Peptide-ionic liquid conjugates for prevention and / or treatment of skin conditions

Abstract

The present application relates to a peptide-ionic liquid conjugate for the prevention and / or treatment of skin conditions. The peptide-ionic liquid conjugate comprises a pharmaceutical cosmetic peptide containing up to 10 amino acids and at least one ionic liquid containing a C1 to C18 saturated or unsaturated hydrocarbon chain substituent. The peptide-ionic liquid conjugates disclosed herein are useful for combating skin conditions, such as melanoma, non-melanoma skin cancer, diabetic foot ulcer, lower limb vein ulcer, stress ulcer, acne, candidiasis, cellulitis, dermatophytosis, erysipelas, folliculitis, impetigo, psoriasis, rosacea or eczema (atopic dermatitis), and the like. As they exhibit potent activity, bactericidal effect, anti-inflammatory and immunomodulatory effect and collagen production inducing effect on antibiotic sensitive strains or gram-positive and gram-negative multi-drug resistant clinical isolates. The present application further discloses topical compositions comprising the peptide-ionic liquid conjugates and uses thereof.

Description

Technical Field

[0001] The present application relates to a peptide-ionic liquid (PIL) conjugate for preventing and / or treating skin disorders, a topical composition comprising the conjugate and uses thereof. Background Art

[0002] Although ionic liquids (ILs) are primarily known for their potential role as “green solvents”, their appeal as organic salts that can be easily customized and easily tuned for a variety of specific purposes (task-specific ILs) is growing. When organic cations are combined with organic or inorganic anions, endless possibilities exist, allowing the production of ILs with different structural, physical, and chemical properties [1], which can be adapted to the needs of a variety of different fields such as materials science [2], biotechnology [3] or biomedicine [4]. Moreover, by utilizing bioactive ions, ILs with relevant biological activities can be produced [5], such as ILs as anticancer agents [6], antimalarial agents [7] and antimicrobial agents [8]. ILs showing broad-spectrum activity against both Gram-negative and Gram-positive bacteria as well as antibiofilm activity have been reported [9]; therefore, ILs are becoming an attractive option to combat antimicrobial resistance, while the world's supply of effective antibiotics, especially against Gram-negative bacteria, is gradually being depleted. In addition, many ILs have also received attention as skin penetration enhancers

[10] , making them particularly attractive for topical applications. ILs that exhibit antimicrobial activity and skin penetration enhancement could be very useful in treating skin conditions, including infectious lesions, as recently demonstrated in an in vivo biofilm-infected wound assay, where ILs were able to kill 95% of bacteria

[11] .

[0003] As life expectancy increases, the prevalence of diabetes, peripheral vascular disease, or conditions that confine the patient to bed is increasing and is often associated with problems such as diabetic foot ulcers (DFUs) and other skin conditions such as complicated skin and soft tissue infections (cSSTIs)

[12] . Although cSSTIs are mainly caused by multidrug-resistant (MDR) bacteria from the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) group, a high incidence of fungi has also been reported in chronic wounds and is associated with both healing time and the formation of mixed biofilms with bacteria; in cSSTIs, Cladosporidium spp. and Candida spp., mainly C. albicans and C. parapsilosis, are described as the most common fungi [13,14]. During the COVID-19 pandemic, the burden of cSSTIs has increased due to reluctance to expose elderly patients to healthcare facilities

[15] and is exacerbated by (i) the spread of MDR pathogens and (ii) the formation of polymicrobial biofilms that are refractory to current antibiotics and the immune system and delay or impair healing.

[0004] In selected cases of cSSTI, treatment requires debridement or incision and drainage, supplemented by antibiotic therapy. Guidelines for the treatment of cSSTI recommend the administration of systemic antibiotics effective against methicillin-resistant Staphylococcus aureus (MRSA) strains, which are among the MDR pathogens prevalent in healthcare settings, followed by a culture-based antibiotic regimen for definitive treatment.

[16] When this regimen fails, amputation becomes the ultimate means of avoiding life-threatening sepsis.

[0005] Resistance to existing antibiotics is increasing rapidly and is now widespread among many different species of Gram-positive and Gram-negative bacteria. Similarly, fungal infections are becoming increasingly difficult to treat due to the development and spread of resistance among pathogenic fungi.

[0006] Peptide-based antimicrobial agents, such as the cyclic lipopeptide daptomycin, offer clinicians an alternative to combat MDR bacteria in the hospital setting, but daptomycin is active only against Gram-positive species, including MRSA (17). However, the polymicrobial nature of most cSSTIs, also involving Gram-negative bacteria and fungi, means that new options are urgently needed to address the emerging post-antibiotic era (18). Although fungal colonization of nonhealing wounds has historically been overlooked (14), Kalan et al. highlighted the importance of identifying not only the bacterial species involved in cSSTIs, but also the fungal species involved (14), namely Cladosporium species and Candida species, including Candida albicans and Candida parapsilosis (19).

[0007] The healing process in skin conditions is often delayed or even impaired due to age- and / or disease-related pathophysiological traits such as high oxidative stress, neuropathy, vasculopathy, poor tissue oxygenation, chronic inflammation, and poor immune response; this is further exacerbated by the presence of microbial pathogens that rapidly form biofilms in the wound bed and are refractory to most existing antibiotics (20). In this context, effective treatment of skin injuries must most often both inhibit infection and promote rapid and proper healing. In this regard, collagen, as a structural protein from the extracellular matrix (ECM), plays an important role in the critical steps of wound healing and closure (21). As collagen is an endogenous protein, it is considered an ideal component for the development of biocompatible and biodegradable wound dressings / biomaterials (22). In addition to collagen itself, collagen-derived / inspired peptides, such as latent collagen peptides (23), are also considered as potential promoters of cell migration and proliferation, inducing fibroblasts to produce new collagen, thereby promoting faster wound healing (24). This has led to the development of so-called cosmeceutical peptides (CPs) and their derivatives for topical application in the treatment of skin disorders, acting by promoting faster skin regeneration. CPs are small peptide fragments produced by proteolysis of ECM macromolecules such as collagen or elastin, and have a variety of potential biomedical applications, including in the cosmetic field (25,26). For example, "pentapeptide-4" (or PP4, amino acid sequence KTTKS) is a widely studied CP that is derived from type I human collagen and is the smallest peptide sequence known to retain a strong ability to stimulate the production of ECM (collagen and fibronectin) (27,28). The N-palmitoylated form of KTTKS, called "palmitoyl pentapeptide-4" or It is used in the cosmetics industry for its ability to produce skin rejuvenation / anti-wrinkle effects, which may be related to its collagen induction effect (29,30).

