Bioinspired peptide for the treatment of melasma

Abstract

The present disclosure is a peptide (Inpén SEQ ID N ° 1) with whitening action for melasma, a cosmetic or dermopharmaceutic composition containing this peptide as active ingredient and the use of this composition in the treatment of melasma or skin hyperpigmentation, developed from a peptide bioinspired from the venom of Brazilian wasps. Melasma is an acquired chronic dermatologic disorder characterized by the emergence of hyperpigmented skin spots. Although it is a common condition, a definitive cure has not been developed so far, due to the complexity of multifactorial causes. Because it is a cosmetic composition, of topic and painless application, the present disclosure can be applied daily, with no need for a specialist in the field of aesthetics, besides not being expensive as the known to date techniques for treating melasma are. The peptide SEQ ID N° 1 acts inhibiting melanogenesis.

Description

BIOINSPIRED PEPTIDE FOR THE TREATMENT OF MELASMAFIELD OF THE DISCLOSURE

[0001] The present disclosure is a compound bioinspired from social wasps’ venom’s peptide named Inpen (SEQ ID N° 1), from Tupiguarani, a Brazilian native tribe’s language, means honey, with whitening effect; a cosmetic or demopharmaceutic composition containing this peptide as active ingredient; and the use of said composition for melasma treatment and skin spots reduction. The composition acts through melanin synthesis inhibition. Because it is a cosmetic composition, of topical, non-invasive, and painless application, it can be used daily, excluding the need for frequent visits to aesthetician clinics or medic specialist. Furthermore, it is inexpensive when compared to known melasma treatments and skin spots whitening known to date.BACKGROUND

[0002] Melasma is a general term used to refer to chronic hyperpigmentation acquired disorders that manifest on the skin through dark irregular skin spots on body parts frequently exposed to sunlight and ultraviolet radiation. The term alludes to melanin, an endogenous polymeric pigment of brown coloring present in some cutaneous cells, which biological role is photoprotection of epidermic cells against damaging effects of sun rays.

[0003] Melanin is produced in melanocytes, cutaneous cells specialized in melanogenesis, majorly located in the epidermis-dermis junction and which morphology is similar to that of dendritic cells. Melanogenesis is the biochemical pathway responsible for melanin synthesis from L-tyrosine and occurs within melanosomes, lysosome derived organelles specialized in synthesizing and storing melanin in melanocytes, in its polymeric forms: eumelanin and pheomelanin. Melanocytes can distribute produced melanin to surrounding keratinocytes through melanosome transport and transfer. Keratinocytes are the most abundant type of cells in the epidermis receiving and directing melanosomes to the perinuclear region, to form a kind of “barrier / shield” surrounding the cellnucleus, to protect the genetic material from damaging effects of ultraviolet light (UV). UV light (comprehending wavelengths of UVA, UVB, and UVC) as well as blue light (consisting of the most energetic wavelengths of visible light) are wavelengths within the solar light spectrum with enough energy to cause damage to DNA as well as other cellular and extracellular structures, both from the dermis and the epidermis (Artzi et al. The pathogenesis of melasma and implications for treatment, vol. 20,11 :3432-3445, 2021, doi: 10.1111 / jocd.14382).

[0004] Such damages can be directly and indirectly induced through two distinct mechanisms: 1) pyrimidine dimers formation and genetic mutations caused by direct absorption of radiation energy by the DNA molecule; 2) free radicals formation, such as reactive oxygen species (ROS), after the radiation interacts with other cytoplasmatic molecules, capable of damaging cellular structures as well as causing chain breakages and oxidative modifications on the DNA molecule (Rajanala et al. Melasma pathogenesis: a review of the latest research, pathological findings, and investigational therapies, vol.25, 10, 2019, doi: 10.5070 / D32510045810).

