Development of an Effective Acne Treatment Based on CBD and Herbal Extracts: Preliminary In Vitro, Ex Vivo, and Clinical Evaluation

Acne vulgaris, the most common form of acne, is characterized by a mixed eruption of inflammatory and noninflammatory skin lesions primarily affecting the face, upper arms, and trunk. The pathogenesis of acne is multifactorial and includes abnormal keratinization and plugging of the hair follicles, increased sebum production, proliferation and activation of Cutibacterium acnes (C. acnes; formerly Propionibacterium acnes, P. acnes), and finally inflammation. Recent studies have found that cannabidiol (CBD) may be beneficial in the treatment of acne. The aim of this study was to explore natural plant extracts that, when combined with CBD, act synergistically to treat acne by targeting different pathogenic factors while minimizing side effects. The first stage of the study investigated the capacity of different plant extracts and plant extract combinations to reduce C. acnes growth and decrease IL-1β and TNFα secretion from U937 cells. The results found that Centella asiatica triterpene (CAT) extract as well as silymarin (from Silybum marianum fruit extract) had significantly superior anti-inflammatory activity when combined with CBD compared to either ingredient alone. In addition, the CAT extract helped potentiate CBD-induced C. acnes growth inhibition. The three ingredients were integrated into a topical formulation and evaluated in ex vivo human skin organ cultures. The formulation was found to be safe and effective, reducing both IL-6 and IL-8 hypersecretion without hampering epidermal viability. Finally, a preliminary clinical study of this formulation conducted on 30 human subjects showed a statistically significant reduction in acne lesions (mainly inflammatory lesions) and porphyrin levels, thereby establishing a tight correlation between in vitro, ex vivo, and clinical results. Further studies must be conducted to verify the results, including placebo-controlled clinical assessment, to exclude any action of the formulation itself.


Introduction
Acne vulgaris, one of the most common human skin diseases, reduces the quality of life of millions of people worldwide. It afects almost everyone between the ages of 15 and 17, with 15-20% of afected individuals experiencing moderate to severe disease [1]. Although acne is principally a disorder of adolescence, current research indicates that the prevalence of acne in adult patients, especially women, is increasing. Te clinical manifestations of acne include noninfammatory lesions (open and closed comedones) and infammatory lesions (papules and pustules) [2]. Major factors in the development of acne include abnormal hyperkeratosis and shedding of the follicular epithelium, increased sebum production, proliferation of the bacteria Cutibacterium acnes (C. acnes; formerly known as Propionibacterium acnes, P. acnes), and fnally infammation. Current guidelines recommend a combination of ingredients and treatments aimed at addressing these diferent pathogenic factors through unique mechanisms of actions. While many options exist, most available acne treatments cause some degree of irritation, leading to low compliance rates [3]. Over the last decade, a rising interest in natural and plant-derived ingredients has led to the discovery and development of new products that provide good efcacy with less irritation, resulting in better compliance and outcomes [4][5][6].
Due to the important role of the endocannabinoid system in the skin, current research has focused on the role of cannabinoids in the treatment of several skin disorders [7]. Recently, cannabidiol (CBD) has been suggested as a new treatment option for acne [8]. In addition to its known anti-infammatory activity, Oláh et al. have shown that CBD can reduce lipolysis and sebocyte proliferation in vitro [9]. Several clinical studies are now underway to evaluate the therapeutic potential of these properties [10].
Te development of cannabinoids as therapeutic agents in dermatology and their use in cosmetic applications have been challenged by limited efcacy data and unclear regulatory and legal guidelines surrounding their use [11]. To circumvent these challenges, our team explored the use of established active molecules to provide synergistic properties, both in efcacy and tolerability, when combined with CBD in the treatment of acne. In this study, the compatibility of these herbal extracts with CBD was evaluated in vitro, ex vivo, and clinically. A statistically signifcant correlation of these fndings using our fnal mixture was demonstrated across all stages of development.

