Ketoconazole-p Aminobenzoic Cocrystal Exhibits a Potent Anti-inammatory Effect on the Skin of BALBc Mice

Fungal infections are a growing global health problem. Therefore, our group has designed and characterized a novel cocrystal formulation starting from ketoconazole and para-amino benzoic acid, named KET-PABA aiming to improve the bioavailability, biocompatibility, and eciency of the parent drug. The cocrystal showed improved physical properties, such as stability in suspension, solubility, as well as antimycotic eciency as compared to ketoconazole. The current study investigated the local possible side effects induced on BALBc mice skin by the application of KET-PABA cocrystal. KET-PABA proved to be safe, without signs of skin sensitization as shown by the mouse ear sensitization test (MEST), or histopathology. KET-PABA induced a potent anti-inammatory effect through the inhibition of proinammatory cytokines such as IL1α, IL1β, IL6 and TNFα, and other inammation promoters such as NRF2, compared to the vehicle. KET-PABA had no effect on the levels of the anti-inammatory cytokine IL10, or proinammatory enzyme COX2 and had minimal effects on the activation of the NFκB pathway. Overall, KET-PABA application induced no sensitization, moreover, it induced an anti-inammatory response. Based on the improved antimycotic effect versus ketoconazole and the anti-inammatory action, KET-PABA cocrystal has the potential to be an ecient drug in the treatment of cutaneous mycosis.


Introduction
Lately, there has been an increased interest in benzoic acids and their derivate, due to their chemical and biological properties. PABA is a precursor for the biosynthesis of folic acid and is a compound present in plants, fungi, and some parasites. PABA is absent in the human body, which does not have the pathway of folic acid (Kluzk et al. 2002).
PABA is also found in food, as part of the vitamin B complex, ensuring an antioxidant effect. PABA has multiple indications in medicine (Crisan et al. 2014) based on its antibacterial, antifungal and antiparasitic properties. Also, PABA has anticoagulant, antimutagenic, brinolytic, immunomodulating, photoprotective effects. It is used in sunscreen creams, where it functions as an UVB shield and it can quench reactive oxygen species generated by UV exposure (Hu et al 1995). It is also used in local anesthetics such as procaine (Mackie et al. 1999). Also, the potassium salt of PABA is used to treat connective tissue diseases such as dermatomyositis and scleroderma (Sharma et al. 2013).
PABA is stable in solid and liquid state, and is soluble in water, in methanol and in ethanol (Kluzk et al. 2002). The aminogroup of PABA ensures the transport of PABA and its derivates, and reduces toxicity (Martin et al. 2020). Several studies that focus on improvement of oral drug delivery and skin targeting of PABA using liquid crystal formulations were published (Kadhum et al. 2016).
However, the topical use of PABA has rendered sensitization reactions, in the form of contact allergic dermatitis, or contact photoallergic dermatitis (Thune et al. 1984, Greenspoon et al. 2013). Also, crossreactivity between PPD and PABA was reported (LaBerge et al. 2011). Since ketoconazole has also been involved in initiating allergic contact dermatitis (Choi et al. 2019), the current study is focused on the evaluation of the local immunological reactions after topical application of the ketoconazole-PABA cocrystal.
In a previous study we showed that the newly synthetized cocrystal ketoconazole with PABA, KET-PABA proved several advantages: safe pharmacological pro le, both in vitro and in vivo, as compared to ketoconazole, with a good crystal stability under ambient conditions, increased solubility, signi cantly enhanced antifungal activity compared to ketoconazole or PABA alone, and an antioxidant effect (Martin et al. 2020).
Therefore, this study aims to evaluate possible local side effects, such as allergic contact dermatitis initiation or another type of local in ammatory reaction, in response to topical application of the cocrystal, as well as ketoconazole and PABA on the skin of BALBc mice. A speci c test (Mouse Ear Swelling Test) was used, combined with histopathology exam and the measurement of pro and antiin ammatory cytokines and in ammation mediators. Overall, the cocrystal proved to be safe on mouse skin, moreover the anti-in ammatory effects of the cocrystal were more important when compared to the parent drug-ketoconazole.
Other solvents and reagents used were of analytical grade, purchased from commercial suppliers.
Characterization of the KET-PABA solid form KET-PABA was prepared, as a white powder sample, by solvent drop grinding (SDG) method performed by mechanical grinding using a Retsch MM400 ball milling (Martin et al. 2020). Its solid form structure was con rmed by comparing the experimental X-ray powder diffraction (XRPD) pattern with the calculated one. The XRPD measurements were performed in the 3-40° range, in steps of 0.02°, at room temperature using a Bruker D8 Advance powder diffractometer with CuKα1 radiation (λ = 1.54056 Å). The calculated XRPD pattern was obtained based on its crystal structure determined from single-crystal X-ray diffraction (Martin et al. 2020 Powder dissolution data for KET-PABA revealed a 10-fold solubility increase in deionized water that determines a 6.7-fold oral bioavailability improvement compared to pure KET. The stability tests performed under accelerated (climatic chamber at 40 °C/75% RH for 6 months) and long-term ambient conditions (12 months) showed no structural changes of the KET-PABA solid form (Martin et al. 2020).

