pharmaceutics

Article Novel and Modified Inhibitor Loaded in Topical Formulations for Psoriasis Management

Andreia Nunes 1, Joana Marto 2,* ,Lídia Maria Gonçalves 2 , Sandra Simões 2, Rita Félix 2, Andreia Ascenso 2 , Francisca Lopes 2 and Helena Margarida Ribeiro 2,*

1 Faculty of Pharmacy, Universidade de Lisboa, 1649-003 Lisbon, Portugal; [email protected] 2 Research Institute for Medicine (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, 1649-003 Lisbon, Portugal; lgoncalves@ff.ulisboa.pt (L.M.G.); ssimoes@ff.ulisboa.pt (S.S.); ritafelix@ff.ulisboa.pt (R.F.); andreiaascenso@ff.ulisboa.pt (A.A.); fclopes@ff.ulisboa.pt (F.L.) * Correspondence: jmmarto@ff.ulisboa.pt (J.M.); [email protected] (H.M.R.)

 Received: 7 March 2020; Accepted: 10 April 2020; Published: 14 April 2020 

Abstract: Human neutrophil elastase (HNE) is a that degrades matrix proteins. An excess of HNE may trigger several pathological conditions, such as psoriasis. In this work, we aimed to synthesize, characterize and formulate new HNE inhibitors with a 4-oxo-β-lactam scaffold with less toxicity, as well as therapeutic index in a psoriasis context. HNE inhibitors with 4-oxo-β-lactam scaffolds were synthesized and characterized by NMR, FTIR, melting point, mass spectrometry and elemental analysis. In vitro cytotoxicity and assays were performed. The compound with the highest cell viability (AAN-16) was selected to be incorporated in an emulsion (AAN-16 E) and in a microemulsion (AAN-16 ME). Formulations were characterized in terms of organoleptic properties, pH, rheology, droplet size distribution, in vitro drug release and in vivo psoriatic activity. All compounds were successfully synthesized according to analytical methodology, with good yields. Both formulations presented suitable physicochemical properties. AAN-16 E presented the most promising therapeutic effects in a murine model of psoriasis. Overall, new HNE inhibitors were synthesized with high and selective activity and incorporated into topical emulsions with potential to treat psoriasis.

Keywords: human neutrophil elastase inhibitors; emulsions; microemulsions; psoriasis; topical application

1. Introduction Psoriasis is a chronic inflammatory skin disease that affects at least 100 million individuals worldwide. It is characterized by red, scaly and erythematous plaques that can be scattered over the entire surface of the body or on a specific area, including the scalp, nails and joints. Psoriasis is considered a non-communicable disease with no cure, and it may progressively worsen with age, depending on the degree of severity [1–3]. The causes of psoriasis are not totally understood, but its pathogenesis is associated with genetic and epigenetic modifications that can be triggered by environmental factors [2,4]. Neutrophil chemokines, such as CXCL1, CXCL2 and CXCL8/IL-8, are usually present in psoriatic skin [5,6]. Despite the effort to find biologic drugs for the treatment of moderate to severe plaque type psoriasis based on the imunopathogenesis of the disease, the current treatment of psoriasis is mostly based on control of symptoms. Currently, phototherapy (ultraviolet light), systemic and topical therapies using corticosteroids, vitamin D analogous, retinoids and dithranol are commonly used [3,4,7]. Nevertheless,

