Evaluation of novel cosmetic shampoo formulations against Malassezia species: Preliminary results of anti‐dandruff shampoo formulations

Malassezia species are common, clinically relevant, and lipid‐dependent yeasts of humans. They are also the leading causes of the dandruff problem of humans, and the azoles are used primarily in their topical and systemic treatment. Resistance to azoles is an emerging problem among Malassezia sp., which indicates the need of new drug assessments that will be effective against dandruff and limit the use of azoles and other agents in treatment. Among them, the efficacy of various combinations of piroctone olamine and climbazole against Malassezia sp. is highly important. Here, we assessed the efficacies of various piroctone olamine and climbazole formulations against Malassezia sp. in comparison with ketoconazole.


| INTRODUC TI ON
Inflammatory or non-inflammatory clinical manifestations due to Malassezia species, which are lipid-dependent yeasts, have been emerging.There are more than 18 described Malassezia sp.today, which are mostly present as commensal members of the skin microbiota, according to the age of the individual.6][7][8] Malassezia sp. is common in the etiology of head and neck dermatitis, seen after puberty as a subtype of atopic dermatitis.Type I hypersensitivity reaction is the expected pathogenic mechanism against Malassezia sp.Similarly, involvement of Malassezia sp. is common in the etiologies of both seborrheic dermatitis and Pityriasis versicolor infestation.
Dandruff and seborrheic dermatitis are common diseases of the scalp that have basically the same histological structure. 9ographical and ethnic differences in the distribution of Malassezia in healthy individuals and as disease agents are reported. 10The incidence of seborrheic dermatitis/dandruff ranges from 30% to 95%, which is due to the lifestyle, habits, climate, and genetic factors, and the causative agents vary according to the location. 2,7,102][13] In dermatological lesions such as pityriasis versicolor, M. sympodialis is considered predominant in the northern and southern United States, while it is also a leading species in the northern hemisphere. 10,14However, it has been observed that dandruff was correlated more with M. restricta, while a lower M. restricta to M. globosa ratio was associated with a healthier scalp.In general, in the etiology of dandruff, a high association with many different Malassezia species within the mycobiome has also been observed. 15Although M. restricta is at the forefront in the etiology of dandruff, it is observed that it is not primarily isolated in Türkiye.
Malassezia sp. is known as the predominant fungi on the mycoflora of the scalp. 4Elevation of unsaturated fatty acid levels is a predisposing factor associated with Malassezia sp.reproduction capacity, while the metabolites of Malassezia sp. may significantly promote dandruff formation. 4,16,17Extreme colonization of Malassezia sp.9][20] New therapeutics have been presented lately for the treatment of Malasseziaassociated skin lesions and dandruff.Effective skin penetration and contact duration are common mechanisms in anti-dandruff shampoos. 18,20Treatment strategies that aim to control hypersensitivity reactions on the skin target primarily the lowering the yeast colonization on the scalp.Such treatment protocols include single or combined applications of antifungals and anti-inflammatory agents.
Alleviation of fungal dysbiosis is also important, especially when the ratios between Malassezia species are varied.Wang et al. 21reported that glycyrrhetinic acid shampoo may be noteworthy as both a therapeutic and maintenance regimen for scalp seborrheic dermatitis as it reduces the number of both M. restricta and M. globosa in addition to classical antifungal treatment options.5][26][27][28][29][30] Zinc pyrithione was also another ingredient of cosmetic shampoos until it was banned by the European Union in 2022.Considering the ban risk also for the other three anti-fungal active compounds, studies are required to confirm not only their efficacy but also their non-hazardous nature to human health.
It was well documented that application of shampoos that contain zinc pyrithione, KTZ, PRT, and CLZ to scalp lowers both Malassezia sp.content and skin flaking, which is a common disorder. 4,31PRT is a common ingredient of various dermatological anti-dandruff formulations. 20,31It has been reported that different topical formulations exhibit different levels of skin penetration for PRT.Its physicochemical structure is still under investigation, but PRT's skin penetration is known to be modified with different excipient and solvents. 32other ingredient, CLZ, has been present in different formulations of anti-dandruff treatment.4][35] Unveiling the safety levels and efficacies of both PRT and CLZ is highly essential, after zinc pyrithione was banned and the treatment options became limited.The aim of the present study was to assess the efficacies of different concentrations of PRT and CLZ on various standard Malassezia species in vitro.1.

