Climbazole Boosts Activity of Retinoids in Skin

Jean Adamus, Li Feng, Stacy Hawkins, Kristopher Kalleberg, Jian-ming Lee* Unilever R&D, Trumbull, CT 06611

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/ics.12390

*Correspondence to: Jian-ming Lee
e-mail: [email protected] Phone: 1-203-381-5721
Fax: 1-203-381-5476

Abstract
OBJECTIVE: To explore if climbazole enhances retinoid-associated biological activities in vitro and in vivo.

METHODS: Primary human dermal fibroblasts (HDFs) were treated from six to 48 hours with either retinoids (retinol, retinyl propionate, retinyl palmitate) alone or in combination with climbazole, then assessed for cellular retinoic acid-binding protein 2 (CRABP2) mRNA expression by RT-qPCR. Next, skin equivalent (SE) cultures were topically treated with a retinol or retinyl propionate, and with or without climbazole, then measured for biological changes in retinoid biomarkers. Lastly, an IRB-approved clinical study was conducted on the outer forearm of 16 subjects to ascertain the effects of low (0.02%) or high (0.1%) levels of retinol, retinyl propionate (0.5%), climbazole (0.5% ) or a combination of retinol (0.02%)
/climbazole (0.5%). Indicators of retinoid activities were measured after 3 weeks.

RESULTS: Treatment of HDFs with retinol or retinyl propionate were unaffected by climbazole but alone, resulted in a significantly (p<0.01) higher sustained CRABP2 mRNA expression than those treated with retinyl palmitate or vehicle control. In SEs, climbazole combined with either retinol or retinyl propionate, boosted retinoid related activity greater than the retinoid only, reflected by a dose-response, down- regulation of loricrin (LOR) and induction of keratin 4 (KRT4) proteins. In vivo, retinol (0.1%) and retinyl propionate (0.5%) significantly increased most evaluated biomarkers, as expected. Low dose retinol or climbazole alone did not increase these biomarkers; however, in combination, significant (p<0.05) increases in retinoid and aging biomarkers were detected.

CONCLUSION: Climbazole boosted retinoid activity both in the SE model, after a combined topic treatment with either retinol or retinyl propionate, and in vivo, in combination with a low level of retinol. Based upon the evidence presented here, we suggest that the topical skin application of climbazole in combination with retinoids, could deliver skin aging benefits more than a less robust retinoid alone.

Introduction

Vitamin A retinol has vital functions in normal cell physiology. In skin, converts to its active form, all- trans retinoic acid (RA), which is critical for skin’s normal development, health, and homeostasis. Retinols are stored as retinyl esters, often called pro-vitamin A or pro-retinol, that hydrolyze to RA upon demand by the cell for a multiplicity of functions [1]. Once formed, RA binds to the cellular retinoic acid-binding protein 2 (CRABP2) protein, a co-activator of transactivation by the RA receptor complex, and is transported into the nucleus where it binds to specific DNA to stimulate expression of a variety of genes, or is further processed to maintain tissue homeostasis [2,3]. Dosing with retinoids induces CRABP2 mRNA expression in skin [4] and in fibroblasts [5], establishing it as a reliable measure of retinoid bioactivity [6].

Retinol and RA are well known to provide skin benefits that help repair damage caused by chronological and photoaging, such as increased epidermal thickness [7, 4], stimulated collagen synthesis and reduced expression of harmful matrix metalloproteinases (MMPs) [8], but are unstable due to oxidative degradation by environmental factors. In addition, it can sometimes cause irritation. Retinyl esters, the cell’s internal storage form of retinol, are more stable, easier to formulate and less irritating. For these reasons, natural and synthetic retinyl esters (palmitate, propionate, etc.) are often used instead of retinol in skin care products, despite their reduced efficacy. Their lower bioavailability is likely due to their initial need to hydrolyze first to retinol, making their conversion slower and less efficient [9, 10, 11].

Modulation of skin cell’s normal RA metabolic pathway can alter its endogenous biological activity [2, 6], potentially achieving excellent skin benefits with a low level of retinol, thus significantly reducing or circumventing the limitations associated with retinol. RA catabolic processes (RA to 4-hydroxylated retinoic acid) are primarily modulated by the cytochrome P450 (CYP) enzymes [12, 13, 14], although not all those detected in skin have been fully characterized [15, 16, 17, 18]. Certain azoles known as RA metabolism blocking agents, or RAMBAs, are recognized modulators of these enzymes [19, 20]. In skin and in vitro, certain RAMBAs, such as liarozole, have been shown to increase RA levels by inhibiting its hydroxylation [21, 22], leading to biological responses similar to those obtained after topical application of RA [19, 23].

Climbazole, a known antifungal, approved and widely used in personal care products, is a weak pan- inhibitor of P450 (CYP) proteins [24, 25]. Important for promoting differentiation and healthy skin condition, climbazole upregulates late-cornified envelope protein and small-proline-rich protein expression in primary keratinocytes [26]. Unlike some other azoles, and important for skin, climbazole protects degradation of vitamin D [27].