Pharmaceutics 2020, 12, 358; doi:10.3390/pharmaceutics12040358 www.mdpi.com/journal/pharmaceutics Pharmaceutics 2020, 12, 358 2 of 19 these therapies have numerous disadvantages. Therefore, it is extremely important to keep searching for new alternatives and discover new active compounds that may be able to manage this disease. Human neutrophil elastase (HNE) is a serine protease with a single polypeptide chain that belongs to the elastase-like serine subfamily, being stored and secreted from polymorphonuclear [8–11]. Its activity depends on a consisting of Ser195, His57 and Asp102, which are all at the of the [10,12,13]. In terms of biological function, HNE has bactericidal, tissue preservative and restorative properties. Moreover, HNE is also involved in inflammation. In a normal inflammatory process, neutrophils release a high concentration of HNE to the extracellular medium, where this enzyme can modulate the immune response through degradation of pro-inflammatory cytokines, reducing the intensity of the inflammation [13–15]. However, if an imbalance between HNE and its endogenous inhibitors occurs during this process, the extracellular HNE in excess can degrade structural proteins of the extracellular matrix, like elastin, proteoglycan, collagen and fibronectin. Hereupon, the excess of HNE may trigger several pathological conditions and tissue injuries inducing chronic inflammatory diseases, such as psoriasis and cystic fibrosis, among others. Accordingly, HNE is a valuable therapeutic target, and the design of new HNE inhibitors able to modulate the proteolytic activity of HNE represents a promising approach for psoriasis, where there is an excess in the production of this serine protease [12]. Under physiological conditions, endogenous HNE inhibitors tightly regulate their activity, avoiding degradation of connective tissue proteins [12,14]. These inhibitors can belong to two different families depending on their tissue location—the and the chelonianins [14,16]—and can be categorized into non-covalent and covalent inhibitors, as well as reversible or irreversible inhibitors [10,17,18]. It is worthy pointing out that elafin, one of the HNE inhibitors, was the first to be isolated from the skin of psoriatic patients. This inhibitor has a second-order constant association of 5 106 M 1 s 1 and is considered a potent reversible inhibitor of this enzyme [14,19]. Over the years, × − · − numerous HNE inhibitors such as 4-oxo-β-lactams, benzoxazinones, diazo compounds or haloketones have been developed [9,20,21]. In this context, it is necessary to consider not only the size and chemical nature of the structure, to decide the route of administration, but also the enzyme selectivity and appropriate pharmacokinetic and pharmacodynamic properties [14,18]. Synthetic HNE inhibitors need to have some peptide character to act as effective drugs, since binding of an aliphatic side chain in the S1 pocket is more favored. Notwithstanding, peptides usually present chemical instability, poor pharmacological profile and low bioavailability. These properties could be improved when low molecular weight drugs are synthesized ( 500 Da) and physicochemical properties (log P = 2–3, ≤ ionization, etc.), drug concentration and diffusion through the skin are optimized [18,22]. Recent studies showed that the structure of β-lactam inhibitors allows a great improvement on the rate of serine acylation since they increase the intrinsic chemical reactivity, when compared to other compounds [10,15,23]. The synthesis of new compounds with a β-lactam structure, such as azetidine-2,4-diones, also known as 4-oxo-β-lactams, were demonstrated to be a very potent and selective class of HNE acylating agents (Scheme1). The structure-activity relationship (SAR) studies performed by Mulchande, J. et al. [24] showed that substituents at C-3 of the four-membered ring are very important for the potency of this inhibitor. Furthermore, these studies indicated that a diethyl substitution in the 4-oxo-β-lactam scaffold, along with N-phenyl derivatization of this structure, selectively improved the activity against HNE [24]. Accordingly, previous studies reported by our group [24–26] also showed that these compounds could be quite promising regarding the inhibition of serine proteases. In particular, ER143 (1) [21] showed high potency and selectivity for HNE but it presented poor solubility and cell viability when tested in immortalized human keratinocyte (HaCaT) cells [21]. Thus, these new HNE inhibitors were synthesized as an elaboration of ER143 (1) (3,3-diethyl-1-(4-(((2-oxo-2H-chromen-7-yl)oxy)methyl)phenyl)-azetidine-2,4-dione), as reported in Scheme1, previously synthesized by our research group [21], and was used as a control [10,15,23]. Pharmaceutics 2020, 12, 358 3 of 19 Pharmaceutics 2020, 12, x FOR PEER REVIEW 3 of 20

SchemeScheme 1.1. Reaction between HNE HNE and and ER143 ER143 (1) (1)..

TopicalTopical delivery delivery systems systems areare used to vehiculate active active substances substances across across the the skin skin barrier barrier,, aaffectingffecting notnot onlyonly thethe drugdrug potency,potency, butbut alsoalso thethe acceptabilityacceptability by the patient [27] [27].. There There are are several several typestypes of of topical topical pharmaceuticalpharmaceutical forms,forms, includingincluding creams, gels, oi ointments,ntments, pastes, pastes, etc. etc. Emulsions Emulsions are are thermodynamicallythermodynamically unstable unstable systems systems composed composed of of two two immiscible immiscible liquids liquids (water (water and and oil) oil) stabilized stabilized by surfactantsby surfactants that that reduce reduce the the interfacial interfacial tension tension between between those those phases. phases. TheThe nature of the the emulsion emulsion is is influencedinfluenced by by the the waterwater andand oiloil ratio,ratio, thethe type and geometry of of the the surfactant surfactant used used in in the the formulation formulation andand the the manufacturingmanufacturing process.process. Emulsions require a a certain certain amount amount of of energy, energy, thus, thus, rotor rotor stators stators are are commonlycommonly used used inin thethe productionproduction ofof emulsionsemulsions allowing the dispersion of of the the droplets droplets [28 [28––30]30].. OverOver time,time, thethe droplets,droplets, throughthrough Brownian movement, tend to to collide collide with with each each other other to to reduce reduce thethe interfacial interfacial area. area. Consequently, Consequently, coalescence coalescence will will occur occur and and continue continue until until full full phase phase separation separation and theand minimum the minimum free energyfree energy (equilibrium) (equilibrium) are reached. are reached. For thisFor reason,this reason, emulsions emulsions are oneare ofone the of mostthe dimostfficult difficult and complex and complex systems systems in the pharmaceutical in the pharmaceutical field to developfield to develop and stabilize, and stabilize, requiring requiring expertise knowledgeexpertise knowledge and experience and experience [29,30]. To [29,30] prepare. To emulsions prepare with emulsions the shelf with life the stability shelf life required stability for arequire pharmaceuticald for a pharmaceutical product (1–3 +productyears), (1 the–3 correct+ years), choice the correct of surfactants, choice of co-surfactants, surfactants, co fatty-surfactants, alcohols, polymersfatty alcohols, (steric polymers stabilization) (steric or solidstabilization) particles or (Pickering solid particles emulsions), (Pickering among emulsions), other excipients, among must other be madeexcipients, [29,31 must]. be made [29,31]. MicroemulsionsMicroemulsions are are considered considered to tobe bedispersed dispersed colloidal colloidal systems systems consisting consisting of two ofliquid two phases liquid phasesand with and a clear with and a clear translucid and translucid macroscopic macroscopic aspect mainly aspect due mainly to their due reduced to their droplet reduced size. dropletThese size.formulations These formulations differ from emulsions differ from in emulsions many aspects, in many such aspects,as macroscopic such as appearance, macroscopic particle appearance, size, particlestability, size, formation stability, and formation composition and composition [32,33]. In [32 addition,33]. In to addition the components to the components mentioned mentioned above, above,microemulsions microemulsions further further require require a higher a higher content content of surfactant of surfactant and and a co a- co-surfactant,surfactant, usually usually a a low low molecularmolecular weight weight alcohol, alcohol, whichwhich may may compromise compromise their safety [33 [33–35]35].. The The formation formation process process of of microemulsionsmicroemulsions isis significantlysignificantly easiereasier since they form spontaneously spontaneously (almost (almost zero zero interfacial interfacial tension) tension) withoutwithout thethe need for for high high energy energy input. input. In practice, In practice, only onlygentle gentle agitation agitation is required is required [29,33,34] [29, which,33,34], whichmakes makes these systems these systems quite attractive quite attractive for thefor industrial the industrial field. field. InIn this this work, work, thethe synthesissynthesis andand fullfull characterizationcharacterization of new new HNE HNE inhibitors inhibitors with with 4 4-oxo--oxo-ββ-lactam-lactam scascaffoldffoldss andand lessless toxicitytoxicity areare reported.reported. After compound selection, selection, two two different different topical topical forms forms are are developed as O/W emulsions and microemulsions further characterized in terms of physicochemical developed as O/W emulsions and microemulsions further characterized in terms of physicochemical properties, in vitro drug release and in vivo antipsoriatic activity. properties, in vitro drug release and in vivo antipsoriatic activity.

