Bioinspired peptide and use for the treatment of periorbital hyperpigmentation (dark circles)

Abstract

The disclosure is a peptide (Iaká (SEQ ID No1) with lightening action for periorbital hyperpigmentation, cosmetic or dermopharmaceutic composition containing this peptide and the use of this composition in the treatment of dark circles. Periorbital hyperpigmentation is a condition that does not add any risks to the patient's health state, however, it can cause aesthetic discomforts and appearance of tiredness. Dark circles can be formed due to anatomy or fat accumulation; due to a very thin skin thickness, allowing visualization of subcutaneous vessels; and, furthermore, due to melanin accumulation in the region. Due to this disclosure being a cosmetic composition, of topic non-invasive application, it can be applied daily without the requirement for an aesthetic specialist, besides not being as expensive as the currently known techniques for dark circles treatment. The peptide acts inhibiting melanogenesis.

Description

BIOINSPIRED PEPTIDE AND USE FOR THE TREATMENT OF PERIORBITAL HYPERPIGMENTATION (DARK CIRCLES)FIELD OF THE DISCLOSURE

[0001] The present disclosure is a compound bioinspired from social wasps'venom peptide named laka, from Tupi-Guarani, a Brazilian native tribe’s language, means ’’clarity” (SEQ ID N° 1 ) with lightening effect, a cosmetic or dermopharmaceutic composition containing this peptide as active ingredient and the use of said composition for periorbital hyperpigmentation treatment or attenuation. The composition acts via 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 dark circles treatments known to date.BACKGROUND

[0002] “Dark circle” is the popular term used to characterize a condition in which the periorbital region (the skin around the eyes) presents darkened appearance when compared to the face’s skin tone. The condition is multifactorial and can be caused by an excess of pigments accumulated in the periorbital region; anatomy of tear grooves; infraorbital fat herniation; thinning of periorbital skin; the presence of blood vessels; and skin sagging and wrinkles.

[0003] Melanin synthesis can be a response to inflammatory processes in the skin, dark circles formed by this process are common in patients with atopic dermatitis or allergic contact dermatitis. In these and in other situations in which an individual habitually rubs the orbital region, post-inflammatory hyperpigmentation can develop. Furthermore, aesthetic procedures within the eye region can lead to inflammatory processes followed by melanin granules accumulation. For these cases, recommended treatment includesanti-inflammatory and / or anti melanogenic application to the affected regions, that can include either infraorbital or eyelid region.

[0004] For cases in which the dark circles are caused by shadows from a deep tear groove, skin sagging, wrinkles, or infraorbital fat herniation, recommended treatments include the application of filling injections, the most popular being hyaluronic acids in appropriate vehicles, or laser treatments which objective is to reduce or eliminate wrinkles and sagginess.

[0005] Finally, in cases of thinness of periorbital skin, this layer becomes translucid, and the darkened effect is due to the visualization of blood vessels and / or muscular structure of the orbital region. In the same way, when there is an increased number of blood vessels in the orbital region, the skin presents darkened appearance. In these cases, the complexity of the treatment is in diminishing the transparency of the skin without increasing its pigmentation.

[0006] Even though dark circles don't represent risks to an individual’s health, they can result in a tired and / or sick appearance. Furthermore, the increasing concern with aesthetic factors stimulates the market and industry to search for practical and cost- effective treatments with better results each time.

[0007] Noted the different causes, it is important to highlight that the treatment for each patient must consider the individual causes. Most treatments nowadays require the patient to go to aesthetician clinics for multiple sessions, sometimes continuously, to obtain satisfactory results. Some auto application products, such as lightening acids or periorbital massagers are recommended in specific cases, however, dealing with a condition in such a delicate area, such applications require an elevated level of care to avoid undesired effects.

[0008] Studies show that there is no specific demographic group with higher tendency to present periorbital hyperpigmentation, affecting in similar way different ages, genders and ethnic groups.

[0009] Accordingly, the present disclosure consists of a treatment option utilizing a compound that is bioinspired in a peptide present in the venom of wasps from the Parachartergus fraternus species with anti-inflammatory action. This peptide was designed to deliver lightening action, confirmed through in vitro and in vivo tests, however, the action mechanisms and pathways through which the peptide acts were not yet elucidated. It is known that the peptide acted in the inhibition of melanogenesis, process that can be related to the appearing of dark circles.

