Photostability Testing of a Third-Generation Retinoid—Tazarotene in the Presence of UV Absorbers

pharmaceutics

Article Photostability Testing of a Third-Generation Retinoid—Tazarotene in the Presence of UV Absorbers

Agata Kryczyk-Poprawa 1,* , István Zupkó 2,3 ,Péter Bérdi 2, Paweł Zmudzki˙ 4 , Justyna Popiół 5 , Bo˙zenaMuszy ´nska 6 and Włodzimierz Opoka 1

1 Department of Inorganic and Analytical Chemistry, Jagiellonian University Medical College, 30-688 Kraków, Poland; [email protected] 2 Department of Pharmacodynamics and Biopharmacy, University of Szeged, 6720 Szeged, Hungary; [email protected] (I.Z.); [email protected] (P.B.) 3 Interdisciplinary Center for Natural Products, University of Szeged, 6720 Szeged, Hungary 4 Department of Medicinal Chemistry, Jagiellonian University Medical College, 30-688 Kraków, Poland; [email protected] 5 Department of Pharmaceutical Biochemistry, Jagiellonian University Medical College, 30-688 Kraków, Poland; [email protected] 6 Department of Pharmaceutical Botany, Jagiellonian University Collegium Medicum, 30-688 Kraków, Poland; [email protected] * Correspondence: [email protected]; Tel.: +48-126-205-480

Received: 7 August 2020; Accepted: 16 September 2020; Published: 22 September 2020

Abstract: Exposure of a drug to UV irradiation could affect its physicochemical properties. Hence, photostability testing is essential for topically administered drugs. Tazarotene, a receptor-selective, third-generation retinoid, is commonly used to treat acne vulgaris and psoriasis. In the present study, an in-depth analysis of the photostability of tazarotene in ethanolic solution in the presence of zinc oxide and/or titanium dioxide as well as benzophenone-type UV filters was performed. Eleven presumed products were derived from the photocatalytic degradation of tazarotene using ultra-performance liquid chromatography–tandem mass spectrometry, and transformation pathways were proposed. The degradation process mainly affected the 4,4-dimethyl-3,4-dihydro-2H-thiopyran moiety. The fragments most susceptible to oxidation were the methyl groups and the sulfur atom. Moreover, in the presence of sulisobenzone, under UV irradiation, tazarotene was subjected to a degradation process, which resulted in two photodecomposition products. In silico studies performed by OSIRIS Property Explorer demonstrated that five of the degradation products could be harmful in terms of the reproductive effects, which are associated with 3,4-dihydro-6-methyl-2H-1-benzothiopyran 1,1-dioxide, while one of them demonstrated potential irritant activity. The cytotoxic properties of the degradation products of tazarotene were assessed by MTT assay on a panel of human adherent cancer cells. Time- and concentration-dependent growth inhibition was evidenced in ovary (A2780) and breast (MDA-MB-231) cancer cell lines. The potential implication of the outcomes of the present research requires further studies mainly concerning the photostability of tazarotene in the topical formulations.

Keywords: tazarotene; photostability studies; photocatalytic degradation; benzophenones; TiO2; ZnO

1. Introduction Tazarotene (ethyl 6-[2-(4,4-dimethyl-2,3-dihydrothiochromen-6-yl)ethynyl]pyridine-3-carboxylate) is a third-generation topical retinoid, which is applied to the skin. It is a prodrug, which undergoes

Pharmaceutics 2020, 12, 899; doi:10.3390/pharmaceutics12090899 www.mdpi.com/journal/pharmaceutics Pharmaceutics 2020, 12, 899 2 of 20 in vivo deesterification in its active form—the cognate carboxylic acid of tazarotene [1]. Tazarotene acid selectively binds to the retinoic acid receptors—RAR-β and RAR-γ [2,3]. This drug is used in the treatment of mild-to-moderate facial acne vulgaris and plaque psoriasis affecting nearly 20% of the body surface [4–6]. Acne vulgaris is a common, chronic skin disease, which affects approximately 90% of humans across the world at some point in their lives. Topical retinoids are currently used in the first-line treatment of the disease. Furthermore, tazarotene is used to treat other skin conditions such as photoaging and facial mottled hyper- and hypopigmentation [7]. It slightly penetrates across the skin, and 10 h after its application, ~2% of the dose reaches the viable epidermis and dermis [8]. Tazarotene gel is also used with narrow-band UVB phototherapy for psoriasis because it enhances the efficiency of the therapy and enables a significant reduction of the irradiation dose [9,10]. Like other topical retinoids, tazarotene could cause photosensitivity reactions—generally mild and reversible phototoxic or photoallergic reactions—and display minor photoirritant potential [1,11,12]. Besides, topical photoprotection is an extremely important area of dermatologic research on skin cancer prevention strategies [13]. A better understanding of the potential interactions between different substances applied simultaneously to the skin, e.g. UV absorbers and active pharmaceutical ingredients, is essential for ensuring the safe and effective administration of drug products [14]. The behavior of these substances is not predictable from independent photostability testing [15,16]. Hence, it is imperative to evaluate the combinations of substances used in the formulations as well as active pharmaceutical ingredients used with cosmetic ingredients [17,18]. The previous investigation of the photostability of terbinafine, in both solutions and formulations in the presence of UV absorbers such as TiO2, ZnO, avobenzone, 3-(4-methylbenzylidene)camphor, octocrylene, benzophenone-1, and benzophenone-2, showed that UV absorbers had a diverse impact on terbinafine stability [19]. It should be emphasized that photostability testing is an integral part of the stability studies included in the Q1A–Q1F Quality Guidelines of The International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH). The guideline concerning the Photostability Testing of New Drug Substances and Products—Q1B was implemented in Europe on 1 December 1996 and in the USA on 1 May 1997 [20]. The photoinstability of a drug could impact its effectiveness and lead to the formation of products with unknown activity [21]. The lack of detailed information about the photostability of tazarotene prompted us to perform in-depth research on this third-generation retinoid. Tazarotene is considered to be photostable; however, it is specifically used by young people who spend a lot of time outside and are exposed to UV irradiation, which increases the frequency of the simultaneous use of UV filters. Hence, there is an urgent need to study the interactions between tazarotene and UV absorbers under UV irradiation. The present study aimed to assess the impact of selected UV absorbers extensively used in sunscreens and cosmetics on the photostability of tazarotene in ethanol solutions under UV irradiation. Benzophenone compounds were chosen due to their widespread employment in cosmetics, photostability, and wide-ranging spectrum protection against not only UVB (290–320 nm) in particular but also UVA (320–400 nm) [22,23]. Out of the twelve benzophenone-type UV absorbers, the following four were selected for this study: benzophenone-1, benzophenone-2, benzophenone-3 (oxybenzone), and benzophenone-4 (sulisobenzone). Additionally, avobenzone, which is one of the most common UVA filters, was investigated. Due to its rapid photodegradation, avobenzone should be included with a stabilization agent—such as octocrylene [24]. Furthermore, an assessment of the effect of TiO2 and ZnO nanoparticles, which could be used as physical UV filters in sunscreens, on the stability of tazarotene under UV irradiation was performed. According to the regulations of the US Food and Drug Administration (FDA), ZnO and TiO2 are used in sun protection products in maximum concentration up to 25% [25]. It is universally accepted that ZnO and TiO2 as semiconductors could be used in heterogeneous photocatalysis [26,27]. Both the oxides display high photocatalytic activity and are chemically and photochemically stable. Nevertheless, there is very little information in the research literature about the potential photocatalytic degradation products of tazarotene. The overarching goal of the conducted in vitro tests was to determine the Pharmaceutics 2020, 12, 899 3 of 20 plausible structures of the photoproducts formed under experimental conditions and to evaluate their cytotoxicity.