Biological studies
All lab animal experiments were approved by ethics committee of the University of Medicine and Pharmacy Cluj-Napoca and the Veterinary Health Directorate, Romania (authorization number 67/ 06 06 2017). The lab animals were humanly treated and sacri ced under anesthesia.

Mice
Animals (6-8 week old female) BALBc mice (Tordesillas et al. 2014) were purchased from Cantacuzino Institute, (Bucharest, Romania). During the experiments, the animals were kept at humidity 65%, 21°C, day/night cycles of 12 h, fed with standard food and water ad libitum. The food of the animals was supplemented with vitamin A (250UI/g) to enhance the sensitivity of the epicutaneous sensitization test (Dunn et al. 1990, Hussain et al. 2011, Tordesillas et al. 2014).

Epicutaneous sensitization test
The test was done according to the MEST protocol. Animals were randomly divided into ve groups, 7/group: group 1-control -vehicle (alcohol 70%), group 2-ketoconazole, group 3 -KET-PABA, group 4-PABA and group 5-DNCB dinitrochlorobenzene (DNCB, Sigma). Animals were anesthetized and abdominal fur was carefully shaved, without abrasion of the skin. All animals were administered subcutaneously, in the right ank 30 µl/mouse of Freund's complete adjuvant (Thermo Scienti c™, Waltham, Massachusetts, United States) to increase the immunogenicity of the tested substance (Stewart et al. 2006). All animals in a group were marked on the back with a nontoxic dye. Then animals were exposed to the tested substances, each in a concentration of 100 µg/ml solved in ethanol 70%. 100 µl solution/mouse was spread thinly on the shaved abdominal skin to dry; solution was applied 1x/day for 6 consecutive days. On day 7, the thickness of the ears was measured with an electronic caliper, which served as the initial ear thickness, then mice were challenged with 50 µl/mouse of the same solution on the left ear pinnae, rechallenge was done on day 14. Ear thickness was measured at 24h and 48h after each ear application. Mice were sacri ced afterwards, and the ear tissue was collected for further investigations.

Histopathology
Samples-left ear pinna were xed in 10% buffered neutral formalin and embedded in para n. Sections were made at 4 mm and the slides were stained by Hematoxylin-Eosin (HE) method. Slides were examined under a microscope Olympus BX 51 and images were taken with an Olympus UC 30 digital camera and processed by a special image acquisition and processing program: Olympus Stream Basic Olympus Stream Basic (Shinjuku, Tokyo, Japan).

ELISA g
Ear sample homogenates were prepared as previously described by David et al. (David et al. 2014). The protein content in tissue homogenates was measured by Bradford method (Noble et al. 2009). IL1α, IL1β and IL6 Quantikine ELISA Immunoassay kit was used. Ear homogenate samples were treated according to manufacturer's instructions; readings were done at 450 nm with correction wavelength set at 540 nm, using an ELISA plate reader.

Western Blot
Lysates (20 µg protein/lane) were separated by electrophoresis on SDS PAGE gels and transferred to polyvinylidene di uoride membranes, using Biorad Miniprotean system (BioRad). Blots were blocked and then incubated with antibodies against: IL10, STAT3, COX2, then further washed and incubated with corresponding secondary peroxidase-linked antibodies. Proteins were detected using Supersignal West Femto Chemiluminiscent substrate and a Gel Doc Imaging system equipped with a XRS camera and Quantity One analysis software (Biorad). β actin was used as a protein loading control.