2.2. MaterialsMaterials andand MethodsMethods

2.1.2.1. MaterialsMaterials

2.1.1.2.1.1. SynthesisSynthesis ofof HNEHNE InhibitorsInhibitors AllAll reagentsreagents andand solventssolvents werewere purchasedpurchased from Sigma-AldrichSigma-Aldrich ( (Munich,Munich, Germany Germany),), Alfa Alfa Aesar Aesar (Heysham,(Heysham, Lancashire,Lancashire, UK)UK) andand TCITCI (Zwijndrecht,(Zwijndrecht, Belgium Belgium)) and and used used without without further further purification. purification. DichloromethaneDichloromethane (DCM) (DCM) andand acetoneacetone werewere dried by distillation from from calcium calcium hydride hydride and and p potassiumotassium carbonate,carbonate, respectively. respectively.

Pharmaceutics 2020, 12, 358 4 of 19

2.1.2. Formulation of HNE Inhibitor Loaded Emulsion and HNE Inhibitor Loaded Microemulsion In this study, the AAN-16 (compound 16) was incorporated in an O/W emulsion and in a microemulsion named “AAN-16 E” and “AAN-16 ME”, respectively. In AAN-16 E, glycerin (Quimidroga, Portugal) was used as a humectant and methylparaben and propylparaben (Nipagin™ M Sodium and Nipasol M Sodium™, Clariant, Muttenz, Switzerland) as preservatives. In addition, hydrogenated lecithin (and) C12-16 alcohols (and) palmitic acid (Biophilic™ H MB, IFF Lucas Meyer Cosmetics, Quebec City, QC, Canada), caprylic/capric triglycerides (Tegosoft® CT, Evonik, Essen, Germany), C10-18 triglycerides (Lipocire™ A SG, Gattefossé, Saint-Priest, France), isopropyl myristate (Tegosoft® M, Evonik, Essen, Germany), oleic acid (Sigma-Aldrich, Munich, Germany), alpha bisabolol (Esperis, Milan, Italy), cetyl alcohol (DS produtos químicos, Cascais, Portugal) were also added to AAN-16 E. On the other hand, C21-C28 alkane (Emosmart ™ C28, Seppic ™, La Garenne-Colombes, France), a plant-based and renewable C15-19 alkane oil (Emogreen ™ L15, Seppic ™, La Garenne-Colombes, France) and caprylic/capric triglycerides (Tegosoft® CT, Evonik, Essen, Germany) were added to AAN-16 ME. In addition, PEG-20 glyceryl triisostearate (Cithrol™ 10GTIS, Croda Inc, Snaith, UK) was used as surfactant and propylene glycol (Mosselman, Ghlin, Belgium) as humectant and co-surfactant, and finally, methylparaben and propylparaben (Clariant, Muttenz, Switzerland) were used as preservatives. Purified water was obtained by inverse osmosis (Millipore, Elix® 3, Merck, Darmstadt, Germany).

2.2. Methods

2.2.1. Synthesis and Characterization of HNE Inhibitors The HNE inhibitors were synthesized according to the literature [21,24,26]. The synthesized compounds were characterized by NMR techniques, FTIR and mass spectrometry, elemental analysis and melting point as described in the Supplementary Materials. Chemical reactions were followed by thin layer chromatography (TLC) using Merck aluminium backed sheets coated with 60 F254 silica gel and visualized under a UV lamp or revealed using iodine, potassium permanganate and vanillin. Compound purification was obtained by flash chromatography using silica gel (0.040–0.063 mm) from Merck (Darmstadt, Germany). NMR experiments were performed on a Bruker 300 ultra-Shield (Bruker, Massachusetts, NE, USA) 1 13 ( H 300 MHz; C 75 MHz) in chloroform-d or acetone-d6. All chemical shifts (δ) were quoted in ppm scale and coupling constants (J) in Hz, and multiplicity was described with the following abbreviations: s = singlet, d = doublet, t = triplet, m = multiplet. FTIR spectra were obtained using a IRAffinity-1 (Shimadzu, Kyoto, Japan). Each compound was previously blended with potassium bromide and pressed with a hydraulic press until a pellet was formed. Melting points (m.p.) were determined using a Bock-Monoscop (Kofler, Berlin, Germany). The Log P was predicted using the ALOGPS 2.1 program [36].