[0008] It is noteworthy that, as mentioned above, the rational selection of the peptide and ionic liquid building blocks to be conjugated can successfully develop a range of products tailored to their intended specific topical uses, which range from severe non-healing skin ulcers (diabetic foot ulcers, pressure ulcers, leg venous ulcers, etc.) to generally milder bacterial or fungal skin infections (acne, atopic dermatitis, candidiasis, cellulitis, dermatophytosis, erysipelas, folliculitis, impetigo, rosacea, etc.), as well as postoperative wounds, general injuries or burns. In addition, considering that peptide-based antimicrobial agents have been widely associated with other activities such as immunomodulatory, antioxidant, anti-aging and anti-tumor effects, our products may also be applied to the topical treatment of autoimmune / inflammatory, aging and cancerous skin conditions such as psoriasis, atopic dermatitis, cutaneous lupus erythematosus, impetigo, skin aging and melanoma, regardless of the presence of secondary infection.

[0009] Recently, the potential of PP4 to promote skin regeneration in the setting of skin damage has attracted attention. Therefore, given the lack of antimicrobial activity of this CP, when the KTTKS sequence was conjugated to an antimicrobial peptide (AMP), a chimeric peptide was generated that exhibited (i) antibacterial activity against reference bacteria and MDR bacteria from clinical isolates; (ii) antibiofilm effects; and (iii) Significant induction of collagen formation (32).

[0010] Further N-terminal modification of the chimeric peptides described above with imidazolium-based ionic liquids provided an equally potent antimicrobial construct that was more stable to enzyme-mediated modification (31). Indeed, ILs have become very attractive for biomedical applications due to their unique physicochemical properties, low cost, and high structural diversity, which allow the synthesis of a wide range of different ILs that can be easily tuned to meet specific requirements, including broad-spectrum activity against bacteria (4) and fungi (33). Recently, alkyl imidazolium-based ILs have been proposed as alternative antibacterial treatments for cSSTIs, with a focus on Gram-positive pathogens (34). In addition, several ILs have been found to improve the skin permeability of drugs (10,35,36,37), including ceftazidime, an antibiotic with poor water solubility and low skin permeability (11).

[0011] The document "Clicking" an Ionic Liquid to a Potent Antimicrobial Peptide: On the Route towards Improved Stability (31) describes an analog (MeIm-3.1-PP4) of peptide 3.1-PP4 (composed of antimicrobial peptide 3.1 and cosmeceutical pentapeptide-4) conjugated to an ionic liquid methylimidazolium at the N-terminus, and its biological application as an antibacterial agent, an antibiofilm agent, and an antifungal agent. This conjugate retains the activity of the unmodified parent chimeric peptide (3.1-PP4) against multidrug-resistant clinical isolates of Gram-negative bacteria, as well as the antibiofilm effect against resistant clinical isolates of Klebsiella pneumoniae, while showing highly improved stability to tyrosinase-mediated modification.

[0012] The document "Disclosure of a Promising Lead to Tackle Complicated Skin and Skin Structure Infections: Antimicrobial and Antibiofilm Actions of PeptidePP4-3.1" (38) describes an analog (MeIm-PP4-3.1) of peptide PP4-3.1 (composed of cosmeceutical pentapeptide-4 and antimicrobial peptide 3.1) conjugated to the ionic liquid methylimidazolium at the N-terminus, and its biological application as an antibacterial agent, antibiofilm agent and antifungal agent. Although the conjugated peptide showed activity in all biological assays, the unmodified parent chimeric peptide (PP4-3.1) stood out for its potent activity against Gram-positive and Gram-negative bacteria, including MDR clinical isolates, and against three clinically relevant Candida species, with an overall performance superior to that of MeIm-PP4-3.1.

[0013] Both of these documents describe peptide analogs with in vitro antibacterial, antibiofilm, and antifungal activity, demonstrating N-terminal modifications of chimeric peptides combining cosmeceutical and antimicrobial sequences. Nonetheless, neither of these documents describes peptide analogs containing quaternary imidazolium moieties with long hydrocarbon chain substituents, nor does any evidence provide that the peptide derivatives reported therein have significant collagen biosynthesis inducing activity. Furthermore, the peptide analog sequences reported in these two documents contain long, highly cationic host defense peptide sequences, which are well known for their direct antimicrobial effects. However, many host defense peptides lack the rapid and effective skin regeneration capabilities that cosmeceutical peptides can offer, and most are too long (greater than 10 amino acids) to allow for efficient dermal and transdermal delivery (39) or cost-effective production on an industrial scale. Furthermore, host defense peptide-resistant strains of Staphylococcus aureus, the most common bacterial pathogen in infected skin lesions, have been reported to have increased membrane surface charge, which reduces the efficiency of the initial electrostatic interaction between the peptide and the bacteria that precedes the bactericidal effect of the peptide through destabilization of the bacterial membrane (40); this implies that previously developed host defense peptide-based constructs may induce the selection of resistant microbial strains. Accordingly, novel antimicrobial constructs derived from small CPs rather than from highly cationic host defense peptides are less likely to induce resistance.

[0014] In 'The Emerging Role of Ionic Liquid-Based Approaches for Enhanced Skin Permeation of Bioactive Molecules: A Snapshot of the Past Couple of Year', Int. J. Mol. Sci., 2021, 22, 11991, Gomes et al. disclosed a review of ionic liquids as skin penetration enhancers, but this review did not disclose technical information related to the present invention.