Statistical methods
The statistical difference between treated and control groups were evaluated by two-way ANOVA, followed by Bonferroni posttest, using GraphPad, the statistical signi cance of the treatments inside a certain group was tested using one way ANOVA and Dunnet's multiple comparison tests; results were considered signi cant for p<0.05. Statistical package used for data analysis was Prism version 4.00 for Windows, GraphPad Software, San Diego, California, USA, www.graphpad.com.

Epicutaneous sensitization
Clinical observation of the treated mice in the vehicle, KET, KET-PABA and PABA groups did not detect any ear surface alteration or swallowing. The animals seemed unaffected by the exposure. However, in the DNCB group, the animal's abdomen became red, and some small crusts appeared on the surface, starting from day 6; on the ears, there was redness, edema and the mice showed a slight sensitivity to touch at 24 and 48h after the challenge and maintained the same aspect after rechallenge. The clinical observations were sustained by the quantitative measurements of the ear thickness (Fig 1.). Vehicle group showed no statistical differences between the measured values (p>0.05, one way ANOVA). Data show that in the groups treated with KET, KET-PABA and PABA there was no signi cant ear swallowing, compared to vehicle. Interestingly KET seemed to diminish ear thickness, compared to the initial value and to the vehicle. There was a strong statistically signi cant difference between the initial ear thickness and the values after challenge and respectively rechallenge (p=0.0009, one way ANOVA). KET-PABA application induced a slight, not signi cant increase in the ear thickness after challenge and at 24h after rechallenging, however, at 48h the values were like those of the vehicle (p>0.05, one way ANOVA). In the PABA group, ear thickness decreased after the challenge and afterwards they slightly increased, however, not signi cantly compared to the initial value (p> 0.05, one way ANOVA). The DNCB group showed a strong statistically signi cant increase in the ear thickness (p<0.0001, one way ANOVA). The most important increase was found after the rechallenge at 24h, afterwards the ear thickness diminished; however, it was still signi cant, when compared to the initial value. There was a strong statistically signi cant difference (p<0.0001) showed by two-way ANOVA between the groups. Bonferroni post test showed signi cant (p<0.001) differences between the DNCB group and all the other groups.

Histopathology analysis
In the DNCB treated group there were signs of moderate in ammation. The ear skin lesions showed epidermal spongiosis, with mild acanthosis, slight dermal edema, and a perivascular dermal in ammatory in ltrate, consisting of a moderate number of lymphocytes and plasma cells (Fig 2.). All these modi cations are consistent with the aspect of contact dermatitis. The epicutaneous sensitization measurements along with the histopathology results prove that contact dermatitis was induced in the positive control group. Therefore, this group can be used for comparison with the other experimental groups. No histological changes were seen in the control -vehicle treated group, Ketoconazole, KET-PABA and in the PABA treated group (Fig 2.).