| Mycological procedures
Three different standard Malassezia strains isolated from the dandruff (Malassezia furfur CBS1878, Malassezia globosa CBS7874, and Malassezia sympodialis CBS9570) were included in the study.All strains were subcultured in mDixon agar medium within 4 days at 33°C.The yeasts were added to sterile saline solution according to 0.5 McFarland level, and mDixon agar medium was spread over these yeasts using a sterile swab.To avoid humidity in the medium, all samples were kept at room temperature for 15 min.Discs having 6 mm of diameter were prepared using Whatman No. 1 filter paper and sterilized at 140°C for an hour.These sterile disks were immersed in the samples for 5 min in non-septic conditions.KTZ (15 μg/disc) was assessed as the positive control in the test with the same method (Table 1). 36Discs were then immersed in Petri dishes with yeast using sterile swabs.All plates were monitorized for 4 days in humid environment at 33°C, and their inhibition zones were checked in mm visually in the end of incubation.All tests were triplicated, and their results were recorded daily.

| Statistical analysis
Statistical analysis was performed with the Minitab® (Ver 16.1.1,Cologne, Germany).One-way analysis of variance (ANOVA) with Dunnett's multiple-comparison tests were implemented to compare the inhibition zone of different Malassezia strains with both the base experimental formula (PO00CL00) and 2% medical shampoo containing KTZ (MS2KTZ).p values of <0.05 were considered as significant.

| RE SULTS
On Day 4, relatively prominent inhibition zones were detected around the discs.The inner zone area was considered as the exact limit of inhibition.Sharp cut-off values were observed by naked-eye evaluation (Figure 1).The inhibition zone of each experimental formula on three Malassezia species were shown in Figure 2. According to the base formulation without PRT and CLZ as active substances; PO01CL00 and PO10CL00 were not different from the base, while the other formulations analyzed were more effective than the base.
The formulation PO10CL01 showed the same effect for M. furfur and M. sympodialis as the medical shampoo containing 2% KTZ, while PO01CL05 showed the same effect for M. furfur (p > 0.05).
For M. globosa, none of the formulations tested was as effective as MS2KTZ (p < 0.05).

| DISCUSS ION
Today, hair shedding and hair thinning are two common problems associated with the dandruff. 37Sunlight, together with commensal microorganisms, are the major environmental factors associated with these problems.It has long been known that seasonal variations and geographical differences may influence the lipid content of the skin. 17,38With or without inflammation, the lipophilic Malassezia genus plays a key role in their pathogenesis, and reduction of these yeasts on the scalp is one of the main therapeutic targets. 3,4,31,37Z, CLZ, and PRT are common agents that are active against TA B L E 1 Studied formulations and their properties.Malassezia sp.; however, the outcomes of their application are mostly based on clinical interpretations since standard antifungal testing is not possible due to the lipophilic nature of Malassezia sp. 4,31,33,34,37 is suggested that testing of not only the physicochemical characteristics but also the antifungal efficacies of therapeutics is crucial.

No
Broth microdilution is mostly preferred for antifungal testing; however, due to the testing of surfactant-like products in dermatological treatment assessments, which may be confusing with microdilution, diffusion-based tests should be chosen. 39It is shown that the standard dilution assays are suitable for the non-lipophylic M. pachydermatis; however, disk diffusion tests may be useful for the lipophilic Malassezia species according to the characteristics of the tested agent in lipophilic conditions, while they are not suitable for all antifungals. 36,40Assessments with KTZ revealed no linear correlation between the disk diffusion and liquid microdilution.In the present study, KTZ levels in each three standard Malassezia sp. was found to be as expected regarding the study of Rojas et al. 36 The disk diffusion method used in the present study as described by Pooja et al. 40 supports the validity of the method of our study.
It was observed that PRT, CLZ, and their combinations with different ratios in the shampoo formulas that were added in different concentrations managed to form inhibition zones under the same conditions.It is possible to claim that the diffusion capacities of tested chemicals may be insufficient to determine true results.
Since there are no disk diffusion test results with PRT and CLZ in the limited literature, it is not possible to compare our results at the moment.Yet, remarkable inhibition zones were determined in all tested Malassezia species against both PRT and CLZ, which may be regarded as sensitive, in our study.Regarding M. furfur and M. sympodialis, different cosmetic shampoo formulations of both PCT and CLZ were found to be as effective as KTZ in the present study, while this was not shown against M. globosa (Figure 2).
Although M. globosa is generally accepted as a causal agent of dandruff, the species distribution of Malassezia sp.varies depending on the geographical location of the host. 2,10,14,41