[0015] In 'Safety Assessment of Tripeptide-1, Hexapeptide-12, their Metal Salts and Fatty Acyl Derivatives, and Palmitoyl Tetrapeptide-7 as Used in Cosmetics', Cosmetic Ingredient Review, 2014, Bergfeld et al. studied the safety of using peptides in cosmetics, but this study did not involve the antimicrobial effect of peptides, nor did it disclose technical information related to the present invention.

[0016] In '"Clicking"an Ionic Liquid to a Potent Antimicrobial Peptide:On the Route towards Improved Stability', International Journal of Molecular Sciences, Vol. 21, No. 17, 2020, Gomes et al. did not disclose a pharmaceutical cosmetic peptide with up to 10 amino acids, but disclosed a 16-amino acid antimicrobial peptide containing both an antimicrobial motif and a pharmaceutical cosmetic motif in its sequence. The present invention is different from this document in that an ionic liquid is covalently linked to a pharmaceutical cosmetic peptide, and this pharmaceutical cosmetic peptide itself has no antimicrobial effect and is independent of the antimicrobial peptide motif.

[0017] The present invention relates only to small (maximum 10 amino acids) CPs, which have been widely used (and patented) as active ingredients in cosmetics for cosmetic applications, but in no case as topical antimicrobial agents, since they lack intrinsic antimicrobial action. Relatedly, the characteristics of the PIL conjugates to which the present invention relates are not simply the sum of the intrinsic properties of each of the two separate building blocks, since simply mixing them (rather than linking them by chemical conjugation) would produce a mixture that is bacteriostatic rather than bactericidal. In addition, the chemical modification of the peptide analogs in the above two documents (31, 38) was performed only at their N-terminus, while the PIL conjugates of the present invention have one or more IL building blocks attached to the N-terminus or the amino acid side chain (e.g., the azido-lysine side chain) or both. Insertions at the amino acid side chain for chemical conjugation of ILs to peptides have never been reported to date. Summary of the invention

[0018] The present invention relates to a peptide-ionic liquid conjugate for preventing and / or treating skin disorders, wherein the peptide-ionic liquid conjugate comprises a cosmeceutical peptide containing up to 10 amino acids and at least one ionic liquid containing a saturated or unsaturated hydrocarbon chain substituent of C1 to C18.

[0019] In one embodiment, the cosmeceutical peptide is a cosmeceutical peptide selected from any one of sequences SEQ.ID NO 1 to 20.

[0020] In one embodiment, the at least one ionic liquid is selected from pyridinium, imidazolium, phosphonium or cholinium ionic liquids.

[0021] In one embodiment, the conjugation between the cosmeceutical peptide and the at least one ionic liquid occurs at the N-terminus of the amino acid of the cosmeceutical peptide.

[0022] In one embodiment, the conjugation between the cosmeceutical peptide and the at least one ionic liquid occurs on the side chains of the amino acids of the cosmeceutical peptide.

[0023] In one embodiment, the conjugation between the cosmeceutical peptide and the ionic liquid occurs at the N-terminus of the amino acids of the cosmeceutical peptide and at the side chains of the amino acids.

[0024] The present application also relates to a topical composition comprising at least one type of peptide-ionic liquid conjugate.

[0025] In one embodiment, the topical composition further comprises nanoparticles, solid lipid nanoparticles, nanostructured lipid carriers (including liposomes or polymer nanoparticles) and / or at least one hydrogel, including a polysaccharide-based hydrogel or a poly(lactic-co-glycolic acid)-based hydrogel.

[0026] In one embodiment, the topical composition is used to prevent and / or treat skin disorders.

[0027] In one embodiment, the skin disorder is a severe non-healing skin ulcer such as diabetic foot ulcers, pressure ulcers, venous leg ulcers; milder bacterial or fungal skin infections such as acne, atopic dermatitis, candidiasis, cellulitis, dermatophytoses, erysipelas, folliculitis, impetigo, rosacea; autoimmune / inflammatory disorders, aging and cancerous skin disorders such as melanoma, non-melanoma skin cancer, psoriasis, cutaneous lupus erythematosus, impetigo, skin aging and melanoma, whether or not there is secondary infection, and also includes the treatment of post-operative wounds, general injuries or burns.

[0028] General Description

[0029] The present application relates to a peptide-ionic liquid (PIL) conjugate suitable for preventing and / or treating skin disorders.

[0030] The peptide-ionic liquid conjugates described herein are a new type of prospective active pharmaceutical ingredients (APIs) suitable for topical compositions to prevent and / or treat skin disorders.

[0031] The present invention relates to conjugates of cosmeceutical peptides (CPs) comprising up to 10 amino acids, forming PIL conjugates with at least one ionic liquid.

[0032] As an example, the direct conjugation of alkyl imidazolium-based ILs to a non-antimicrobial pentapeptide-4 (PP4) having the sequence SEQ.ID 1 (a cosmeceutical peptide of up to 10 amino acids: KTTKS) was investigated to see if it would provide a novel peptide-based construct that, despite not containing an antimicrobial peptide (AMP) motif, exhibits collagenogenic and antimicrobial effects. To this end, three different alkyl imidazolium-based ILs were chemically modified to introduce the alkyne moiety required for subsequent conjugation with different azide derivatives of PP4. As reported and discussed herein, seven different IL-PP4 conjugates were prepared and their antibacterial, antifungal, and collagenogenic properties were investigated.

[0033] These conjugates showed potent activity against antibiotic-susceptible strains or multidrug-resistant clinical isolates of Gram-positive and Gram-negative bacterial species belonging to the so-called "ESKAPE" group of pathogens. Notably, their antibacterial activity was retained in simulated wound exudate, which suggests that they will be effective in the environment of a real wound bed. In addition, their induction of collagen formation in vitro was consistent with that of Equivalent or stronger. In summary, IL-PP4 plays a triple role in vitro in antibacterial, antifungal and collagen formation induction. These findings provide a solid foundation for advancing IL-PP4 conjugates as promising leads for future development of topical prevention and / or treatment of skin disorders.

[0034] Further studies are included to incorporate the PIL conjugates into suitable nanoformulations to reduce toxicity, improve resistance to proteolytic degradation and retain the active pharmaceutical ingredient at the intended site of action (wound bed, area of ​​injury).