[0010] Patent database searches showed documents describing compositions for dark circles treatment comprehending peptides, such as document BR 11 2022 014944 1 A2, which describes a peptide that acts inhibiting SNARE complex formation; document BR 112023022421-7 A2 discloses about a composition of recombinant collagen and peptides formed by the lather’s fragments in a formulation applied as skin care; document BR 11 2023 024114 6 A2, refers to a cosmetic formulation containing a peptide, vitamin C, a moisturizing agent and an antioxidant agent for senescence signs control, such as freshness loss, sagginess, wrinkles and spots, also promoting dark circles lightening. Other dark circles treatments were also found, however, these do not refer to peptide uses: document BR 10 2020 020434 A2 refers to a non-toxic composition, in which the active ingredient is a floral extract for treating conditions that affect the eye region, such as dark circles and periorbital edema; in a similar way, document US8741357B2 is about a cosmetic or dermopharmaceutic composition containing Euglena gracilis algae extract; also, document CH718846A2 describes a composition that associates biochemical components for treatment and prevention of skin spots on the face and scalp, including dark circles.

[0011] However, the composition as well as the peptide presented in the current document presents a different sequence of amino acids from those found during the background search, thus representing an unpublished to composition to date. The composition described in this document acts through melanin synthesis inhibition, possibly resulting in dark circle attenuation.BRIEF DESCRIPTION OF THE FIGURES

[0012] Figure 1 illustrates the percentage of cellular viability after laka (SEQ ID N° 1 ) sample treatment.Figure 2 shows the percentage of melanocyte inhibition after laka (SEQ ID N° 1 ) sample treatment in comparison with positive and negative controls.Figure 3 illustrates the subjective efficacy results of the placebo group on clinical trials.Figure 4 illustrates the subjective efficacy results of the laka (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 laka (SEQ ID N° 1 ) sample daily use on clinical trials.DETAILED DESCRIPTION OF THE INVENTION

[0013] The present disclosure consists of a cosmetic or dermopharmaceutic composition containing the bioinspired peptide laka (SEQ ID N° 1 ) and its use for periorbital hyperpigmentation reduction and elimination.

[0014] The peptide of the present disclosure (SEQ ID N° 1 ) presents theoretical mass of 1617.07 Da (degree of purity >95%) and consists of the following amino acid sequence H-lle-Phe-Gly-Leu-Leu-Ala-Lys-Leu-Gly-Phe-Leu-Lys-Lys-Gly-Leu-NH2(IFGLLAKLGFLKKGL-NH2). It was bioinspired and developed from peptides identified on social wasp Parachartergus fraternus’s venom.

[0015] 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. Clique aqui para inserir texto.

[0016] The formulation containing the peptide consists of water, peptide laka (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.

[0017] Clique aqui para inserir texto. 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=34, CEP / Plataforma Brazil number 6.679.726) with the goal of evaluating the safety profile (dermatologic acceptability) and subjective clinical efficacy of laka (SEQ ID N° 1). Besides, the cosmetic appreciability of laka (SEQ ID N° 1 ) was assessed during the clinical trials.

[0018] The technology can be better comprehended from the examples, non-limiting, that follow:EXAMPLES OF EMBODIMENTS OF THE INVENTION

[0019] EXAMPLE 1 - IN VITRO BIOLOGICAL ASSAYS UTILIZING EUCARYOTIC DERMIC CELL CULTURE SYSTEMS

[0020] Evaluating the in vitro cytotoxic potential of the peptide laka (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.

[0021] laka (SEQ ID N° 1 ) sample preparation: Starting solution preparation conditions: the sample was diluted to a 10% (w / v) in culture medium;

[0022] Preparation of concentrations: serial dilution into 5 decreasing concentrations using a 10-fold dilution series;

[0023] Solubilized sample aspect: totally solubilized.

[0024] Control groups preparation:

[0025] Control group: supplemented cell culture medium;

[0026] Positive control group: Sodium Lauril Sulfate diluted in supplemented cell culture medium (1 OOpg / ml);

[0027] Incubation and cell viability assessment: solutions containing the control, positive control and sample groups were applied to cell cultures, 100pL 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.

