Peptide-Ionic Liquid Conjugates for the Prevention and / or Treatment of Skin Diseases

Abstract

This application relates to peptide - ionic liquid conjugates for the prevention and / or treatment of skin diseases. The peptide - ionic liquid conjugate comprises a cosmeceutical peptide containing up to 10 amino acids, and at least one ionic liquid containing a C1 - C18 saturated or unsaturated hydrocarbon chain substituent. The peptide - ionic liquid conjugates disclosed herein are suitable for skin diseases such as, for example, melanoma, non - melanoma skin cancer, diabetic foot ulcer, venous leg ulcer, pressure ulcer, acne, candidiasis, cellulitis, dermatophytosis, erysipelas, folliculitis, impetigo, psoriasis, rosacea, or eczema (atopic dermatitis), etc., because they exhibit strong activity, bactericidal - type action, anti - inflammatory action and immunomodulatory action against both Gram - positive and Gram - negative antibiotic - sensitive strains or multi - drug resistant clinical isolates, as well as an action of inducing collagen production. This application further discloses a topical composition containing the peptide - ionic liquid conjugate and its use.

Description

Technical Field

[0001] The present application relates to peptide - ionic liquid (PIL) conjugates for the prevention and / or treatment of skin diseases, topical compositions containing the same, and their use.

Background Art

[0002] Ionic liquids (ILs) are mainly known for their potential role as "green solvents", but are becoming increasingly attractive as task - specific ILs, organic salts that can be easily customized and tuned for a variety of specific purposes. Combining organic cations with organic or inorganic anions offers infinite possibilities, enabling the generation of ILs [1] with diverse structural, physical, and chemical properties that can meet the requirements of various fields such as materials science [2], biotechnology [3], or biomedicine [4]. Furthermore, by utilizing bioactive ions, it is possible to generate ILs [5] that exhibit related bioactivities, such as anti - cancer agents [6], anti - malaria agents [7], and antibacterial agents [8]. ILs with broad - spectrum activity against both Gram - negative and Gram - positive bacteria, as well as ILs with anti - biofilm activity, have been reported [9]. Thus, especially in the world where there is a shortage of effective antibiotics against Gram - negative bacteria, ILs are emerging as attractive alternative substances to combat antibiotic resistance. Furthermore, many ILs have attracted attention as skin penetration enhancers

[10] and are particularly attractive for topical application. ILs with both antibacterial activity and skin penetration - promoting effects may be very useful for the treatment of skin diseases including infected wounds, as demonstrated recently in an in vivo biofilm - infected wound assay where ILs were able to kill 95% of bacteria

[11] .

[0003] Among these, the prevalence of diseases such as diabetes, peripheral vascular disease, or immobility due to bedridden status has been increasing along with the average lifespan, and is often associated with problems such as diabetic foot ulcers (DFU) and other skin diseases such as complicated skin and soft tissue infections (cSSTI)

[12] . Although most cSSTIs are caused by multidrug-resistant (MDR) bacteria of the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) group, a high prevalence of fungi in chronic wounds has also been reported, which is related to both the healing time and the formation of mixed biofilms with bacteria. Cladosporidium spp. and Candida spp., mainly C. albicans and C. parapsilosis, have been reported to be the most prevalent fungi in cSSTI [13,14]. The burden of cSSTI increased during the COVID-19 pandemic because it discouraged elderly patients from seeking medical treatment in healthcare facilities

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

[0004] In cases specific to cSSTI, treatment requires debridement or incision and drainage, in addition to antibiotic therapy. Guidelines for the treatment of cSSTI recommend administering a systemic antibiotic effective against methicillin-resistant Staphylococcus aureus (MRSA) strains, which are one of the MDR pathogens spreading in healthcare facilities, and then implementing an antibiotic treatment program based on culture evaluation for final treatment

[16] . If this fails, amputation emerges as the last resort to avoid life-threatening sepsis.

[0005] Resistance to available antibiotics is increasing rapidly and is now widespread among many different species of Gram - positive and Gram - negative bacteria. Similarly, fungal infections are becoming more difficult to treat because resistant strains are emerging and spreading among pathogenic fungi.

[0006] Peptide - based antibacterial agents such as daptomycin, a cyclic lipopeptide, offer clinicians options for dealing with MDR bacteria in the hospital environment, but daptomycin is only active against Gram - positive species, including MRSA (17). However, most cSSTIs are polymicrobial, including Gram - negative bacteria and fungi, and new options are urgently needed to address the post - antibiotic era (18). Fungal colonization of non - healing wounds has been traditionally overlooked (14), but Kalan et al. have emphasized the importance of identifying not only bacterial but also fungal species involved in cSSTIs, namely Cladosporidium spp. and Candida spp., including C. albicans and C. parapsilosis (19) (14).