[0035] The main advantages of the PIL conjugates of the present invention are:

[0036] 1.CP has been used in cosmetics for advanced skin care due to its rapid skin regeneration ability and safety on human skin;

[0037] 2. The small size of CP (maximum 10 amino acids) facilitates its cost-effective production and skin penetration without the use of unusual / expensive skin and transdermal delivery enhancement methods (e.g., electroporation, iontophoresis, sonophoresis, microneedles, etc.);

[0038] 3. In fact, CP has been produced on an industrial scale according to current good manufacturing practices (cGMP);

[0039] 4. Widely reported ILs with both antimicrobial and skin penetration abilities, namely, but not limited to, those based on imidazolium and pyridinium; for example, cetylpyridinium chloride is an antibacterial IL used in mouthwashes, toothpastes, and throat / nasal sprays that combines microbicidal action with skin penetration ability (41);

[0040] 5. The possibility of using rapid and direct chemoselective conjugation between CP and IL building blocks by means of click chemistry, namely, but not limited to, copper(I)-catalyzed azide-alkyne coupling (CuAAC);

[0041] 6. Potentially offer new products that can be fine-tuned to not only reflect the sum of the properties of their individual structural units but also to exhibit new properties (e.g., immunomodulation), thereby expanding their promising therapeutic (and cosmetic) applications beyond the management of skin infections and wound healing (i.e., topical use for autoimmune skin conditions such as psoriasis or eczema). BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to make the present application more easily understood, drawings representing preferred embodiments are attached in the appendix; however, these drawings are not intended to limit the technology disclosed herein.

[0043] Figure 1 - Route to obtain the target IL-PP4 conjugate. (A) Synthesis of alkyne derivatives of IL: (i) 1.1 equivalents of C 16 Im, C 14Im or MeIm was reacted with 1.0 equivalent of propargyl bromide (80% in toluene) at 40°C for 24 hours. (B) Synthesis of azido derivatives of KTTKS and their coupling with alkynyl IL via CuAAC: (ii) 5 equivalents of Fmoc-protected amino acid, 10 equivalents of N-ethyl-N,N-diisopropylamine (DIEA) and 5 equivalents of O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) were placed in N,N-dimethylformamide (DMF) and reacted at room temperature (rt) for 1 hour; (iii) 20% piperidine in DMF was added and reacted at rt for 15 minutes; (iv) 5 equivalents of azidoacetic acid, 10 equivalents of DIEA and 5 equivalents of HBTU were added and reacted at rt for 1 hour; (v) 10 equivalents of DIEA, 10 equivalents of 2,6-lutidine, 1 equivalent of copper (I) bromide, 1 equivalent of sodium L-ascorbate and 1 equivalent of Pr-MeIm, Pr-C 16 Im or Pr-C 14 Im was placed in DMF:acetonitrile (ACN) (3:1 v / v) and reacted at room temperature for 24 hours; (vi) trifluoroacetic acid (TFA) / triisopropylsilane (TIS) / distilled water (95:25:2.5 v / v / v) was added.

[0044] Figure 2 - Structures and corresponding molecular weights (in Da) of IL-PP4 conjugates. Amino acids are represented by single-letter codes as defined by the IUPAC-IUBMB Guide to Nomenclature and Symbolism for Amino Acids and Peptides, except for amino acid residues whose side chains are coupled to ionic liquids by click chemistry, in which case the complete modified structure is shown.

[0045] Figure 3 -Use Sircol TM Kit, at 5 μM C 16 -KTTKS-OH KTTK(C 16 Im)S and C 16 Collagen synthesis of human dermal fibroblasts (HDF) was measured in the presence of Im-KTTKS. Data are presented as mean ± SEM (3 independent experiments, triplicates) of collagen amount (% of control); *p<0.05, **p<0.01. DETAILED DESCRIPTION

[0046] The preferred embodiments of the present application will now be described in detail with reference to the accompanying drawings. However, they are not intended to limit the scope of the present application.

[0047] The present invention relates to a peptide-ionic liquid conjugate of a cosmeceutical peptide (CP) and at least one ionic liquid, wherein the cosmeceutical peptide comprises up to 10 amino acids to form a PIL conjugate.

[0048] In one embodiment, the pharmaceutical cosmetic peptide is selected from but not limited to PP4 (SEQ.ID 1), KVK, PKEK (SEQ.ID 2), GEKG (SEQ.ID 3), GHK, copper tripeptide-1, manganese tripeptide-1, palmitoyl tripeptide-1, tripeptide-5, palmitoyl tripeptide-5, tetrapeptide-3 (SEQ.ID 4), pentapeptide-3 (SEQ.ID5), pentapeptide-18 (SEQ.ID 6), hexapeptide-11 (SEQ.ID 7), acetyl hexapeptide-3 (SEQ.ID 8), hexapeptide-10 (SEQ.ID 9), hexapeptide-12 (SEQ.ID 10), palmitoyl hexapeptide-12, acetyl octapeptide-1 (SEQ.ID 11), SA1-III (SEQ.ID 12), and lipospondin.

[0049] In one embodiment, the at least one ionic liquid is selected from but not limited to, comprising C1 to C 18 In one embodiment, the ionic liquid is selected from different ionic liquid families, namely, but not limited to, pyridinium (e.g., hexadecylpyridinium), imidazolium (e.g., 1-alkyl-3-methylimidazolium), phosphonium (e.g., trihexyltetradecylphosphonium) or cholinium ionic liquids.

[0050] The conjugation between the cosmeceutical peptide and at least one ionic liquid occurs at the N-terminus of the amino acids in the peptide sequence and / or at the side chains of specific amino acids, namely, but not limited to, lysine, arginine and histidine side chains.