In ammatory markers
Pro-in ammatory cytokines IL1α, IL1β and IL6 from the treated mouse ear tissue homogenates were determined by ELISA (Fig 3.).
IL1α and IL1β levels were slightly decreased by KET, KET-PABA and PABA, not signi cantly compared to vehicle. The most important decrease was in the case of KET-PABA. In the DNCB group, there was an important, signi cant increase compared to the vehicle and all the other groups (Fig 3.). There were signi cant differences between the groups as shown by one way ANOVA (p=0.0001 for IL1α and IL1β). IL6 was only decreased by KET-PABA (Fig 3.), while the other substances increased it, however, the only signi cant increase was for DNCB. One way ANOVA showed signi cant differences between the groups (p=0.011).
The anti-in ammatory cytokine IL10 level was measured by Western Blot. IL10 showed a lower level in the mice treated with KET-PABA compared to controls, although not signi cantly. The other groups showed a slight increase, but only DNCB was signi cant (Fig 4.). These modi cations were not signi cant between the groups (p=0,074, one way ANOVA) The level of COX2, a proin ammatory enzyme was found strongly increased by DNCB, as expected, leading to the local in ammation (Fig 4.). In the other groups the modi cations were not signi cant, with a slight increase in the ketoconazole and PABA group and decrease in the KET-PABA group. The difference between the groups treated with ketoconazole and KET-PABA were signi cant (p=0.012).
Overall, one-way ANOVA showed that these modi cations were signi cant between the treated groups (p < 0.0001).
TNFα was signi cantly increased in the case of ketoconazole and DNCB, showing a proin ammatory effect of these substances (Fig 4.). Moreover, there was a signi cant difference between the levels of TNFα in the groups treated with ketoconazole versus KET-PABA (p=0.03) and the one-way ANOVA showed a strongly signi cant difference between the groups (p< 0.0001).
NRF2 level showed a similar trend with that of COX2, it was increased slightly by ketoconazole and PABA and decreased by KET-PABA (Fig 4.). There was a signi cant difference between the effects of ketoconazole and KET-PABA on NRF2 expression (p=0.0007). Overall, the groups showed a signi cantly different effect on NRF2 (p < 0.0001, one-way ANOVA).
NFκB was statistically signi cant increased by exposure to ketoconazole and DNCB. There was a signi cant difference between the groups, as showed by one way ANOVA (p< 0.0001). The level of the phosphorylated, active form -pNFκB was increased in all treated groups, compared to controls (Fig 4.).
The effect was signi cant only in the case of DNCB. One way ANOVA showed a weak signi cance, p=0.0108, between the groups. The ratio of activated form pNFκB / versus total protein NFκB was increased by KET-PABA and PABA exposure, however not signi cantly compared to the vehicle. This is explained by the lower level of NFκB protein in the two groups and the higher level in the ketoconazole and DNCB groups. The ratio pNFκB /NFκB showed signi cant variation between groups (p=0.0023, one way ANOVA).