Cell, Bacteria, and Skin Organ
Cultures. U937 cells were grown and maintained in RPMI-1640 supplemented with 10% fetal calf serum and 1% streptomycin/penicillin. Te cells were maintained at 5% CO 2 in a humidifed incubator at 37°C. For passaging, the cells were diluted in fresh media at a ratio of 1 : 4. Prior to the experiment, the cells were seeded at 300,000 cell/ml (150 μl pr 96-well plates) and diferentiated by PMA (phorbol 12-myristate 13-acetate) into a macrophage-like phenotype, an observation similarly reported in other studies [12]. After 24 hours, the adhered cells were treated as indicated. C. acnes was grown in a modifed and reinforced clostridial broth medium with reduced levels of oxygen.
Human skin samples were obtained from 40-to 60year-old healthy women undergoing aesthetic abdominal surgery, after signing an informed consent form. Te experiments were conducted with the approval of the IRB Committee of the Soroka Medical Center, Beer Sheva, Israel (#0258-19-SOR, approval protocol scrc20016; 29.06.2020). Te tissue was processed and maintained in an air/liquid interface with the dermal side submerged, as previously detailed [13,14].

Viability
Determination. U937 cells and skin tissue epidermal viability were monitored by the MTT assay. Following treatment, the cells were washed, and the cellular indicator was added (150 μl of 0.5 mg/mL (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dissolved in PBS) to the 96-well plates. Te plates were then incubated for 1 hour at 37°C, after which the solution was aspirated. One hundred and ffty microliters of isopropanol was then added to the solution to extract the formazan dye. Te absorbance was recorded at 570 nm using a plate reader (TECAN, f200 Infnite, Switzerland). Epidermal viability was determined similarly following heat separation of the epidermis (1 min., 56°C) and its transfer to the 96-well plates.

Infammation Induction and Cytokine Quantifcation.
Te cells were seeded at 300,000 cell/ml (150 μl pr 96-well plates) and diferentiated by PMA (phorbol 12-myristate 13acetate, 10 ng/ml) into a macrophage-like phenotype. Ten, the cells were treated without or with 1 μg/mL in the absence or presence of the diferent treatments listed below. In addition, dexamethasone (10 μM) was used as a positive control. After diferent treatments, the spent medium from the cell and tissue cultures was centrifuged (to remove particles) and the supernatant (100 μl) was aliquoted and stored at −20°C until used. Human ELISA tests were performed according to the manufacturer's instructions (Biolegend, San Diego, CA).

MIC (Minimum Inhibitory Concentration) Assay.
To evaluate the impact of the diferent treatments on C. acnes, a stock of frozen bacteria was removed from a −80°C freezer and thawed into a U-shaped falcon tube containing 4 ml of the modifed, reinforced clostridial broth medium at 37°C under reduced oxygen levels (anaerobic chamber). After 3 hr, the bacteria were inoculated into agar plates (100 μl/10 cm plate) and grown under reduced oxygen levels. A single colony was grown in 4 ml media to a midlog phase (O.D. 0.5; 600 nm, approx. 3 hr). Ten, the bacteria were diluted to O.D. 0.05 in the absence or presence of diferent treatments. Bacterial growth, determined by absorbance (O.D. 600), was monitored kinetically using a microplate reader (Infnite f200, TECAN), heated to 37°C. Ampicillin, at 10 μg/ml, was used as a positive control. ) and skin Fitzpatrick phototype II to IV. All enrolled participants and/or their parent/legal guardian (for minor subjects) provided written, informed consent and willingly agreed to comply with study requirements.

Exclusion Criteria.
Subjects were excluded from the study if 1 or more of the following treatments were used: oral retinoids (within 6 months prior), topical retinoid treatment or any facial aesthetic or medical treatments (within 1 month prior), and excessive UV exposure (within 1 month prior). Pregnant women, breastfeeding women, or women planning a pregnancy during the clinical study were also excluded. Participants with medical conditions or those receiving treatments that, according to the investigator's judgment, could compromise the safety of the participant or interfere with outcomes of the study were also excluded. Furthermore, subjects with a planned or expected major surgical procedure during the clinical study were not included. Table 1. Tirtythree subjects aged 15-40 with mild to moderate acne were enrolled in the study, with three withdrawals (#5 on D0, #19 on D28 and #11 on D56). Data from the remaining 30 subjects were included in our analysis.