[0051] In one embodiment, the PIL conjugate is used to treat and / or prevent skin disorders ranging from severe non-healing skin ulcers such as diabetic foot ulcers, pressure ulcers, venous leg ulcers, etc., to generally milder bacterial or fungal skin infections such as acne, atopic dermatitis, candidiasis, cellulitis, dermatophytosis, erysipelas, folliculitis, impetigo, rosacea, etc., and also includes autoimmune / inflammatory disorders, aging and cancerous skin disorders such as melanoma, non-melanoma skin cancer, psoriasis, cutaneous lupus erythematosus, impetigo, skin aging and melanoma, whether or not there is secondary infection. It can also be used to treat postoperative wounds, general injuries or burns.

[0052] The present application also relates to a topical composition comprising at least one type of PIL conjugate.

[0053] In one embodiment, the topical composition comprising the PIL conjugate is used to treat and / or prevent skin disorders ranging from severe non-healing skin ulcers such as diabetic foot ulcers, pressure ulcers, venous leg ulcers, etc., to generally milder bacterial or fungal skin infections such as acne, atopic dermatitis, candidiasis, cellulitis, dermatophytosis, erysipelas, folliculitis, impetigo, rosacea, etc., and also includes autoimmune / inflammatory disorders, aging and cancerous skin disorders such as melanoma, non-melanoma skin cancer, psoriasis, cutaneous lupus erythematosus, impetigo, skin aging and melanoma, whether or not there is secondary infection. It can also be used to treat postoperative wounds, common injuries or burns.

[0054] In one embodiment, the topical composition further comprises nanoparticles, including but not limited to solid lipid nanoparticles, nanostructured lipid carriers (liposomes or polymer nanoparticles) and / or at least one hydrogel, including but not limited to polysaccharide-based hydrogels or poly(lactic-co-glycolic acid)-based hydrogels.

[0055] It has been previously shown that by combining the amino acid sequence of AMP with that of the well-known non-antimicrobial cosmetic peptide PP4, it is possible to generate a chimeric peptide with both antimicrobial and collagen formation-inducing effects (32). It was further shown that when an imidazolium IL was coupled to the N-terminus of the chimeric peptide by a CuAAC “click” approach, resistance to enzyme-mediated modification was conferred on the peptide, while the potent antimicrobial activity was retained (31).

[0056] Based on these findings, the study disclosed in the present application was aimed at exploring whether the dual activities of antimicrobial and collagen formation induction are retained by removing the AMP sequence and directly coupling at least one IL to the amino acid sequence of the cosmeceutical peptide PP4.

[0057] The findings disclosed in this application strongly suggest that direct conjugation between IL and other cosmeceutical peptides containing up to 10 amino acids will have the same properties observed in the examples disclosed herein.

[0058] In the experimental examples below, the PP4 peptide will be referred to by its amino acid sequence, ie, KKTKS.

[0059] The antibacterial activity of the novel construct IL-KKTKS presented herein was evaluated against reference bacterial strains, and the results obtained (as shown in the Examples section) allowed the advancement of several structure-activity relationships (SARs) with respect to: (i) the site of IL insertion, since the covalent grafting was located at the N-terminus or the Lys1 / Lys4 side chains of the KTTKS sequence, and (ii) the length of the alkyl substituent in the imidazole ring, which varied between one (methyl or Me), fourteen (tetradecyl or C14), and sixteen (hexadecyl or C16) carbons. Thus, the antibacterial activity (i) increased with increasing length of the alkyl substituent in the IL moiety [KTTK(C14Im)S vs. KTTK(C16Im)S], and (ii) decreased in all conjugates carrying methyl-substituted imidazolium ILs, regardless of other structural features.

[0060] In addition, the MIC values ​​of the parent building blocks KTTKS and [C16 M1Im][Br], and their non-covalent equimolar mixture (denoted as KTTKS:[C16 M1Im][Br](1:1)) were determined to assess the importance of covalent conjugation. The non-covalent mixture KTTKS:[C16 M1Im][Br](1:1) showed similar MIC values ​​to [C16 M1Im][Br] alone, confirming that the IL building block is the main factor causing the observed activity of the mixture, as expected.

[0061] Interestingly, when the MIC values ​​of the non-covalent mixture KTTKS:[C16 M1Im][Br](1:1) are compared with those of the covalent conjugates KTTK(C16Im)S and C16Im-KTTKS, it is found that the covalent conjugation clearly favors the activity against Gram-negative bacteria, but the effect on Gram-positive bacteria is not as great. However, while the non-covalent mixture is bacteriostatic against Gram-positive species at MIC values, the covalent conjugate is bactericidal at these concentrations. These results suggest that the antibacterial activity of the covalent conjugates KTTK(C16Im)S and C16Im-KTTKS is modulated not only by the IL building block but also by its conjugation to the peptide. Therefore, biophysical studies will be conducted to further explore the mechanism of action of the IL-KTTKS conjugate. The conjugates KTTK(C16Im)S and C16Im-KTTKS also showed potent activity against MDR clinical isolates of Gram-positive and Gram-negative bacteria, more active than the reference antibiotic ciprofloxacin.

[0062] This is a relevant finding considering that the three clinical isolates tested refer to bacterial species belonging to the “ESKAPE” group of pathogens, which covers life-threatening nosocomial pathogens, namely Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species. The ability of “ESKAPE” pathogens to evade the action of currently available antibiotics is one of the major medical threats of our time, especially for Gram-negative bacteria, against which the world’s effective options are gradually running out.

[0063] Additionally, in simulated wound exudate (SWF), the conjugates showed either retained (C16Im-KTTKS) or slightly reduced (KTTK(C16Im)S) antibacterial activity against S. aureus. These preliminary observations using SWF, which simulates wound exudate, are of interest because they suggest that IL-peptide conjugates, especially C16Im-KTTKS, may be more stable in the wound environment compared to analogs in which the peptide is not protected at the N-terminus.

[0064] The IL-KTTKS conjugates described herein also showed interesting antifungal properties, particularly against Candida parapsilosis. This is one of the most common non-albicans Candida species in the genus Candida and is considered an important nosocomial pathogen of concern as they have been reported to be associated with cases of sepsis and cSSTI. In this regard, the antifungal activity of the conjugates was evaluated and MIC values ​​as low as 2.4 and 2.7 μM were observed against Candida parapsilosis and as low as 4.7 and 5.7 μM against Candida albicans.