[0007] The healing process of skin diseases is often delayed or even inhibited due to pathophysiological characteristics associated with aging and / or diseases, such as high oxidative stress, neuropathy, angiopathy, weak tissue oxygenation, chronic inflammation, weak immune response, etc. This scenario is further exacerbated by the establishment of microbial pathogens that rapidly form biofilms on the wound bed and are refractory to the action of most current antibiotics (20). In such cases, effective treatment of skin lesions must, in most cases, suppress infection while promoting rapid and accurate healing. In this regard, collagen, as a structural protein derived from the extracellular matrix (ECM), plays an important role in important steps of wound healing and closure (21). Since collagen is an endogenous protein, it is considered a desirable component for the development of wound dressings / biomaterials with biocompatibility and biodegradability (22). In addition to collagen itself, peptides derived from / inspired by collagen, such as cryptic collagen peptides (23), have also been considered as potential substances that can promote cell migration and proliferation, which can induce new collagen production in fibroblasts and, as a result, accelerate wound healing (24). This has motivated the development of so-called cosmeceutical peptides (CPs) and their derivatives, which are topically applied for the treatment of skin diseases and act by promoting skin regeneration. CPs are small peptide fragments resulting from the proteolysis of ECM macromolecules such as collagen or elastin and have potential in various biomedical applications including cosmetics (25, 26). For example, "pentapeptide-4" (or PP4, amino acid sequence KTTKS) is a widely studied CP, derived from type I human collagen, and is the smallest peptide sequence known to possess a strong ability to stimulate ECM (collagen and fibronectin) production (27, 28). The N-palmitoylated form of KTTKS, known as "palmitoyl pentapeptide-4" or Matrixyl® (registered trademark), is used in the cosmetics industry for its ability to cause skin rejuvenation / anti-wrinkle effects and is presumably related to its collagen production-inducing action (29, 30).

[0008] It should be noted that, as described above, by appropriately selecting the peptide to be conjugated and the ionic liquid building block, various products tailored to specific local applications can be developed, ranging from severe non-healing skin ulcers (such as diabetic foot ulcers, pressure ulcers, venous leg ulcers, etc.) to usually mild bacterial or fungal skin infections (acne, atopic dermatitis, candidiasis, cellulitis, dermatophytosis), in addition to postoperative wounds, general injuries and burns. Furthermore, considering that peptide-based antibacterial agents are widely associated with other effects such as immunomodulatory, antioxidant, anti-aging, and antitumor effects, the products of the inventors of the present application are likely to be applicable to the topical treatment of autoimmune / inflammatory, aging, and cancerous skin diseases such as psoriasis, atopic dermatitis, cutaneous erythematosus, impetigo, skin aging, and melanoma, regardless of the presence or absence of secondary infections.

[0009] Recently, it has been noted that PP4 may promote skin regeneration in the context of skin lesions. That is, since this CP has no antibacterial action, conjugating the KTTKS sequence with an antibacterial peptide (AMP) results in a chimeric peptide that exhibits (i) antibacterial activity against reference bacteria and MDR bacteria of clinical isolates, (ii) antibiofilm action, and (iii) collagen production-inducing action comparable to Matrixyl (registered trademark) (32).

[0010] When the aforementioned chimeric peptide is further N-terminally modified with an imidazolium-based ionic liquid, a similarly potent antibacterial construct with improved stability against enzyme-mediated modification is obtained (31). In fact, ILs are becoming very attractive in biomedical applications due to their unique physicochemical properties, low cost, and high structural diversity that allows for the facile synthesis of a wide variety of ILs that can be easily tailored to meet specific requirements such as broad-spectrum activity against bacteria (4) and fungi (33). Recently, alkylimidazolium-based ILs have been proposed as an alternative antibacterial treatment for cSSTIs specialized for Gram-positive pathogens (34). In addition, several ILs have been found to improve the skin permeability of drugs containing ceftazidime (11), an antibiotic with low water solubility and low skin permeability (10, 35, 36, 37).

[0011] The paper "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 the cosmetic pentapeptide-4) with an ionic liquid methylimidazolium attached to the N-terminus, and its biological applications as an antibacterial, antibiofilm, and antifungal agent. This conjugate showed much improved stability against modification via tyrosinase while retaining the activity against multi-drug resistant clinical isolates of Gram-negative bacteria and the antibiofilm action against resistant clinical isolates of Klebsiella pneumoniae of the unmodified parent chimeric peptide (3.1-PP4).

[0012] The literature "Disclosure of a Promising Lead to Tackle Complicated Skin and Skin Structure Infections: Antimicrobial and Antibiofilm Actions of Peptide PP4-3.1" (38) describes an analog (MeIm-PP4-3.1) in which an ionic liquid methylimidazolium is conjugated to the N-terminus of peptide PP4-3.1 (composed of cosmetic pentapeptide-4 and antimicrobial peptide 3.1), as well as its biological applications as an antibacterial, antibiofilm, and antifungal agent. The conjugated peptides showed activity in all biological assays, but the unmodified parent chimeric peptide (PP4-3.1) stood out with strong activity against Gram-positive and Gram-negative bacteria, including MDR clinical isolates, and three clinically relevant Candida fungi, and its overall performance was superior to that of MeIm-PP4-3.1.

[0013] Both documents describe peptide analogs having antibacterial, antibiofilm, and antifungal activity in vitro, and show N-terminal modifications of chimeric peptides combining a sequence for cosmetics and a sequence for antibacterial use. Nevertheless, these documents do not describe peptide analogs containing a quaternary imidazolium moiety with a long hydrocarbon chain substituent, nor is there any demonstration in the reports in the literature regarding the significant collagen biosynthesis inducing activity of the peptide derivatives. Also, considering the well-known direct antibacterial action, the peptide analog sequences reported in these two documents contain long and highly cationic host defense peptide sequences. However, many host defense peptides lack the rapid and potent skin regeneration ability obtainable with cosmeceutical peptides, and most are too long (more than 10 amino acids) for efficient skin administration and transdermal administration (39) as well as cost-effective production on an industrial scale. Furthermore, it has been reported that host defense peptide-resistant strains of Staphylococcus aureus, the most common bacterial pathogen in infected skin lesions, have an increased membrane surface charge, and the efficiency of the initial peptide-bacterial electrostatic interaction prior to the bactericidal action of the peptide due to destabilization of the bacterial membrane is reduced (40). This means that constructs based on previously developed host defense peptides may induce the selection of resistant microbial strains. On the other hand, the new antibacterial constructs are derived from small CPs rather than highly cationic host defense peptides and thus have a low potential to induce resistance.