2. Materials and Methods

2.1. Reagents Tazarotene (ethyl 6-[2-(4,4-dimethyl-2,3-dihydrothiochromen-6-yl)ethynyl]pyridine-3-carboxylate) was purchased from Sigma-Aldrich (St. Louis, MO, USA). High-performance liquid chromatography (HPLC)-grade methanol, acetonitrile, and formic acid (98%) were purchased from J.T. Baker (Phillipsburg, NJ, USA). Ethanol absolute for HPLC 99.8% was obtained from POCH (Gliwice, Poland). ≥ 1 Water (quadruple-distilled) with a conductivity of less than 1 µS cm− was prepared using the S2-97A2 distillation apparatus (ChemLand, Stargard Szczecin, Poland). Zinc oxide (nanopowder <100 nm particle size) and titanium (IV) oxide (anatase, nanopowder < 25 nm particle size, 99.7% trace metals basis) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The UV absorbers—benzophenone-1, benzophenone-2, benzophenone-3, benzophenone-4, avobenzone, and octocrylene—were obtained from Sigma-Aldrich (St. Louis, MO, USA).

2.2. Reagent Solutions Preparation

1 The stock solution of tazarotene was prepared at a concentration of 0.2 mg mL− in a 50-mL volumetric ﬂask by dissolving the weighted portion of the standard substance in ethanol and was then stored at 2–8 ◦C for no longer than three days. For the method validation, six solutions with tazarotene 1 concentrations of 0.01, 0.025, 0.05, 0.1, 0.15, and 0.2 mg mL− were prepared. For the assessment of photocatalytic degradation, suspensions of TiO2, ZnO, or TiO2/ZnO (1:1, w/w) in ethanol were prepared before each experiment by adding 100 mg of oxide to 5 mL of ethanol and were then sonicated in an ultrasonic bath for 3 min. The solutions of organic UV absorbers were made at a concentration of 1 1 mg mL− .

2.3. Photostability Assessments The investigated solutions were placed in quartz Petri dishes with Ø 50 12 mm (Hornik, Poznań, × Poland), which were protected against evaporation with parafilm. The irradiation experiments were carried out in a solar light simulator (Suntest CPS+, Atlas, Germany) which included an optical filter cutting off wavelengths shorter than 290 nm and an infrared-block filter to minimize the thermal 2 2 effects. The samples were irradiated for 1 h at 500 W m− (cumulative dose of UV radiation 218 kJ m− ). For photostability analyses of tazarotene in ethanol, 2 mL of the investigated solution at a final 1 concentration of 0.1 mg mL− was irradiated. Photocatalytic degradation tests were performed by adding 1 mL of tazarotene solution, 0.5 mL of one of the prepared suspensions of TiO2, ZnO, or TiO2/ZnO along with the right amount of ethanol to achieve the final concentration of tazarotene. After the stipulated time of irradiation, the quartz dishes were withdrawn, and the samples were filtered through 0.45 µm syringe filters before the ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) analysis. Analogously prepared samples, which were protected with aluminum foil before irradiation, were used as the dark control. For UV absorbers, analogously prepared solutions containing 1 mL of 0.2 mg 1 mL− tazarotene solution, 0.8 mL of ethanol, and 0.2 mL of benzophenone-type UV absorber solution in ethanol or 0.1 mL of avobenzone +0.1 mL of octocrylene were exposed to UV irradiation in a solar light simulator. Pharmaceutics 2020, 12, 899 4 of 20

2.4. In Vitro Cytotoxicity Assays

2.4.1. Investigated Solutions The stock solution of tazarotene in ethanol was prepared in a 10-mL flask at a concentration of 10 mM. The prepared samples containing 2 mL of the above solution with the additional 10 mg of TiO2/ZnO (1:1, w/w) were placed into quartz dishes. The samples were irradiated for 0.5, 1, and 2 h in a solar light simulator. Later, the samples were filtered through a 0.45 µm filter and subjected to cytotoxicity testing.

2.4.2. Cell Lines and Cell Growth Inhibitory Assay Human cancer cell lines isolated from breast (MDA-MB-231), cervix (HeLa), and ovary (A2780) cancers were purchased from European Collection of Cell Cultures (ECCAC, Salisbury, UK). Cells were cultivated in minimal essential medium supplemented with 10% fetal bovine serum, 1% antibiotic–antimycotic mixture, and nonessential amino acids. All media and supplements were obtained from Lonza Group Ltd. (Basel, Switzerland). Near-confluent cells were seeded onto a 96-well microplate at a density of 5000 cells/well, and after overnight standing, new medium containing the tested substance was added. After incubation for 72 h at 37 ◦C in humidified air containing 5% CO2, the viability of the cells was determined by the addition of 20 µL of MTT 1 (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5 mg mL− ) solution. After a 4h contact period, the medium was removed, and the precipitated formazan crystals were dissolved in 100 µL of dimethyl sulfoxide (DMSO) during a 60 min period of shaking. Finally, the produced formazan was assayed at 545 nm, using a microplate reader utilizing wells with untreated cells as control [28]. All in vitro experiments were carried out on two microplates with at least five parallel wells.

2.5. In Silico Toxicity Prediction OSIRIS Property Explorer was used to predict mutagenicity, tumorigenicity, irritation, and reproductive eﬀects of tazarotene and its eleven photocatalytic degradation products [29]. The investigated structures were used to predict the toxicity risks related to the speciﬁc risk category.

