluence of 70-80%.
  • the cells were harvested and resuspended in 70% KSFM and 30% CnT-Prime 3D Barrier medium (CellnTec, Bern, Switzerland).
  • the preparation of small unilamellar vesicles (SUVs) and large unilamellar vesicles (LUVs) was performed as described before 5 .
  • the lipid 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) (Avanti Polar Lipids, Alabaster, USA) was dried under high vacuum and the film was rehydrated by vortexing in 50 mM phosphate buffer (50 mM phosphate buffer, 150 mM NaCI pH 7.4) with or without protein.
  • POPC lipid 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine
  • rhCDSN as well as rhCDSN:GFP a concentration of 500 pg/ml was used.
  • the lipid concentration of the stock preparation was 20 mM, if not otherwise stated.
  • SUVs (average diameter 28 nm) were generated by sonication of the lipid suspension for 25 min using a titanium tip ultra sonicator.
  • LUVs were prepared by 35 extrusion steps through two polycarbonate filters with a pore size of 100 nm or 200 nm using a MiniExtruder (Avestin, Ottawa, Canada).
  • the cationic lipopeptide P2K12 (SEQ ID NO. 20) dissolved in buffer was added to the liposomal suspension to modify the surface of the vesicles. This dipalmitoylated lysine- rich lipopeptide spontaneously incorporates into the outer lipid layer of liposomes 5 .
  • /p ) of the SUV preparations was 20/1, for LUVs of a diameter of about 100 nm or 200 nm the ratio was 1000/1.
  • the liposomal size distribution was analyzed by dynamic light scattering (DLS) on a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). To check the stability of the preparations the change in the size was analyzed at different temperatures (4 °C, room temperature, 37°C) and over a period of one month.
  • Keratinocytes were seeded onto poly-L-lysine coated coverslips placed in a 35 mm petri dish in a density of 5 x 10 4 cells per well in KSFM medium supplemented with 10 ng/ml EGF, 50 mg/ml BPE (all from Invitrogen, Darmstadt, Germany). The cells were incubated for 2 days at 37 °C and 5% C0 2 in humidified conditions. Keratinocyte differentiation was performed by incubation for 48 h with medium supplemented with 1.5 mM CaCI 2 .
  • Carboxyfluorescein and rhodamine were excited with a 200 mW argon laser at 488 nm and a 15 mW HeNe laser at 543 nm, respectively.
  • CellMaskTM Deep Red plasma membrane stain was excited at 633 nm with a HeNe laser. Pictures were taken with a BP505-530 bandpass filter and a LP650 cutoff filter, respectively. [00111] Cell viability assay
  • Cationic peptides might be highly toxic. Therefore, the cytotoxicity of the P2K12-decorated liposomes was measured using the neutral red uptake assay 27 .
  • keratinocytes were seeded with a density of 2 x 10 4 cells per well 48 h prior to the experiment into a 96 well plate and were cultivated at 37 °C and 5% C0 2 . The medium was removed, and cells were exposed to P2K12 associated liposomes for 1 h at increasing concentrations (lipid concentration ranged from 50 mM to 2000 mM, peptide concentration ranged from 0.05 pM to 2 pM).
  • HBSS Hank's balanced salt solution
  • DMEM Dulbecco ' s modified Eagle Medium
  • Topical toluidine blue penetration assay on human epidermal equivalents To determine the integrity of the outside-in skin barrier in HEEs, an existing test for mouse models based on toluidine blue was modified for our specific use on in-vitro models. For this purpose, the HEEs were first topically washed with 300 pi dPBS. The PBS was removed and the equivalents were fixed using a methanol series (25% MetOH, 50% MetOH, 75% MetOH, 100% MetOH, 75% MetOH, 50% MetOH, 25% MetOH, each 300 pi for 1 min) and washed again with 300 pi dPBS.