[0014] A review of ionic liquids as skin permeation enhancers is disclosed in Gomes et al.'s '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, but no technical information relevant to the present invention is disclosed.

[0015] 'Safety Assessment of Tripeptide-1, Hexapeptide-12, their Metal Salts and Fatty Acyl Derivatives, and Palmitoyl Tetrapeptide-7 as Used in Cosmetics' by Bergfeld et al., Cosmetic Ingredient Review, 2014, is a study on the safety of using peptides in cosmetics, but it does not touch on the antibacterial effects of peptides and does not disclose technical information related to the present invention.

[0016] '“Clicking” an Ionic Liquid to a Potent Antimicrobial Peptide: On the Route towards Improved Stability' by Gomes et al., International Journal of Molecular Sciences, vol. 21, n. 17, 2020, does not disclose cosmeceutical peptides up to 10 amino acids and is a 16 - amino - acid antibacterial peptide containing both antibacterial and cosmetic motifs within the sequence. What the present invention differs from this literature is that it covalently attaches an ionic liquid to a cosmeceutical peptide that itself has no antibacterial effect without relying on the antibacterial peptide motif.

[0017] The present invention only refers to small (up to 10 amino acids) CPs that are already widely used (and patented) as active ingredients in cosmetics but have no essential antibacterial action and thus have no examples of use as topical antibacterial agents. In this context, the characteristic of the PIL conjugate referred to in the present invention is not merely the sum of the properties inherent in each of the two individual building blocks, because simply mixing them (instead of binding them through chemical conjugation) results in a mixture that has bacteriostatic rather than bactericidal action. Furthermore, the chemical modification of the peptide analogs in the above two documents (31, 38) is carried out only at the N-terminus, while in the PIL conjugate of the present invention, one or more IL building blocks are attached to either or both of the N-terminus or the amino acid side chain (e.g., azide-lysine side chain). The chemical conjugation of IL to the peptide by insertion into the amino acid side chain has not been reported so far.

Summary of the Invention

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

[0019] In one embodiment, the cosmeceutical peptide is selected from cosmeceutical peptides selected from any of SEQ ID NOs: 1 - 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 acids of the cosmeceutical peptide.

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

[0023] In one embodiment, the conjugation between the cosmeceutical peptide and the ionic liquid occurs on the N-terminus of the amino acid of the cosmeceutical peptide and on the side chain of the amino acid.

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

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

[0026] In one embodiment, the topical composition is for use in the prevention and / or treatment of skin diseases.

[0027] In one embodiment, the skin diseases are severe non-healing skin ulcers such as diabetic foot ulcers, pressure ulcers, venous leg ulcers; mild bacterial or fungal skin infections such as acne, atopic dermatitis, candidiasis, cellulitis, dermatophytosis, erysipelas, folliculitis, impetigo, scabies; autoimmune / inflammatory diseases, aging and cancerous skin diseases such as melanoma, non-melanoma skin cancer, psoriasis, cutaneous erythematosus, contagious impetigo, skin aging, and melanoma, regardless of the presence or absence of secondary infection, treatment of postoperative wounds, general injuries or burns.

[0028] (Summary) The present application relates to a peptide-ionic liquid (PIL) conjugate suitable for the prevention and / or treatment of skin diseases.

[0029] The peptide - ionic liquid conjugates described in this specification are a new type of future active pharmaceutical ingredient (API) suitable for topical compositions for preventing and / or treating skin disorders.

[0030] The present invention relates to a conjugate of a cosmeceutical peptide (CP) containing up to 10 amino acids and at least one ionic liquid, which forms a PIL conjugate.

[0031] As an example, for the direct coupling of an alkylimidazolium - based IL and a non - antibacterial pentapeptide - 4 having SEQ ID NO: 1 (cosmeceutical peptide with up to 10 amino acids: KTTKS), in order to understand whether a new type of peptide - based construct that shows collagen - production - inducing and antibacterial effects can be obtained despite not having an antibacterial peptide (AMP) motif, three different alkylimidazole - based ILs were chemically modified and an alkyne moiety necessary for subsequent coupling with different azide derivatives of PP4 was introduced. As reported and discussed herein, seven different IL - PP4 conjugates were prepared and studied for their antibacterial, antifungal, and collagen - production - inducing properties.

[0032] These conjugates show strong activity against both antibiotic - sensitive and multi - drug - resistant clinical isolates of Gram - positive and Gram - negative bacteria belonging to the so - called "ESKAPE" group of pathogens. Notably, these antibacterial activities are maintained in simulated wound fluid, suggesting effective action in an actual wound bed. Furthermore, their in vitro collagen - production - inducing effects are comparable to or stronger than those of Matrixyl®. Overall, IL - PP4 exhibits three functions in vitro: antibacterial, antifungal, and collagen - production - inducing. These findings provide a solid basis for advancing IL - PP4 conjugates as promising leads for the development of topical prevention and / or treatment methods for skin diseases.