2.6. Ultra-Performance Liquid Chromatography–Tandem Mass Spectrometry Analysis The UPLC–MS/MS system equipped with a Waters ACQUITY® UPLC® (Waters Corporation, Milford, MA, USA) and a Waters TQD mass spectrometer (electrospray ionization (ESI) mode tandem quadrupole) was used for the analysis. The investigated compound was analyzed on a Acquity UPLC BEH (bridged ethyl hybrid) C18 column (2.1 100 mm, 1.7 µm), comprising an Acquity UPLC BEH × C18 VanGuard precolumn (2.1 5 mm, 1.7 µm) eluted under gradient conditions using 95% to 0% × 1 of eluent A over 10 min, at a flow rate of 0.3 mL min− . The column temperature was maintained at 40 ◦C. Eluent A was water/formic acid (0.1%, v/v), while eluent B was acetonitrile/formic acid (0.1%, v/v). Chromatograms were acquired using a Waters eλ PDA detector. The concentration (%i) of tazarotene after its degradation was calculated as the quotient of the peak area (Ai) to the sum of all P P peak areas ( A) on chromatograms according to the formula, %i = (Ai/( A) 100). In the case of × organic UV absorbers, the individual peak areas of the analyzed UV filters were not included in the total peak area. Chromatograms were recorded using the Waters eλ PDA detector. The spectra were 1 analyzed at 200–700 nm with 1.2 nm resolution and a sampling rate of 20 points s− . MS detection settings of Waters TQD mass spectrometer were as follows: source temperature—150 ◦C, desolvation 1 1 temperature—350 ◦C, desolvation gas flow rate—600 L h− , cone gas flow—100 L h− , capillary potential—3.00 kV, and cone potential—30 V. Nitrogen was used for both nebulizing and drying gases. The optimum collision energy was determined to be 50 eV. The ion spectra were obtained by scanning from 50 to 350 m/z. MassLynx V 4.1 (Waters) software was used for data acquisition. PharmaceuticsPharmaceutics 20202020,, 1212,, 899x 55 of 2020

2.7. Statistical Analysis 2.7. Statistical Analysis Statistical analyses were performed using Statistica v. 12 (StatSoft) and GraphPad Prism (GraphPadStatistical 6 Software, analyses wereSan Diego, performed CA, USA). using StatisticaFor analysis v. 12 of (StatSoft) cell death, and two-way GraphPad analysis Prism of (GraphPad variance 6with Software, Tukey’s San multiple Diego, CA, comparisons USA). For analysistest was ofcarrie cell death,d out. two-wayDifferences analysis were ofconsidered variance with statistically Tukey’s multiplesignificant comparisons in comparison test with was untreated carried out. controls Differences at p ≤ 0.05. were considered statistically significant in comparison with untreated controls at p 0.05. ≤ 3. Results and Discussion 3. Results and Discussion 3.1. Method Validation 3.1. Method Validation The main objective of applying the UPLC method was to confirm its applicability for the The main objective of applying the UPLC method was to confirm its applicability for the assessment assessment of tazarotene in the presence of its degradation products. The developed method was of tazarotene in the presence of its degradation products. The developed method was suitable for suitable for determining tazarotene and its degradation products in the tested ethanolic solutions. determining tazarotene and its degradation products in the tested ethanolic solutions. The specificity of The specificity of the method was evaluated through the assessment of blank chromatograms, the method was evaluated through the assessment of blank chromatograms, chromatograms of the pure chromatograms of the pure standard substance, and chromatograms of tazarotene after degradation. standard substance, and chromatograms of tazarotene after degradation. Furthermore, the resolution of Furthermore, the resolution of the investigated benzophenone-type UV filters, avobenzone, the investigated benzophenone-type UV filters, avobenzone, octocrylene, and tazarotene was verified. octocrylene, and tazarotene was verified. The chromatographic separation of the other selected UV The chromatographic separation of the other selected UV filters and tazarotene was satisfactory, both filters and tazarotene was satisfactory, both before and after UV irradiation (Figure 1). The results before and after UV irradiation (Figure1). The results were validated for linearity, precision, and were validated for linearity, precision, and accuracy [15]. Least squares linear regression was accuracy [15]. Least squares linear regression was performed to statistically evaluate the relationship performed to statistically evaluate the relationship between the peak areas and the concentration of between the peak areas and the concentration of bexarotene. The linearity was determined for the bexarotene. The linearity was determined for the concentration range from 0.01 to 0.2 mg mL−1, with concentration range from 0.01 to 0.2 mg mL 1, with a correlation coefficient and a determination a correlation coefficient and a determination −coefficient (R2) of 0.9991 and 0.9982, respectively. The coefficient (R2) of 0.9991 and 0.9982, respectively. The Shapiro–Wilk test was used to validate the Shapiro–Wilk test was used to validate the normality assumption; the significance value (p = 0.17151) normality assumption; the significance value (p = 0.17151) was greater than 0.05, and hence, the residuals was greater than 0.05, and hence, the residuals were considered to be normally distributed. The were considered to be normally distributed. The precision determined from the analysis of the six precision determined from the analysis of the six replicates of tazarotene solutions at a single replicates of tazarotene solutions at a single concentration of 0.1 mg mL 1 was lower than 1.29% concentration of 0.1 mg mL−1 was lower than 1.29% (%RSD), and the− recovery rate at three (%RSD), and the recovery rate at three concentration levels (80%, 100%, and 120%) ranged from 97.5% concentration levels (80%, 100%, and 120%) ranged from 97.5% to 101.90%. to 101.90%.

Figure 1. Cont.