Discussion
The current study aims to evaluate the possible unwanted reactions of the cocrystal, when applied to the skin. Considering the sensitizing potential of the two active components (ketoconazole and PABA), local skin testing was performed with each individual component, and with the cocrystal compound using the Mouse Ear Swelling Test (MEST). MEST protocol evaluates contact sensitization by a quantitative method and data generated by this test are approved by US FDA (Gad et al. 1986). This was followed by the histopathology tissue assessment and measurement of pro and anti-in ammatory cytokines and in ammation mediators from the ear tissue by ELISA and Western-Blot to have a better image of the underlining mechanisms. Different studies showed bene ts of ketoconazole in many skin disorders: primarily as an e cient topical antimicotic drug, but also in the treatment of androgenetic alopecia by inhibiting 5α-reductase; the treatment of atopic dermatitis and psoriasis by decreasing Malassezia colonization, which is considered a trigger factor (Choi et al. 2019), by anti-in ammatory effect, due to inhibition of 5-lipoxygenase, and by restoring the skin barrier due to inhibition of hyperproliferation; the treatment of acne by decreasing the sebum levels, due to inhibition of androgenesis.
Ketoconazole has also anti-parasitic properties, with indication in the treatment of cutaneous leishmaniasis, and anti-bacterial properties, with indication in the treatment of staphylococcus blepharitis and folliculitis (Choi et al. 2019).
In the present study we evaluated the sensitizing potential of three compounds -KET, PABA and KET-PABA, the last one resulted by co crystallization. The study was performed on BALBc mice because their skin is immunologically like that of humans. This is a commonly used model for epicutaneous sensitization tests of newly developed substances (Tordesillas et al. 2014).
To study the contact sensitization, several animal models have been used, initially the guinea pig and then the mouse. Of these, the most validated test is mouse ear swelling test (MEST) (Garrigue et al. 1994). MEST is based on the method described by Gad and al. and by Thorne for evaluating the potential to cause sensitization in mice, to different substances (Gad et al. 1986, Thorne et al. 1991).
In time, the initial protocol was improved to increase the sensitivity of the test for detection of contact sensitizers. The factors used to increase the sensitivity were the removal of the stratum corneum by tapestripping, the stimulation of the immune system of the animals by increasing reactivity due to vitamin oral A supplementation, and Freund's adjuvant injection, methods that have been reported to have an effect of stimulating epicutaneous sensitization to moderately allergenic substances (Stewart et al. 2006, Tordesillas et al. 2014, Dunn et al. 1990). The advantages of this method turned out to be effective, less expensive, and objective and the data collected more accurate for predicting the response in humans (Gad et al. 1986).
Therefore, in our test we used oral vitamin A supplementation and Freund's adjuvant, to increase the test sensitivity. Moreover, a positive control group using DNCB and a negative control, using vehicle alone were employed to have reference points for the assessment of the developing of an in ammatory reaction.
The MEST test was negative for all tested substances, and shown no epicutaneous sensitization of mice to KET, PABA, and KET-PABA. Interestingly, KET seemed to diminish ear thickness, compared to the initial value and the vehicle, although the difference was not statistically signi cant.
The histopathology results (epidermal spongiosis, with mild acanthosis, slight dermal edema, and a perivascular dermal in ammatory in ltrate) con rmed that contact dermatitis was induced in the positive control group (DNCB). This group was effective as a positive control for contact sensitization and served as a reference for the other test groups. No histological changes were seen in the vehicle and in the other test groups.
Evaluation of contact sensitization is necessary for the development of a new drug as part of the biocompatibility testing and relies on in vitro and in vivo tests in animals. Contact sensitization is a T-cell mediated (type IV) delayed hypersensitivity reaction which results from exposure and sensitization of a genetically susceptible host to an allergen, followed by re-exposure to that allergen that will trigger an in ammatory reaction (Garrigue et al. 1994).
There are two phases in the development of contact sensitization-the sensitization phase and the elicitation phase. In the sensitization phase, haptens, or the unprocessed allergens, penetrate the skin and then are taken up by the dendritic cells. The dendritic cells then migrate via lymphatics to the regional lymph nodes where they present the leucocyte antigen-complex to naive antigen-speci c T lymphocytes.
This process leads to expansion of these T cells, that will immigrate into circulation and then to the skin. The migration of dendritic cells from the skin to the lymph nodes is regulated by different factors, including the cytokines (Sebastiani et al. 2002). In allergic contact dermatitis the in ammatory in ltrate comprises CD4+ and CD8+ T lymphocytes, monocytes, and dendritic cells, and in early phase, neutrophils are also present. Leukocyte attraction is controlled by cytokines. (Sebastiani et al. 2002). The in ammation is mediated by members of IL1 family and other cytokines. (Lauritanoa et al. 2020). The IL1 family includes two molecules, IL1α and IL-1 , responsible for regulating immune reactions and the in ammatory response (Lauritanoa et al. 2020). The important role of these molecules in contact hypersensitivity was demonstrated in studies of mice de cient in IL1α and IL1 genes, and it has been showed that the IL1α has a more critical role. IL1 is produced by monocytes, macrophages, Langerhans cells and dendritic cells, and IL1α is released by keratinocytes. (Ho et al. 2019).
Also, studies showed in an animal model of allergic contact dermatitis that alternatively activated macrophages accumulate in the skin, and these macrophages can exacerbate the contact hypersensitivity, by producing high levels of IL1 , IL6, and TNFα. (Suzuki et al. 2017). IL6 is a well characterized cytokine, and it is known that acts as a pro-in ammatory cytokine.
In our study KET-PABA had the most important anti-in ammatory effect exerted by the stronger decrease of IL1α and IL1 , compared to the vehicle and to KET and PABA and the inhibition of IL6.
In contact hypersensitivity in mice, mast cells may also attenuate the in ammation by producing antiin ammatory cytokines, such as IL10 (Lauritanoa et al. 2020). Another source of IL10 are the epidermal keratinocytes. IL10 acts on monocytes, macrophages, and dendritic cells, inducing inhibition of expression of class II MHC and inhibit the production of T cell-stimulating cytokines, such as IL1, IL6 and IL12. (Ho et al. 2019). Studies showed that the absence of IL10 predispose mice to exaggerated contact sensitivity responses. In our model, KET-PABA had no effect on IL10, while KET, PABA and DNCB increased it. This correlated with the decrease of all the other pro-in ammatory cytokines such as IL1α and β, IL6, or the lack of augmentation of the other in ammatory promoters: COX2, TNFα and NRF2. The data suggest that the increase of the IL10 was no longer necessary, since IL10 is secreted to alleviate and modulate an in ammatory state. In the presence of KET-PABA, the treated skin showed a normal status, similar to that of the vehicle for most of the in ammatory molecules or even the inhibition of the IL1 and IL6 compared to the vehicle. Overall, data showed a signi cant anti-in ammatory effect exerted by the local application of the KET-PABA. This effect could have additional bene cial clinical applications in the case of the treatment of a cutaneous mycotic infection especially in the presence of an acute in ammatory reaction to the pathogen.