Given the important role that retinoids play in skin function, and the potential to augment that activity, models that could best reflect their potency and efficacy were utilized to investigate whether climbazole enhances retinoid activity in vitro and in vivo.

Materials and methods

Monolayer HDF cell culture and treatments

For monolayer cell experiments, low passage (P2) primary human dermal fibroblasts (HDFs) cells from three different female donors (two 30-40 years old and one 55 years old, or older) (purchased from ThermoFisher, Waltham, MA), were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum, in a 37°C humidified incubator containing 5% CO2.

Prior to treatment, cells were grown to subconfluence in 6-well polystyrene, flat bottom cell culture plates. At this time, complete media was exchanged for non-supplemented media and the plates were returned to the incubator overnight. The following day, the media was removed and replaced with supplemented media containing, in duplicate wells, either 1 µM retinol (Sigma-Aldrich, St Louis, MO), 3 µM retinyl propionate (BASF, Tarrytown, NY or UniProma, Cardiff, Wales, UK), 3 µM retinyl palmitate (BASF, Tarrytown, NY) with, or without, 6 µM climbazole (Sigma-Aldrich, St Louis, MO). Control cultures were dosed with 0.1% ethanol vehicle (ETOH), or left untreated. No additional treatments or media exchanges were provided. Cells were collected from 6 hours to 48 hours after dosing by removing the media and rinsing wells twice with cold phosphate buffered saline (PBS). Plates were stored at -80°C until further processed.

RNA extraction and real time RT-PCR

Total cellular RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA), quantified and reverse transcribed using High Capacity RNA-to-cDNA Kit (Applied Biosystems Life Technologies, Carlsbad, CA) according to the manufacturers’ protocols. Duplicate amplification reactions containing 10 ng mRNA each were analyzed by the ViiA7 Real Time PCR System using TaqMan Fast Advanced Master Mix and TaqMan® Gene Expression Assays for CRABP2 (Hs00275636_m1) according to the manufacturer’s protocol (Applied Biosystems Life Technologies, Carlsbad, CA). B2M (Hs99999907_m1) and/or RPL37 (Hs02340038_g1) primers, similarly purchased, were used as endogenous controls. Real- Time qPCR was progressed for 40 cycles, each cycle consisting of 95°C for 1 second followed by 60°C for 20 seconds. Treated samples were normalized to controls and the mean and SD calculated, with fold change gene expression calculated as 2^ -∆∆Ct. Significance was determined by a two-tailed, paired t- test.

SE cell culture, treatments and collection

SEs were assembled and grown in 24 mm Transwell inserts (Corning Life Sciences, Tewksbury, MA), according to published protocols [28,29]. Briefly, primary human epidermal keratinocytes (NHEKs) were expanded in EpiLife® medium supplemented with Human Keratinocyte Growth Supplement (ThermoFisher, Waltham, MA). For each full thickness culture, approximately 300,000 NHEKs were applied to the top of a 2 mg/ml collagen (Sigma-Aldrich, St. Louis, MO, USA) matrix containing 75,000 neonatal primary HDFs. Cultures were grown submerged for 3 days in a low calcium medium, for 3 days in high calcium medium, then raised to the air-liquid interface. Four day after air exposure, triplicate cultures were dosed topically for one hour each day, for four days, with 80 µL of either retinol (50 µM, 100 µM) or or retinyl propionate (150 µM, 300 µM), with or without climbazole (300 µM, 600 µM). Controls were treated with either 0.1% ETOH, or left untreated. Any remaining liquid was removed after one hour to maintain surface dryness. Samples were harvested 8 days post air exposure. From each SE culture, a 6 mm biopsy was collected. The biopsied area was split evenly into two halves, one-half for IHC and the other half left unused in these experiments. The epidermal area remaining outside the biopsied area, but within the contained culture area, was detached from the dermal component and used for western blot analysis.

SE Immunohistochemistry (IHC) and quantification

One half of the SE 6 mm biopsy was formalin-fixed, paraffin-embedded (FFPE), sectioned and either counterstained with hematoxylin and eosin (H&E) by AML Labs (Baltimore, MD), or left unstained for antibody treatment [29].
Unstained sections were evaluated for specific protein with diluted cytokeratin (KRT4) primary antibody Clone EP1599Y (Abcam, Cambridge, MA) at 1:1000 using SuperPicture™ 3rd Gen IHC Detection Kit (ThermoFisher, Waltham, MA), with antigen retrieval. IR Dye 800 conjugated, affinity purified secondary antibodies (Rockland Immunochemicals Inc., Limerick, PA) were used at 1:10,000 dilution. Images were obtained at 20X magnification by microscopy, and quantitation of H&E (pixel count) and KRT4 protein

expression (summed intensity) was completed using in-house software. Mean and SD were calculated, and paired t-tests used to determine significance.

