In vitro, ex vivo, and clinical evaluation of anti‐aging gel containing EPA and CBD

Skin aging manifestation, such as coarse wrinkles, loss of elasticity, pigmentation, and rough‐textured appearance, is a multifactorial process that can be exacerbated by air pollution, smoking, poor nutrition, and sun exposure. Exposure to UV radiation is considered the primary cause of extrinsic skin aging and accounts for about 80% of facial aging. Extrinsic skin aging signs can be reduced with demo‐cosmetic formulations. Both cannabidiol (CBD) and eicosapentaenoic acid (EPA) have been previously suggested as potent active dermatological ingredients.


Aims:
The objective of the current research was to evaluate the compatibility of both agents in the prevention and treatment of skin aging.First, the impact of both agents was assessed using standard photoaging models of UV-induced damage, both in vitro (HaCaT cells) and ex vivo (human skin organ culture).Then, a clinical validation study (n = 33) was performed using an optimized topical cream formulation tested at different time points (up to Day 56).
Results: EPA was found to potentiate the protective effects of CBD by reducing the secretion of prostaglandin E2 (PGE 2 ) and interleukin-8 (IL-8), two primary inflammatory agents associated with photoaging.In addition, a qualitative histological examination signaled that applying the cream may result in an increase in extracellular matrix (ECM) remodeling following UV radiation.This was also evidenced clinically by a reduction of crow's feet wrinkle area and volume, as well as a reduction of fine line wrinkle volume as measured by the AEVA system.The well-established age-dependent subepidermal low-echogenic band (SLEB) was also reduced by 8.8%.
Additional clinical results showed significantly reduced red spots area and count, and an increase in skin hydration and elasticity by 31.2% and 25.6% following 56 days of cream application, respectively.These impressive clinical results correlated with high satisfaction ratings by the study participants.
Discussion and Conclusions: Collectively, the results show a profound anti-aging impact of the developed formulation and strengthen the beneficial derm-cosmetic properties of CBD-based products.

| INTRODUC TI ON
The skin is a vital homeostatic organ that copes with both its internal and external surroundings. 13][4] However, the effects of accelerated extrinsic aging can be prevented, minimized, and even sometimes reversed with the use of dermo-cosmetics formulations. 1veral physiologic changes have been linked to photoaging, including the breakdown of collagen and elastin, induction of matrix metalloproteinases (MMPs), and local inflammation.Collectively, these may cause clinical signs of aging, such as an increased area and volume of the crow's feet (peri-ocular wrinkling), fine lines and wrinkles, pigmentation, skin inflammation, and reduced skin hydration, and elasticity.
][7][8][9][10][11][12][13] For instance, Ikarashi et al. demonstrated that the topical application of 1% CBD for 14 days increased skin moisture in HR-1 hairless mice by upregulating aquaporin-3. 14In addition, CBD can reduce ROS formation in an NRF2-Hemoxigenase-1-dependent manner in keratinocytes, 15 a key signaling pathway in the endogenous skin defense mechanism. 16D signaling has also been linked to hair growth, and its application has been shown to improve atopic dermatitis and contact dermatitis via its anti-inflammatory and anti-pruritic effects (Reviewed recently by Baswan et al. 6 ).
Omega-3 long-chain unsaturated fatty acids have also shown beneficial cosmetic and therapeutic activities.Eicosapentaenoic acid (EPA) supplementation showed to reduce cyclooxygenase-2 activity and the concomitant generation of prostaglandin-E2 (PGE 2 ), which acts as a significant proinflammatory mediator upon UV injury. 17In addition, EPA was previously shown to reduce UVinduced IL-8 secretion in HaCaT keratinocyte cell line. 18Clinically, 10 weeks of oral supplementation of EPA in healthy volunteers attenuated the production of proinflammatory lipids formation after UVB exposure. 19llectively, both CBD and EPA showed promising cosmetic potential.The objective of the current research was to develop and assess the anti-aging efficacy of a topical cream formulation based on both active ingredients.In addition, the efficacy was evaluated in three separate systems to monitor in vitro, ex vivo, and clinical correlation to further support non-animal replacement options in the development of new formulations.CO 2. Cells were passaged routinely at 80%-90% confluency and seeded in all experiments at 500 000 cells/mL.Human skin samples were obtained from 40-to 60-year-old healthy women undergoing aesthetic abdominal surgery.Informed consent was obtained before sample collection.The experiments were conducted with the approval of the IRB Committee (#0258-19-SOR, approval protocol scrc20016; 29 June 2020).The tissue was processed and maintained in an air/liquid interface with the dermal side submerged in DMEM, as previously reported in detail. 20