2.2.2. Biological Assays of Synthesized HNE Inhibitors

Enzymatic Inhibition Assays and In Vitro Cytotoxicity Fluorometric assays for HNE inhibition activity were conducted as previously described [37]. Briefly, the assays were carried out in 200 µL assay buffer (0.1 M HEPES pH 7.5 at 25 ◦C) containing 20 µL of 0.17 µM HNE in assay buffer, 155 µL of assay buffer and 5 µL of each concentration of the tested inhibitors. The reaction was initiated by the addition of 20 µL of fluorogenic substrate at 200 µM (MeO-Suc-Ala-Ala-Pro-Val-AMC), and the inhibition activity was monitored for 30 min at 25 ◦C on a microplate reader (FLUOstar Omega; BMG Labtech, Baden-Württemberg, Germany) (excitation wavelength 380 nm and emission 460 nm). Inhibitor stock solutions were prepared in DMSO. Controls were performed as follows: (1) enzyme; (2) substrate; (3) enzyme with DMSO and (4) positive Pharmaceutics 2020, 12, 358 5 of 19 control with Sivelestat sodium salt hydrate. Assays were performed in triplicate and presented as log of inhibitor concentrations versus the percentage of activity. IC50 values were determined by non-linear regression analysis in GraphPad PRISM® 5 software. Compounds 1, 16 and 17 were evaluated for their ability to inhibit other human serine proteases, namely , , and in 200 µL reaction volumes at 25 ◦C, as follows: 0.05 M Tris-HCl and 0.138 M NaCl at pH 8.0, 30 or 2 nM human pancreas of each protease and kallikrein respectively, and 50 or 100 µM substrate (Z-Gly-Gly-Arg-AMC.HCl for trypsine; Suc-Ala-Ala-Pro-Phe-7-amino-4-methylcoumarin for chymotrypsin; Z-Gly-Gly-Arg-AMC.HCl for urokinase; H-Pro-Phe-Arg-AMC acetate salt for kallikrein, from Bachem, Switzerland). For all serine proteases, the activity was measured at excitation and emission wavelengths of 360 nm and 460 nm, respectively, in a microplate reader (FLUOstar Omega, BMG Labtech, Germany). The IC50 was determined by non-linear regression as previously published [37]. The cell viability was evaluated with the endpoint MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl- 2H-tetrazolium bromide, Sigma Aldrich) assay performed on a human keratinocyte cell line, HaCaT (Cell line Service GmbH, Eppelheim, Germany) [38]. These cells were seeded in sterile flat bottom 96 well tissue culture plates in RPMI culture medium, supplemented with 10% FBS, 100 units of penicillin G (sodium salt), 100 µg of streptomycin sulfate and 2 mM L-glutamine and incubated at 37 ◦C and 5% CO2. Cell viability was assessed after 24 h of incubation of HaCaT cells with 50 µM concentration of each sample. DMSO diluted in culture medium was the negative control, whereas sodium dodecyl sulfate at 1.0 mg/mL was the positive one. After exposure, the culture medium was replaced by medium containing 0.5 mg/mL MTT. The cells were further incubated for 3 h. After removing the media, the intracellular formazan crystals were solubilized and extracted with DMSO. After 15 min at room temperature, the absorbance was measured at 570 nm in a microplate reader (FLUOstar Omega). The cell viability (%) compared to control cells was calculated by [Absorbance] sample/[Absorbance]control 100, culture medium with the same amount of DMSO as the samples used as control. × 2.2.3. HNE Inhibitor Topical Formulations and Characterization Studies

Solubility Studies According to biological studies of synthesized HNE inhibitors, the selected compound to incorporate in final pharmaceutical formulations was compound 16 (2-((4-(3,3-diethyl-2,4- dioxoazetidin-1-yl)benzyl)thio)-N-(3-((7nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)propyl)benzo[d]oxazole- 5-carboxamide). To perform the solubility studies, the oily phases of AAN-16 ME and AANN-16 E were both saturated with 1 mg of compound 16 under stirring at room temperature. The solubility was measured by a fluorescence method (excitation 380 nm; emission 460 nm) at 25 ◦C on a microplate reader (FLUOstar Omega, BMG Labtech GmbH, Germany).

Preparation of HNE Inhibitor Loaded Emulsion and HNE Inhibitor Loaded Emulsion Microemulsion

The AAN-16 E was prepared by a hot method (80 ◦C), adding the oily phase previously melted, where the compound 16 was dissolved, to the aqueous phase under stirring using an Ultra Turrax® (IKA® T25 Digital, Staufen, Germany) at 7000 rpm for 1 min. On the other hand, the AAN-16 ME was prepared by dissolving the compound 16 in the oily phase with subsequent addition of the aqueous phase under stirring with a glass rod at room temperature. All formulations were carried out using a digital scale (VWR, Portugal) and their composition is shown in Table1. Pharmaceutics 2020, 12, 358 6 of 19

Table 1. Qualitative and quantitative composition of formulations.