SE western blotting and protein quantification

Total protein lysate was isolated from the treated epidermis remaining outside the SE biopsy area and quantitated by protein assay (ThermoFisher, Waltham, MA). Five (5) µg protein from each sample was electrophoresed on 4-20% gradient gels (BioRad Laboratories, Inc., Hercules, CA) and western blotted as previously described previously [29]. Evaluation of loricrin (LOR) protein was determined using primary rabbit polyclonal antibody (Abcam, Cambridge, MA) at 1:3000 dilution. The signal intensity of migrated protein bands of interest were quantitated and calculated as integrated intensity (I.I.) K Counts mm2 (LI- COR Odyssey, Lincoln, NE). Migrated bands were normalized to actin (C-2) mouse monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX) at 1:1000 dilution. IR Dye 800 conjugated, affinity purified secondary antibodies used were obtained from Rockland Immunochemicals, Inc. (Limerick, PA). Mean, SD and LOR/actin ratios were calculated, and paired t-tests were used to determine significance.

Clinical efficacy study design, treatment and analyses methods

Sixteen healthy females, ages 40-60 years, Fitzpatrick Skin Type I-III, provided informed consent to participate in an IRB-approved randomized, double-blinded forearm study. The study was performed in accordance with the declaration of Helsinki principles. Subjects were recruited to have a moderate to severe degree of overall photodamage on their outer forearms. Test products (20 µl) were applied under closed patches for 24 hours to outer forearms, with 24-hour recovery gaps, over a time course of 21 days for a total of nine patch applications. Test formulations included 0.5% climbazole, 0.5% retinyl propionate, two different doses of retinol (0.02% or 0.1%), a combination of 0.02% retinol/0.5% climbazole, and a vehicle control. All formulations were in a simple PEG400-base vehicle. Eight millimeter (8 mm) Finn chambers on Scanpor® with paper inserts (Allerderm Laboratories, Phoenix, AZ)

were used for patch application [30]. On the last day of the study, a licensed board-certified dermatologist obtained full thickness 3 mm punch biopsies from each site.
The excised 3 mm biopsies were immediately placed into 10% neutral buffered formalin for five hours and processed overnight, then paraffin embedded the next day. Five (5) µM thick sections were stained with H&E. IHC development steps were performed by the IntelliPath FLX system (Biocare Medical Inc., Concord, CA) except for antigen retrieval, as needed. Diluted primary antibodies were applied to slides for a 2 hour incubation period: CRABP2 (1:1000) (Bethyl Laboratories, Montgomery, TX), KRT4 (1:200) (Epitomics, Burlingame, CA), Ki-67 (1:10,000) (ThermoFisher, Waltham, MA), and procollagen-I (PC-1) (1:1000) (Millipore, Billerica, MA). Vulcan Fast Red chromagen stain (Biocare Medical Inc., Concord, CA) was applied for 15 minutes to all clinical slides, washed in running water, and then left to air dry before being cover-slipped. Image acquisition was performed on a Zeiss Axioplan2 microscope with a Nuance multispectral camera (PerkinElmer, Waltham, MA), at 20X magnification.

The raw data obtained after IHC imaging was transformed to the logarithmic base 10 scale for statistical analysis with the exception of Ki-67, where cell counts were calculated. Analysis of variance (ANOVA) model was employed, in which subjects acted as random block effect and test product as a fixed effect. The p-value was calculated to account for multiple tests using a Bonferroni Holm multiplicity correction. Clinical results are reported as the geometric mean with 95% confidence intervals.

Results

Climbazole did not boost retinoid-stimulated CRABP2 mRNA synthesis in primary HDFs

Certain retinoids are known to provide skin benefits at the dermal level in vivo [31], and their potency has been established in HDFs, as a surrogate for skin [4, 32]. Our initial experiment investigated the potency of two of the more commonly used retinyl esters (propionate, palmitate) as compared to the benchmark, retinol. Once potency of the tested retinoids was confirmed, the potential boosting effect of climbazole on retinoid activity was explored.

CRABP2 mRNA is a known measure of retinoid potency and activity, and both retinol [5,6] and retinyl esters [33] increase its expression in vitro and in vivo in a concentration dependent manner. To our knowledge, the potency of these particular retinyl esters has not been explored compared to each other in HDFs, utilizing CRABP2 as a biomarker. CRABP2 mRNA was measured in three different primary HDF donor cells. Cells were treated once with either 3 µM retinyl propionate, 3 µM retinyl palmitate, or the benchmark 1µM retinol, and with or without climbazole. The ratio of the dosing concentrations of the retinyl esters was reflective of the bioequivalent of retinol in vivo [11].
Under these conditions, the response of the two retinyl esters was dissimilar. In all donor cells, a significant and sustained increase in the expression of CRABP2 mRNA was detected after treatment with retinol and retinyl propionate, but not with retinyl palmitate (Fig. 1). This included all time points from 6 hours to 48 hours after treatment, during which cell morphology and growth appeared normal. We did not observe any boosting effect with climbazole in these experiments (data not shown). Given the limitations of this model, further analysis with retinol and retinyl propionate was progressed to the more relevant skin equivalent model.