Pharmaceutics 2020, 12, 899 6 of 20 Pharmaceutics 2020, 12, x 6 of 20

Figure 1. High-performance liquid chromatography chromatograms of tazarotene (TAZ) following Figure 1. High-performance liquid chromatography chromatograms of tazarotene (TAZ) following UV irradiation in the presence of selected UV absorbers: (A) tazarotene standard, (B) tazarotene with UV irradiation in the presence of selected UV absorbers: (A) tazarotene standard, (B) tazarotene with sulisobenzone (BP-4), (C) tazarotene with TiO2 and ZnO (TP- degradation products of tazarotene), sulisobenzone (BP-4), (C) tazarotene with TiO2 and ZnO (TP- degradation products of tazarotene), (D) tazarotene with benzophenone-2 (BP-2). (D) tazarotene with benzophenone-2 (BP-2). 3.2. Identification of Photocatalytic Degradation Products of Tazarotene 3.2. Identification of Photocatalytic Degradation Products of Tazarotene The impact of UV irradiation on tazarotene photostability in the presence of TiO2 and/or ZnO nanoparticlesThe impact was of assessed. UV irradiation The photocatalytic on tazarotene degradation photostability products in the of presence tazarotene of wereTiO2 identifiedand ZnO withnanoparticles the help was of the assessed. UPLC–MS The/ MSphotocatalytic analysis and degradation supported products with fragmentation of tazarotene patterns were identified obtained withfrom the MS /helpMS experiments.of the UPLC–MS/MS Table1 presents analysis theand proposed supported structures with fragmentation of the degradation patterns products, obtained fromand Table MS/MS2 depicts experiments. the proposed Table 1 fragmentation presents the prop patternsosed ofstructures tazarotene of the and degradation its degradation products, products. and TableIt was 2 found depicts that the the proposed degradation fragmentation process mainly patterns affected of tazarotene the 4,4-dimethyl-3,4-dihydro-2 and its degradation Hproducts.-thiopyran It wasmoiety. found The that fragments the degradation most susceptible process main to oxidationly affected were the the4,4-dimethyl-3,4-dihydro-2 methyl groups and theH sulfur-thiopyran atom. Themoiety. oxidation The fragments of methyl most groups susceptible led to the to degradation oxidation were products—TP-1–TP-5, the methyl groups TP-7, and the TP-9, sulfur and TP-10.atom. The oxidation of methyl the sulfur groups atom led to sulfoxideto the degrada and sulfonetion products—TP-1–TP-5, moiety was observed TP-7, for TP-9, the degradation and TP-10. productsThe oxidation TP-1–TP-8, of the ultimatelysulfur atom resulting to sulfoxide in the eliminationand sulfoneof moiety the sulfur-containing was observed for moiety, the degradation as observed productsfor products TP-1–TP-8, TP-9 and ultimately TP-10. The resulting elimination in the of theelimination ester moiety of the was sulfur-containing observed only for moiety, the minor as product—TP-11.observed for products TP-6 TP-9 and and TP-8 TP-10. were The the eliminat earliestion appearing of the ester products moiety and was dominant observed only in terms for the of quantity.minor product—TP-11. TP-6 has also beenTP-6 regardedand TP-8 aswere a hypothetical the earliest productappearing of products peroxide and oxidation dominant or photolytic in terms ofexposure quantity. of TP-6 tazarotene has also [30 been]. In regarded 2020, a publication as a hypoth aboutetical the product degradation of peroxide chemistry oxidation of tazarotene or photolytic was published,exposure of in tazarotene which eleven [30]. In degradation 2020, a publicatio productsn about of the the investigated degradation drug chemistry were characterized of tazarotene was [31]. published,Forced degradation in which studies eleven included degradation the photolytic products degradationof the investigated of tazarotene drug inwere the followingcharacterized solutions: [31]. Forced degradation studies included the photolytic degradation of tazarotene in the following 0.1 M HCl, 0.1 M NaOH, or CH3CN:H2O (80:20, v/v), and tazarotene in the solid state. TP-6 was one solutions:of the presumed 0.1 M HCl, products 0.1 M of NaOH, photolysis or CH in3CN:H acid and2O (80:20, neutral v/v conditions.), and tazarotene The oxidative in the solid degradation state. TP- 6 was one of the presumed products of photolysis in acid and neutral conditions. The oxidative of tazarotene performed in 3% H2O2 at room temperature gave rise to two products with the mass degradationof protonated of forms— tazarotenem/z performed368.1308 and in 3% 384.1258, H2O2 at corresponding room temperature to TP-6 gave and rise TP-8, to two respectively products with [31]. Analysisthe massof of the protonated synthesis andforms— degradationm/z 368.1308 process and of tazarotene384.1258, corresponding revealed four impurities, to TP-6 and including TP-8, respectivelytransformation [31]. to TP-6Analysis and TP-8of the through synthesis oxidation and degradation [4]. Exposure process of tazarotene of tazarotene to 1.2 million revealed lux hours four impurities,resulted in theincluding degradation transformation of 8.85% ofto theTP-6 initial and quantityTP-8 through with oxidation the formation—mainly [4]. Exposure Imp-B,of tazarotene that is TP-6to 1.2 [32 million]. We did lux not hours identify resulted tazarotenic in the acid, degradation the active metaboliteof 8.85% of of the tazarotene, initial quantity which is thewith major the formation—mainlydegradation product Imp-B, formed that during is TP-6 hydrolysis [32]. We did [4,26 not,33 identify]. Tazarotenic tazarotenic acid does acid, not the accumulate active metabolite in the of tazarotene, which is the major degradation product formed during hydrolysis [4,26,33]. Tazarotenic acid does not accumulate in the body over time. The concentrations of tazarotenic acid in the plasma of patients with psoriasis were found to be 0.45 ± 0.78 μg L−1 (after 0.05% tazarotene