| UVB-induced damage models
To investigate the in vitro and ex vivo ability of the tested ingredients, both the HaCaT cells and the human skin tissue were subjected to UVB (VL-6.M lamp, UVB mediumwave emission spectrum 280-350 nm, emission peak 312 nm, Spectroline).Prior to irradiation, the cells and tissue were moved to PBS containing 6-well plated.
Immediately after UVB stimuli (25 and 350 mJ/cm 2 , for HaCaT and skin, respectively), the tissue was applied topically 3 μg/cm 2 of placebo cream or formulation, whereas the cell culture media of the cells were added with CBD (10 μg/mL; Echo Pharmaceuticals), EPA (10 μg/mL KD pharma), or both (1:1 ratio).Sunscreen spry (SPF-15; skingard careline) and PABA (0.02 mg/mL; Merck, Israel) were used as positive control to block UVB radiation in the tissue and cell cultures, respectively.All treatments were applied after UVB stimuli for 24 h, excluding the positive control that was applied 30 min prior of irradiation.The topical formulation (used in both the ex vivo organ culture evaluation and clinically) was prepared by Echo Pharmaceuticals.Supplement to the standard base formulation (placebo), (0.1%) CBD, (0.1%) fish oil eicosapentaenoic acid (EPA), and Salvia miltiorrhiza root extract (Labiatae; 0.1%; Draco Natural Products) shown in preliminary screening to have high compatibility with the agents (data not shown).

K E Y W O R D S
anti-aging cream, cannabidiol, dermo-cosmetic, eicosapentaenoic acid, ex vivo human skin organ culture

| Epidermal and cellular viability
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Merck Israel) assay was employed in both HaCaT cells and the skin organ culture.For the latter, after treatments, the epidermal side that was in close contact with the formulation was washed with phosphate-buffered saline (PBS) and separated from the dermis (1 min., 56°C).Then, it was transferred to 96-well plates containing 150 μL of 0.5 mg/mL MTT dissolved in PBS.After 1 h of incubation, the epidermal sheets were transferred to new wells and submerged in 150 μL of isopropanol to elute the dye.Absorbance was recorded at 570 nm using a plate reader (TECAN, f200 Infinite, Switzerland).To determine HaCaT cell viability, a similar methodology was used; the medium was aspirated, 150 μL of MTT solution was added, and incubated for 1 h.The dye was eluted with an equal volume of isopropanol after the removal of the MTT solution by aspiration.

| IL-8 and PGE 2 quantification
The spent media from the cells and skin organ cultures was centrifuged, aliquoted, and stored at −80°C until use.The supernatant was evaluated with enzyme-linked immunosorbent assay (ELISA).IL-8 (Biolegend) and PGE 2 (Enzo Life Sciences) levels were quantified according to the manufacturer's instructions.

| Collagen and elastin evaluation
After treatment, skin explants were washed with PBS and fixed with 4% formaldehyde for 1 h at room temperature.The samples were transferred to 15 mL tubes containing 70% ethanol and kept at 2-8°C.The skin samples were dehydrated with increasing concentrations of ethanol and embedment.Paraffin sections were prepared, pasted on slides, and stained with Masson trichrome according to the manufacturer's specifications (Sigma-Aldrich).
Slides were mounted, and images were captured using the Zeiss inverted microscope Axio Observer 7 and the Moticam 5 + Camera.

| Clinical evaluation
The study was performed according to the Declaration of Helsinki principles.The first product application was carried out by the responsible researcher at the research center and continued at home.The protocol and test conditions were reviewed by the Internal Review Board (opinion no 6949/2021) and the standard protocol was submitted to the Ethical Commission (from January 4, 2021).Inclusion and exclusion criteria are shown in Table 1.

| Study population
Table 2 summarizes the study population.Thirty-four subjects aged 45-65 were enrolled in the study.Only one participant initiated the study (first application) but was unable to complete the study and was excluded (final n = 33).Phototype was assessed according to Fitzpatrick, established on the principle of a first 30-to 40-min sun exposure after the winter or a period without exposure of an equivalent duration whereas previous medical history was checked to evaluate skin sensitivity.