Climbazole increased retinoid responses in the SE

The ability of climbazole to influence retinoid activity was next investigated in SE cultures via phenotypic and biological changes. No significant increased thickness in the viable epidermis was detected as determined by H&E staining and image analysis in all SEs treated topically with either retino or retinyl propionatel; however, with addition of climbazole, we observed a marginal increase (Fig. 2A, B, left).
KRT4 is a sensitive biomarker of retinoid activity in vitro [34, 35] and in vivo [36], and provides a stronger response to retinoids in our model than CRABP2, based on prior observed staining. In the SE, retinol induced KRT4 expression as determined in FFPE sections after staining (Fig. 2A, right) and measurement (Fig. 2B, right). This response was significantly stronger with the addition of climbazole. A similar, but less intense, response was seen with retinyl propionate.

LOR is a cornified cell envelope protein expressed in late terminally differentiated epidermal cells, and decreased LOR expression reflects an increase in retinoid activity in vitro [37, 38]. Protein lysates were isolated from the SE epidermis, processed by western blot and quantitated for LOR protein. The results are presented in Fig. 2C. Both retinol and retinyl propionate treatments decreased LOR protein in the SE model in a concentration dependent manner, indicating increased retinoid activity. Importantly, SE cultures treated with a combination of retinoid/climbazole decreased LOR protein significantly more than either the individual treatment or the vehicle control.
Climbazole plus low level retinol induced biological and retinoid biomarkers in vivo

An in vivo clinical study was performed, based upon the SE results, to confirm that climbazole boosted retinol activity. Retinoids, with or without climbazole, were applied to human skin under patch for three weeks and biopsies were evaluated. Results shown in Fig. 3 (A, F) demonstrate that the application of 0.1% retinol and 0.5% retinyl propionate significantly (p<0.05) induced viable epidermal thickness compared to the vehicle control. Treatment with 0.02% retinol/ 0.5% climbazole appeared to increase viable epidermal thickness slightly but did not reach significance from the vehicle control.
In the basal layer of healthy normal human skin epidermis, Ki-67 protein staining identifies actively proliferating cells and is indicative of the level of epidermal proliferation. As compared to the vehicle control, a significant (p<0.05) increase of 2- to 3-fold Ki-67 staining in the basal layer was seen in skin samples treated with 0.1% retinol as well as in skin treated with the 0.02% retinol/ 0.5% climbazole combination (Fig. 3B, 3F). The combination was significantly (p<0.05) higher than its individual components, which did not show significant differences from the vehicle control. Treatment with 0.5% retinyl propionate showed a higher magnitude (cell counts) than the vehicle control, but this was not significant. The combination 0.02% retinol/ 0.5% climbazole treatment was not statistically significant from skin treated with 0.1% retinol or 0.5% retinyl propionate.

It is well established that CRABP2 protein expression increases in skin dosed with retinol [4]. In the current study, higher amount of retinol (0.1%), and retinyl propionate (0.5%) indeed induced significant (p<0.05) expression of CRABP2 compared to the vehicle alone (Fig. 3C, 3F). The lower level of retinol (0.02%) or the 0.5% climbazole did not induce significant expression of CRABP2. However, after

treatment with the combination of 0.02% retinol/0.5% climbazole, CRABP2 expression was significantly (p<0.05) induced as compared to both the vehicle control, 0.02% retinol or climbazole alone, strongly indicating that climbazole functions as a robust retinol booster in skin. As expected, the level of CRABP2 expression was low in the vehicle treated control.
KRT4 protein staining significantly (p<0.05) increased after application of 0.02% retinol/0.5% climbazole compared to either the vehicle control or to its individual components, 0.02% retinol or 0.5% climbazole (Fig. 3D, 3F). A significant (p<0.05) increase in expression was also seen after 0.1% retinol treatment and after treatment with 0.5% retinyl propionate, with less intense staining in the latter treatment. This is consistent with the SE results, where an increase in KRT4 was also observed.
Dermal fibroblast procollagen-1 (PC-1) synthesis is a well-recognized indicator of dermal extracellular matrix modification. In the dermis it declines with age, but its expression can be augmented after retinol treatment [8]. PC-1 protein staining (Fig. 3E, 3F) clearly demonstrated that a combination of 0.02% retinol/ 0.5% climbazole synergistically and significantly (p<0.05) induced this marker as compared to its individual components (0.02% retinol or 0.5% climbazole) and to the vehicle control. Higher amounts of retinol (0.1%), and retinyl propionate (0.5%) also increased the level of PC-1 protein expression significantly (p<0.05) as compared to the vehicle control. The combination treatment of 0.02% retinol/
0.5% climbazole did not show any significant difference versus the higher amount of retinol (0.1%) or retinyl propionate (0.5%).