[0065] Nevertheless, as observed in the antibacterial activity assay, the non-covalent mixture and the parent IL were more effective than the IL-KTTKS conjugate against Candida species, suggesting that the antifungal activity of the covalent conjugate is modulated not only by the IL building block but also by its conjugation to a peptide (lacking antifungal activity). Therefore, further biophysical studies will be conducted to elucidate the possible mechanism of action of the IL-KTTKS conjugate against Candida species.

[0066] Assessment of the cytotoxicity of IL-KTTKS conjugates is obviously important in itself, not only to check their selectivity but also because toxic effects are often associated with ILs, depending on, for example, the length of the cation alkyl chain or the specific ion used.

[0067] The parent IL [C16 M1Im][Br] and its covalent equimolar mixture with the peptide KTTKS: [C16 M1Im][Br] (1:1) were significantly toxic to the human cell lines tested. Thus, the covalent conjugation of the IL to the peptide imparted on the one hand antimicrobial activity to the peptide building block that was otherwise lacking such activity and on the other hand lower cytotoxicity compared to the parent IL.

[0068] The conjugates C16Im-KTTKS and KTTK(C16Im)S were further evaluated for their ability to induce collagen production in human dermal fibroblasts in vitro. No significant difference between the two conjugates was observed, indicating that changing the side chain of Lys4 did not affect the collagenogenesis-inducing behavior of the peptide.

[0069] In summary, the findings of the disclosed embodiments are unprecedented and remarkable because they provide evidence by proposing a pair of PIL conjugates, C16Im-KTTKS and KTTK(C16Im)S, which have antibacterial, antifungal and collagen formation inducing activities in vitro, and the latter is actually similar to the KTTKS peptide-based cosmeceutical ingredients. Moreover, the insertion site of IL did not significantly affect the overall in vitro properties of the conjugate [C16Im-KTTKS vs. KTTK(C16Im)S], although N-terminal conjugation seemed to better preserve the antibacterial effect of the conjugate in SWF and improve collagen synthesis in human dermal fibroblasts.

[0070] Further studies are planned to incorporate the PIL into nanoformulations and / or hydrogels, which will reduce toxicity, improve resistance to proteolytic degradation, and retain the active pharmaceutical ingredient at the intended site of action (wound bed, area of ​​injury). In addition, since the IL-KKTKS conjugate will be used to treat cSSTIs, which are primarily polymicrobial infections, the antimicrobial activity of IL-KTTKS against polymicrobial cultures will be further investigated. This will allow us to select the best IL-KTTKS-based formulation to advance in vivo studies.

[0071] Considering that KTTKS and other small cosmeceutical peptides are already produced on an industrial scale as ingredients in cosmetic products, these findings reveal the value of IL-CP conjugates, which could serve as a promising start for the future development of cost-effective topical formulations for the prevention and / or treatment of skin conditions, ranging from mild to severe skin conditions such as cSSTIs.

[0072] Example

[0073] 1. Synthesis of target conjugate

[0074] The route to the target IL-KTTKS conjugate began with the synthesis of alkyne-modified imidazolium IL ( Figure 1 -A). According to the method of Hu et al. (42), 1-methyl-imidazole (Me-IM), 1-tetradecyl-imidazole (C14-Im) and 1-hexadecyl-imidazole (C16-Im) were reacted with propyne bromide ( Figure 1 -A) to provide three target imidazolium ILs, namely propargyl-MeIm (Pr-MeIm), propargyl-C 14 Im(Pr-C 14 Im) and propargyl-C 16 Im(Pr-C 16 Im). The structures of these ILs are 1 H-NMR, 13 The results were confirmed by C-NMR and ESI-IT MS.

[0075] In parallel, PP4 (amino acid sequence KTTKS) derivatives that were easily modified were generated by solid phase peptide synthesis (SPPS) to provide different final IL-KTTKS conjugates ( Figure 2 ), which differ in: (a) the propargyl-imidazolium building block used, (b) the site of insertion of the latter (N-terminus, side chains of either or both lysine residues), and (c) the length of the spacer between the imidazolium moiety and the N-terminus of the peptide. For this purpose, firstly according to Figure 1 Steps ii and iii in -B assembled the PP4 sequence and conveniently protected lysine (Fmoc-Lys(Boc)-OH) or azido-lysine (Fmoc-Lys(N3)-OH) building blocks were inserted at the corresponding position of the sequence, depending on the site required for the subsequent introduction of the imidazolium moiety by the "click" copper(I)-catalyzed alkyne-azide cycloaddition reaction (CuAAC). To generate peptides modified at the N-terminus, sequences with two native Lys residues were assembled and cleaved by coupling azidoacetic acid (step iv, Figure 1 -B) is further extended to generate a 2-carbon (ethyl) spacer between the N-terminal lysine and the imidazolium moiety to be incorporated by CuAAC. Next, the desired propargyl-imidazolium IL (step v, Figure 1 -B) and our previously reported CuAAC conditions (31), all precursor azido-peptides were subjected to this click reaction on the resin. Figure 1-B) and after purification of the crude conjugate thus obtained by reverse phase preparative high performance liquid chromatography (RP-HPLC), all resulting IL-KTTKS conjugates were isolated with high purity (>95%) and their expected molecular weights were confirmed by ESI-IT-MS.

[0076] In addition to the target conjugates, reference cosmeceutical peptides were assembled by SPPS following a recently reported procedure. (C 16 -KTTKS-OH), its C-terminal carboxamide analog (C 16 -KTTKS-NH2) and native PP4 (KTTKS) (32). For palmitoylated peptides, after assembling the complete amino acid sequence of PP4, palmitic acid (C 16 ). Then, the crude peptide was obtained by acid cleavage from the solid support and purified by RP-HPLC. The final peptide was obtained with high purity and its molecular weight was confirmed by ESI-IT MS.