| Methods
Study participants were instructed to apply the study cream (ECHO-A-01) at Librium to their facial skin two times a day (morning and evening) and massage it in until completely absorbed (mean daily usage was 1.17 ± 0.34 g).The first application for each patient was performed at the institute in order to demonstrate the appropriate application technique.Wrinkle count, volume depth, and roughness were assessed with this method.

| Ultrasonography assessment
A Dermascan C ultrasound system (Cortex Technology) with a special modified 20 MHz ultrasound probe was used to measure facial skin (malar area).This was performed during study initiation and termination.

| VISIA-CR red areas image analysis
VISIA-CR imaging system is a clinical research, standardized imaging photographic system that takes high-resolution photos with a standard light IntelliFlash, a cross polarized flash, a parallel polarized flash, and under an ultraviolet lighting.The analysis software allows a definition of red region (Malar area), and the calculation of the areas and number of the parameters.

| Assessment of skin hydration
The measurements were performed with a Corneometer CM825 probe connected to a Cutometer dual MPA 580 (Courage & Khazaka).Hydration content is obtained by an electrometric system which measurement of capacitance.This allowed the calculation of the water dielectric constant.The measuring probe has an interdigital grid of gold-covered electrodes.It is covered by a low dielectric vitrified material.Therefore, there is no galvanic contact between the electrode and skin surface.A constant application pressure was applied on the skin surface through a spring system.
As a result of the probe design, the system (electrode, superficial parts of the stratum corneum and epidermis) behaves as a capacitor.Measurements were obtained in arbitrary units as reference to a factory standard.

| Assessment of elasticity and firmness
Skin biomechanical evaluation was performed with a Cutometer® dual MPA 580 using a 2 mm probe.This system was used to measure the elasticity of the upper skin layers with negative pressure, which deformed the skin mechanically.Negative pressure was created by (450 mbar) drawing the skin into the aperture of the probe for 2 s and later released for 2 s.Within the probe, the penetration depth is determined by a non-contact optical measuring system.The resistance of the skin to the negative pressure (firmness) and its ability to return into its original position (elasticity) are displayed as curves (penetration depth in mm/time) in real-time during the measurement.This measurement is able to retrieve information about the elasticity and mechanical properties of the skin surface.

| Assessment of efficacy (self-assessment)
The subjects answered questionnaires at the second and final visits (D28 ± 2 and D56 ± 3).Responses looked to assess cosmetic improvements and efficacy of the product.These questionnaires were pre-defined according to the category and target market of the test product.

| Statistical analysis
Statistical calculations were performed using SPSS 23 (IBM).Values are presented as average of three replicates, and standard errors of the mean (SEM) are provided.Significant differences between values were analyzed using the unpaired t-test.Difference of *p < 0.05 was set as statistically significant.