Discussion

Topical application of retinol can improve the signs of chronological and photoaging, but it is unstable and can induce skin irritation. Retinyl esters are widely used in cosmetics due to their higher formulation stability and lower irritation; however, increased amounts or longer application times are needed to achieve skin benefits. Our aim in these studies was to establish if climbazole enhances retinoid- associated biological activities in vitro or in vivo. The results presented here provide strong evidence that climbazole boosts retinoid activity in the SE model and in skin.

With aging, fibroblast activity decreases but with retinol treatment, this activity can be improved [8]. Initially, retinyl ester (propionate, palmitate) potency was explored as an alternative for retinol, using adult primary HDFs. Here, retinyl propionate was as effective as retinol in increasing CRABP2 mRNA expression when used in equivalent physiological levels, whereas retinyl palmitate was not. This suggests that the processing of retinyl ester to retinol is faster than that for retinyl palmitate, indicating that retinyl propionate is a more potent activator of CRABP2. This is not surprising, as retinyl esters can be diverse in size and although similarly processed, hydrolyze at different rates depending on the length of their fatty acid chain (41). Unlike retinyl palmitate, both retinol and retinyl propionate induced CRABP2 expression to higher sustained levels in vitro from six to 48 hours. This is in line with studies that indicate that retinoid expression is stable for several days [5, 4].

A boosting effect with climbazole was not seen in treated fibroblasts. This particular model may not be optimal for observing this type of effect, which is supported by multiple studies in which monolayer culture may not always reflect the biological changes seen in vivo. For that reason, further analysis was progressed to the more relevant skin equivalent model.
The results obtained in the SE substantiate, for the first time, that climbazole in combination with a retinoid augments retinoid activity in vitro, as compared to retinoid alone. This is verified by the dose- responsive protein expression changes in retinoid markers (LOR, KRT4) observed in the SE model after topical retinol/climbazole treatment. Similar responses were observed after treatment with retinyl propionate/climbazole, but were somewhat less intense, likely due to the ester’s need to process to retinol. This in agreement with existing reports, where treatment with retinyl esters resulted in biological and phenotypic changes in skin and skin cells, but are sometimes less intense than that seen with retinol [9, 39, 40]. Viable epidermal thickness did not increase significantly in this model as compared to the controls. It is possible that a higher dosage or longer treatment would be necessary for perceiving any differences.
Preliminary SE studies determined that ~50 µM retinol was required to induce biomarker effects when topically dosed. The higher amount used for retinyl propionate is supported by established parameters of their physiological bioequivalence in vivo, where thrice-fold retinyl ester corresponds to a single entity of

retinol [11]. Climbazole dosing accounted for its cytotoxic threshold and relevance to in vivo treatment [26].
The results from the clinical study also demonstrated that climbazole is a retinoid booster. When climbazole was combined with a low level of retinol, the effects observed for most of the biomarkers tested were similar to that of a higher level of retinol (0.1%) (benchmark), or retinyl propionate (0.5%), and significantly better than climbazole alone, low level retinol alone, or vehicle control. To our understanding, this is the first time that climbazole has been demonstrated as a retinoid booster in human skin. In fact, analysis of most of the clinical endpoints tested indicated that there was no statistical difference between the combination of 0.02% retinol/ 0.5% climbazole and the 0.1% retinol or 0.5% retinyl propionate treatments, where all delivered significant outcomes for most endpoints tested.
In this study, treatment with 0.02% retinol was used in vivo for achieving marginal retinoid responses after retinoid/climbazole topical application so that climbazole’s boosting effects could be better observed. This was based on earlier reports, where an increased response in CRABP2 expression was observed with 0.025 % retinol treatment, and epidermal thickness significantly increased with 0.05% retinol treatment [4, 43]. Although we did not see a definitive response here in CRABP2 expression with 0.02% retinol alone, we believe it is in line with previous observations. It is likely that the differences between these studies (e.g., dosing regimen, body site, vehicle formulation) would plausibly account for these discrepancies.

Both epidermal and dermal components of skin were impacted after treatment with the retinol/climbazole combination. Two notable effects seen in this clinical study include 1) the stimulation of epidermal basal layer proliferation (Ki-67), providing strong evidence that a combination of retinol plus climbazole impacts the epidermis, and 2) increased PC-1 synthesis, indicating putative remodeling of dermal extracellular matrix. Here, the combination treatment appeared to trend higher than 0.1% retinol, and 0.5% retinyl propionate for the level of PC-1 protein; however, additional studies will be need to confirm these results and to better understand its impact in the dermis.

