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-inflammatory cytokines and inflammation mediators from the ear tissue by ELISA and Western-Blot to have a better image of the underlining mechanisms.
Topical ketoconazole is the first-line therapy, according to current guidelines, for seborrheic dermatitis and pityriasis versicolor (Choi et al. 2019), but it is also approved by the United States FDA for the treatment of candidiasis and dermatophyte infections.
Different studies showed benefits of ketoconazole in many skin disorders: primarily as an efficient 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-inflammatory 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 tape-stripping, 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 inflammatory 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 significant.
The histopathology results (epidermal spongiosis, with mild acanthosis, slight dermal edema, and a perivascular dermal inflammatory infiltrate) confirmed 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 inflammatory 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-specific 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 inflammatory infiltrate 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 inflammation is mediated by members of IL1 family and other cytokines. (Lauritanoa et al. 2020). The IL1 family includes two molecules, IL1α and IL-1ꞵ, responsible for regulating immune reactions and the inflammatory response (Lauritanoa et al. 2020). The important role of these molecules in contact hypersensitivity was demonstrated in studies of mice deficient in IL1α and IL1ꞵ genes, and it has been showed that the IL1α has a more critical role. IL1ꞵ 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 IL1ꞵ, IL6, and TNFα. (Suzuki et al. 2017). IL6 is a well characterized cytokine, and it is known that acts as a pro-inflammatory cytokine.
In our study KET-PABA had the most important anti-inflammatory effect exerted by the stronger decrease of IL1α and IL1ꞵ, 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 inflammation by producing anti-inflammatory 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-inflammatory cytokines such as IL1α and β, IL6, or the lack of augmentation of the other inflammatory 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 inflammatory state. In the presence of KET-PABA, the treated skin showed a normal status, similar to that of the vehicle for most of the inflammatory molecules or even the inhibition of the IL1 and IL6 compared to the vehicle. Overall, data showed a significant anti-inflammatory effect exerted by the local application of the KET-PABA. This effect could have additional beneficial clinical applications in the case of the treatment of a cutaneous mycotic infection especially in the presence of an acute inflammatory reaction to the pathogen.
NFκB signaling pathway is involved in inflammation, and TNFα, IL1β, IL6, COX2, and NOS2 are biomarkers for inflammation, 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 inflammation in the skin. TNFα induces inflammation by activation of cytokines IL6 and IL1β, and by increasing the influx of inflammatory 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 inflammation in the skin. NFκB regulates the expression of many genes that are involved in the initiation of the inflammatory 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-specific 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 fibroblasts (HDFa) (Martin et al. 2020).
This effect correlated with the increase of the IL6. Overall data show that KET had a slight pro-inflammatory effect, but it was modulated by the presence of the increased anti-inflammatory cytokine Il10 which led to the suppression of the inflammation. 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 inflammatory reaction.
NRF2 plays a major role in the control of both phases of ACD: sensitization and elicitation. During the sensitization phase, NRF2 influence the irritation potency of the contact sensitizers, and, has the role of signaling the danger (Ali et al. 2013). NRF2 deficiency leads to an increased inflammatory response during the elicitation phase, suggesting that NRF2 may also play a role in the regulation of inflammation 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 inflammatory 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 p65-mediated CBP deprivation as well as p65-induced Keap1 nuclear translocation. Nrf2 and NFκB are regulated antagonistically: if NRF2 predominates, it decreases inflammation and oxidative stress through activation of antioxidant enzymes; if NFκB predominates, it leads to pro-inflammatory 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 proinflammatory environment. KET- PABA had no effect on NFκB level. Interestingly, in the case of the activated form, p NFκB, the effects were only statistically significant in the case of DNCB.
NFκB is a key transcription factor of M1 macrophages and induces the transcription of pro-inflammatory mediators such as IL6, TNFα, and COX2 (Wang et al. 2014).
Prostaglandins play a key role in the generation of the inflammatory response. They are produced during the acute phase of the inflammation. 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 inflammatory stimuli, and is the most important source of prostaglandin synthesis. It has been suggested that COX2 have a dual role in the inflammatory process, initially contributing to the onset of inflammation 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-inflammatory 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 difficult 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 finding 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 beneficial 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 profile 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.