| RE SULTS AND D ISCUSS I ON
Recent studies suggest that both CBD and EPA may possess favorable dermo-cosmetic action. 9,21To evaluate if the combination of the two may protect from skin aging, in vitro, ex vivo, and clinical evaluations were performed.First, a standard photoaging model was employed to evaluate UVB-induced damage in vitro. 22,23 were measured (Figure 1).These biomarkers were selected as both were shown to play a key in UVB-induced inflammation. 24The results show that CBD alone was capable to attenuate both immune mediators.These results parallel previous studies that demonstrated the anti-inflammatory actions of CBD. 9 Importantly, EPA supplementation potentiated the effects of CBD, and increased the inhibitory potential.Interestingly, prior studies have shown that EPA can reduce inflammatory processes and also reduce COX-2 expression. 96][27][28] Therefore, the anti-inflammatory effects shown above were validated in the ex vivo human skin culture.First, the impact on epidermal viability was assessed.As shown in Figure 2, UVB reduced epidermal viability by ~40%.Importantly, combined CBD and EPA topical formulation salvaged the tissue viability, which was comparable to the level of naïve not-irradiated tissue.This effect was not due to non-active ingredients in the formula, as clearly shown by the lack of effect in the placebo formula (Figure 2A).Correlating to the in vitro results, the topical formulation reduced both cytokines dramatically.A qualitative histological analysis was also performed and revealed a marked increase in the ECM, restoring its morphology to a normal architecture (Figure 2D) in the same experimental systems.Both wrinkle lines and volume adjacent to the lateral epicanthal folds found significant improvement.Collectively, these results showed a clear reduction in aging signs.
In addition to the AEVA evaluation, Sub-Epidermal Low echogenic band (SLEB) was also monitored by ultrasound as an additional method to monitor aging.Nearly all patients showed high satisfaction with the product results based on questionnaire responses (Figure 5).The results obtained from the in-vitro, ex vivo and clinical evaluations suggests a significant anti-aging of the CBD-EPA cream.CBD-based production has gained increasing interest in the last few years due to its favorable cosmetic activities. 29Most notably, the combination of CBD with EPA produces a potent synergistic effect.Inflammatory responses induced by UV are mostly achieved through various mediators, such as nitric oxide (NO) and prostaglandin E2 (PGE 2 ).PGE 2 is a well-known inhibitor of collagen synthesis in fibroblasts. 30,31erefore, UV-induced PGE2 may contribute to collagen deficiency in chronically UV-irradiated skin.Aging-associated increase in PGE 2 production in the skin may have pathophysiologic implications for the aging phenomena, such as skin wrinkling and aging-associated changes.Additional cytokines and chemokines involved in aging, such as IL8, promote skin inflammation.IL8 levels are increased in aged skin and following pollution. 32The results support the notion that the EPA-CBD cream anti-aging signs correlate with its anti-inflammatory action. 33CBD-enriched ointment has also been clinically evaluated, showing reduce skin inflammation and increase skin hydration. 34Of note, topical application of CBD has also emerging medicinal properties, which includes improvement in atopic dermatitis, reduced pruritis, anti-acne action as well as improved wound-healing (reviewed in Baswan et al.6).The combination approach taken here CBD was recently evaluated by Few et al. 10 In their study, CBD, combined with 0.2% retinol was found to be effective in several age-related markers, including an improvement in surface roughness and wrinkles manifestation.

| SUMMARY
The results of this study demonstrate the efficacy of the newly developed gel in reducing age-related manifestations.In addition, a high correlation was found between the in vitro, ex vivo, and clinical outcome.These provide evidence that an easy transformation can be obtained from the results in the human skin organ culture model

CO N FLI C T O F I NTER E S T S TATEM ENT
Dr. Rozenblat is a consulate of Eco pharmaceuticals.The company had sponsored the manuscript but did not direct the research nor had any impact on data acquisition and conclusions.

2 | 2 . 1 |
MATERIAL S AND ME THODS In vitro and ex vivo evaluation 2.1.1 | Cell and skin organ cultures Unless specified, all materials were from biological industries (Beit-HaEmek).HaCaT cells were grown and maintained in Dulbecco's modified eagle medium (DMEM; high glucose) supplemented with 10% fetal bovine serum and 1% Penicillin-Streptomycin-Amphotericin B Solution in a humidified incubator at 37°C and 5% Three-D images of skin topography were obtained by a combination of stereo cameras and a fringe projection system (AEVA-HE TA B L E 1 Inclusion and exclusion criteria.Inclusion criteria • Female subjects aged 45-65 with signs of aging (wrinkles and/or fine lines) and skin Fitzpatrick phototype II-IV.• Willingly agree to provide written consent and comply with study requirements.Exclusion criteria • Cutaneous marks on the experimental areas, which could interfere with the assessment of skin reactions (pigmentation disorders, scars, thick or excess hair, freckles and nevi in great quantity, sunburn or inflammatory conditions of the facial skin, including atopic dermatitis, psoriasis, and rosacea) • Known allergy or hypersensitivity to any of the ingredients in the test product or one of its excipients.• Received any facial aesthetic treatments or oral retinoids within the last 6 months or anticipated during the study period.• Excessive UV light (natural or artificial) 1 month prior to inclusion.• Pregnant or breastfeeding or planning to become pregnant during the study period.• History of skin cancer.• Presence of medical conditions or receiving medical treatment and/or medical or surgical history of conditions, which, in the opinion of the investigator, could compromise the safety of the study participant or affect the outcome of the study.• Undergone a bilateral or unilateral mastectomy with lymph node removal or a bilateral axillary lymph node removal within the last year.• History of immune deficiency or autoimmune disease.• History of asthma or diabetes and currently receiving treatment.device;Eotech, France).A fringe standard was projected on the skin and detected by the dual cameras of the optical system.The 3D effect was calculated by the deflection in the fringes, which represent a qualitative and quantitative skin profile.The stereocombination of the camera images produced the 3D result.