Although we did not detect a substantial increase in H&E staining with the combination treatment, there was significant proliferation stimulation, as observed with Ki-67 staining. This implies that increasing viable epidermal thickness may be a matter of dynamics of the response, where a more prolonged treatment might be more optimal for observation of certain phenotypic modifications, particularly in vivo.
Due to the limited number of biopsy samples permitted, we were unable to investigate retinyl propionate/climbazole treatment combinations in this clinical study. Current results suggest that we might expect a similar outcome and additional studies will be required to confirm.
Inhibition of retinol esterification and RA oxidation pathways in skin results in the elevation of intracellular levels of RA, which can be observed by looking at specific biological endpoints related to retinoid activity [15, 42, 44]. We do not currently know the precise mode of action for climbazole. Although it is an inhibitor of P450 enzymes, and a certain subset of these enzymes (e.g., CYP26A1, etc.) affect RA processing, it was not known whether climbazole would have an effect in skin when combined with a retinoid. Climbazole alone did not elicit observable effects in the SE (not shown) or in vivo, indicating that climbazole cannot stimulate retinoid activity without an external source of retinoid, similar to what has been observed with other azoles [22]. As a P450 inhibitor, climbazole may function to increase retention of endogenous levels of RA by inhibiting its degradation. It might be reasonable to suggest that climbazole blocks the regulated feedback of RA catabolism, thereby increasing the intracellular retention of endogenous RA as a secondary influence on the RA pathway rather than by direct participation.

Based upon these collective results, improved biomarkers of retinoid activity and skin aging were observed using of a combination of climbazole with retinol or retinyl propionate in the SE model, and in combination with lower levels of retinol, in vivo.
Taken together, these results clearly demonstrate, for the first time, that climbazole, a safe and widely used ingredient in personal care, is an effective booster of retinoid activity both in vitro and in vivo. This opens up the opportunity to develop superior skin care products using a lower level of retinol, or retinyl esters, with a safer and more stable profile. We suggest that the combination of climbazole with retinoids

may provide antiaging skin benefits; therefore, clinical studies are required to determine consumer benefits.

Acknowledgements

We thank Stella Arcella for her technical contributions, Jessica Carlson for her clinical contributions and Mei-Fen Yeh for her statistical analyses. All authors to this paper are employees of Unilever R&D and state no conflict of interest. Funding for the work presented here was provided by Unilever R&D.

References

1.Kurlandsky, S.B., Duell, E.A., Kang, S., Voorhees J.J. and Fisher G.J. Auto-regulation of retinoic acid biosynthesis through regulation of retinol esterification in human keratinocytes. J Biol Chem. 271(26), 15346-15352 (1996).

2.Fisher, G.J., and Voorhees, J.J. Molecular mechanisms of retinoid actions in skin. FASEB 10(9), 1002-1013 (1996).

3.Roos, T.C., Jugert, F.K., Merk, H.F. and Bickers D.R. Retinoid metabolism in the skin. Pharmacol Rev. 50, 315–333 (1998).

4.Kang, S., Duell, E.A., Fisher, G.J., Datta, S.C., Wang, Z-Q., Reddy, A.P., Tavakkol, A., Yi, J.Y. and Griffiths, C.E.M., Elder, J.T. and Voorhees, J.J., Application of retinol to human skin in vivo induces epidermal hyperplasia and cellular retinoid binding proteins characteristic of retinoic acid but without measurable retinoic acid levels or irritation. J Invest Dermatol. 105(4), 549-556 (1995).

5.Aström, A., Tavakkol, A., Pettersson, U., Cromie, M.A., Elder, J.T. and Voorhees, J.J. Molecular cloning of two human cellular retinoic acid-binding proteins (CRABP): Retinoic acid-induced expression of CRABP-II but not CRABP-I in adult human skin in vivo and in skin fibroblasts in vitro. J Biol Chem. 266(26), 17662-17666 (1991).

6.Elder, J.T., Cromie, M.A., Griffiths, C.E., Chambon, P. and Voorhees, J.J. Stimulus-selective induction of CRABP-II mRNA: A marker for retinoic acid action in human skin. J Invest Dermatol. 100(4), 356-359 (1993).
7.Fisher, G.J., Esmann, J., Grifliths, C.E., Talwar, H.S., Duell, E.A., Hammerberg, C., Elder, J.T., Karabin, G.D., Nickoloff, B.J., Cooper, K.D. and Voorhees, J.J. Cellular, immunologic and biochemical characterization of topical retinoic acid-treated human skin. J Invest Dermatol. 96, 699–707 (1991).

8.Varani, J., Warner, R.L., Gharaee-Kermani, M., Phan, S.H., Kang, S., Chung, J., Wang, Z., Datta, S.C., Fisher, G.J. and Voorhees, J.J. Vitamin A antagonizes decreased cell growth and elevated collagen-degrading matrix metalloproteinases and stimulates collagen accumulation in naturally aged human skin. J Invest Dermatol. 114(3), 480-486 (2000).