[0005] In this context, melanin acts as a redox photoprotection agent, absorbing energy from UVA, UVB and blue light rays, as well as indirectly scavenging ROS generated by UV light induced oxidative stress in the skin. The efficiency of this physiological response has a quantitative and qualitative character, once it depends on the rates of production and distribution of melanin, as well as on the produced type of melanin polymer: eumelanin has photoprotection and antioxidant actions greater than pheomelanin. However, damage is not restricted to DNA and intracellular epidermic structures: chronic exposure to UV light, mainly to the UVA range rays (which have highest skin penetrance), also results in extracellular matrix damage, basal membrane rupture and photoaging of other skin components. Still, the physiological response of some skin phenotypes can be inadequate due to eumelanin / pheomelanin ratio or due to other genetic factors resulting in damage accumulation.

[0006] Therefore, damage accumulation in given regions can result in local tissue homeostasis loss, compensating mechanisms activation, and triggers immune repair mechanisms, resulting in an environment favorable to the emergence of chronic disorders like melasma.

[0007] Melasma affects majorly women, in the age range of 30 to 40 years old. Individuals with darker skin tones, within IV-VI phototypes, exposed to intense UV radiation are also moresusceptible to the disorder. Within the various clinical characteristics presented by patients, literature highlights the following risk factors: genetic predisposition, frequent exposure to intense sun radiation, pregnancy, hormonal therapies, chronic skin inflammation, chronic drug use, thyroid disorders, phototoxic and antiepileptic medicine use, ovarian tumors, and chronic exposure to air pollution.

[0008] The main feature is the presence of irregular skin spots that vary from light to dark brown, on body areas frequently exposed to sunlight, like arms, neck, and, especially on the face. Histologically, manifests on the epidermis, dermis and dermic extracellular matrix.

[0009] Despite the availability of multimodal treatments, including various topic agents, systemic agents and laser therapies, results for melasma treatment are usually disappointing. To this date, no therapeutic approach has revealed entirely effective. At the same time, routine treatments can lead to a range of adverse effects, such as skin barrier role impairment, inflammation, post- inflammatory pigmentation, scaring and depigmentation (Wang et al. The Application of Skin Care Product in Melasma Treatment, vol. 14: 1165-1171. 2021,doi:10.2147 / CCID.S323748).

[0010] Therefore, the present disclosure consists of a treatment option for Melasma and cutaneous spots through a simple cosmetic or dermopharmaceutic composition, of topical, non-invasive and painless application, that can be applied daily and without the presence of a medical specialist, being efficient for Melasma treatment. The composition of the present disclosure also acts inhibiting the melanogenesis in a non-invasive way. Advantageously, the active ingredient of this composition, a bioinspired peptide (SEQ ID NO 1 - named Inpen), can be activated by the skin, therefore, the composition can be presented as a cream, serum, or other pharmaceutic form of topical application for easy daily use, sparing time that would have been necessary for the patient to be present at a specialized clinic and also of the linked risks. Besides being a simple dermo facial treatment, less onerous and that can be linked to higher patient adhesion when compared to medic procedures previously mentioned.

[0011] Patent database searches showed documents describing compositions for Melasma treatment comprehending peptides, for example: BR1020190118393 A2, about a composition with the Densiskin polypeptide; the document FR2944796A1 about a composition with a hydrolyzed peptide enriched with arginine, lysine and glycine residues to prevent or treat hyperpigmentation;the document US2003166570A1 describes a 13 amino acid residues derived from human hormones; documents WO20166034541, EP2990072 and WO2016034541 report inventions formed by whitening anti melanogenic peptides consisting of 3 to 15 amino acid residues with in tandem repeated 3 amino acid sequences: EPL, PLN, LNN, NNL, NLQ, LQV, QVA, VAV, AVK; documents EP3800193 and US20220169678 report a whitening composition for multiple clinical uses, characterized by 3 peptides of 6, 7 and 11 amino acid residues; documents US20120100086 and US 20120020904 report a composition formed by a hydrolyzed peptide enriched with bioactive peptides with depigmenting and whitening effects, the bioactive portion is composed by peptides with at least one aspartic acid residue, one cysteine, and one arginine; document US20240066086A1 describes a composition formed by tri and tetra peptides with melanin inhibiting agents; document EUA20230391825A1 describes an addition of a palmitate chain to the decapeptide- 12 in order to avoid transcutaneous penetration, with anti-inflammatory and antisenescence; document EUA20230312649A1 describes peptides capable of regulating melanogenesis, either inhibiting or enhancing melanin synthesis.