Pharmaceutics 2020, 12, 899 7 of 20 body over time. The concentrations of tazarotenic acid in the plasma of patients with psoriasis were found to be 0.45 0.78 µg L 1 (after 0.05% tazarotene gel) and 0.83 1.22 µg L 1 (after 0.1% tazarotene ± − ± − gel) [8]. The maximumPharmaceutics average 2020, 12, x plasma concentrations of tazarotenic acid after topical application7 of 20 of Pharmaceutics 2020, 12, x 1 7 of 20 0.1% of tazarotenePharmaceutics cream on2020,the 12, x face were less than 0.25 µg L− [34]. It is metabolized to sulfoxide7 of 20 and Pharmaceuticsgel) and 0.83 2020 , 12± , 1.22x μg L−1 (after 0.1% tazarotene gel) [8]. The maximum average plasma7 of 20 more polar compoundsgel)Pharmaceutics and and0.83 2020 eliminated, ±12 , 1.22x μg L from−1 (after the 0.1% body tazarotene via urine gel) and [8]. feces The [3maximum,8]. The bindingaverage plasma to7 of plasma 20 gel)concentrations and 0.83 of± tazarotenic1.22 μg L− 1acid (after after 0.1% topical tazarotene application gel) of [8].0.1% The of tazarotenemaximum cream average on theplasma face gel)concentrations and 0.83 of± tazarotenic1.22 μg L −1acid (after after 0.1% topical tazarotene application gel) of [8].0.1% The of tazarotenemaximum cream average on theplasma face proteins, which playsconcentrationsweregel) andless a key 0.83than role of±0.25 tazarotenic1.22 in μ thegμ Lg− distribution,1 L[34]. −acid1 (after It after is 0.1%metabolized topical elimination, tazarotene application to sulfoxidegel) and of [8].0.1% therapeutic andThe of moretazarotenemaximum polar eﬀ ectivenesscream compoundsaverage on theplasma of faceand drugs, wereconcentrations less than of0.25 tazarotenic μg L−1 [34]. acid It after is metabolized topical application to sulfoxide of 0.1% and of moretazarotene polar cream compounds on the faceand was greater thanwereeliminatedconcentrations 99% less [8]. than from After of 0.25the tazarotenic topical bodyμg L via−1 administration,[34]. urine acid It afterand is metabolizedfeces topical [3,8]. tazaroteneapplication The to binding sulfoxide of and 0.1%to plasmaand tazarotenic of moretazarotene proteins, polar acid cream whichcompounds metabolize playson the a andfacekey via eliminatedwere less than from 0.25the bodyμg L via−1 [34]. urine It and is metabolizedfeces [3,8]. The to binding sulfoxide to plasmaand more proteins, polar whichcompounds plays a andkey eliminatedrolewere in less the distribution, thanfrom 0.25the body μg elimination, L via−1 [34].urine It and andis metabolizedfeces therapeuti [3,8]. Thec effectivenessto binding sulfoxide to plasmaofand drugs, more proteins, was polar greater which compounds than plays 99% a andkey[8]. oxidation to sulfoxides,roleeliminated in the sulfones,distribution, from the body and elimination, via other urine and polarand feces therapeuti metabolites [3,8]. Thec effectiveness binding [8,35 to]. plasmaof It drugs, appears proteins, was greater that which therethan plays 99% are a key[8]. some roleAftereliminated in topical the distribution, from administration, the body elimination, via tazaroteneurine and and feces therapeutiand tazarotenic[3,8]. Thec effectiveness binding acid metabolize to plasmaof drugs, viaproteins, was oxidation greater which tothan playssulfoxides, 99% a key[8]. similarities betweenAfterrole in topical thethe distribution, known administration,in elimination, vivo tazarotenemetabolic and andtherapeuti pathways tazarotenicc effectiveness acid of tazarotene metabolize of drugs, via and was oxidation greater those tothan postulated sulfoxides, 99% [8]. for Aftersulfones,role in topical the and distribution, administration, other polar elimination, metabolites tazarotene and [8,35]. andtherapeuti tazarotenicIt appearsc effectiveness that acid there metabolize ofare drugs, some via was similaritiesoxidation greater to thanbetween sulfoxides, 99% the[8]. sulfones,After topical and administration, other polar metabolites tazarotene [8,35]. and tazarotenicIt appears that acid there metabolize are some via similaritiesoxidation to between sulfoxides, the its in vitro photocatalyticsulfones,knownAfter topical in and vivo degradation. administration,other metabolic polar pathwaysmetabolites tazarotene In both of [8,35].tazarotene the and cases,tazarotenicIt appears and the those that acid sulfoxide postulatedthere metabolize are (TP-6)some for via its similarities oxidation in and vitro sulfone photocatalytic to between sulfoxides, (TP-8) the of knownsulfones, in andvivo other metabolic polar pathways metabolites of [8,35].tazarotene It appears and those that postulatedthere are some for its similarities in vitro photocatalytic between the tazarotene wereknowndegradation.sulfones, identiﬁed. in andvivo In othermetabolic both polar the cases,pathways metabolites the sulfoxide of [8,35].tazarotene (TP-6)It appears and and those sulfonethat postulatedthere (TP-8) are someof for tazarotene its similarities in vitro were photocatalytic between identified. the degradation.known in vivo In metabolic both the cases,pathways the sulfoxide of tazarotene (TP-6) and and those sulfone postulated (TP-8) of for tazarotene its in vitro were photocatalytic identified. degradation.known in vivo In metabolic both the cases,pathways the sulfoxide of tazarotene (TP-6) and and those sulfone postulated (TP-8) of for tazarotene its in vitro were photocatalytic identified. degradation. In bothTable the 1. cases, Proposed the sulfoxidestructures of(TP-6) the degr andadation sulfone products (TP-8) ofof tazarotene. tazarotene were identified. degradation.Table 1. Proposed In bothTable the structures1. cases, Proposed the structuressulfoxide of the degradation of(TP-6) the degr andadation sulfone products products (TP-8) of oftazarotene.of tazarotene. tazarotene were identified. Table 1. Proposed structures of the degradation products of tazarotene. Table 1. Proposed structuresFragmentation of the degr adation products of tazarotene. CompoundCompound RT (min) RT(min) [MTable+H+ 1.][M+H Proposed+ Fragmentation] structuresFragmentation of the Ions degr adation products of tazarotene. Structure Compound RT(min) [M+H+] FragmentationIons Structure Compound RT(min) [M+H+] FragmentationIons Structure Compound RT(min) [M+H+] FragmentationIons Structure Compound RT(min) [M+H+] Ions Structure Ions

192.1,192.1, 204.1, 204.1, 310.1, 310.1, TP-1 3.52TP-1 3.52 412.1 412.1 192.1, 204.1, 310.1, TP-1 3.52 412.1 366.1,192.1,366.1, 204.1, 384.1 384.1 310.1, TP-1 3.52 412.1 192.1,366.1, 204.1, 384.1 310.1, TP-1 3.52 412.1 192.1,366.1, 204.1, 384.1 310.1, TP-1 3.52 412.1 366.1, 384.1 366.1, 384.1

TP-2 3.94 428.1 382.1, 400.1 TP-2 3.94TP-2 3.94 428.1 428.1 382.1,382.1, 400.1 400.1 TP-2 3.94 428.1 382.1, 400.1 TP-2 3.94 428.1 382.1, 400.1 TP-2 3.94 428.1 382.1, 400.1

192.1, 282.1, 296.1, TP-3 4.80 414.1 192.1, 282.1, 296.1, TP-3 4.80 414.1 192.1,192.1, 282.1,368.1, 282.1, 296.1,396.1 296.1, TP-3 4.80TP-3 4.80 414.1 414.1 192.1,368.1, 282.1, 396.1 296.1, TP-3 4.80 414.1 368.1,192.1,368.1, 282.1, 396.1 396.1 296.1, TP-3 4.80 414.1 368.1, 396.1 368.1, 396.1

178.1, 204.1, 294.1, TP-4 5.43 430.1 178.1, 204.1, 294.1, TP-4 5.43 430.1 178.1,312.1, 204.1,356.1, 294.1,384.1 TP-4 5.43 430.1 178.1,178.1,312.1, 204.1, 204.1,356.1, 294.1, 294.1,384.1 TP-4 5.43TP-4 5.43 430.1 430.1 178.1,312.1, 204.1,356.1, 294.1,384.1 TP-4 5.43 430.1 312.1,312.1, 356.1, 356.1, 384.1 384.1 312.1, 356.1, 384.1

308.0, 336.1, 350.1, TP-5 6.00 414.1 308.0, 336.1, 350.1, TP-5 6.00 414.1 308.0,368.1, 336.1, 396.1 350.1, TP-5 6.00 414.1 308.0,368.1, 336.1, 396.1 350.1, TP-5 6.00 414.1 308.0,308.0, 336.1,368.1, 336.1, 350.1,396.1 350.1, TP-5 6.00TP-5 6.00 414.1 414.1 368.1, 396.1 368.1,368.1, 396.1 396.1

Pharmaceutics 2020, 12, 899 8 of 20

Table 1. Cont.