[0033] Further research is envisioned to incorporate the PIL conjugate into appropriate nanopharmaceuticals to reduce toxicity, improve resistance to proteolysis, and retain the active pharmaceutical ingredient at the intended site of action (wound bed, lesion).

[0034] The main advantages of the PIL conjugate of the present invention are as follows: 1. The rapid skin regenerative ability by CP, which is already used in cosmetics for advanced skin care, and the safety of use on human skin; 2. Cost-effective production and the small size of CP (up to 10 amino acids), which favorably acts on skin penetration ability and eliminates the need to use important / expensive skin delivery and transdermal delivery promotion methods (such as electroporation, iontophoresis, sonophoresis, microneedles, etc.); 3. The fact that CP is already produced on an industrial scale according to current Good Manufacturing Practice (cGMP); 4. The antibacterial ability and skin permeation ability of widely reported ILs, i.e., imidazolium-based and pyridinium-based ILs, but not limited to these. For example, cetylpyridinium chloride is a bactericidal IL used in mouthwashes, toothpastes, throat / nose sprays, and has both bactericidal action and skin penetration ability (41); 5. The possibility of using rapid and simple chemoselective conjugation between CP and IL building blocks by a click chemistry approach, i.e., copper(I)-catalyzed azide-alkyne coupling (CuAAC), but not limited to this approach; 6. The potential to expand the scope of therapeutic (and cosmetic) applications beyond the management of skin infections and wound healing (i.e., topical use for autoimmune skin diseases such as psoriasis and eczema), as it can provide new products that can not only reflect the sum of the characteristics of each building block but also be finely tuned to exhibit new characteristics (such as immunomodulation).

[0035] To facilitate the understanding of the present application, figures representing preferred embodiments are attached to the annex, which are not intended to limit the technology disclosed in this specification.

Brief Description of the Drawings

[0036]

Figure 1

Figure 2

Figure 3

Mode for Carrying Out the Invention

[0037] Next, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, these are not intended to limit the scope of the present application.

[0038] The present invention relates to a peptide - ionic liquid conjugate of a cosmeceutical peptide (CP) containing up to 10 amino acids and at least one ionic liquid, which is a conjugate forming a PIL conjugate.

[0039] In one embodiment, the cosmeceutical peptide is selected from, but not limited to, PP4 (SEQ ID NO: 1), KVK, PKEK (SEQ ID NO: 2), GEKG (SEQ ID NO: 3), GHK, copper tripeptide - 1, manganese tripeptide - 1, palmitoyl tripeptide - 1, tripeptide - 5, palmitoyl tripeptide - 5, tetrapeptide - 3 (SEQ ID NO: 4), pentapeptide - 3 (SEQ ID NO: 5), pentapeptide - 18 (SEQ ID NO: 6), hexapeptide - 11 (SEQ ID NO: 7), acetyl hexapeptide - 3 (SEQ ID NO: 8), hexapeptide - 10 (SEQ ID NO: 9), hexapeptide - 12 (SEQ ID NO: 10), palmitoyl hexapeptide - 12, acetyl octapeptide - 1 (SEQ ID NO: 11), SA1 - III (SEQ ID NO: 12), and liposponjin.

[0040] In one embodiment, at least one ionic liquid is C1 - C 18Selected from, but not limited to, any ionic liquid containing a saturated or unsaturated hydrocarbon chain substituent. In one embodiment, the ionic liquid is selected from a variety of ionic liquid families, namely pyridinium (e.g., cetylpyridinium), imidazolium (e.g., 1-alkyl-3-methylimidazolium), phosphonium (e.g., trihexyltetradecylphosphonium), but not limited to these ionic liquid families, or choline ionic liquids.

[0041] Conjugation between a cosmeceutical peptide and at least one ionic liquid occurs at the N-terminus of the amino acid and / or the side chains of specific amino acids in the peptide sequence, namely lysine, arginine, histidine side chains, but not limited to these.

[0042] In one embodiment, the PIL-conjugate is for use in the treatment and / or prevention of skin diseases, ranging from severe non-healing skin ulcers such as diabetic foot ulcers, pressure ulcers, venous leg ulcers, to mild bacterial or fungal skin infections such as acne, atopic dermatitis, candidiasis, cellulitis, dermatophytosis, erysipelas, folliculitis, impetigo, rosacea, etc., regardless of the presence or absence of secondary infections, to autoimmune / inflammatory diseases, aging and cancerous skin diseases such as melanoma, non-melanoma skin cancer, psoriasis, cutaneous erythematosus, contagious impetigo, skin aging, and melanoma. It can also be applied to the treatment of postoperative wounds, general injuries and burns.

[0043] This application also relates to a topical composition comprising at least one PIL conjugate.

[0044] In one embodiment, a topical composition containing a PIL-conjugate is for use in the treatment and / or prevention of skin diseases ranging from severe non-healing skin ulcers such as diabetic foot ulcers, pressure ulcers, and venous leg ulcers to usually mild bacterial or fungal skin infections such as acne, atopic dermatitis, candidiasis, cellulitis, dermatophytosis, erysipelas, folliculitis, impetigo, and rosacea, and including melanoma, non-melanoma skin cancer, psoriasis, cutaneous erythematosus, contagious impetigo, skin aging, and autoimmune / inflammatory diseases, aging and cancerous skin diseases such as melanoma, regardless of the presence or absence of secondary infections. It can also be applied to the treatment of postoperative wounds, general injuries and burns.