[0012] However, the composition as well as the peptide described by the present invention presents a different amino acid sequence of those listed above, thus consisting of an unpublished composition. The composition described in the present document acts through melanin synthesis inhibition, resulting in decreased skin spots and in melasma treatment.DESCRICAO SUCINTA DAS FIGURAS

[0013] Figure 1 illustrates the percentage of cellular viability after Inpen (SEQ ID N° 1) sample treatment.Figure 2 shows the percentage of melanocyte inhibition after Inpen (SEQ ID N° 1) sample treatment.Figure 3 illustrates the subjective efficacy results of the placebo group on clinical trials.Figure 4 illustrates the subjective efficacy results of the Inpen (SEQ ID N° 1) treated group on clinical trials.Figure 5 demonstrates the cosmetic appreciability after 30+ / - days of a placebo sample daily use on clinical trials.Figure 6 demonstrates the cosmetic appreciability after 30+ / - days of an Inpen (SEQ ID N° 1) sample daily use on clinical trials.DETAILED DESCRIPTION OF THE DISCLOSURE

[0014] The present diclosure consists of a cosmetic ou dermopharmaceutic composition containing the bioinspired peptide Inpen (SEQ ID N° 1) and its use for Melasma reduction and elimination.

[0015] he peptide of the present disclosure (SEQ ID N° 1) presents theoretical mass of 1746,90 Da (degree of purity >95%) and consists of the following amino acid sequence H-Ile-Phe-Thr- Leu-Leu-Ala-Lys-Phe-Leu-Lys-Ala-Asp-Val-Ala-Leu-NH2 (IFWLLAKFLKADVAL-NH2). It was bioinspired and developed from peptides identified on social wasp Parachar ter gus fratemus’s venom.

[0016] Other than that, for the present disclosure, the sequence resulted from exchanges of the amino acid Isoleucine for the chemically similar amino acid Leucine and vice-versa can be considered.

[0017] The formulation containing the peptide consists of water, peptide Inpen (SEQ ID N° 1) from 0,01% to 0,0001% and / or peptides obtained from the exchange of amino acid Isoleucine for Leucine and vice-versa on the SEQ ID N° 1 structure.

[0018] In vitro cytotoxicity studies were conducted, considering eucaryotic dermatologic cell cultures, as well as melanocyte melanin synthesis inhibition assays with the peptide SEQ ID N° 1. Clinical trials were conducted with volunteers (n=32, CEP / Plataforma Brazil number 6.679.740) with the goal of evaluating the safety profile (dermatologic acceptability) and subjective clinical efficacy of Inpen (SEQ ID N° 1). Besides, the cosmetic appreciability of Inpen (SEQ ID N° 1) was assessed during the clinical trials.

[0019] The technology can be better comprehended from the examples, non -limiting, that follow:INVENTION CONCRETIZATION EXAMPLES

[0020] EXAMPLE 1 - IN VITRO BIOLOGICAL ASSAYS UTILIZING EUCARYOTIC DERMIC CELL CULTURE SYSTEMS

[0021] Evaluating the in vitro cytotoxic potential of the peptide Inpen (SEQ ID N° 1) on Balb / c 3T3 clone 31 cell culture kept in DMEM (Dulbecco’s Modified Eagle’s Medium) with supplement addition, in incubator at 37OC and 5% CO2 and manipulated within a laminar flow cabinet. For this study, cells were distributed through 96 well plates on the previously described conditions.

[0022] Inpen (SEQ ID N° 1) sample preparation: Starting solution preparation conditions: the sample was diluted to a 10% (w / v) in culture medium;

[0023] Preparation of concentrations: serial dilution into 5 decreasing concentrations using a 10- fold dilution series;

[0024] Solubilized sample aspect: totally solubilized.