Formulations (%, w/w) Disperse Phase Ingredients (INCI) AAN-16 E AAN-16 ME Water 72.79 5.97 Hydrogenated lecithin (and) C12-16 6.00 - alcohols (and) palmitic acid Aqueous phase Glycerin 5.00 - PEG-20 glyceryl triisostearate - 40.27 Propylene glycol - 5.97 Methylparaben 0.18 0.11 Propylparaben 0.02 0.01 Caprylic/capric triglycerides 2.50 15.90 C15-19 alkane - 15.90 C21-C28 alkane - 15.90 C10–18 triglycerides 4.00 - Oily Phase Isopropyl myristate 5.00 - Oleic acid 1.00 - Alpha bisabolol 1.00 - Cetyl alcohol 2.50 - Compound 16 0.01 0.01

The composition of both emulsions and microemulsions was selected by taking previous studies performed by our group into account (data not shown). In those studies, several topical delivery systems (emulsions and microemulsions) were developed, optimized and characterized. Different humectants and preservatives were studied as well as the influence of different concentrations of cetyl alcohol on the final properties of the O/W emulsions. Oils containing different carbon chains were incorporated into microemulsions to mimic the skin lipid layer. At the end, the formulations with the most suitable physicochemical and sensorial properties and higher stability were chosen.

Characterization Studies of HNE Inhibitor Loaded Emulsion and HNE Inhibitor Loaded Microemulsion The pH values were determined at room temperature using a potentiometer (Mettler Toledo, Columbus, OH, USA) 24 h after the production of each formulation. To test the physical stability of AAN-16 E, an accelerated stability test based on centrifugation (Heraeus Sepatech, Hanau, Germany) for 10 min at 3000 rpm was used. The AAN-16 ME physical stability was evaluated by observing the macroscopic appearance (translucent or opaque) of each formulation immediately after preparation. The rheological characteristics of the formulations were examined at high shear rates using a continuous shear technique and oscillation technique in the viscoelastic region. These experiments were performed with a controlled stress Kinexus Lab+ Rheometer (Malvern, UK) using cone geometry (truncated cone angle 4◦ and radius 40 mm). All measurements were performed at room temperature. The shear rate method was carried out using a destructive measurement, where the shear stress of 1 1 each formulation was obtained by increasing the shear rate from 0.1 s− to 100 s− and using 8 samples per decade. First, in the oscillatory method, an amplitude sweep test was performed where the shear strain ranged between 0.01 and 100 Hz, and the frequency was set at 1 Hz. Then, a frequency sweep test was performed with a shear strain of 0.1 and 5% for AAN-16 E and AAN-16 ME respectively, and a frequency range between 0.01 and 10 Hz. Ten samples per decade were evaluated in each method. The mean droplet sizes of emulsions and microemulsions were determined using Malvern Mastersizer (with Hydro 2000S module) and Malvern Zetasizer Nano (Kassel, Germany), respectively. To visualize the internal structure of AAN-16 E, an optical fluorescence microscope with video camera (Bresser, Rhede, Germany) was used. Pharmaceutics 2020, 12, 358 7 of 19

In Vitro Release Studies In vitro release studies were performed using vertical Franz diffusion cells (receptor volume: 4 mL, release area: 1 cm2) with synthetic membranes (Tuffryn® 0.45 µm, hydrophilic polysulfone membrane filter; Pall Corporation (East Hills, NY, USA)) and silicone membranes (Sil-Tec, reff 500-2, 0.002”; Technical Products Inc. of Georgia (Lawrenceville, GA, USA). The samples were evenly applied (0.2 to 0.4 g of formulation correspondent to 0.02 to 0.04 mg of compound (16) on the surface of the membrane in the donor compartment, and sealed with Parafilm® (Bemis Company, Inc, Neenah, WI, USA) to prevent water evaporation. Water:ethanol (50:50 w/w) with 2.5% of PEG-40 Hydrogenated Castor Oil was used as a receptor phase (approx. solubility: 0.39 mg/mL). The study was performed for 6 h at 32–37 ◦C under stirring, and each sample was collected (200 µL) with an interval of one hour. The amount of compound 16 released was analyzed as previously described on a Fluorescence Microplate Reader FLUOstar Omega (BMG Labtech GmbH, Germany), and the data was expressed as the cumulative released drug amount as a function of time. The data obtained was computed using DDSolver [39] (Excel-plugin module) and fitted to different kinetic models, such as: zero order, first order, the Higuchi model and the Korsmeyer–Peppas model [40,41].

In Vivo Antipsoriatic Activity The experiments were conducted using female BALB/c mice at 6 weeks of age provided by Charles River Laboratories (Écully, France). Animals were housed in polypropylene cages at ambient temperature (20–24 C), relative humidity (55 5%), 12 h’ light/dark cycle and given ◦ ± standard diet and water ad libitum. All animal experiments were performed according to the animal welfare organ of the Faculty of Pharmacy, University Lisbon, approved by the competent national authority (Direção-Geral de Alimentação e Veterinária—DGAV) and in accordance with the EU Directive (2010/63/EU), the Portuguese laws (DL 113/2013, 2880/2015, 260/2016 and 1/2019) and all relevant legislation. The in vivo studies regarding the imiquimod (IMQ)-induced psoriasis-like inflammation model were performed according to Pivetta et al. [42]. The psoriasis-inflammation evaluation criteria were assessed, based on the clinical Psoriasis Area and Severity Index (PASI), as previously described [42]. Erythema, scaling and thickening were scored from 0 to 4 (0: none; 1: slight; 2: moderate; 3: marked; 4: very marked). In the first day, all mice were shaved in the dorsum and kept in individual cages throughout the experiment. Animals were randomly selected, and five groups of animals were tested:

Group 1 (n = 5) was treated with 60 mg of Dermovate® (GSK, Algés, Portugal) (containing 0.05% • clobetasol propionate) as a positive control of inflammation inhibition; Group 2 (n = 4) was treated with 60 mg of emulsion placebo; • Group 3 (n = 4) was treated with 60 mg of microemulsion placebo; • Group 4 (n = 5) was treated with 60 mg of AAN-16 E; • Group 5 (n = 5) was treated with 60 mg of AAN-16 ME; • Group 6 (n = 2) was used for control of non-induced and non-treated skin. • All tested formulations were spread over the mice back skin with a spatula in an average amount of 26.7 mg/cm2. The two placebo formulations were used as control, since they present the same composition of AAN-16 E and AAN-16 ME, without any active compound. Five hours after the treatment, 60 mg of Aldara® cream (Meda AB, Solna, Sweden) containing 5% of imiquimod was applied on the back skin. This treatment lasted for 5 consecutive days. Apart from the 1st day, the animals were always subjected to daily visual inspection and measurement of body weight and skin thickness, which was measured in the application zone using a digital calliper (Fisherbrand, Leicestershire, UK). On the 6th day, the mice were sacrificed, and their dorsal skin exposed to the test compounds was preserved in a 10% formalin solution and sent for histopathological analysis. The psoriasis-inflammation evaluation criteria were assessed according to the clinical Psoriasis Area Pharmaceutics 20202020,, 1212,, 358x FOR PEER REVIEW 8 of 20 19

Severity Index (PASI) as previously described [42]. Erythema, scaling and thickening were scored andfrom Severity 0 to 4 (0: Index none; (PASI) 1: slight; as previously 2: moderate; described 3: marked; [42]. 4: Erythema, very marked) scaling. and thickening were scored fromF 0ormalin to 4 (0:- none;fixed 1:tissues slight; were 2: moderate; trimmed 3:into marked; longitudinal 4: very sections. marked). Samples were then paraffin- embedded,Formalin-fixed sectioned tissuesat 4 µm wereand stained trimmed with intoheamtoxylin longitudinal and eosin. sections. Lesions Samples were evaluated were thenby a parapathologistffin-embedded, blinded sectionedto experimental at 4 µ m groups and stained. Measurements with heamtoxylin were performed and eosin. in slides Lesions digitally were evaluatedscanned in by the a NanoZoomerSQ pathologist blinded with to NDP.view2 experimental software groups. (H Measurementsamamatsu, Japan were) corresponding performed in slidesto the digitallymean value scanned obtained in the from NanoZoomerSQ 7 to 12 different with points NDP.view2 for epidermis software and (Hamamatsu, dermis layers. Japan) corresponding to the mean value obtained from 7 to 12 different points for epidermis and dermis layers. 2.2.4. Statistical Analysis 2.2.4. Statistical Analysis One-way analysis of variance (ANOVA) and the Tukey–Kramer post-hoc multiple comparison test wasOne-way used to analysis identify of the variance significant (ANOVA) differences and the between Tukey–Kramer the groups post-hoc, and were multiple performed comparison using testGraphPad was used PRISM to identify® 5 software. the significant An alpha dierrorfferences of 5% between was chosen the groups,to set the and significance were performed level. Results using ® GraphPadwere expressed PRISM as mean5 software. ± standard An deviation. alpha error of 5% was chosen to set the significance level. Results were expressed as mean standard deviation. ± 3. Results and Discussion 3. Results and Discussion 3.1. Synthesis of 4-oxo-β-Lactams 3.1. Synthesis of 4-oxo-β-Lactams 4-oxo-β-lactams, and in particular ER143 (1), showed high potency and selectivity for HNE. 4-oxo-β-lactams, and in particular ER143 (1), showed high potency and selectivity for HNE. However, they presented some toxicity when tested in HaCaT cells. To improve efficacy and decrease However, they presented some toxicity when tested in HaCaT cells. To improve efficacy and decrease cytotoxicity, we synthesized additional analogues of ER143 (1) with varied physicochemical cytotoxicity, we synthesized additional analogues of ER143 (1) with varied physicochemical properties. properties. As depicted in Scheme 2, the 4-oxo-β-lactam core was synthesized by reaction of As depicted in Scheme2, the 4-oxo- β-lactam core was synthesized by reaction of diethylmalonyl diethylmalonyl dichloride with an appropriate aniline. The target compound 6 was prepared through dichloride with an appropriate aniline. The target compound 6 was prepared through radical radical bromination of intermediate 5 using NBS and benzoyl peroxide. Compound 8 was bromination of intermediate 5 using NBS and benzoyl peroxide. Compound 8 was synthesized by synthesized by a cyclization reaction between aniline 7 and diethylmalonyl dichloride, and a cyclization reaction between aniline 7 and diethylmalonyl dichloride, and subsequently, debenzylated subsequently, debenzylated using a palladium and hydrogen atmosphere to yield the carboxylic acid using a palladium and hydrogen atmosphere to yield the carboxylic acid derivative compound 9. derivative compound 9.

Scheme 2 2.. SynthesisSynthesis of compound 6, 8 and 9 .. Reagents Reagents and and conditions: conditions: ( (i)i) pp--toluidine,toluidine, NEt NEt33,, DCM, DCM, r.t; r.t;

(ii)(ii) NBS, benzoylbenzoyl peroxide,peroxide, ACN,ACN, reflux;reflux; (iii)(iii) IntermediateIntermediate 77,, NEtNEt33,, DCM,DCM, r.t;r.t; (iv)(iv) HH22, PdPd/C,/C, MeOH.

