Abstract

In response to the growing consumer demand for highly effective anti-aging products, Science4Beauty LLC obtained a recombinant conotoxin μ-TIIIA in E. coli strain and developed a novel cosmetic formulation. The main active ingredient, recombinant conotoxin μ-TIIIA, has the ability to block Nav 1.4 voltage-gated sodium channels, thereby reducing muscle contractions and the appearance of dynamic wrinkles. Company comprehensive approach aims to address multiple aspects of skin ageing, offering a product – the MIORELAXANT MAGIC cream, not only provides immediate improvements in skin texture and wrinkle appearance but also significantly prolongs the action of botulinum toxin. The 60 volunteers suffering from facial wrinkles around the lateral corners of the eyes, randomly divided into two groups – patients who underwent treatment with botulinum toxin in the area of the circular muscles of the eyes, they used the tested cosmetic products (Study Cream and Placebo) for 6 months after the treatment. The conducted research indicates a clinically significant use of MIORELAXANT MAGIC primarily in enhancing/extending the effects of aesthetic treatments using botulinum toxin and reducing skin seborrhea. Moreover, this product shows strong, positive and statistically significant effect on improving hydration, elasticity and skin tone, which justifies its use in a wide group of the population.

Keywords:Recombinant conotoxin TIIIA; Recombinant peptide; Anti-wrinkle effect; Botulinum toxin; Skin elasticity; Skin colour; Skin seborrhea

Introduction

Conotoxins are found in nature in venom of sea snails of the Conidae family and are used to immobilize and paralyses prey. These snails inhabit coral reefs, and most are found in tropical and subtropical waters such as the South China Sea, the coast of Australia and the Pacific Ocean. There are about 700 species of cone snails and all of them are venomous. They are usually classified into three groups according to their feeding habits: worm hunters, mollusk hunters, and fish hunters. Conotoxins have evolved to target specific ion channels, receptors, and transporters in prey, leading to rapid immobilization. The primary mechanism of action of conotoxins involves the blockade or modulation of ion channels, particularly voltage-gated sodium, potassium, and calcium channels, as well as ligand-gated ion channels such as nicotinic acetylcholine receptors. Conotoxins are small peptides present in the cone snail venom that have evolved with the purpose to capture prey and defend against predators. They are a structurally and functionally highly diverse class of bioactive molecules that are highly selective for various ligand- and voltage-gated ion channel subtypes. Conotoxins have proven to be a useful tool for studying the ligand-binding domain of ion channels. Moreover, they can be used as therapeutic agents to target specific ion channels and glucose transporters. Ion channels are specialized cell membrane proteins that transportations across the membrane. Ion channels are widely expressed in both excitable cells (neurons, muscles) and other cells (renal tubules, epithelial cells) [1-3].

Conotoxins themselves, on the other hand, are microproteins usually less than 40 amino acid residues in length, which enables their heterologous, recombinant expression. The formation of (often multiple) disulfide bonds in a highly precise arrangement is particularly common among conotoxins and generally serves to stabilize the main bioactive conformation, critical for potency and selectivity, but also to enhance proteolytic resistance [4]. Venom of each individual cone snail contains up to 100 different peptides, each of which has a distinctive role when injected into the target. It is the cumulative effect of the individual peptides that makes the venom lethal to the prey. Based on their specificity, conotoxins have been grouped into several classes. Omega-conotoxins target and block voltage-sensitive Ca2⁺ channels, thereby inhibiting the neurotransmitter release. Alpha- and psi-conotoxins target and block nicotinic acetylcholine (ACh) receptors, causing ganglion and neuromuscular blockade. Mu (μ)- and delta-conotoxins block voltage- sensitive Na⁺ channels in muscle. Kappa-conotoxin blocks voltage-sensitive K⁺ channels, and these can also cause increased neuronal excitability. Gamma-conotoxin targets voltage-sensitive, non-specific cation channels, and sigma-conotoxin antagonizes the serotonin 5HT3 receptor [5,6]. Conotoxins are extremely specific biological probes that offer scientists a tool to understand and distinguish between closely related receptors. The simplicity of conotoxins has made them valuable in advancing neuroscience research, and consequently in development of drugs and cosmetic applications. Many diseases, such as epilepsy, schizophrenia, Tourette syndrome, Parkinson’s disease and sclerosis, are associated with malfunctioning of ligand- and voltage-gated channels. Conotoxins have proven to be very promising because they are relatively small, potent, selective antagonists and agonists of specific cell membrane channel proteins, which can be used as great cosmetic and pharmaceutical tools [7,8].