[0045] In one embodiment, the topical composition further comprises nanoparticles including solid lipid nanoparticles, nanostructured lipid carriers (liposomes or polymer nanoparticles) but not limited thereto, and / or at least one hydrogel including polysaccharide-based hydrogels or poly(lactic-co-glycolic acid)-based hydrogels but not limited thereto.

[0046] It has been previously demonstrated (32) that it is possible to produce a chimeric peptide having both antibacterial action and collagen production-inducing action by combining the amino acid sequence of AMP and the amino acid sequence of the well-known non-antibacterial cosmeceutical peptide PP4. Furthermore, it has been shown that when imidazolium IL is coupled to the N-terminus of the chimeric peptide via a CuAAC "click" approach, such strong antibacterial activity is maintained and peptide resistance to enzyme-mediated modification is conferred (31).

[0047] Based on these findings, the study disclosed in the present application investigated whether two types of activities, antibacterial activity and collagen production-inducing activity, are maintained by removing the AMP sequence and directly coupling at least one IL to the amino acid sequence of the cosmeceutical peptide PP4.

[0048] The findings disclosed in this application strongly suggest that direct conjugation between IL and other cosmeceutical peptides containing up to 10 amino acids would have the same properties as those observed in the examples disclosed herein.

[0049] In the following experimental examples, the PP4 peptide is referred to by its amino acid sequence, i.e., KKTKS.

[0050] The antibacterial activity of the new construct, IL-KKTKS, represented herein was evaluated against reference strains, and based on the results obtained (shown in the Examples section), several structure-activity relationships (SARs) could be advanced as follows: (i) the IL insertion site, the covalent graft was either at the N-terminus of the KTTKS sequence or on the side chains of Lys1 / Lys4, and (ii) the length of the alkyl substituent of the imidazole ring, which varied between 1 carbon (methyl or Me), 14 (tetradecyl or C14), and 16 (hexadecyl or C16). Thus, the antibacterial activity increased as (i) the length of the alkyl substituent of the IL moiety increased [KTTK(C14Im)S vs KTTK(C16Im)S], and (ii) decreased in all conjugates with methyl-substituted imidazolium IL, regardless of other structural features.

[0051] Furthermore, the MIC values were also measured for the parent building blocks KTTKS and [C16 M1Im][Br], as well as their non-covalent equimolar mixtures shown as KTTKS:[C16 M1Im][Br] (1:1), to evaluate the importance of covalent conjugation. The non-covalent mixture KTTKS:[C16 M1Im][Br] (1:1) showed MIC values similar to those of [C16 M1Im][Br] alone, confirming that the IL building block was the main cause of the activity observed in the mixture, as expected.

[0052] Interestingly, when comparing the MIC values of the non-covalent mixture KTTKS: [C16M1Im][Br] (1:1) with those of the covalent conjugates KTTK(C16Im)S and C16Im-KTTKS, the covalent conjugation is clearly beneficial for the activity against Gram-negative bacteria, but less so against Gram-positive bacteria. Furthermore, while the non-covalent mixture is bacteriostatic with MIC values against Gram-positive bacterial species, the covalent conjugates are bactericidal at these concentrations. These results suggest that the antibacterial activity of the covalent conjugates KTTK(C16Im)S and C16Im-KTTKS is regulated not only by the IL building blocks but also by the conjugation with the peptide. Therefore, biophysical studies will be conducted to further explore the mechanism of action of the IL-KTTKS conjugate. The KTTK(C16Im)S and C16Im-KTTKS conjugates also showed strong activity against MDR clinical isolates of Gram-positive and Gram-negative bacteria and exhibited higher activity than the reference antibiotic ciprofloxacin.

[0053] This is a relevant finding considering that the three clinical isolates tested are bacterial species belonging to the "ESKAPE" pathogen group, which includes life-threatening nosocomial infection pathogens, namely Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. The ability of "ESKAPE" pathogens to escape from the action of currently available antibiotics is one of the major threats in modern medicine, and there are few effective options left worldwide, especially for Gram-negative bacteria.

[0054] Furthermore, these conjugates were found to retain (C16Im-KTTKS) or slightly decrease (KTTK(C16Im)S) antibacterial activity against S. aureus in simulated wound fluid (SWF). These preliminary observations using SWF that mimics wound exudate suggest that IL-peptide conjugates, especially C16Im-KTTKS, may be more stable in the wound environment compared to analogs where the N-terminus of the peptide is not protected, which is relevant.

[0055] The IL-KTTKS conjugates described herein also showed interesting antifungal properties, particularly against C. parapsilosis. This is one of the most common species among Candida other than C. albicans and is considered an important nosocomial pathogen of concern as its involvement in cases of sepsis and cSSTI has been reported. From this perspective, the antifungal activity of the conjugates was evaluated, and the observed MIC values were low, at 2.4 μM and 2.7 μM against C. parapsilosis and 4.7 μM and 5.7 μM against C. albicans.