[0028] 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 to the control. Samples are considered cytotoxic when they cause 30% or more reduction on cell viability.

[0029] 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 laka (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%.

[0030] EXEMPLE 2 - BIOLOGICAL IN VITRO ASSAYS COMPREHENDING EUCARYOTIC DERMIC CELLS SYSTEMS FOR MELANOCYTE MELANIN PRODUCTION ASSAY

[0031] 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.

[0032] 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 %.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] Melanin granule production was assessed quantitatively for assessment and comparison of the melanin synthesis inhibition between samples.

[0037] 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, laka (SEQ ID N° 1 ) showed reduction of 19,2% (±0,85) on melanin granule count, statistically different from control group (p<0,001), as shown in figure 2.

[0038] EXAMPLE 3 - DERMATOLOGICAL ACCEPTABILITY ASSESSMENT

[0039] For human safety and efficacy of laka (SEQ ID N° 1) use evaluation, a clinical trial was performed with 34 volunteers, both male and female, from 18 to 34 years of age and phototypes II to IV, that used a product containing the peptide for 30±2 days.

[0040] Initial medical evaluation was performed ate the time of participants inclusion to confirm that no clinical signs incompatible with participant inclusion were observed.

[0041] 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.

[0042] 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.

[0043] Results were assessed as follows:

[0044] 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, dragging, cooling, heating, etc.); were classified 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.

[0045] 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 to the product was investigated.

[0046] No participant related discomfort sensations, and no clinical signs were detected after product use. Therefore, supporting the “dermatologically tested” appeal.

[0047] EXAMPLE 4 - SUBJECTIVE CLINICAL EFFICACY ASSESSMENT

[0048] For subjective clinical efficacy assessment of this product, clinical trials were performed with 34 volunteers, both male and female, from 18 to 34 years of age, phototypes II to IV, that used a product containing the peptide for 30±2 days.

[0049] 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:

[0050] After 30±2 days of placebo product use, 41 % of participants showed improvement on the infraorbital fat pads, 35% of participants showed improvement on dark circles, and 47% of participants showed improvement on hydration of the region, as shown in figure 3.

[0051] After 30±2 days of using product containing the peptide SEQ ID N°1 , 65% of participants showed improvement on infraorbital fat pads, 65% of participants showed improvement on dark circles, and 61 % showed improvement on hydration of the region, as shown in figure 4.

[0052] EXAMPLE 5 - COSMETIC APPRECIABILITY (PARTICIPANTS OPINION)

[0053] Participants were instructed to answer a questionaire containing qustions and possible answers listed bellow, after 30 ± 2 days of investigational products use (placebo and product containing the SEQ ID N° 1 peptide).

[0054] The volunteers’ perception was assessed through a questionaire which questions could be answered with “yes” or “no”:- Did the product leave a more even skin tone on the periorbital area?- Did the product lighten dark circles?- Did the product brighten the periorbital skin?- Did the product soften tiredness signs arround the eyes?- Did you notice reduction on swelling around the eyes?- Did the product increase hydration on your periorbital skin?- Did you notice improvement on skin texture?- Does the product deliver a good result?- Would you buy this product?

[0055] Figure 5 shows the results after 30±2 days of placebo product use, namely: 53% felt a more even skin tone on the periorbital region; 53% stated that the product lightened dark circles; 59% identified that the periorbital region was brighter; 53% felt improvement on tiredness signs around the eyes; 53% noticed reduction on swelling around the eyes; 59% felt an increase on periorbital skin hydration; 53% stated that the skin texture did improve; 53% said that the product delivers a good result; and 47% stated that they would buy the product.

[0056] Figure 6 shows the results after 30±2 days of use of the product containing SEQ ID N°1 peptide, namely: 82% felt a more even skin tone on the periorbital region; 88% stated that the product lightened dark circles; 94% identified that the periorbital region was brighter; 88% felt improvement on tiredness signs around the eyes; 82% noticed reduction on swelling around the eyes; 94% felt an increase on periorbital skin hydration; 76% stated that the skin texture did improve; 82% said that the product delivers a good result; and 82% stated that they would buy the 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 for dark eye circles treatment.

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 for dark eye circles treatment, acting as melanogenesis inhibitor.

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