| CON CLUS ION
According to the results of this study, climbazole and piroctone olamine combination seem to be an effective option for a cosmetic shampoo with high antifungal/anti dandruff efficacy.New in vitro and in vivo studies are required to further support the efficacy of climbazole and piroctone olamine combinations.

A
total of nine formulations were assessed in the study.One of them was a shampoo base without any active agents.Two active agents (piroctone olamine [PRT] and/or climbazole [CLZ]) were combined to form a total of seven formulations in the base shampoo.In addition, a K E Y W O R D S climbazole, dandruff, hair treatment, Malassezia sp., piroctone olamine medical shampoo was included in the study.The base shampoo consisted of: water, sodium laureth sulfate, cocamidopropyl betain, glycerin, sodium chloride, decyl glucoside, parfum, benzyl alcohol, carbomer, glycol distearate, menthol, dimethiconol, TEA-dodecylbenzenesulfonate, guar hydroxypropyltrimonium chloride, cocamide MEA, methylchloroisothiazolinone, methylisothiazolinone.All materials were added to water and dissolved at room temperature.A carbomer resin was used for thickening.The process is completed by adjusting the pH between 5.2 and 6.0.The medical anti-dandruff shampoo was purchased and used for the study.The details of the formulated shampoos are listed in Table

F I G U R E 2
Inhibition zone diameter of the tested samples of the strains in the study.PO10CL01 for (**) Malassezia sympodialis, and both PO01CL05 and PO10CL01 are as effective as medical 2% ketoconazole (MS2KTZ) formulation on (*) Malassezia furfur (*, ** = p > 0.05).while the inhibition was no different than the one in Sample 6 (PO01CL01; PRT 0.1% -CLZ 0.1%) combination.These results are comparable with Sample 9 (MS2KTZ; KTZ 2% w/w Shampoo), which contains only KTZ.Considering the Malassezia sp.studied, PRT alone (PO01CL00, PO10CL00) had an inhibitory effect, but less than the effect of CLZ alone (PO00CL01, PO00CL05).The combination forms had a remarkable inhibitory effect on all three Malassezia sp.Previously, it has been shown that 2% climbazole shampoo lowered the number of Malassezia sp. on the dogs.42Clinical efficacy was reported with combination formulas, as well.31,34KTZ is an old antifungal agent of the azole group; it has long been sold in the market worldwide as a commercial medical shampoo.There is a common resistance to the azole group antifungals in systemic Malassezia infections.However, this is not yet common among the Malassezia sp.isolated from the skin,[43][44][45][46] although gene mutations have recently been demonstrated in M. globosa and M. sympodialis, which were associated with lower sensitivity to azole-group antifungals.46Development of new cosmetic shampoos that contain PRZ-CLZ combination is expected to lower the use of azole group antifungals.This will also prevent the spread of azole resistance in the community as well.The decision of European Union Regulatory Board about the prohibition of zinc pyrithion in antidandruff shampoo formulations is highly important as it creates an urgent need worldwide to develop new commercial products with available, effective, and non-restricted active ingredients against Malassezia sp.In the present study, CLZ and PRT, which are permitted by EU in the maximum concentrations of 0.5% and 1%, respectively, for rinse-off products (Regulation (Ec) No. 1223/2009 of the European Parliament and of the Council), were found to be effective against Malassezia sp.Further studies are warranted to confirm these findings.