Pharmaceutics 2020, 12, x + 8 of 20 CompoundPharmaceutics RT (min) 2020, 12 [M, x+ H ] Fragmentation Ions Structure 8 of 20 Pharmaceutics 2020, 12, x 8 of 20 Pharmaceutics 2020, 12, x 8 of 20 Pharmaceutics 2020, 12, x 8 of 20 Pharmaceutics 2020, 12, x 8 of 20 Pharmaceutics 2020, 12, x 8 of 20 280.1,280.1, 296.1, 296.1, 308.2, 308.2, TP-6 6.30TP-6 6.30 368.1 368.1 280.1, 296.1, 308.2, TP-6 6.30 368.1 280.1,340.1 296.1,340.1 308.2, TP-6 6.30 368.1 280.1, 296.1,340.1 308.2, TP-6 6.30 368.1 280.1, 296.1,340.1 308.2, TP-6 6.30 368.1 280.1, 296.1,340.1 308.2, TP-6 6.30 368.1 280.1, 296.1,340.1 308.2, TP-6 6.30 368.1 340.1 340.1

294.1, 338.0, 366.1, TP-7 6.62 430.1 294.1,294.1, 338.0, 338.0, 366.1, 366.1, TP-7 6.62TP-7 6.62 430.1 430.1 294.1,384.1, 338.0, 412.1 366.1, TP-7 6.62 430.1 384.1,294.1,384.1, 338.0, 412.1 412.1 366.1, TP-7 6.62 430.1 294.1,384.1, 338.0, 412.1 366.1, TP-7 6.62 430.1 294.1,384.1, 338.0, 412.1 366.1, TP-7 6.62 430.1 294.1,384.1, 338.0, 412.1 366.1, TP-7 6.62 430.1 384.1, 412.1 384.1, 412.1

TP-8 6.97 384.1 356.1 TP-8 6.97 384.1 356.1 TP-8 6.97TP-8 6.97 384.1 384.1 356.1 356.1 TP-8 6.97 384.1 356.1 TP-8 6.97 384.1 356.1 TP-8 6.97 384.1 356.1 TP-8 6.97 384.1 356.1

262.1, 280.1, 269.1, TP-9 7.47 368.1 262.1, 280.1, 269.1, TP-9 7.47 368.1 262.1, 280.1,340.1 269.1, TP-9 7.47 368.1 262.1,262.1, 280.1, 280.1,340.1 269.1, 269.1, TP-9 7.47TP-9 7.47 368.1 368.1 262.1, 280.1,340.1 269.1, TP-9 7.47 368.1 262.1,340.1 280.1,340.1 269.1, TP-9 7.47 368.1 262.1, 280.1,340.1 269.1, TP-9 7.47 368.1 340.1

340.1

252.1, 280.1, 294.1, TP-10 8.49 366.1 252.1, 280.1, 294.1, TP-10 8.49 366.1 252.1, 280.1,338.1 294.1, TP-10 8.49 366.1 252.1, 280.1,338.1 294.1, TP-10 8.49 366.1 252.1,252.1, 280.1, 280.1,338.1 294.1, 294.1, TP-10TP-10 8.49 8.49 366.1 366.1 252.1, 280.1,338.1 294.1, TP-10 8.49 366.1 252.1,338.1 280.1,338.1 294.1, TP-10 8.49 366.1 338.1

338.1

222.1, 266.1, 294.1, Tazarotene 9.28 352.1 222.1, 266.1, 294.1, Tazarotene 9.28 352.1 222.1,308.2, 266.1, 324.1 294.1, Tazarotene 9.28 352.1 222.1,308.2, 266.1, 324.1 294.1, Tazarotene 9.28 352.1 222.1,308.2, 266.1, 324.1 294.1, Tazarotene 9.28 352.1 222.1,222.1, 266.1,308.2, 266.1, 294.1,324.1 294.1, Tazarotene 9.28 352.1 308.2, 324.1 Tazarotene 9.28 352.1 222.1, 266.1, 294.1, Tazarotene 9.28 352.1 308.2,308.2, 324.1 324.1 308.2, 324.1

TP-11 9.60 280.1 136.1, 156.1, 236.1 TP-11 9.60 280.1 136.1, 156.1, 236.1 TP-11 9.60 280.1 136.1, 156.1, 236.1 TP-11 9.60 280.1 136.1, 156.1, 236.1 TP-11 9.60 280.1 136.1, 156.1, 236.1 TP-11 9.60 280.1 136.1, 156.1, 236.1 TP-11 9.60 280.1 136.1, 156.1, 236.1 TP-11 9.60 280.1 136.1, 156.1, 236.1

Pharmaceutics 2020, 12, 899 9 of 20 Pharmaceutics 2020, 12, x 9 of 20

Table 2. Recommended fragmentation patterns. Table 2. Recommended fragmentation patterns.

TazaroteneTazarotene

Pharmaceutics 2020, 12, 899 10 of 20

Pharmaceutics 2020, 12, x Table 2. Cont. 10 of 20 Pharmaceutics 2020, 12, x 10 of 20 TP-1 TP-1

TP-2 TP-2

Pharmaceutics 2020, 12, 899 11 of 20

Pharmaceutics 2020, 12, x Table 2. Cont. 11 of 20 Pharmaceutics 2020, 12, x 11 of 20 TP-3 TP-3 TP-3

TP-4 TP-4

Pharmaceutics 2020, 12, 899 12 of 20

Pharmaceutics 2020, 12, x Table 2. Cont. 12 of 20 Pharmaceutics 2020, 12, x 12 of 20 TP-5 TP-5 TP-5

TP-6 TP-6 TP-6

Pharmaceutics 2020, 12, 899 13 of 20

Pharmaceutics 2020, 12, x Table 2. Cont. 13 of 20 Pharmaceutics 2020, 12, x 13 of 20 TP-7 TP-7 TP-7

TP-8 TP-8

Pharmaceutics 2020, 12, 899 14 of 20

Pharmaceutics 2020, 12, x Table 2. Cont. 14 of 20 Pharmaceutics 2020, 12, x 14 of 20 TP-9 TP-9 TP-9