The μ-TIIIA conopeptide, derived from the venom of the marine cone snail Conus tulipa exhibits myorelaxant activity through specific blockade of skeletal muscle Nav (Nav1.4) channels. By inhibiting these channels, TIIIA prevents nerve impulses that lead to muscle contractions, producing a local muscle relaxant effect similar to botulinum toxin (Botox). This mechanism is particularly beneficial in reducing the appearance of dynamic wrinkles, such as those around the eyes and forehead, which are caused by repeated muscle movements. This property can be used in cosmetics for daily anti-wrinkle therapy [9-11]. In summary, conotoxin TIIIA represents an exciting frontier in cosmetic science, offering a novel approach to skin care and anti-aging. Its unique mechanism of action, coupled with advancements in cosmetic formulation, could pave the way for new and improved beauty products. Given this background, Science4Beauty LLC (S4B) company developed a brand-new formulation of cream with TIIIA conotoxin as the key ingredient, registered under the name Miorealaxant Magic. The active ingredient – TIIIA conotoxin was obtained by genetic engineering methods in the prokaryotic expression system of Escherichia coli. TIIIA peptide is a proprietary ingredient obtained in the scientific laboratory of the S4B company. S4B company has filed a patent application for the method of producing TIIIA conotoxin in E. coli strain (EP 23195429.8) and for the MIORELAXANT MAGIC cream formulation (EP 23195443.9). In response to consumer demand for highly effective anti-aging products, and based on the established physiology of skin ageing, S4B obtained a recombinant conotoxin, and developed a new cosmetic product to provide immediate and long-term antiaging effects. In addition to main active ingredient – recombinant conotoxin TIIIA, low molecular weight hyaluronic acid, and natural oils were included to provide extreme hydration and nourishment of the skin; for longer-term effects, we combined the biomimetic peptides, flax seed extract, inulin and arbutin, which lightens discolorations. Full ingredient list is included in Table 1. The goal of these studies was to demonstrate that the MIORELAXANT MAGIC cream significantly prolongs the action of botulinum toxin.

Materials and Methods

This prospective comparative study was performed between July 2023 and March 2024 and was approved by the Bioethics Committee at the District Medical Chamber in Gdańsk on June 27, 2023 (protocol code KB-G2/2023). All the patients who participated in this study signed informed written consent forms. The active substance of the MIORELAXANT MAGIC cream is TIIIA conotoxin, a 22- amino acid peptide produced by genetic engineering in Escherichia coli cells using recombinant DNA technology obtained in the scientific laboratory of the S4B company.

Cream preparation protocol

Two cream formulas were prepared for the sake of the study. First the complete formula of cream containing conotoxin (Study Cream) which was the differentiation, and second - placebo formula (Placebo) which contained all the ingredients except conotoxin. Full ingredient lists were included in Table 1. The cream is prepared by hot emulsification method - well known method to prepare O/W emulsions.

Treatment Protocol

The study involved 60 volunteers (58 women and 2 men, aged 30 to 68; average age 43.5) suffering from facial wrinkles around the lateral corners of the eyes, randomly divided into two groups: Study group (30 patients: 29 women and 1 man; average age 44.6 years): patients who underwent treatment with BOTOX (20 units in total) in the area of the circular muscles of the eyes in the area responsible for the occurrence of wrinkles around the lateral corner of the eye, then they used the tested cosmetic product for 6 months after the treatment; Control group (30 patients: 29 women and 1 man; average age 42.4 years) - patients who will undergo treatment with BOTOX (20 units in total) (Allergan Pharmaceuticals Ireland, Castlebar Road, Westport, County Mayo, Ireland) in the circular muscles of the eyes in the area responsible for the occurrence of wrinkles around the lateral corner of the eye and then They will use a placebo for 6 months after the procedure.