To improve solubility solubility of of compound compound 6,6 derivative, derivative 12 12waswas synthesized synthesized as follows as follows (Scheme (Scheme 3). The3). Thecarboxylic carboxylic acid acid derivative derivative 12 was12 was prepared prepared as asdepicted depicted in inScheme Scheme 3,3 ,starting starting with the cyclizationcyclization reaction between 3-amino-4-hydroxybenzoic3-amino-4-hydroxybenzoic acid acid 10 anandd potassium ethyl xanthate to form the benzoxazole 1111,, which which then then reacted reacted with with compound compound6 in 6 the in presence the presence of potassium of potassium carbonate carbonate to obtain to theobtain target the compoundtarget compound12. 12.

Pharmaceutics 2020, 12, 358 9 of 19 Pharmaceutics 2020, 12, x FOR PEER REVIEW 9 of 20 Pharmaceutics 2020, 12, x FOR PEER REVIEW 9 of 20

SchemeScheme 3 3.. SynthesisSynthesis of compound 12. Reagent and conditions: ( (i)i) K KSS2COEtCOEt,, EtOH:H EtOH:H22OO (6:1), (6:1), reflux; reflux; Scheme 3. Synthesis of compound 12. Reagent and conditions: (i) KS2COEt, EtOH:H2O (6:1), reflux; ((ii)ii) K K22COCO33, ,acetone, acetone, 20min.; 20min.; ( (iii)iii) Intermediate Intermediate 66, ,acetone, acetone, reflux. reflux. (ii) K2CO3, acetone, 20min.; (iii) Intermediate 6, acetone, reflux. ToTo allow identificationidentification and quantificationquantification of 4-oxo-4-oxo-β--lactamslactams in in biological assays, assays, a a synthetic To allow identification and quantification of 4-oxo-β-lactams in biological assays, a synthetic scheme was devised to incorporate a fluorophore in inhibitors 9 and 12 (Scheme4). First, intermediate schemescheme was was devised devised to to incorporate incorporate a afluorophore fluorophore in in inhibitorsinhibitors 9 andand 12 12 (Scheme (Scheme 4). 4). First, First, intermediate intermediate 14 was synthesized by reacting amine 13 with NBD-Cl. Removal of the Boc protecting group with 14 14was was synthesized synthesized by by reacting reacting amine amine 13 13 with with NBDNBD--Cl. Removal ofof thethe Boc Boc protecting protecting group group with with trifluoroacetictrifluoroacetictrifluoroacetic acid acid acid in in in dry dry dry dichloromethane dichloromethane yielded yieldedyielded intermediateintermediate 151515, ,, whichwhich which was was was then then then converted converted converted to to to compoundscompoundscompounds 1616 16 andand and 1717 17,, by by, by an an an amide amide amide coupling couplingcoupling reaction reaction with with 1212 andandand 999,, , respectively,respectively, respectively, in in in the the the presence presence presence of of of 1 13 TBTUTBTU and and DIPEA. DIPEA. Compounds Compounds 66 ,,6 8, ,,8 9, ,9, 12, 12, ,16 16 and and 1717 werewere fully characterized by byby 1 H1H and and 13 C13C-C-NMRNMR-NMR and and and massmassmass spectrometry. spectrometry. spectrometry.

SchemeScheme 4. Synthesis4. Synthesis of of compound compound16 16and and17 .17 Reagents. Reagents and and conditions: conditions: (i) (i) NBD-Cl, NBD-Cl, MeOH, MeOH, r.t; r.t; (ii) (ii) TFA; Scheme 4. Synthesis of compound 16 and 17. Reagents and conditions: (i) NBD-Cl, MeOH, r.t; (ii) (iii)TFA; Compound (iii) Compound12, TBTU, 12, DIPEA,TBTU, DIPEA, DCM, DCM, 0 ◦C; (iv)0 °C; Compound (iv) Compound9, TBTU, 9, TBTU, DIPEA, DIPEA, DCM, DCM, 0 ◦C. 0 °C TFA; (iii) Compound 12, TBTU, DIPEA, DCM, 0 °C; (iv) Compound 9, TBTU, DIPEA, DCM, 0 °C ER143ER143 (1) (1)was was also also synthesized synthesized as as described described elsewhereelsewhere [21] [21] to to be be used used as as a acontrol. control. ER143 (1) was also synthesized as described elsewhere [21] to be used as a control. 3.2.3.2. Biological Biological Assays Assays of of Synthesized Synthesized HNE HNE InhibitorsInhibitors 3.2. Biological Assays of Synthesized HNE Inhibitors EnzymaticEnzymatic Inhibition Inhibition Assays Assays and and In In Vitro Vitro Cytotoxicity Cytotoxicity EnzymaticToT understando understand Inhibition the Assaysthe activity activity and of of In synthesized synthesized Vitro Cytotoxicity compoundscompounds against against HNE, HNE, enzymatic enzymatic inhibition inhibition assays assays werewereT assessedo understandassessed as as shown shown the activity in in Table Table of2. synthesized2. compounds against HNE, enzymatic inhibition assays were assessed as shown in Table 2.