  • Filaggrin antibody were marked with polyclonal goat anti mouse, and CDSN as well as claudin 1 with polyclonal goat anti rabbit immmunoglobulins/biotinylated antibodies (DAKO Cytomation) 1:100 and detected by dichlorotriazinylaminofluorescein (DTAF) conjugated Streptavidin antibodies (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) 1:100 for 30 min. Fluorescence microscopy was performed with an Axioskop 2 microscope. Images were taken with an AxioCam HR camera and Axiovision 4.8.2 software (Carl Zeiss, Jena, Germany).
  • Transglutaminase 1 is a key enzyme involved in the assembly of the cornified cell envelope which is essential for the barrier function of the epidermis.
  • TG1 activity assay was performed as described before 26 . Briefly, unfixed cryosections were saturated for 30 min with 1% BSA in PBS. Subsequently the sections were incubated for 90 min with 0.1 mM TG1 substrate biotinyl cadaverine (Molecular Probes, Leiden, Netherlands). In the presence of calcium ions (5 mM) the substrate will be incorporated by TG1 into the cornified cell envelope under physiological conditions (100 mM Tris/HCI pH 7.4). Incubation with 5 mM EDTA served as negative control (data not shown). Afterwards sections were incubated for 30 min with (DTAF) streptavidin antibody as described above.
  • Example 1 Recombinant expression and purification of human CDSN The successful cloning of the CDSN expression constructs pASK- ⁇ BA45+-CDSNA33 and pASK-IBA45+-CDSNA33-CopGFP was verified by DNA sequencing (data not shown). Both constructs were expressed in E. coli and purified by Ni-charged immobilized metal affinity chromatography. The protein purification and the buffer exchange were analyzed by SDS-PAGE and immunoblotting with a CDSN specific antibody.
  • CDSNA33-His results in a protein with a molecular mass of approximately 55 kDa ( Figure 15), whereas the CDSNA33- GFP-His fusion protein has a molecular mass of 75 kDa (not shown).
  • Example 2 Development of a liposome-based delivery system for recombinant human CDSN
  • the uppermost layer of the skin represents a protective barrier against many factors including chemical and biological substances. Therefore, the application of substances requires specific delivery systems to reach their specific target sites.
  • ligand-tagged liposomes associated with the peptide P2K12 which mediates accumulation at the cell membrane, were used to transport rhCDSN.
  • Example 3 Carrier characteristics and toxicity
  • LUVs were stored at different temperatures (4 °C, 25 °C and 37 °C) for up to 28 days ( Figure 1C and 1D). Immediately after preparation, on the 3rd day and then every week, the liposome size distribution and the vesicle diameter were checked by DLS. At all storage temperatures, the vesicle diameter slightly decreased from the day of preparation to day 3 but remained constant from day 3 until day 28. The preparations are stable over weeks.
  • Example 4 Liposomal localization on primary keratinocytes
  • FIG. 2A shows that the micelle forming lipopeptide is located at the plasma membrane of keratinocytes while LUVs without peptide are found neither at the plasma membrane nor inside the keratinocytes ( Figure 2B). Based on this result the localization of P2K12 associated SUVs and LUVs was investigated. Interestingly, SUVs are strongly internalized into the keratinocytes ( Figure 2C) whereas empty and rhCDSN loaded P2K12-modified LUVs are localized at the plasma membrane ( Figure 2D and 2E).
  • the keratinocytes undergo a special differentiation process that results in a changed structure and altered protein composition.
  • CDSN is synthesized in the stratum granulosum and its target site is the transition area of the stratum granulosum to the stratum corneum. Since keratinocytes are differentiated in these layers the localization studies were also carried out with differentiated keratinocytes to confirm the binding to the plasma membrane after this process.
  • Figures 10 to 14 show that liposomes with an average size of 28 nm enter in the inner part of the cell of keratinocytes and accumulate in the cell plasma, while liposomes with an average size of 73 nm, 89 nm, 106 nm, and 193 nm do not accumulate in the cell plasma, but at and/or in the cell membrane.