NFκB signaling pathway is involved in in ammation, and TNFα, IL1β, IL6, COX2, and NOS2 are biomarkers for in ammation, which can be induced by dermatitis (Li et al. 2016).
The cytokines, particularly tumor necrosis factor alpha (TNFα), which is secreted by epidermal keratinocytes, plays an important role in in ammation in the skin. TNFα induces in ammation by activation of cytokines IL6 and IL1β, and by increasing the in ux of in ammatory cells via induction of expression of adhesion molecules such as endothelial leukocyte adhesion molecule 1 and intercellular adhesion molecule 1 (ICAM1) on neighboring endothelial cells (Zhang et al. 2014).
TNFα can also activate the nuclear factor κB (NFκB) signaling pathway, leading to chronic in ammation in the skin. NFκB regulates the expression of many genes that are involved in the initiation of the in ammatory response. Also, the synthesis of cytokines, such as TNFα, IL1β, IL6, and IL8, and the expression of cyclooxygenase 2 (COX2) are mediated by NFκB (Tak et al. 2001). During the sensitization phase, dendritic cells are activated, migrate to the draining lymph nodes, and induce the priming of hapten-speci c T cells (Kissenpfennig et al. 2005). NRF2 is induced in response to contact sensitizers in dendritic cells and keratinocytes (Emter et al. 2010).
In our study, NRF2 has shown an important increase in the DNCB group, suggesting the activation of the sensitization phase, in the presence of the substance, along with the other markers like COX2 and the cytokines. In the KET and PABA groups, NRF2 also showed a slight increase, correlated with the COX2 and TNFα increases. Interestingly, in the KET group, TNFα showed a stronger induction, an effect observed by us in vitro, on endothelial (HUVEC) cells but not on dermal broblasts (HDFa) (Martin et al. 2020).
This effect correlated with the increase of the IL6. Overall data show that KET had a slight proin ammatory effect, but it was modulated by the presence of the increased anti-in ammatory cytokine Il10 which led to the suppression of the in ammation. This rendered the negative MEST result as well as the lack of morphopathology changes in the ear tissue. In the KET-PABA group, there was no NRF2 response, which suggests that the substance did not elicit any in ammatory reaction.
NRF2 plays a major role in the control of both phases of ACD: sensitization and elicitation. During the sensitization phase, NRF2 in uence the irritation potency of the contact sensitizers, and, has the role of signaling the danger (Ali et al. 2013). NRF2 de ciency leads to an increased in ammatory response during the elicitation phase, suggesting that NRF2 may also play a role in the regulation of in ammation through a defect in regulatory T cells function (Josefowicz et al. 2012).
The immune response which appears in allergic contact dermatitis involves the oxidative and in ammatory pathways. The NRF2/Keap1 pathway is a major regulator of cellular oxidative and electrophilic stress, and is activated in the different skin innate immune cells including epidermal Langerhans cells and dermal dendritic cells, but also in keratinocytes.
Both Nrf2 and NFκB pathways are activated by oxidative stress. NRF2 antagonizes NFκB activation through IKK proteasomal activation and HO-1 activity end-products. NFκB downregulates NRF2 via p65mediated CBP deprivation as well as p65-induced Keap1 nuclear translocation. Nrf2 and NFκB are regulated antagonistically: if NRF2 predominates, it decreases in ammation and oxidative stress through activation of antioxidant enzymes; if NFκB predominates, it leads to pro-in ammatory mediators' secretion and maintains the oxidative stress (Helou et al. 2019).
In the present study, there is a strong NFκB induction in the ketoconazole and DNCB groups, further leading to a proin ammatory environment. KET-PABA had no effect on NFκB level. Interestingly, in the case of the activated form, p NFκB, the effects were only statistically signi cant in the case of DNCB.
NFκB is a key transcription factor of M1 macrophages and induces the transcription of pro-in ammatory mediators such as IL6, TNFα, and COX2 ).
Prostaglandins play a key role in the generation of the in ammatory response. They are produced during the acute phase of the in ammation. Prostaglandin production depends on the activity of enzymes that contain both cyclooxygenase and peroxidase activity and which exist as distinct isoforms referred to as COX1 and COX2.
COX2 is induced by in ammatory stimuli, and is the most important source of prostaglandin synthesis. It has been suggested that COX2 have a dual role in the in ammatory process, initially contributing to the onset of in ammation and later in supporting resolution of this process (Ricciotti et al. 2011).
In our study, KET-PABA induced the decrease of COX2 and IL6, compared to the vehicle, while KET and PABA slightly increased them. This is consistent with the inhibition of the NRF2 and IL1 and the lack of NFkB activation and suggests an intrinsic anti-in ammatory effect of the cocrystal on the skin of BALBc mice. In our study the contact sensitization test was negative for all substances tested -Ketoconazole, PABA, and cocrystal compound KET-PABA. Knowing the allergenic potential of PABA, reported in the literature (Aronson 2016, Glaser 2016) multiple, high-dose skin applications were used, but no contact allergy was found.
However, the allergenic potential of PABA is very small, and an extremely large number of cases are needed for validation.
Compared to animals, in humans the sensitivity is more variable, and it is highly di cult to predict what proportion of the human population will be sensitized. The compound obtained by co crystallization (KET-PABA) is composed by two active components, KET and PABA, each of them having different properties.
A negative nding does not guarantee that this compound will not be a sensitizer in humans, although we expect it to be at most a weak sensitizer.
Another problem that could raise is the cross-sensitization, meaning that one component of the compound is not distinguished as different by the educated lymphocytes.
Because PABA is widely used in cosmetics, the number of users is high, and the possibility of allergies is obviously increased with the number of applications. Another aspect of PABA use is the occurrence of phototoxic reactions. Because PABA is used in creams as a sunscreen, these reactions can occur as an important part of the reported skin reactions. On the other hand, since PABA functions as a sunscreen, the cocrystal might have a bene cial role in protecting ketoconazole against UV induced degradation and skin phototoxic reactions. However, these hypotheses were not tested in our study. In the current research, the mice were not exposed to UV, so phototoxic reactions were not studied. Further studies are needed to test the appearance of this type of adverse reactions prior to complete the safety pro le of the substance. In fact, KET-PABA has been synthesized for a possible use as an antifungal product with topical application, which does not involve concomitant exposure to ultraviolet radiation.