TP-10 TP-10

Pharmaceutics 2020, 12, 899 15 of 20

Pharmaceutics 2020, 12, x Table 2. Cont. 15 of 20

TP-11 TP-11 Pharmaceutics 2020, 12, x 15 of 20

TP-11

3.3. UV Irradiation of Tazarotene in the Presence of Organic UV Absorbers

3.3. UV Irradiation of Tazarotene in the Presence of Organic UV Absorbers The impact of UV irradiation on tazarotene stability in the presence of benzophenone-type UV The impact of UV irradiation on tazarotene stability in the presence of benzophenone-type UV 3.3.filters UV was Irradiation assessed. of Benzophenone-1,Tazarotene in the Presence benzopheno of Organicne-2, UVbenzophenone-3, Absorbers and benzophenone-4 were filters was assessed. Benzophenone-1, benzophenone-2, benzophenone-3, and benzophenone-4 were selected for the study. Besides, the combination of avobenzone and octocrylene was also tested in this The impact of UV irradiation on tazarotene stability in the presence of benzophenone-type UV selectedexperiment. for the study. This is Besides, a popular the combination combination of UV of avobenzone filters widelyand used octocrylene in sunscreens. was To also balance tested thein this filters was assessed. Benzophenone-1, benzophenone-2, benzophenone-3, and benzophenone-4 were experiment. This is a popular combination of UV filters widely used in sunscreens. To balance the selectedinstability for of the avobenzone, study. Besides, octocrylene the combination acts as of aavobenzone photostabilizer and octocrylene and UV absorber. was also testedTwo ofin thisthe instabilityexperiment.investigated of avobenzone, Thisbenzophenones—oxybenzone is a popular octocrylene combination acts as a anof photostabilizer dUV sulisobenzone—with filters widely and used UV peakin absorber. sunscreens. absorption Two To ofλ maxbalance the equal investigated the to benzophenones—oxybenzoneinstability286 and 324 of nm avobenzone, have been andapprovedoctocrylene sulisobenzone—with by theacts FDA. as a Accordingphotostabilizer peak to absorption the andFDA UVregulations,λmax absorber.equal the toTwo 286maximum of and the 324 nm recommended concentrations of benzophenone-3, benzophenone-4, avobenzone, and octocrylene are have beeninvestigated approved benzophenones—oxybenzone by the FDA. According an tod thesulisobenzone—with FDA regulations, peak the absorption maximum λmax recommended equal to concentrations2866%, and10%, 324 3%, of nm benzophenone-3,and have 10%, been respectively approved benzophenone-4, [25].by the Figure FDA. 2 Accordingdisplays avobenzone, the to therelative FDA and peak regulations, octocrylene areas of the tazarotene are maximum 6%, 10%, to 3%, illustrate the impact of UV absorbers on its photostability under experimental conditions. Tazarotene and 10%,recommended respectively concentrations [25]. Figure of benzophenone-3,2 displays the relativebenzophenone-4, peak areas avobenzone, of tazarotene and octocrylene to illustrate are the was stable in all cases, excluding sulisobenzone, where a slight degradation was observed. Two impact6%, of 10%, UV absorbers 3%, and 10%, on itsrespectively photostability [25]. Figure under 2 experimentaldisplays the relative conditions. peak areas Tazarotene of tazarotene was stableto in illustrateadditional the peaks impact were of UVidentified absorb oners theon itschromatogr photostabilityams (Figure under experimental 1B), and two conditions.peaks in the Tazarotene spectrum all cases, excluding sulisobenzone, where a slight degradation was observed. Two additional peaks waswere stable at m/z in412.18 all cases, (TP-1) excluding and 368.18 sulisobenzon (TP-6). The degradatione, where a slightprocess degradation occurred through was observed. the oxidation Two were identified on the chromatograms (Figure1B), and two peaks in the spectrum were at m/z 412.18 additionalof the sulfur peaks atom were toidentified sulfoxide on theand chromatogr the subsequentams (Figure oxidation 1B), and of twothe peaksmethyl in thegroups. spectrum No (TP-1)werephotodegradation and 368.18at m/z 412.18 (TP-6). products(TP-1) The and degradation were 368.18 observed (TP-6). process in The all degradationthe occurred dark control through process samples, occurred the oxidationwhich through indicate of the the the oxidation sulfur stability atom to sulfoxideof tazarotenethe and sulfur the in subsequent atomthe absence to sulfox oxidationof UVide irradiation and of the under methylsubsequent the groups. applied oxidation conditions. No photodegradation of the methyl groups. products No were observedphotodegradation in all the dark products control were samples, observed which in all indicate the dark the control stability samples, of tazarotene which indicate in the the absence stability of UV irradiationof tazarotene under thein the applied absence conditions. of UV irradiation under the applied conditions.

Figure 2. The relative peak area of tazarotene following UV irradiation in the presence of UV absorbers: BP-1 (benzophenone-1), BP-2 (benzophenone-2), BP-3 (benzophenone-3), BP-4 (benzophenone-4), and AVO + OCT (avobenzone + octocrylene). FigureFigure 2. The 2. relative The relative peak areapeak ofarea tazarotene of tazarotene following followi UVng irradiationUV irradiation in the in presencethe presence of UV of absorbers:UV

BP-1 (benzophenone-1),absorbers: BP-1 (benzophenone-1), BP-2 (benzophenone-2), BP-2 (ben BP-3zophenone-2), (benzophenone-3), BP-3 (benzophenone-3), BP-4 (benzophenone-4), BP-4 and (benzophenone-4), and AVO + OCT (avobenzone + octocrylene). AVO + OCT (avobenzone + octocrylene).

Pharmaceutics 2020, 12, 899 16 of 20 Pharmaceutics 2020, 12, x 16 of 20

3.4. Cytotoxic Risk Assay Figure3 3 depictsdepicts thethe relativerelative peakpeak areaarea ofof tazarotenetazarotene andand itsits photocatalyticphotocatalytic degradationdegradation productsproducts presented in chromatograms following 0.5, 0.5, 1, 1, and and 2 2 h of UV irradiation of the examined solutions. solutions. The leading products present in all chromatogramschromatograms were TP-6,TP-6, TP-8,TP-8, and TP-10.TP-10. After After 2 2 h of UV irradiation ofof the the investigated investigated solutions, solutions, all the all identiﬁed the identified phototransformation phototransformation products ofproducts tazarotene of weretazarotene noted were on chromatograms. noted on chromatograms.

Figure 3. AverageAverage relative peak areas of tazarotene and its main photocatalytic degradation products