[0056] Furthermore, as observed in the antibacterial activity assays, non-covalent mixtures and the parent IL were more potent against Candida spp. than the IL-KTTKS conjugate. This suggests that the antifungal activity of the covalent conjugate is regulated not only by the IL building blocks but also by conjugation with the peptide (which has no antifungal activity). Therefore, further biophysical studies will be conducted to shed light on the possible mechanism of action of the IL-KTTKS conjugate against Candida species.

[0057] Evaluating the cytotoxicity of the IL-KTTKS conjugate is important in itself to check for selectivity, but also because of the toxic effects often associated with ILs, depending on factors such as the length of the cationic alkyl chain and the specific ions used.

[0058] The covalent equimolar mixture of parent IL [C16 M1Im][Br] and its peptide, KTTKS:[C16 M1Im][Br] (1:1), showed significant toxicity against the human cell lines tested. Thus, covalent conjugation of IL to peptides confers antibacterial activity on peptide building blocks that otherwise have no such activity and reduces cytotoxicity compared to the parent IL.

[0059] For conjugates C16Im-KTTKS and KTTK(C16Im)S, the activity of inducing collagen production by human dermal fibroblasts in vitro was further evaluated. Both compounds were shown to be equivalent to the standard cosmetic Matrixyl® and to be more active than the control. No significant difference was observed between the two conjugates, suggesting that changing the side chain of Lys4 has no effect on the ability of the peptide to induce collagen production.

[0060] Overall, the findings regarding this example are unprecedented and surprising. By advancing several PIL conjugates, C16Im-KTTKS and KTTK(C16Im)S, it was confirmed that they have antibacterial, antifungal, and collagen production-inducing activities in vitro, and the latter actually rivals the cosmetic ingredient Matrixyl® based on the KTTKS peptide. Furthermore, the insertion site of IL does not significantly affect the overall in vitro properties of the conjugate [C16Im-KTTKS vs KTTK(C16Im)S], but N-terminal conjugation seems to better maintain the antibacterial action of the conjugate in SWF and improve collagen synthesis by human dermal fibroblasts.

[0061] As a further study, it is envisioned to incorporate PIL into nanopharmaceuticals and / or hydrogels, which would reduce toxicity, improve resistance to proteolysis, and retain the active pharmaceutical ingredient at the intended site of action (wound bed, lesion). Furthermore, since the IL-KKTKS conjugate is mainly applied to the treatment of cSSTI, which is mainly a polymicrobial infection, the antibacterial activity of IL-KTTKS against polymicrobial media will be further studied. This will enable the selection of an optimal IL-KTTKS-based formulation and its progression to in vivo testing.

[0062] Considering that KTTKS and other small cosmeceutical peptides are already produced on an industrial scale as raw materials for cosmetics, these findings clarify the value of IL-CP conjugates as a promising starting point for the future development of cost-effective topical formulations for the prevention and / or treatment of skin diseases ranging from mild to severe such as cSSTI.

Example

[0063] 1. Synthesis of the target conjugate The route to the target IL-KTTKS conjugate started with the synthesis of alkynyl-modified imidazolium ILs (Figure 1-A). 1-Methyl-imidazole (Me-IM), 1-tetradecyl-imidazole (C14-Im), and 1-hexadecyl-imidazole (C16-Im) were reacted with propargyl bromide according to Hu et al. (Figure 1-A)(42), and three target imidazolium ILs of propargyl-MeIm (Pr-MeIm), propargyl-C 14 Im (Pr-C 14 Im) and propargyl-C 16 Im (Pr-C 16 Im) were obtained. The structures of these ILs were confirmed by 1 H-NMR, 13 C-NMR, and ESI-IT MS.

[0064] In parallel, derivatives of PP4 (amino acid sequence KTTKS) conveniently modified were generated by solid-phase peptide synthesis (SPPS), and diverse final IL-KTTKS conjugates (Figure 2) were obtained that differed in (a) the propargyl-imidazolium building block used, (b) the insertion site 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, first the PP4 sequence was assembled according to steps ii and iii of Figure 1-B, and the conveniently protected lysine (Fmoc-Lys(Boc)-OH) or azido-lysine (Fmoc-Lys(N3)-OH) building blocks were inserted at respective positions of the sequence according to the desired site for subsequent introduction of the imidazolium moiety via the "click" copper(I)-catalyzed alkyne-azide cycloaddition reaction (CuAAC). To generate peptides with modified N-termini, sequences with two native lysine residues were assembled and further extended through coupling of azidoacetic acid (step iv, Figure 1-B) to obtain a two-carbon (ethyl) spacer between the N-terminal lysine and the imidazolium moiety incorporated via CuAAC. Next, this click reaction was performed on-resin for all precursor azidopeptides using the desired propargylimidazolium IL (step v, Figure 1-B) and the CuAAC conditions previously reported by the inventors (31). After acidolytic cleavage (step vi, Figure 1-B) and purification of the thus-obtained crude conjugate by reverse-phase preparative high-performance liquid chromatography (RP-HPLC), all of the resulting IL-KTTKS conjugates were isolated with high purity (above 95%) and the molecular weights predicted by ESI-IT-MS were confirmed.