Methods.
Study participants were instructed to apply a thin layer of the study cream (ECHO-A-01) to any spot (red or not) and any imperfection on the skin 2 or 3 times a day (morning, noon, and evening (before bedtime)) on clean skin. Instrumental efcacy data were collected on days D0, D28, D42, and D56 and analyzed using the Wilcoxon signed-rank test. All the calculations were performed using SPSS 23 (IBM) with a 95% confdence interval. High-resolution photographs were taken at each visit using the VISIA-CR imaging system, a standardized clinical imaging and image analysis system that uses a standard light (IntelliFlash), a cross-polarized fash, and a parallel, polarized fash under ultraviolet lighting. Te software allows a region of interest to be defned and calculated using numbered parameters and quantify porphyrin levels.

In Vitro Screening for CBD-Based Herbal Composition.
Two initial in vitro screening models were used to evaluate the compatibility and possible synergistic action of herbal mixtures: LPS-induced infammation of U937 cells and the growth inhibition of C. acnes. First, the impact of CBD (10 μg/ml) alone was evaluated in these systems and is demonstrated in Figures 1(a)-1(c). Exposure to CBD signifcantly decreased the secretion of both TNFα and IL-1β (Figure 1(a)) in the in vitro LPS-induced infammatory model. Concentrations of 5 and 10 μg/ml of CBD also reduced C. acnes growth in a comparable manner to ampicillin, which was used as a positive control (Figure 1(c)). Tese results complement previous fndings and reports, suggesting CBD as a potential treatment for acne. Olah et al. demonstrated that CBD inhibits lipogenesis in sebocytes stimulated with either arachidonic acid or a combination of linoleic acid and testosterone. In addition, CBD reduced cell proliferation via the activation of transient receptor potential vanilloid 4 channels [9]. A recent review also shed light on other infammatory disorders that can be mitigated by CBDbased treatment, including allergic contact dermatitis, psoriasis, acne, scleroderma, and dermatomyositis [15]. In addition, a study dating back to 1976 found that CBD may display antibacterial properties [16], and a more recent, comprehensive study by Blaskovich et al. found that CBD can reduce growth of several bacteria, including highly resistant Staphylococcus aureus, Streptococcus pneumoniae, and Clostridioides difcile. Te authors also concluded that membrane impairment is the primary bactericidal mechanism of CBD [17]. Our studies confrm both the anti-infammatory and antibacterial properties of CBD, thereby supporting its use in the treatment of acne. However, as acne is a multifactorial skin condition, we sought to create a more comprehensive treatment formulation by adding other already existing chemicals with unique mechanisms of action to act synergistically with CBD to enhance its activity but at a lower dose in order to reduce local side efects and improve tolerability. Terefore, the efcacy of subefective concentrations of CBD when used alone or combined with seven commercially available herbal extracts was investigated (preliminary screen, data not shown). As shown in Figure 1(b), only two extracts were Evidence-Based Complementary and Alternative Medicine  found to be highly compatible with CBD, the Centella asiatica triterpene (CAT) extract and silymarin extracted from Silybum marianum fruit. Te combination of either extract with CBD potentiated its efect in reducing LPSinduced infammation. Moreover, the CAT extract enhanced the growth arrest of C. acnes when combined with CBD (Figure 1(d)). Te CAT extract is well known for its therapeutic efect mainly due to the presence of madecassoside, asiaticoside, madecassic acid, and asiatic acid [18][19][20][21]. Our study shows that CAT potentiates the mitigating efect of subtherapeutic concentrations of CBD. Since the combination was active in two separate models, it is possible that it increases the bioavailability of CBD. Further studies are required to understand the mechanism of action underlining this phenomenon. Te Silybum marianum fruit extract had been repeatedly shown to modulate the infammatory system [20,21] and therefore can be benefcial in the treatment of several skin disorders, including the infammatory lesions of acne vulgaris. Our study identifed a synergistic efect between CBD and silymarin on two cytokines (Figure 1(b)).