[0025] Control groups preparation:

[0026] Control group: supplemented cell culture medium;

[0027] Positive control group: Sodium Lauril Sulfate diluted in supplemented cell culture medium (lOOpg / ml);

[0028] Incubation and cell viability assessment: solutions containing the control, positive control and sample groups were applied to cell cultures, lOOpL per well in quadruplicate for each group, followed by 24 hours of incubation in 37OC and 5% CO2 incubator. After cells were washed, the cellular viability was assessed with neutral red indicator, using the volume of 100 pL per well. After incubation, the reaction was developed and absorbance was determined at 540 nm.

[0029] Results were assessed using the Microsoft Excell software. Control group were normalized to 100% and the viability percentage for positive control and test groups were calculated in relation

[0030] to the control. Samples are considered cytotoxic when they cause 30% ou more reduction on cell viability.

[0031] Figure 1 shows the cell viability percentage found after treatment with the sample in comparison to the control in a 10-fold dilution series between 10% and 0,001%. For Inpen (SEQ ID N° 1), no cytotoxicity was observed in any of the concentrations tested. Positive control group showed a cellular viability percentage of 36,4%.

[0032] EXEMPLE 2 - BIOLOGICAL IN VITRO ASSAYS COMPREHENDING EUCARYOTIC DERMIC CELLS SYSTEMS FOR MELANOCYTE MELANIN PRODUCTION ASSAY

[0033] In this study melanocyte cultures were employed, cultivated in DMEM (Dulbecco’s Modified Eagle’s Medium) with supplement addition, the incubator was kept at 37OC and 5% CO2 and manipulated within laminar flow cabinet. Initially, a cytotoxicity assay was performed to determine safe concentrations for whitening potential assessment.

[0034] Samples preparation: sample condition in starting solutions: sample was diluted to a 10% concentration in cellular culture medium; Solubilized sample aspect: totally solubilized; non- cytotoxic concentration assessed: 0,1%.

[0035] Control groups preparation: basal control group was done in cell culture medium and positive control group was in cellular culture medium added by Kojic acid.

[0036] On the first day after plaquing, the culture medium was removed and samples were added as described previously. For quantitative assessment, cells were trypsinized for cell clump formation. To this cell clump, a NaOH solution was added for melanin pigment extraction. Next, absorbance at 475nm was measured.

[0037] Basal control group was normalized to 100% and other groups were compared to it. Absorbance results were assessed using software Graph Pad Prism 5. Statistical analysis for groups comparison was done through One-way Anova and statistical significance level considered less than 0,05.

[0038] Melanin granule production was assessed quantitatively for assessment and comparison of the melanin synthesis inhibition between samples.

[0039] For sample efficacy analysis, the control group was normalized to 100% (±3,91) and other groups were compared to it. As positive control, Kojic acid was used for being a known whitening agent. Melanin granule count was reduced by 43,5% (±2,1) for the positive control. Inpen (SEQ ID N° 1) showed reduction of 29,33% (±4,2) on melanin granule count, statistically different from control group (p<0,001), as shown in figure 2.

[0040] EXAMPLE 3 - DERMATOLOGICAL ACCEPTABILITY ASSESSMENT

[0041] For human safety and efficacy of Inpen (SEQ ID N° 1) use evaluation, a clinical trial was performed with 32 volunteers, both male and female, from 36 to 65 years of age and phototypes II to IV, that used a product containing the peptide for 30 days.

[0042] Initial medical evaluation was performed ate the time of participants inclusion to confirm that no clinical signs uncompatible with participant inclusion were observed.

[0043] After 30 ± 2 days of product use, participants returned to the medical evaluation institution for final medical assessment of clinical signs observed and feelings of discomfort and acceptability were questioned.

[0044] Medical assessment data were registered on the research notebook. The medic was available during the entire duration of the trial for evaluation of possible adverse events.