[0065] In addition to the target conjugate, the reference cosmeceutical peptide Matrixyl® (C 16 -KTTKS-OH), its C-terminal carboxamide analog (C 16-KTTKS-NH2), and native PP4 (KTTKS) were also assembled by SPPS according to the recently reported procedure (32). For the palmitoylated peptide, after assembling the entire amino acid sequence of PP4, palmitic acid (C 16 ) was coupled. Then, acidolytic cleavage from the solid support was performed to obtain the crude peptide, which was purified by RP-HPLC. The final peptide was obtained with high purity, and its molecular weight was confirmed by ESI-IT MS.

[0066] 2. In vitro antibacterial activity The antibacterial activity of the IL-KTTKS conjugate was evaluated in vitro against reference bacterial strains (American Type Culture Collection, ATCC). The minimum inhibitory concentration (MIC) was measured against Gram-positive bacteria (S. aureus, E. faecalis) and Gram-negative bacteria (E. coli, P. aeruginosa) according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) (43). The obtained MIC values are shown in Table 1. It should be noted that the reference peptides C 16 -KTTKS-NH2 and C 16 -KTTKS-OH were soluble in water and dimethyl sulfoxide (DMSO), respectively, but both precipitated when diluted in cation-adjusted Mueller-Hinton broth (MHB2), a medium recommended by the CLSI guidelines, thus preventing the measurement of the MIC values of these reference peptides. The data in Table 1 show, as expected, that the peptide KTTKS alone has no significant antibacterial activity and that the MIC values of [C 16 M1Im][Br]IL are consistent with those previously reported (44). Interestingly, all conjugates having 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 throughout the structure. On the other hand, the methyl substituent of the imidazolium ring was replaced with a tetradecyl group (C 14 ) or a hexadecyl group (C 16) Replacement with it led to an improvement in antibacterial activity, with the MIC values ranging from 6.45 to 52.6 μg / mL. Thus, antibacterial activity was added to the parent KTTKS peptide. KTTK(C 16 Im)S and C 16 Im-KTTKS showed the strongest antibacterial activity and reflected two different conjugation positions. Therefore, both of these peptides were further investigated by measuring their MIC against S. epidermidis, S. pyogenes (both Gram-positive), and K. pneumoniae (Gram-negative). They were chosen because of their high abundance on the skin (S. epidermidis)(45), their association with cSSTI (S. pyogenes)(46 - 48), and their relationship with the so-called "ESKAPE" pathogens (K. pneumoniae)(49). The non-covalent mixture of the parent peptide KKTKS and the [C 16 M1Im][Br] ionic liquid showed MIC values equivalent to those of [C 16 M1Im][Br] alone.

[0067]

Table 1

[0068] The antibacterial activities of the best couples of conjugates, namely C16Im-KTTKS and KTTK(C16Im)S, and that of the reference antibiotic ciprofloxacin were evaluated against MDR clinical isolates of K. pneumoniae (KP010), S. aureus (SA007), and P. aeruginosa (PA004). The MIC values thus obtained are shown in Table 2, indicating that all conjugates maintained the antibacterial activity observed against ATCC susceptible bacterial strains. In this regard, these conjugates were clearly more active against MDR isolates than the reference antibiotic ciprofloxacin. For example, the MIC value of C16Im-KTTKS against SA007 was nearly 10 times higher than that of ciprofloxacin.

[0069]

Table 2

[0070] C 16 The antibacterial activities of Im-KTTKS and KTTK(C 16 Im)S were also evaluated against S. aureus (ATCC 29213) within SWF(50) to confirm whether they were maintained in the medium. The MIC values were obtained by repeating three independent experiments three times in both SWF medium and MHB medium (Table 3), and the antibacterial activity in SWF was 16 the same as that in MHB for Im-KTTKS, and it was suggested that KTTK(C 16 Im)S showed a two-fold lower MIC with a decrease.

[0071]

Table 3

[0072] 3. In vitro antifungal activity The antifungal activities of the best couples of the IL-KTTKS conjugates, their parent building blocks, and their respective non-covalent 1:1 mixtures were all evaluated against three Candida species, namely Candida albicans (ATCC 90028), Candida glabrata (ATCC 90030), and Candida parapsilosis (ATCC 22019). The MIC values were measured according to the protocol of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (51 - 53) and are shown in Table 4. KTTK(C 16 Im)S and 16 both conjugates of Im-KTTKS showed equal activity against all Candida spp., with MIC values of 2.4 - 5.4 μM. Both peptides were two-fold more active against C. parapsilosis than the other two Candida species. In relation, the non-covalent mixture KTTKS:[C16 M1Im][Br](1:1) exhibited potent activity against all Candida spp., being as potent as the parent IL alone, and both showed seven-fold the activity of the reference antifungal drug fluconazole.

[0073]

Table 4

[0074] 4. Toxicity to HFF-1 and HaCaT cells KTTK(C 16 Im)S and C 16 The cytotoxicity of the Im-KTTKS conjugate was evaluated in human foreskin fibroblasts (HFF-1) and human immortalized keratinocytes (HaCaT). The results shown in Table 5 are expressed as the conjugate concentration that causes 50% inhibition of cell growth (IC 50 ). As expected from previous reports (54), both the parent peptide sequence KTTKS and the reference cosmetic Matrixyl® (C 16 -KTTKS-OH) did not show toxicity up to 100 μM to the tested cell lines. On the other hand, the parent IL[C 16 M1Im][Br] and its equimolar mixture with PP4, KTTKS:[C 16 M1Im][Br](1:1), showed significant toxicity. Interestingly, covalent conjugation of the peptide to IL resulted in an intermediate situation where the conjugate was more toxic than the peptide alone but clearly less toxic than the IL alone or the non-covalent mixture with the peptide.