9.Duell, E.A., Kang, S. and Voorhees, J.J. Unoccluded retinol penetrates human skin in vivo more effectively than unoccluded retinyl palmitate or retinoic acid. J Invest Dermatol. 109(3), 301-305 (1997).

10.Green, C., Orchard, G., Cerio, R. and Hawk, J.L.M. A clinicopathological study of the effects of topical retinyl propionate cream in skin photoageing. Clin Exp Dermatol. 23, 162-167 (1998).

11.Trumbo, P., Yates, A.A., Schlicker, S. and Poos, M. Dietary reference intakes: vitamin A, vitamin K, arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon, vanadium, and zinc. J Am Diet Assoc. 101(3), 294-301 (2001).

12.Van Wauwe, J.P., Coene, M.C., Goossens, J., Van Nijen, G., Cools, W. and Lauwers, W. Ketoconazole inhibits the in vitro and in vivo metabolism of all-trans-retinoic acid. J Pharmacol Exp Therapeut. 245(2), 718-722 (1988).

13.Vanden Bossche, H., Willemsens, G. and Janssen, P.A.J. Cytochrome-P450-dependent metabolism of retinoic acid in rat skin microsomes: inhibition by ketoconazole. Skin Pharmacol. Physiol. 1, 176-185 (1988).

14.Miller, W.H. The emerging role of retinoids and retinoic acid metabolism blocking agents in the treatment of cancer. Cancer. 83, 1471–1482 (1998).

15.White, J.A., Beckett-Jones, B., Guo, Y.D., Dilworth, F.J., Bonasoro, J., Jones, G. and Petkovich, M. cDNA cloning of human retinoic acid-metabolizing enzyme (hP450RAI) identifies a novel family of cytochromes P450 (CYP26). J Biol Chem. 272(30), 18538-18541 (1997).

16.Smith, G., Wolf, C.R., Deeni, Y.Y., Dawe, R.S., Evans, A.T., Comrie, M.M., Ferguson, J. and Ibbotson, S.H. Cutaneous expression of cytochrome P450 CYP2S1: individuality in regulation by therapeutic agents for psoriasis and other skin diseases. Lancet. 361, 1336-1343 (2003).

17.Heise, R., Mey, J., Neis, M.M., Marquardt, Y., Joussen, S., Ott, H., Wiederholt, T., Kurschat, P., Megahed, M., Bickers, D.R. and Merk, H.F. Skin retinoid concentrations are modulated by CYP26A1 expression restricted to basal keratinocytes in normal human skin and differentiated 3D skin models. J Invest Dermatol. 126(11), 2473-2480 (2006).

18.Kedishvili, N.Y. Enzymology of retinoic acid biosynthesis and degradation Thematic Review Series: Fat-Soluble Vitamins: Vitamin A. J Lipid Res. 54(7), 1744-1760 (2013).

19.Verfaille, C.J., Borgers, M. and Van Steensel, M.A. Retinoic acid metabolism blocking agents (RAMBAs): a new paradigm in the treatment of hyperkeratotic disorders. J Dtsch Dermatol Ges. 6, 355– 364 (2008).

20.Thatcher, J.E. and Isoherranen, N. The role of CYP26 enzymes in retinoic acid clearance. Expert Opin Drug Metab Toxicol. 5(8), 875-886 (2009).

21.Van Wauwe, J., Van Nyen, G., Coene, M.C., Stoppie, P., Cools, W., Goosens, J., Borghgraef, P. and Janssen P.A. Liarozole, an inhibitor of retinoic acid metabolism, exerts retinoid-mimetic effects in vivo. J Pharmacol Exp Ther. 261, 773–778 (1992).

22.Kang, S., Duell, E.A., Voorhees, J.J. and Kim, K.J. Liarozole inhibits human epidermal retinoic acid 4-hydroxylase activity and differentially augments human skin responses to retinoic acid and retinol in vivo. J Invest Dermatol. 107(2), 183-187 (1996).

23.Pavez Loriè, E., Cools, M., Borgers, M., Wouters, L., Shroot, B., Hagforsen, E., Törmä, H. and Vahlquist, A. Topical treatment with CYP26 inhibitor talarozole (R115866) dose dependently alters the expression of retinoid-regulated genes in normal human epidermis. Br J Dermatol. 160, 26–36 (2009).

24.Kobayashi, Y., Suzuki, M., Ohshiro, N., Sunagawa, T., Sasaki, T., Tokuyama, S., Yamamoto, T. and Yoshida, T. Climbazole is a new potent inducer of rat hepatic cytochrome P450. J Toxicol Sci. 26(3), 141-150 (2001).

25.Kobayashi, Y., Suzuki, M., Ohshiro, N., Sunagawa, T., Sasaki, T., Oguro, T., Tokuyama, S., Yamamoto, T. and Yoshida, T. Induction and inhibition of cytochrome P450 and drug-metabolizing enzymes by climbazole. Biol Pharm Bull. 25(1), 53-57 (2002).