F I G U R E 1
In vitro evaluation of CBD and EPA on photoaging.HaCaT cells were treated without or with CBD, EPA or both and irradiated with 25 mJ/cm 2 UVB.After 24 h, cell viability was measured by MTT (A and B).Concomitantly, PGE 2 (C and D) and IL-8 (E and F) levels were evaluated in the spent media.n = 3 (three experiments), */# p < 0.05 show significant difference in comparison to untreated control cells or UVB-stimulated cells, respectively.P.C, positive control (PABA sunscreen).
Due to the significant in vitro and ex vivo results, clinical evaluation of CBD-and EPA-containing cream was performed on 33 healthy volunteers with age-related signs.The results presented in F I G U R E 2 Ex vivo evaluation of CBD and EPA on photoaging.The skin tissues were irradiated with UVB (350 mJ/cm 2 ), washed and treated topically without or with the newly developed formula.After 24 h, epidermal viability (A), PGE2 (B), and IL-8 (C) levels and histological examination (D) were performed.n = 3 (three experiments), */# p < 0.05 show a significant difference in comparison to untreated control cells or UVB-stimulated tissue, respectively.Placebo, cream without the active compounds; P.C, positive control (sunscreen; SPF = 15).

F
I G U R E 3 CBD-EPA containing formulation reduce skin aging in a clinical trial.EVA system was employed to determine skin topography following the treatment.(A and C) depicts the results of crow's feet area, respectively.(B and D) depict volume of fine lines.E depicts a representative image of the analysis.n = 33 *p < 0.05 in comparison to the basal state at study initiation."D0, 28, 56" -day 0 (study initiation), and after 28 and 56 days.

Figure 3
Figure 3 show a gradual time-dependent improvement of wrinkles.

Figure 4
demonstrates that a dramatic improvement in SLEB was found following 56 days of the treatment.Concomitantly, F I G U R E 4 CBD-EPA formulation improves skin-aging-related manifestations.(A) SLEB analysis was employed to determine skin aging following the treatment.(B) shows the quantification of red spots and (C) depicts a representative image of a subject.Skin properties are summarized in (D and E).n = 33 *p < 0.05 in comparison to the basal state at study initiation."D0, 28, 56"-Day 0 (study initiation), and after 28 and 56 days of treatment.
VISIA-CR evaluation was quantified and revealed a 15% reduction after 56 days of treatment (Figure4Band a representative image shown in Figure4C).These clinical results correlate with the antiinflammatory action of the cream shown in the in-vitro and ex vivo evaluation.To further determine the efficacy of the cream, skin elasticity, hydration, and firmness were evaluated as well.The results are presented in Figure4D,E, demonstrating a significant improvement in each category.In addition, the clinical results demonstrate a timedependent improvement in skin elasticity, skin hydration, firmness as well in the reduction in red sots.Thus, long application duration is recommended to fully harness the therapeutic potential of the gel.

(
ex vivo) to the clinical outcome.The increase in skin hydration and elasticity, as well as in wrinkle formation of the developed EPA-CBD cream correlates with recent findings on the anti-aging and beneficial cosmetic usage of the agents.AUTH O R CO NTR I B UTI O N S R.G., G.C, and T.A. performed the research.G.C., N.O.S., E.S., and S.R. designed the research study.N.O.S., E.S., and T.A contributed essential tools to the research.G.C., J.J, M.P., and E.S. analyzed the data.G.C., J.J., M.P., and S.R. wrote the paper.All authors revised the manuscript.FU N D I N G I N FO R M ATI O N This study was supported by an ADSSC faculty grant and by ECO Pharmaceutics.G.C, R.G, N.O.S are partially supported by the Ministry of Science and Technology (580458776).

F I G U R E 5
Product satisfaction summary.The overall impression of the volunteers is depicted, following a self-assessment questionnaire after 26 (left) and 56 days (right).
Study population.
HaCaT keratinocytes cells were stimulated with UVB, and prostaglandin E2 (PGE 2 ) and IL-8 TA B L E 2