Inclusion criteria

To qualify patients for the study, the following inclusion criteria were established:
• age 18 - 80 years,
• good general health
• recognition of the presence of facial wrinkles around the lateral canthus of the eye
• consent to participate in the study and acceptance of its course.

Exclusion criteria

To ensure the safety and accuracy of clinical trials involving MIORELAXANT MAGIC cream, it was essential to establish exclusion criteria. These criteria helped to identify and exclude participants who may be at risk of adverse reactions or whose participation could skew the study results. Here are the exclusion criteria for this studies: allergy (hypersensitive) to botulinum neurotoxin type A complex or any of the other ingredients of this medicine (human albumin complex, sodium chloride), infection in the planned area of the procedure, skin and connective tissue diseases (e.g. systemic lupus, collagenopathy, cutaneous porphyria), active or frequently recurring Herpes simplex infection (herpes), taking medications that may affect the skin condition (including antibiotics tetracyclines, immunosuppression, i.e. cortisol and its derivatives, anticoagulants, i.e. dipyridamole and coumarin derivatives) for a period of up to 6 months back, immunodeficiency diseases (including active HIV infection)
• pregnancy and breastfeeding
• hypertension
• unregulated diabetes
• vitiligo or disorders of melanin production i.e. hypermelanosis,
• tattoos on areas undergoing treatment
• constant intake of anti-inflammatory drugs,
• tendency to scarring..

After the doctor’s visit included in the study, all study participants received 20 Allergan units of the drug BOTOX by intramuscular injection in the area of facial wrinkles around the lateral corners of the eyes (10 units per side), in accordance with the manufacturer’s guidelines included in the summary of product characteristics. Immediately before this treatment, all patients underwent cytometric measurements of facial skin hydration and elasticity (Multi Skin Test Center 1000, manufacturer: Courage + Khazaka, Germany) and medical photographic documentation was made (FotoMedicus; manufacturer: ELFO, Poland). Cutometric measurements and medical photographic documentation were repeated on the 180th day after the procedure. On the day of the visit, patients received six packages of MIORELAXANT MAGIC (intended for use twice a day for a period of six months) depending on the randomly determined group membership - the product contained or did not contain the active ingredient (conotoxin). All study participants were asked to send survey data and self-taken facial photographs (without facial expressions and while smiling) via a dedicated ICT system after 30, 60, 90, 105, 120, 135, 150 and 165 days of using the product. It was planned for the 180th day after the procedure follow-up visit to the medical center. The patient was not informed about belonging to the study or control group in order to blind the sample.

Analysis of skin reaction

The skin reaction was assessed based on the International Contact Dermatitis Research Group (ICDRG) consensus [12-15] (Figure 1).

Analysis of survey research

Analysis of the course of the study

The study participants were obliged to provide feedback on the course of the study in the following areas:
• occurrence of swelling lasting up to 7 days (possible answers: did not occur; 1-2 days; 3-4 days; 5-
• 6 days; 6-7 days);
• occurrence of swelling longer than 7 days (possible answers: none; very small swelling; small swelling; medium swelling; large swelling);
• occurrence of skin redness (possible answers: did not occur; 1-2 days; 3-4 days; 5-6 days; 6-7 days);
• occurrence of skin itching (possible answers: did not occur; 1-2 days; 3-4 days; 5-6 days; 6-7 days);
• occurrence of skin burning (possible answers: did not occur; 1-2 days; 3-4 days; 5-6 days; 6-7 days);
• occurrence of a feeling of skin numbness (possible answers: did not occur; 1-2 days; 3-4 days; 5-6 days; 6-7 days).
• In addition, study participants added their own observations in the form of any comments. Answers in the basic part of the study were provided by study participants after 30 days of using the product.