Ex Vivo Validation of the Compounds.
A topical formulation containing 1% CBD, 1% CAT, and 1% silymarin was prepared and evaluated in the ex vivo human skin organ culture (hSOC). Te fnal formulation contained additional 0.5% salicylic acid, which was foreseen to facilitate the therapeutic action of the mixture through several mechanisms [22,23]. Te hSOC system was used repeatedly for both efcacy and safety evaluation, as it emulated the intact tissue [24][25][26]. Te results presented in Figure 2 show that the formulation containing the natural mixture (written as "formula" in Figure 2) was well tolerated by the skin and did not reduce epidermal viability (determined by the MTT method). Although LPS stimulation increased the secretion of IL-6 and IL-8, the cytokines were subsequently blocked by the formula, an efect comparable to that of dexamethasone. Both IL-6 and IL-8 are the main cytokines known to be induced by LPS in the ex vivo system [13]. Importantly, both cytokines have been suggested as possible therapeutic targets to acne vulgaris [27][28][29]. Terefore, their attenuation, as exhibited in our study, may make our formula clinically relevant as a potent anti-infammatory formulation. Te formula showed a statistically signifcant reduction in acne lesions, mainly infammatory. A reduction of 31.8% in total infammatory lesions was shown following 28 days, 38.2% following 42 days, and 70.9% following 56 days ( Figure 3), suggesting that the formula's potent antiinfammatory activity has signifcant therapeutic potential in infammatory acne. Te topical application signifcantly reduced acne severity, shown as a reduction in the Investigator's Global Assessment (IGA) score. In addition, a marked reduction in porphyrin levels (count and area) suggested C. acnes inhibition. Representative images are presented in Figure 4. It should also be noted that the overall rate of volunteer satisfaction was extremely high throughout the trial.

Conclusions
Te data presented here demonstrate the efcacy of a newly developed, natural topical formulation in the treatment of acne. Since several pathological pathways underlie the physiology of acne, a treatment that targets several mechanisms of action is advantageous. Te topical formulation presented in this study showed signifcant ability in reducing infammation and eliminating C. acnes. Te ex vivo results show a marked impact of the developed formulation in comparison to untreated tissue. However, the addition of salicylic acid may have further enhanced the efcacy of the formulation, due to its keratolytic activity, and as a result, it may have exhibited positive outcomes in the treatment of acne. Tus, a major limitation of this study is the lack of formulation without CBD and Silybum marianum fruit extract to exclude indirect efects of other compounds. Tus, placebo-controlled evaluation must be performed to validate the study. A signifcant correlation was shown between in vitro, ex vivo, and clinical tests, where the inhibition of infammation and C. acnes growth were the main mechanisms of action exhibited by our formulation. Te study also demonstrates a transformation factor of 1 : 1000 (∼10 μg/ml to 1%) when converting from in vitro concentrations to ex vivo and clinical ones. Te selected concentration in the formulation was determined after a preliminary evaluation determining that the formulation was safe and well tolerated by the tissue on one hand and efective on the other one (data not shown). Tis fnding is also supported by previous fndings, transforming data obtained in HaCaT keratinocyte cells to ex vivo experiments [28]. Te study has several limitations: in vitro screening was aimed to fnd CBDcompatible agents, and thus, low levels of the herbal extracts were used. In addition, the signifcant results obtained clinically were compared to their basal state, as typically performed in cosmetic trials, with no placebo control used. However, the massive reduction in infammatory acne lesions, as well as porphyrin levels, clearly stands out and was foreseen based on the dual in vitro and ex vivo data.

Data Availability
All clinical data are available upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.