  • Example 5 In vitro penetration studies The penetration of a substance or a carrier system into the skin is of central importance for the development of a topical therapy. Since the site of action of CDSN is the stratum corneum or the upper stratum granulosum, HEEs were used in this study to investigate the penetration properties of the P2K12 modified LUVs. HEEs were treated two times for 3 h (day 3 and 5) with empty as well as with rhCDSN:GFP loaded P2K12 associated LUVs. For P2K12 associated LUVs we found a strong and continuous accumulation at the keratinocytes of the stratum corneum and a slightly pericellular accumulation at the upper stratum granulosum (Figure 5A). The studies with the encapsulated rhCDSN:GFP also showed an accumulation in these layers, but the distribution of the protein was more scattered and not as homogenous (Figure 5B).
  • Comparative Example 5a In vitro penetration studies with brain endothelia cells and aortic endothelial cells
  • Example 6 Functional analysis of liposomal encapsulated recombinant human CDSN
  • CDSN deficient HEEs were treated twice, as described above, with P2K12 associated LUVs loaded with rhCDSN (15 pg protein/1.13 cm 2 ). While untreated CDSN deficient HEEs were completely negative for CDSN in immunofluorescence (Figure 6B), the treated HEEs show a distinct CDSN staining in the area of the upper stratum granulosum ( Figure 6C). However, the staining was weaker and not as pericellular as in normal (CDSN+/+) HEEs ( Figure 6A).
  • CDSN deficient HEEs that were treated with CDSN LUVs show thin, but continuous structures of stratum corneum (Figure 7C) comparable to positive control HEEs which were generated with normal (CDSN+/+) keratinocytes and showed a well-structured epidermal layer ( Figure 7A).
  • Figure 7B In untreated CDSN deficient HEEs the stratum corneum is either absent or detached ( Figure 7B).
  • CDSN deficient HEEs treated with empty P2K12 associated LUVs Figure 7D
  • treated with PBS Figure 7E
  • Topical enzyme-replacement therapy restores transglutaminase 1 activity and corrects architecture of transglutaminase-1-deficient skin grafts.
  • Corneodesmosomes and corneodesmosin From the stratum corneum cohesion to the pathophysiology of genodermatoses. Eur. J. Dermatology 21, 35-42.
  • Corneodesmosin a component of epidermal corneocyte desmosomes, displays homophilic adhesive properties. J. Biol. Chem. 277, 5024-5029.

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Abstract

La présente invention concerne un système de vecteur à base de liposomes dans lequel les liposomes comprennent au moins un lipide (L), au moins un composé actif (AC) et au moins un peptide vecteur (VP). Ledit au moins un peptide vecteur (VP) comprend au moins une séquence vectorielle (VS) : (GKKKK)n (SEQ ID NO : 1), dans laquelle n est un nombre entier compris entre 1 et 8 et au moins un groupe acyle, le groupe acyle étant un groupe répondant à la formule : R-C(O)-, dans laquelle R représente un alkyle en (C4-C20) ou un groupe alkylène en (C4-C20). Les liposomes ont un diamètre moyen situé dans la plage allant de 65 à 1 000 nm. En outre, la présente invention concerne l'utilisation du système de vecteur à base de liposomes en vue d'une utilisation en médecine, en particulier en vue d'une utilisation dans le traitement ou la prévention de syndromes cutanés de pelage (PSS), la maladie de Netherton, le syndrome de SAM, le psoriasis vulgaire, l'eczéma, l'impétigo, l'eczéma microbien, l'eczéma microbien associé à la dermatomycoses, le syndrome de NISCH, l'alopécie androgénétique, l'alopécie en aires, les immunodermatoses bulleuses et l'épidermolyse bulleuse, le raffermissement de la peau et les affections cutanées sèches. De plus, l'invention concerne des compositions pharmaceutiques comprenant le système de vecteur à base de liposomes.
PCT/EP2020/076395 2019-09-23 2020-09-22 Système de liposomes comprenant un lipide, un composé actif et un peptide vecteur WO2021058463A1 (fr)

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US20150140066A1 (en) * 2012-03-21 2015-05-21 National University Corp. Hokkaido University Carrier for intracellular delivery of functional protein

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