Satisfaction analysis

Analysis of satisfaction survey results: patient satisfaction with the anti-wrinkle effect (without mimics) (Table 2)

The study participants were obliged to provide feedback on their satisfaction with the use of the tested product in terms of:
• anti-wrinkle effect (without facial expressions) on a scale from 1 to 5 (where 1 = I’m not satisfied; 2 = I am rather dissatisfied with; 3 = I am moderate satisfied; 4 = I am satisfied; 5 = I am very satisfied) (Figure 2);
• anti-wrinkle effect (when smiling) on a scale from 1 to 5 (where 1 = no jI am satisfied; 2 = I am rather dissatisfied with; 3 = I am moderate satisfied; 4 = I am satisfied; 5 = I am very satisfied);
• assessment of improvement in skin quality (elasticity) on a scale from 1 to 5 (where 1 = worsening; 2 = no change; 3 = moderate improvement; 4 = satisfactory improvement; 5 = definite improvement); (Table 3)

• assessment of skin tone improvement on a scale from 1 to 5 (where 1 = no improvement; 2 = slight improvement; 3 = moderate improvement; 4 = satisfactory improvement; 5 = definite improvement);
• assessment of the reduction of seborrhea on a scale from 1 to 5 (where 1 = did not decrease seborrhea; 2 = 2 = slight improvement; 3 = moderate improvement; 4 = satisfactory improvement; 5 = seborrhea has significantly decreased) (Figure 3)

In addition, study participants added their own observations in the form of any comments. Responses in the basic part of the study were provided by study participants after 30, 60, 90, 105, 120, 135, 150, 165 and 180 days of using the product.

Cutometric evaluation

The cutometric evaluation was performed using the Courage + Khazaka Multi Skin Test Center MC1000 device under standard conditions of temperature (22-23°C) and air humidity (55-60%). The procedure for preparing the patient for the examination has also been standardised (make-up removal without the use of alcohol, performed no later than an hour before the examination). The measurements were performed by appropriately trained personnel in the area of the skin of the treated area and the use of the tested formulations. Skin elasticity and hydration were assessed on a scale of 1-100 (where 1: worst possible result - 100: best possible result). Three point-by-point measurements were taken each time to obtain an average result. The results were recorded in the observation card and then subjected to statistical analysis.

Expert rating

The expert opinion was conducted by an independent team of five researchers who are experts in the field of dermatology and/ or aesthetic medicine with a minimum of five years of experience. The experts were shown photos of patients taken immediately before the procedure and after 30, 60, 90, 105, 120, 135, 150, 165 and 180 days of using the product. The experts were informed about the fact that the patient had received an intramuscular injection of botulinum toxin in the treatment area and about the time point of taking the photos, but were not informed about the use of the tested formulations by the study participants. The assessment was made in the form of an anonymous survey in which experts assessed the aesthetic effect of using botulinum toxin around the lateral corner of the eye on a scale from 1 to 5 where 1 = no noticeable result; 2 = slight result; 3 = moderate result; 4 = good result; 5 = very good result).

Results

Results of skin reaction analysis

Among the patients belonging to the research group (who underwent intramuscular administration of 20 units of BOTOX to the orbicularis oculi muscles and then using MIORELAXANT MAGIC cream with recombinant conotoxin TIIIA) a single episode of questionable reaction was recorded lasting no longer than 24 hours. Among the patients belonging to the control group (who underwent intramuscular administration of 20 units of BOTOX to the orbicularis oculi muscles and then using MIORELAXANT MAGIC cream without conotoxin) a single episode of mild reaction was recorded lasting no longer than 72 hours. It is not possible to clearly attribute the cause of the observed skin reactions regarding the triggering factor (administration of botulinum toxin or use of the test subject product), however, taking into account the transient nature of the episodes and their occurrence in during the first 24 hours after botulinum toxin injection, that is performing this procedure seems to be a more likely cause of the occurrence recorded episodes.