[0045] Results were assessed as follows:

[0046] Discomfort sensations: participants were questioned about discomfort sensations felt in parallel with the clinical exam. Discomfort sensations related were described in relation to its nature (for example: burning, itching, prurience, drawing, cooling, heating, etc.); were described in relation to the intensity as: light, moderate, intense; in relation to the location; and in relation to the duration; finally, causal link to the tested product was verified.

[0047] Clinical signs: were classified according to the presence of clinical signs such as edema, papules, blisters, vesicle, erythema and nothing to declare and causal link of reactions to the product was investigated.

[0048] No participant related discomfort sensations, and no clinical signs were detected after product use. Therefore, supporting the “dermatologically tested” appeal.

[0049] EXAMPLE 4 - SUBJECTIVE CLINICAL EFFICACY ASSESSMENT

[0050] For subjective clinical efficacy assessment of this product, clinical trials were performed with 32 volunteers, both male and female, from 36 to 65 years of age, that used a product containing the peptide for 30 days.

[0051] Volunteers were assessed at the beginning of the study (DO) and after 30 ± 2 days of at home product use (D30) using the following parameters:

[0052] After 30 ± 2 days of placebo product use, 38% of participants showed improvement on region skin spots and 44% of participants showed improvement on skin uniformity (Figure 3).

[0053] After 30 ± 2 days of product containing the peptide use, as illustrated in figure 3, 69% of participants showed improvement on region skin spots and 63% of participants showed improvements on skin uniformity (Figure 4).

[0054] EXAMPLE 5 - COSMETIC APPRE CIABILITY (PARTICIPANTS OPINION)

[0055] Participants were instructed to answer a questionaire containing qustions and possible answers listed bellow, after 30 ± 2 days of investigational products use.

[0056] A cosmetic appreciability questionaire was applied. On the questionaire, some participant’s perceptions were assessed from aswers “yes” or “no”. The applied questions are listed bellow.- Did you feel a more even skin tone?- Did the product lighten skin spots?- Did the product lighten melasma spots?- Does the product have anti-melasma action?- Did the product hydrate your skin?- Did you notice better skin texture?- Did you notice better general skin quality and appearance?- Does the product deliver a good result?- Would you buy this product?

[0057] Figure 5 shows results after 30 ± 2 days of placebo product use, namely: 50% felt a more even skin tone; 50% stated that the product lightened skin spots; 44% stated that the product reduced melasma spots; 44% said that the product has anti-melasma action; 63% stated that the product hydrated the skin; 63% noticed better skin texture; 44% noticed better appearance and general skin quality; 44% said that the product delivers a good result; and 44% said they would buy this product.

[0058] Figure 6 shows results after 30 ± 2 days of Inpen (SEQ ID N° 1) use, namely: 81% felt a more even skin tone; 88% stated that the product lightened skin spots; 81% stated that the product reduced melasma spots; 81% said that the product has anti-melasma action; 88% stated that the product hydrated the skin; 88% noticed better skin texture; 81% noticed better appearance and general skin quality; 88% said that the product delivers a good result; and 44% said they would buy this product.

Claims

AMENDED CLAIMS received by the International Bureau on 22 August 2025 (22.08.2025)1. A peptide consisting of SEQ ID NO: 1.

2. Use of the peptide according to claim 1 in the preparation of a cosmetic or dermopharmaceutical composition for the treatment of melasma and / or the attenuation of skin spots.

3. A cosmetic or dermopharmaceutical composition comprising:• from 0.0001% to 0.01% by weight of the peptide of claim 1,• water, and• 1,3 -butylene glycol.

4. A cosmetic or dermopharmaceutical composition comprising:• from 0.0001% to 0.01% by weight of the peptide of claim 1,• 1,3 -butylene glycol, and• a cosmetic base serum.

5. Use of the cosmetic or dermopharmaceutical composition according to claim 3 or 4 for topical application to the skin in the treatment of melasma or skin spots, by inhibiting melanogenesis.

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