identifiedidentiﬁed in the tested solution solutionss after 0.5, 1, 1, or 2 h of UV irradiation in the presence of TiO2/ZnO./ZnO. TAZ—tazarotene; TP-3,TP-3, TP-5, TP-5, TP-6, TP-6, TP-8, TP-8, and TP-10—photocatalytic and TP-10—photocatalytic degradation degradation products ofproducts tazarotene. of tazarotene. The cell growth-inhibitory activities of tazarotene following its photocatalytic degradation in experimentalThe cell conditionsgrowth-inhibitory were determined activities inof vitrotazarotenagainste following cervical (HeLa), its photocatalytic breast (MDA-MB-231), degradation and in experimentalovary (A2780) conditions cancer cell were lines determined by means in of vitro MTT against assays. cervical Cells were (HeLa), treated breast with (MDA-MB-231), 3, 10, and 30 andµM ovarysolution (A2780) after UV cancer irradiation cell lines (T0—without by means of irradiation, MTT assays. T1—0.5 Cells h,were T2—1 treated h, and with T3—2 3, 10, h). and The results30 μM solutionare shown after in Figure UV irradiation4. The in vitro(T0—withoutcytotoxicity irradiation, risk evaluation T1—0.5 based h, T2—1 on the h, photocatalytic and T3—2 h). degradation The results areof tazarotene shown in indicated Figure the4. The antiproliferative in vitro cytotoxicity action atthe risk concentration evaluation ofbased 30 µM. on A2780 the photocatalytic cells exhibited degradationsubstantial growth of tazarotene inhibition indicated by 30 µ theM tazarotene antiproliferative without action irradiation at the followedconcentration by abiphasic of 30 μM. action A2780 in cellsterms exhibited of the irradiation substantial time. growth Samples inhibition of lower by 30 concentrations μM tazarotene (3 without and 10 µirradiationM) resulted followed in a modest by a biphasicincrease ofaction proliferation. in terms T3of solutionsthe irradiation generally time. more Samples eﬀectively of lower inhibited concentrations cell proliferation (3 and than 10 otherμM) resultedsamples, in which a modest indicated increase that of antiproliferative proliferation. T3 metabolites solutions couldgenerally be generated more effectively during irradiationinhibited cell in proliferationa time-dependent than manner.other samples, The behavior which of HeLaindicated cells that was uniqueantiproliferative with a concentration-dependent metabolites could be generatedaction of tazarotene during irradiation with no irradiation in a time-dependent which was substantiallymanner. The lessbehavior after 0.5–2of HeLa h of cells irradiation. was unique with a concentration-dependent action of tazarotene with no irradiation which was substantially less after 0.5–2 h of irradiation.

Pharmaceutics 2020, 12, 899 17 of 20 Pharmaceutics 2020, 12, x 17 of 20

FigureFigure 4.4. Changes in thethe cellcell viabilityviability ofof HeLa,HeLa, A2780,A2780, andand MDA-MB-231MDA-MB-231 cancercancer cellscells causedcaused byby thethe solutionssolutions ofof tazarotenetazarotene following following photocatalytic photocatalytic degradation. degradation. Cells Cells were were treated treated with with 3, 10, 3, and10, and 30 µ M30 solutionsμM solutions of tazarotene of tazarotene after UVafter irradiation UV irradiation (T0—without (T0—without irradiation, irradiation, T1—0.5 T1—0.5 h of irradiation, h of irradiation, T2—1 h ofT2—1 irradiation, h of irradiation, and T3—2 and h ofT3—2 irradiation): h of irradiation): * p < 0.05, *p ** < p0.05,< 0.01, **p and< 0.01, *** andp < ***0.001.p < 0.001. 3.5. In Silico Toxicity Predictions 3.5. In Silico Toxicity Predictions In the next step of the study, the OSIRIS Property Explorer server was deployed to calculate In the next step of the study, the OSIRIS Property Explorer server was deployed to calculate the the toxicities of tazarotene and all its identified photodecomposition products generated under toxicities of tazarotene and all its identified photodecomposition products generated under experimental conditions. Table3 presents the outcomes of the toxicity risk assessment. Five degradation experimental conditions. Table 3 presents the outcomes of the toxicity risk assessment. Five products—TP-2, TP-3, TP-4, TP-7, and TP-8—could be harmful in terms of the reproductive degradation products—TP-2, TP-3, TP-4, TP-7, and TP-8—could be harmful in terms of the eﬀects, which is associated with 3,4-dihydro-6-methyl-2H-1-benzothiopyran 1,1-dioxide, while one of reproductive effects, which is associated with 3,4-dihydro-6-methyl-2H-1-benzothiopyran 1,1- them—TP-10—demonstrated potential irritant activity. dioxide, while one of them—TP-10—demonstrated potential irritant activity.

Table 3. Toxicity risk assessment of the photocatalytic degradation products of tazarotene (TP-1–TP-11) Table 3. Toxicity risk assessment of the photocatalytic degradation products of tazarotene (TP-1–TP- performed using OSIRIS Property Explorer. 11) performed using OSIRIS Property Explorer. Compound Mutagenic Tumorigenic Irritant Reproductive Eﬀective Compound Mutagenic Tumorigenic Irritant Reproductive Effective TazaroteneTazarotene -- - - - TP-1TP-1 -- - - - +/− TP-2TP-2 -- - - +/ TP-3 - - - +/− TP-3 - - - +/ TP-4 - - - +/− TP-4 - - - +/ TP-5 - - - - − TP-5TP-6 -- - - - TP-7 - - - +/− TP-8 - - - +/− TP-9 - - - - TP-10 - - +/− - TP-11 - - - - (-) None, (+/−) Medium

Pharmaceutics 2020, 12, 899 18 of 20

Table 3. Cont.

Compound Mutagenic Tumorigenic Irritant Reproductive Effective TP-6 - - - - TP-7 - - - +/ − TP-8 - - - +/ − TP-9 - - - - TP-10 - - +/ - − TP-11 - - - - (-) None, (+/ ) Medium. − 4. Conclusions The presented results show the significance of the photostability studies for topical agents applied to the skin. Tazarotene, a receptor-selective, third-generation retinoid, is commonly used as monotherapy and included in combination therapy for acne vulgaris and psoriasis. This study revealed the impact of the presence of UV absorbers on the photostability of tazarotene in ethanol solution under UV irradiation. The photodecomposition of tazarotene in the presence of TiO2, ZnO, and sulisobenzone was observed under the applied experimental conditions. Furthermore, the presumable structures of photocatalytic degradation and photodegradation products of tazarotene were identified for the first time. The in silico toxicity testing of degradation products indicated their theoretical toxic reproductive effects and irritant activity. The photostability studies provided valuable information to optimize the manufacturing process as well as to ensure the safe use of medications by patients. It is worth mentioning that UV filters act as photostabilizers but could also contribute to the degradation of active pharmaceutical ingredients under UV irradiation. Therefore, further efforts should be taken to verify the potential impact of UV irradiation on different compositions of substances used in topical preparations.

Author Contributions: Conceptualization: A.K.-P.; methodology: A.K.-P., I.Z. and P.Z.;˙ software: P.Z.,˙ J.P. and P.B.; validation: P.Z.˙ and A.K.-P.; formal analysis: A.K.-P., I.Z. and B.M.; investigation: A.K.-P., P.Z.˙ and P.B.; data curation: A.K.-P., I.Z., P.Z.˙ and P.B.; writing—original draft preparation: A.K.-P. and I.Z.; writing—review and editing: A.K.-P. and B.M.; supervision: W.O. All authors have read and agreed to the published version of the manuscript. Funding: The authors acknowledge the support of the National Science Center, Poland (grant no. 2018/02/X/NZ7/01016) and Ministry of Human Capacities, Hungary (20391-3/2018/FEKUSTRAT). Conﬂicts of Interest: The authors declare no conﬂict of interest.

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