Survey research analysis

Analysis of the course of the study

Among the patients belonging to the research group (who underwent intramuscular administration of 20 units of BOTOX to the orbicularis oculi muscles and then using MIORELAXANT MAGIC cream with conotoxin) noted:
• a single episode of skin redness lasting 1-2 days;
• two episodes of numbness in the area of the procedure lasting 1-2 days.

Among the patients belonging to the control group (who underwent intramuscular administration of 20 units of BOTOX to the orbicularis oculi muscles and then using MIORELAXANT MAGIC cream without conotoxin) researchers noted:
• a single episode of swelling localised in the area of the procedure lasting 3-4 days;
• three episodes of skin itching in the area of the treatment lasting 1-2 hours days;
• two episodes of skin burning in the area of the treatment lasting 1-2 hours days;
• a single episode of numbness in the area of the procedure lasting 1-2 days;
• a single episode of numbness in the area of the procedure lasting 6-7 days.

It is not possible to clearly attribute the cause of the observed reactions to establish the triggering factor (administration of botulinum toxin or use of the tested product), however, taking into account the transient nature of the episodes and their occurrence over time the first 24 hours after botulinum toxin injection, and also specific character associated with several days of swelling or a feeling of numbness, that is performing this procedure seems to be a more likely cause of the occurrence recorded episodes.

Satisfaction analysis

Analysis of satisfaction survey results: patient satisfaction with the anti-wrinkle effect (without mimics) (Table 2), (Figure 2)
Analysis of satisfaction survey results: patient satisfaction with the anti-wrinkle effect (during mimics) (Table 3), (Figure 3),
(Image 1, Image 2, Image 3, Image 3, Image 4, Image 5, Image 6) Analysis of satisfaction survey results: skin elasticity (Table 4),
(Figure 4)
Analysis of satisfaction survey results: skin color (Table 5), (Figure 5)
Analysis of satisfaction survey results: skin seborrhea (Table 6), (Figure 6)
Analysis of satisfaction survey results: experts satisfaction (PGAIS) (Table 7), (Figure 7)
Analysis of cutometric measurements: skin elasticity (Table 8), (Figure 8)
Analysis of cutometric measurements: skin hydration (Table 9), (Figure 9)

Discussion

The impact of the tested formulations on the anti-wrinkle effects the impact of the tested formulations on satisfaction with the effects of the treatment using of botulinum toxin in the area of wrinkles of the lateral angle of the eye showed significant results statistically differences between the research group (using the tested formulation containing conotoxin) and the control group (used the tested conotoxin-free formulation). In terms of assessing the anti-wrinkle effect at rest (without facial expressions), the difference with satisfaction 30 days after the procedure was rated by patients as: level of +3% in favor of the conotoxin formulation, and the difference in the level of satisfaction increased over time reaching a value of +16.8% in favor of the formulation containing conotoxin. In terms of assessing the anti-wrinkle effect while making facial expressions, the difference with satisfaction 30 days after the procedure was rated by patients as: level of +11.2% in favor of the formulation with conotoxin, and the difference in the level of satisfaction increased over time, reaching a value of +23.3% in favor of the formulation containing conotoxin. These results are confirmed by the assessment of a team of independent experts who made the overall difference aesthetic effect of administering botulinum toxin to the wrinkles of the lateral corner of the eye rated +3.8% in favor of the conotoxin formulation 30 days after the treatment and +39% in favor of the conotoxin-containing formulation after 180 days of use tested formulations.