Conclusions
The current study investigated the local possible side effects induced on the skin of BALBc mice by the application of KET-PABA cocrystal. The previously characterized co-crystal showed improved physical properties, such as stability in suspension, solubility, as well as antimycotic e ciency as compared to the parent drug. According to our data, KET-PABA application proved to be safe, without any sensitization effects showed by the MEST study, or the histopathological exam. KET-PABA induced a potent antiin ammatory effect through the inhibition of proin ammatory cytokines such as IL1α, IL1β, IL6 and TNFα, and other proin ammatory inducers such as NRF2. KET-PABA had no effect on the levels of the anti-in ammatory cytokine IL10, or proin ammatory enzyme COX2 and had minimal effects on the activation of the NFκB pathway. Overall, KET-PABA application led to a better anti-in ammatory response when compared to ketoconazole. Based on the improved antimycotic effect and anti-in ammatory action, KET-PABA cocrystal has the potential to be an e cient drug in the treatment of cutaneous mycosis. Mouse ear swelling test (MEST). The measurements of the ear thickness (% of initial value) for each group are presented (mean ± SD, n=7); C24h, C48h = challenge at 24h, respectively 48h from ear skin application, R24h and R48h = rechallenge at 24h respectively 48h from ear skin applications; *** = p< 0.0001 compared to the vehicle; #= p<0.05, ###=p<0.0001 between the different time points. Last panel presents the distribution values of the ear thickness within the groups, *= p< 0.05 and ***= p< 0.0001.  ELISA measurement of pro-in ammatory interleukins IL1α, Il1β and IL6. Data are presented as mean ± SD, n=3, * = p< 0.05, ***= p< 0.0001, compared to vehicle; #=p<0.05, ###= p<0.0001, compared to other groups.