[0077] 2. In vitro antibacterial activity

[0078] The antimicrobial activity of the IL-KTTKS conjugate against reference bacterial strains (American Type Culture Collection, ATCC) was evaluated in vitro. The minimum inhibitory concentration (MIC) against Gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa) was determined according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) (43). The obtained MIC values ​​are shown in Table 1. It is worth noting that the reference peptide C 16 -KTTKS-NH2 and C 16 -KTTKS-OH were soluble in water and dimethyl sulfoxide (DMSO), respectively, but both precipitated when diluted in the CLSI guideline-recommended medium, cation-adjusted Mueller-Hinton broth (MHB2), thus preventing the determination of MIC values ​​for these reference peptides. As expected, the data in Table 1 show that the peptide KTTKS alone lacks significant antibacterial activity and [C 16 The MIC values ​​of M1Im][Br]IL were consistent with those reported previously (44). Interestingly, all conjugates bearing a methylimidazolium (MeIm) unit were inactive against the bacterial species tested, even at the highest concentration used, regardless of the number or position of the MeIm moiety in the overall structure. Conversely, replacing the methyl substituent in the imidazolium ring with a tetradecyl (C 14 ) or hexadecyl (C 16), resulting in an increase in antibacterial activity, with MIC values ​​ranging from 6.45 μg / mL to 52.6 μg / mL, thus adding antimicrobial activity to the parent KTTKS peptide. 16 Im)S and C 16 Im-KTTKS showed the strongest antibacterial activity, reflecting the two different conjugation positions, so these two peptides were further studied by determining their MICs against Staphylococcus epidermidis (S. epidermidis), Streptococcus pyogenes (S. pyogenes) (all Gram-positive), and Klebsiella pneumoniae (Gram-negative). These bacteria were chosen because of their abundance in the skin (S. epidermidis) (45), association with cSSTI (S. pyogenes) (46-48), and relationship with the so-called “ESKAPE” pathogens (K. pneumoniae) (49). The parent peptide KKTKS and [C 16 The non-covalent mixture of M1Im][Br] ionic liquids exhibits similar 16 M1Im][Br] equivalent MIC value.

[0079] Table 1: MIC values ​​(n=3) of IL-KTTKS conjugates against Gram-negative and Gram-positive bacteria (ATCC reference strains) in μM (expressed as μg / mL).

[0080]

[0081]

[0082] The best pair of conjugates, C16Im-KTTKS and KTTK(C16Im)S, were further evaluated for antibacterial activity against MDR clinical isolates of Klebsiella pneumoniae (KP010), Staphylococcus aureus (SA007), and Pseudomonas aeruginosa (PA004), as well as the reference antibiotic ciprofloxacin. The MIC values ​​so obtained are shown in Table 2 and indicate that both conjugates retain their antibacterial activity observed against susceptible ATCC bacterial strains. In relation to this, the conjugates were significantly more active than the reference antibiotic ciprofloxacin against MDR isolates; for example, against SA007, the MIC value obtained for C16Im-KTTKS was nearly 10 times higher than that for ciprofloxacin.

[0083] Table 2: MIC values ​​of C16-Im-KTTKS and KTTK(C16Im)S against Gram-positive and Gram-negative MDR clinical isolates (n=3) in μM (expressed as μg / mL).

[0084]

[0085] C was also evaluated in SWF (50) 16 Im-KTTKS and KTTK(C 16 The antibacterial activity of C. aureus (ATCC 29213) was measured to examine whether it was maintained in this medium. The MIC values ​​in SWF and MHB medium were obtained in three independent experiments performed in triplicate (Table 3) and showed that for C. 16 Im-KTTKS showed the same antibacterial activity in SWF as in MHB, whereas for KTTK (C 16 Im)S, however, was reduced, and was two times lower in terms of MIC.

[0086] Table 3: MIC [MBC] values ​​of C16Im-KTTKS and KTTK(C16Im)S against Staphylococcus aureus (ATCC29213) in MHB and SWF (n=3), units are μg / mL.

[0087]

[0088] 3. In vitro antifungal activity

[0089] The best pair of IL-KTTKS conjugates, their parent building blocks, and the corresponding non-covalent 1:1 mixtures were evaluated for antifungal activity against three species of the genus Candida, namely Candida albicans (ATCC 90028), Candida glabrata (ATCC 90030), and Candida parapsilosis (ATCC 22019). The MIC values ​​were determined according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) protocol (51-53) and are shown in Table 4. The two conjugates KTTK (C 16 Im)S and C 16 Im-KTTKS was equally active against all Candida species, with MIC values ​​ranging from 2.4 to 5.4 μM. Both peptides were two-fold more active against Candida parapsilosis than against the other two Candida species. 16 M1Im][Br](1:1) showed potent activity against all Candida species, equivalent to the parent IL alone, and both were seven times more active than the reference antifungal drug fluconazole.

[0090] Table 4. MIC values ​​of the best performing conjugates, their parent building blocks and the corresponding 1:1 non-covalent mixtures against ATCC Candida species (n=2) in μM (expressed as μg / mL).

[0091]

[0092] 4. Toxicity to HFF-1 and HaCaT cells

[0093] Evaluated KTTK(C 16 Im)S and C 16 Cytotoxicity of Im-KTTKS conjugate to human foreskin fibroblasts (HFF-1) and human immortalized keratinocytes (HaCaT). The results are shown in Table 5, expressed as the concentration of conjugate that causes 50% cell growth inhibition (IC 50 As expected from previous reports (54), the parent peptide sequence KTTKS and the derived reference cosmeceuticals (C 16 -KTTKS-OH) showed no relevant toxicity to any of the cell lines tested at concentrations up to 100 μM. 16 M1Im][Br] and its covalent equimolar mixture with PP4 KTTKS:[C 16 M1Im][Br](1:1) was significantly toxic. Interestingly, covalent conjugation of this peptide to IL produced an intermediate state, as the conjugate was more toxic than the peptide alone but significantly less toxic than the IL alone or its non-covalent mixture with the peptide.