The impact of tested formulations on skin elasticity

Both tested formulations, those containing conotoxin and those without conotoxin showed positive statistically significant results on impact on skin elasticity both in satisfaction surveys and in machine (cutometric) measurements. Average patient satisfaction with the impact on skin elasticity of both tested formulations was approximately 4.1, which is slightly higher than the answer “satisfactory improvement”. Detailed elasticity measurements showed statistically significant increase in measurement values by +8.8% in the case of the formulation containing conotoxin and by 8.0% in the case of the conotoxin-free formulation after 180 days application. The impact of tested formulations on skin hydration both tested formulations, those containing conotoxin and those without conotoxin showed positive statistically significant results on impact on skin hydration in machine (cutometric) measurements. Detailed skin hydration measurements showed an increase in measurement values by +15.6% in the case of a formulation containing conotoxin and by 17.1% in the case of a formulation conotoxin-free after 180 days of use.

The impact of tested formulations on skin tone

Both tested formulations received an average rating above neutral in patients’ evaluation satisfaction rating, but only in the case of the tested formulation containing conotoxin, the result showed statistical significance. In this group, the result statistically significantly different from the neutral satisfaction assessment appears from the 60th day use of the tested formulation, reaching the highest significance on days 120 and 150.

Impact of tested formulations on skin seborrhea

The group testing the formulation containing conotoxin showed a tendency to increase satisfaction in relation to seborrhea control and its statistical significance from the 105th day using the formulation, achieving an average satisfaction level of 3.8/5 after 180 days of application. In the group testing the formulation without conotoxin, no statistically significant results on the influence of the tested formulation on the ant seborrheic effect were found.

Conclusions

The tested MIORELAXANT MAGIC product showed a very high safety profile on skin reaction tests and in the results of the test during the first 30 days application. Isolated episodes of skin reactions, affecting less than 5% patients appear to be caused by botulinum toxin injection rather than use tested product. Also, rare episodes of itching, burning and numbness of the skin should be associated with intramuscular administration of the drug, especially considering the fact that they occurred in most cases in the control group, i.e. in patients using it MIORELAXANT MAGIC cream without conotoxin. In studies on the effect of the conotoxin contained in MIORELAXANT MAGIC on effectiveness of botulinum toxin administration to reduce wrinkles of lateral angle of the eye, a high, statistically significant effect of the conotoxin contained in the tested substances to increase satisfaction with the treatment performed and to prolongation of the positive aesthetic effect of botulinum toxin injection was demonstrated. The patients’ own observations were confirmed by the assessment of an independent panel of experts.

The tested formulations, regardless of the conotoxin content, showed positive and statistically significant impact on improving skin elasticity. The patients’ high rating remained confirmed by independent machine measurements. No dependencies between content conotoxin in the tested formulations or its absence and the level of effect indicates the impact of the MIORELAXANT MAGIC product base (not active ingredient in the form of conotoxin) used in this matter. A similar, although higher impact of the tested formulations was demonstrated in the context of changes in skin hydration. Again, as with elasticity changes, the obtained results indicate the impact of the base of the tested product, not conotoxin as an active ingredient. The perception of improvement in skin tone was shown to be positive in the treatment group tested formulation containing conotoxin, which indicates the importance of this ingredient in improving patients’ skin tone. In assessing the impact of the tested formulations on the control of skin seborrhea, there is no doubt a significant effect of the conotoxin contained in MIORELAXANT MAGIC on the reduction of skin seborrhea was demonstrated. This effect does not occur in the control group using the formulation without conotoxin. The conducted research indicates a clinically significant use of MIORELAXANT MAGIC primarily in enhancing/extending the effects of aesthetic treatments using botulinum toxin and reducing skin seborrhea. Moreover, this product shows strong, positive and statistically significant effect on improving hydration, elasticity and skin tone, which justifies its use in a wide group of the population.

Author Contributions

Investigation - DM; AMP; MJ; PK; AP; AM; TC; AS; Methodology - DM; AMP; MJ; AS; AM; WG; AP; KD; Writing - DM; MJ; PK; AP; Review & editing - TC; AMP; DM; PK; MJ;

Institutional Review Board Statement

The study was approved by the Institutional Review Board (or Ethics Commitee) of the Bioethics Committee at the District Medical Chamber in Gdańsk (protocol code KB-G2/2023) on 27 June 2023.

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