26.Pople, J.E., Moore, A.E., Talbot, D.C.S., Barrett, K.E., Jones, D.A. and Lim, F.L. Climbazole increases expression of cornified envelope proteins in primary keratinocytes. Int J Cosmetic Sci, 36(5), 419-426 (2014).

27.Rawlings, A.V. Biotechnology in Skin Care (III): Skin Aging. Cosmetic Science and Technology Series. (29), 163-200 (2006).

28.Margulis, A., Zhang, W. and Garlick, J.A. In vitro fabrication of engineered human skin, in: Epidermal Cells: Methods and Protocols, Turksen K (Ed), Humana Press, Totowa, NJ, 61-70 (2005).

29.Aho, S., Harding, C.R., Lee, J.M., Meldrum, H. and Bosko, C.A. Regulatory role for the profilaggrin N-terminal domain in epidermal homeostasis. J Invest Dermatol. 132(10), 2376-238 (2012).

30.Fischer, T. and Maibach, H. Finn chamber patch test technique. Contact Dermatitis. 11, 137–140 (1984).

31.Varani, J., Mitra, R.S., Gibbs, D., Phan, S.H., Dixit, V.M., Mitra, R., Wang, T., Siebert, K.J., Nickoloff, B.J. and Voorhees, J.J. All-trans retinoic acid stimulates growth and extracellular matrix production in growth-inhibited cultured human skin fibroblasts. J Invest Dermatol. 94(5), 717-723 (1990).

32.Elder, J.T., Kaplan, A., Cromie, M.A., Kang, S. and Voorhees, J.J. Retinoid induction of CRABP II mRNA in human dermal fibroblasts: use as a retinoid bioassay. J Invest Dermatol, 106(3), 517-521 (1996).

33.Kim, H., Kim, B., Kim, H., Um, S., Lee, J., Ryoo, H. and Jung, H. Synthesis and in vitro biological activity of retinyl retinoate, a novel hybrid retinoid derivative. Bioorg Med Chem. 16(12), 6387-6393 (2008).

Climbazole

34.Virtanen, M., Sirsjö, A., Vahlquist, A. and Törmä, H. Keratins 2 and 4/13 in reconstituted human skin are reciprocally regulated by retinoids binding to nuclear receptor RARα. Exp Dermatol, 19, 674-81 (2010).

35.Pavez Loriè, E., Chamcheu, J.C., Vahlquist, A. and Törmä, H. Both all-trans retinoic acid and cytochrome P450 (CYP26) inhibitors affect the expression of vitamin A metabolizing enzymes and retinoid biomarkers in organotypic epidermis. Arch Dermatol Res. 301, 475-485 (2009).

36.Virtanen, M., Törmä, H. and Vahlquist, A. Keratin 4 upregulation by retinoic acid in vivo: A sensitive marker for retinoid bioactivity in human epidermis. J Invest Dermatol 114(3), 487-493 (2000).

37.Magnaldo, T., Bernerd, F., Asselineau, D. and Darmon, M. Expression of loricrin is negatively controlled by retinoic acid in human epidermis reconstructed in vitro. Differentiation. 49(1), 39-46 (1992).

38.Brown, L.J., Geesin, J.C., Rothnagel, J.A., Roop, D.R. and Gordon, J.S. Retinoic acid suppression of loricrin expression in reconstituted human skin cultured at the liquid-air interface. J Invest Dermatol. 102(6), 886-890 (1994).

39.Erling, T. Skin treatment with two different galenical formulations of retinyl palmitate in humans. J Appl Cosmetol. 11, 71-76 (1993).

40.Bissett, D.L. and Johnson, M.B. Cosmetic anti-aging ingredients, in: Farage MA, Miller KW, Maibach HI (Eds.), Textbook of Aging Skin, Springer-Verlag, Berlin Heidelberg, 1069-1078 (2010).

41.Rigtrup, K.M. and Ong, D.E. A retinyl ester hydrolase activity intrinsic to the brush border membrane of rat small intestine. Biochemistry. 31(11), 2920-2926 (1992).

42.Van Wauwe, J.P., Coene, M.C., Goossens, J., Cools, W. and Monbaliu, J. Effects of cytochrome P- 450 inhibition on the in vivo metabolism of all trans-retinoic acid in rats. J Pharmacol Exp Ther. 252, 365– 369 (1990).

43.Duell, E.A., Derguini, F., Kang, S., Elder, J.T., Voohrees, J.J. Extraction of human epidermis treated with retinol yields retro-retinoids in addition to free retinol and retinyl esters. J Invest Dermatol 107, 178- 182 (1996).

44.Nelson, C.H., Buttrick, B.R. and Isoherranen, N. Therapeutic potential of the inhibition of the retinoic acid hydroxylases CYP26A1 and CYP26B1 by xenobiotics. Curr Top Med Chem. 13(12), 1402 (2013).