[0075] 5. In vitro collagen production The purpose of conjugating antibacterial IL with collagen production-inducing peptide was to obtain a simple construct that could exert both antibacterial and skin reconstruction effects. Therefore, for the two most excellent IL-KTTKS conjugates, their ability to promote collagen production by human dermal fibroblasts (HDF) in vitro was further tested. This was evaluated using the Sircol® kit assay method, in which the amount of newly formed collagen in the ECM precipitated in the cell culture medium plated in microplates was solubilized in acidic medium and then quantified through a collagen standard curve according to the procedure of the Sircol® kit assay method (55). The assay was performed under different conditions for comparison, namely in the presence of the reference cosmetic Matrixyl® (positive control - C 16 -KTTKS-OH), in the presence of the test conjugate KTTK(C 16 Im)S and C 16 Im-KTTKS, and in the absence of all peptides (negative control). The data shown in Figure 3 indicate that both conjugates induce more collagen production by HDF cells compared to the negative control. No significant difference was observed between the two conjugates or between the KTTK(C 16 Im)S conjugate and the reference Matrixyl®, demonstrating that the collagen production-inducing ability of Matrixyl® is not affected by introducing imidazolium IL to the Lys side chain of the peptide sequence.

[0076] Of course, this specification is not limited to the embodiments shown herein, and those with average knowledge in this field can provide many possibilities for its modification without departing from the general idea defined by the claims. The above-described preferred embodiments can clearly be combined with each other. The following claims further define the preferred embodiments.

[0077] (Sequence) SEQ ID NO: 1 = PP4 SEQ ID NO: 2 = PKEK Sequence number 3 = GEKG Sequence number 4 = Tetrapeptide-3 Sequence number 5 = Pentapeptide-3 Sequence number 6 = Pentapeptide-18 Sequence number 7 = Hexapeptide-11 Sequence number 8 = Acetylhexapeptide-3 Sequence number 9 = Hexapeptide-10 Sequence number 10 = Hexapeptide-12 Sequence number 11 = Acetyloctapeptide-1 Sequence number 12 = SA1-III Sequence number 13 = KVK Sequence number 14 = GHK Sequence number 15 = Copper tripeptide-1 = Cu(II)-GHK Sequence number 16 = Manganese tripeptide-1 = Mn(II)-GHK Sequence number 17 = Palmitoyl tripeptide-1 = C16-GHK (Palmitoyl derived from palmitic acid, CH3-(CH2)14-COOH) Sequence number 18 = Palmitoyl tripeptide-5 = C16-KVK Sequence number 19 = Palmitoyl hexapeptide-12 = C16-VGVAPG Sequence number 20 = Liposponjin = Elaidyl-KFK (Elaidyl derived from elaidic acid, HOOC-(CH2)7-CH=CH-(CH2)7-CH3)

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Claims

1. A peptide - ionic liquid conjugate for the prevention and / or treatment of skin diseases, comprising a cosmeceutical peptide containing at most 10 amino acids and at least one ionic liquid containing a C1 - C18 saturated or unsaturated hydrocarbon chain substituent.

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

3. The peptide - ionic liquid conjugate according to Claim 1 or 2, 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 Claims 1 - 3, wherein the conjugation between the cosmeceutical peptide and the at least one ionic liquid occurs at the N - terminus of the amino acids of the cosmeceutical peptide.

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

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

7. The skin diseases are severe non - healing skin ulcers such as diabetic foot ulcers, pressure ulcers, venous leg ulcers; mild bacterial or fungal skin infections such as acne, atopic dermatitis, candidiasis, cellulitis, dermatophytosis, erysipelas, folliculitis, impetigo, rosacea; autoimmune / inflammatory diseases, aging - related and cancerous skin diseases such as melanoma, non - melanoma skin cancer, psoriasis, cutaneous erythematosus, infectious impetigo, skin aging, and melanoma, with or without secondary infection, and the treatment of postoperative wounds, general injuries or burns. The peptide - ionic liquid conjugate according to any one of Claims 1 - 6.

8. A topical composition comprising at least one type of the peptide - ionic liquid conjugate according to any one of Claims 1 - 7.

9. The topical composition according to claim 8, further comprising at least one hydrogel comprising nanoparticles, solid lipid nanoparticles, liposomes or polymer nanoparticles, and / or a polysaccharide-based hydrogel or a poly(lactic-co-glycolic acid)-based hydrogel.

10. The topical composition according to any one of claims 8 to 9, comprising the peptide-ionic liquid conjugate according to any one of claims 1 to 6 for use in the prevention and / or treatment of skin diseases.

11. The topical composition according to claim 10, wherein the skin disease is a severe non-healing skin ulcer such as a diabetic foot ulcer, a pressure ulcer, or a venous leg ulcer; a mild bacterial or fungal skin infection such as acne, atopic dermatitis, candidiasis, cellulitis, dermatophytosis, erysipelas, folliculitis, impetigo, or scabies; an autoimmune / inflammatory disease, an aging and cancerous skin disease such as melanoma, non-melanoma skin cancer, psoriasis, cutaneous erythematosus, infectious impetigo, skin aging, and melanoma, regardless of the presence or absence of secondary infection, the treatment of postoperative wounds, general injuries or burns.

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