[0094] 5. In vitro collagen production

[0095] The aim of conjugating an antimicrobial IL to a peptide that induces collagen formation was to provide a simple construct that could exert both antimicrobial and skin remodeling effects. Therefore, the two best IL-KTTKS conjugates were further tested for their ability to promote collagen production by human dermal fibroblasts (HDFs) in vitro. This was assessed using a kit-based assay in which the amount of newly formed collagen in ECM deposited in microplate cell cultures was solubilized in acidic medium and then The kit assay procedure (55) was quantified using a collagen standard curve. For comparison, the assay was performed under different conditions, i.e., under a reference cosmeceutical. (Positive Control – C 16 -KTTKS-OH), test conjugate KTTK (C 16 Im)S and C 16In the presence of Im-KTTKS, and in the absence of any peptide (negative control). Figure 3 The data presented in show that both conjugates induced HDF cells to produce more collagen compared to the negative control. 16 Im)S conjugates and references No significant differences were observed between The ability to induce collagen formation was not affected by the introduction of imidazolium IL at the Lys side chain of the peptide sequence.

[0096] Of course, this specification is not limited in any way to the implementation forms proposed herein, and anyone with a basic understanding of the art can provide many possibilities for modification thereof without departing from the general concept defined by the claims. The above preferred implementation forms can obviously be combined with each other. The following claims further define the preferred implementation forms.

[0097] Sequence Listing

[0098] SEQ ID NO 1=PP4

[0099] SEQ ID NO 2=PKEK

[0100] SEQ ID NO 3=GEKG

[0101] SEQ ID NO 4 = Tetrapeptide-3

[0102] SEQ ID NO 5 = Pentapeptide-3

[0103] SEQ ID NO 6 = Pentapeptide-18

[0104] SEQ ID NO 7=Hexapeptide-11

[0105] SEQ ID NO 8 = Acetyl Hexapeptide-3

[0106] SEQ ID NO 9=Hexapeptide-10

[0107] SEQ ID NO 10 = Hexapeptide-12

[0108] SEQ ID NO 11 = Acetyl Octapeptide-1

[0109] SEQ ID NO 12 = SA1-III

[0110] SEQ ID NO 13=KVK

[0111] SEQ ID NO 14 = GHK

[0112] SEQ ID NO 15 = Copper tripeptide-1 = Cu(II)-GHK

[0113] SEQ ID NO 16 = Manganese tripeptide-1 = Mn(II)-GHK

[0114] SEQ ID NO 17 = Palmitoyl Tripeptide-1 = C16-GHK (palmitoyl is from palmitic acid, CH3-(CH2)14-COOH)

[0115] SEQ ID NO 18 = Palmitoyl tripeptide-5 = C16-KVK

[0116] SEQ ID NO 19 = Palmitoyl hexapeptide-12 = C16-VGVAPG

[0117] SEQ ID NO 20 = lipospondin = lipospondin-KFK (lipospondin is derived from lipospondin acid,

[0118] HOOC-(CH2)7-CH=CH-(CH2)7-CH3)

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Claims

1. A peptide-ionic liquid conjugate for preventing and / or treating skin disorders, wherein the peptide-ionic liquid conjugate comprises a cosmeceutical peptide and at least one ionic liquid, wherein the cosmeceutical peptide comprises up to 10 amino acids, and the at least one ionic liquid comprises a saturated or unsaturated hydrocarbon chain substituent of C1 to C18.

2. The peptide-ionic liquid conjugate according to the preceding claim, wherein the cosmeceutical peptide is selected from any one of the sequences SEQ. ID NOs 1 to 20.

3. The peptide-ionic liquid conjugate according to any one of the preceding claims, wherein the at least one ionic liquid is selected from pyridinium, imidazolium, phosphonium or cholinium ionic liquids.

4. The peptide-ionic liquid conjugate according to any one of the preceding claims, wherein the conjugation between the cosmeceutical peptide and the at least one ionic liquid occurs at the N-terminus of the amino acid of the cosmeceutical peptide.

5. The peptide-ionic liquid conjugate according to any one of claims 1 to 3, wherein the conjugation between the cosmeceutical peptide and the at least one ionic liquid occurs on the side chains of the amino acids of the cosmeceutical peptide.

6. The peptide-ionic liquid conjugate according to any one of claims 1 to 3, wherein the conjugation between the cosmeceutical peptide and the ionic liquid occurs at the N-terminus of the amino acids of the cosmeceutical peptide and at the side chains of the amino acids.

7. A peptide-ionic liquid conjugate according to any one of the preceding claims, wherein the skin disorder is a severe non-healing skin ulcer such as diabetic foot ulcer, pressure ulcer, venous leg ulcer; a less severe bacterial or fungal skin infection such as acne, atopic dermatitis, candidiasis, cellulitis, dermatophytosis, erysipelas, folliculitis, impetigo, rosacea; autoimmune / inflammatory disorders, aging and cancerous skin disorders such as melanoma, non-melanoma skin cancer, psoriasis, cutaneous lupus erythematosus, impetigo, skin aging and melanoma, in the absence or presence of secondary infection, treatment of post-operative wounds, general injuries or burns.

8. A topical composition comprising at least one peptide-ionic liquid conjugate of the type described in any one of the preceding claims.

9. The topical composition according to the preceding claim, wherein the topical composition further comprises nanoparticles, solid lipid nanoparticles, nanostructured lipid carriers including liposomes or polymer nanoparticles, and / or at least one hydrogel including a polysaccharide-based hydrogel or a poly(lactic-co-glycolic acid)-based hydrogel.

10. A topical composition according to any one of claims 8 to 9, comprising a peptide-ionic liquid conjugate as described in any one of claims 1 to 6, for use in preventing and / or treating skin disorders.

11. A topical composition according to claim 10, wherein the skin disorder is a severe non-healing skin ulcer such as diabetic foot ulcer, pressure ulcer, venous leg ulcer; a less serious bacterial or fungal skin infection such as acne, atopic dermatitis, candidiasis, cellulitis, dermatophytosis, erysipelas, folliculitis, impetigo, rosacea; autoimmune / inflammatory disorders, aging and cancerous skin disorders such as melanoma, non-melanoma skin cancer, psoriasis, cutaneous lupus erythematosus, impetigo, skin aging and melanoma, treatment of post-operative wounds, general injuries or burns in the absence or presence of secondary infection.

Images