Pharmaceutics 2020, 12, 358 10 of 19 Pharmaceutics 2020, 12, x FOR PEER REVIEW 10 of 20 PharmaceuticsPharmaceutics 20 202020, 1, 21,2 x, xFOR FOR PEER PEER REVIEW REVIEW 1010 of of 20 20 PharmaceuticsPharmaceutics 20 2020,20 12, ,1 x2 ,FOR x FOR PEER PEER REVIEW REVIEW 10 10of 20of 20 Pharmaceutics 2020, 12, x FOR PEER REVIEW 10 of 20 TableTable 2. 2.Enzymatic Enzymatic inhibition inhibition assays. assays. TableTable 2. 2. Enzymatic Enzymatic inhibition inhibition assays. assays. TableTable 2. Enzymatic2. Enzymatic inhibition inhibition assays. assays. CompoundsTable 2. Enzymatic Structure inhibition assays. IC 50 SD (nM) Compounds Structure IC50 ± SD (nM) CompoundsCompounds StructureStructure ± ICIC5050 ± ±SD SD (nM (nM) ) CompoundsCompounds StructureStructure ICIC50 ±50 SD± SD (nM (nM) ) Compounds Structure IC50 ± SD (nM) 6 6 0.64 0.030.64 ± 0.03 6 6 0.640.64 ± ±0.03 0.03 6 6 ± 0.640.64 ± 0.03± 0.03

6 0.64 ± 0.03

ER 143 (1) 0.42 ± 0.09 ERER 143 143ER (1) (1)143 (1) 0.42 0.090.420.42 ± ±0.09 0.09 ERER 143 143 (1) (1) ± 0.420.42 ± 0.09± 0.09 ER 143 (1) 0.42 ± 0.09

8 0.96 ± 0.13 8 8 8 0.96 0.130.960.96 ± ±0.13 0.13 8 8 ± 0.960.96 ± 0.13± 0.13

8 0.96 ± 0.13

9 14.71 ± 5.55 9 9 14.7114.71 ± ±5.55 5.55 9 9 9 14.71 14.715.5514.71 ± 5.55± 5.55 9 ±14.71 ± 5.55

12 0.38 ± 0.05 1212 0.380.38 ± ±0.05 0.05 1212 12 0.38 0.050.380.38 ± 0.05± 0.05 12 ±0.38 ± 0.05

By analyzing these data, 4-oxo-β-lactam was revealed to be essential for this activity, as IC50 ByBy analyzing analyzing these these data, data, 4 -4oxo-oxo-β-β-lactam-lactam was was r evealedrevealed to to be be essential essential for for this this activity activity, ,as as IC IC5050 ByBy analyzing analyzing these these data, data, 4- oxo4-oxo-β-lactamβ-lactam was was revealed revealed to tobe be essential essential for for this this activity activity, as, asIC IC50 50 valuesvaluesBy analyzingwere were obtained obtained these at atdata, a asub sub 4-oxonanomolar-nanomolar-β-lactam range, range, was rexcept evealedexcept for for to compound compoundbe essential 9 .9 for.Thus, Thus, this it itactivity was was possible possible, as IC 50to to valuesBy analyzingwere obtained these at data, a sub 4-oxo---nanomolarβ-lactam wasrange, revealed except to for be compound essential for 9 this.. Thus,Thus, activity, itit waswas as IC possiblepossible50 values toto confirmvalues the were importance obtained ofat athe sub β--lactamnanomolar ring range,in the exceptstructure for ofcompound these molecules 9. Thus,, allowing it was possible a great to valueswereconfirmconfirm obtained were the the obtainedimportance atimportance a sub-nanomolar at aof ofsub the the-nanomolar β β- range,lactam-lactam except ring range,ring in forin theexcept the compound structure structure for compound9 .of of Thus, these these it molecules9 wasmolecules. Thus, possible it, ,wasallowing allowing to confirmpossible a agreat great theto confirmconfirm the the importance importance of ofthe the β- lactamβ-lactam ring ring in inthe the structure structure of ofthese these molecules molecules, allowing, allowing a greata great confirmimprovementimprovement the importance on on the the rate rate of of ofthe serine serine β-lactam acylation, acylation, ring asin as confirmedthe confirmed structure in in [26] of[26] .these . molecules, allowing a great importanceimprovementimprovement of the onon βthethe-lactam raterate ofof ring serineserine inthe acylation,acylation, structure asas of confirmedconfirmed these molecules, inin [26][26].. allowing a great improvement on improvementFrom these onresul thets, rate none of ofserine the synthesized acylation, as compounds confirmed inwith [26] the. oxo-β-lactam moiety could be improvementthe rateFromFrom of serine these these on the acylation,resul resul ratets,ts, noneof none asserine confirmedof of the acylation,the synthesized synthesized in [ 26as]. confirmed compounds compounds in [26] with with. the the oxo oxo-β-β-lactam-lactam moiety moiety could could be be FromFrom these these resul results,ts, none none of ofthe the synthesized synthesized compounds compounds with with the the oxo oxo-β-lactamβ-lactam moiety moiety could could be be excluded,excluded,From theseand and thus resulthus a ts, acell cell none viability viability of the assay synthesizedassay was was carried carried compounds out out with with with HaCat HaCat the cells oxocells- toβ to- lactamchoose choose moietythe the compounds. compounds. could be excluded,From theseand thus results, a cell none viability of the assay synthesized was carried compounds out with with HaCat the cells oxo- totoβ-lactam choosechoose moietythethe compounds.compounds. could be excluded,All compounds and thus a presented cell viability lower assay cell was viability carried out than with the HaCat negative cells control to choose (DMSO the compounds.), except excluded,excluded,AllAll compoundsand and compounds thus thus a cell presented presentedviability assay lower lower was cell cell carried viability viability out thanwith than HaCat the the negative negative cells to choosecontrol control the (DMSO (DMSO compounds.),), except except AllAll compounds compounds presented presented lower lower cell cell viability viability than than the the negative negative control control (DMSO (DMSO), ), e xcept except compoundscompounds 9 9and and 12 12 (> (>50%)50%) ( Figure(Figure 1 )1.) .The The lower lower cell cell viability viability of of compound compound 6 6might might be be due due to to the the compoundscompoundsAllAll compounds compounds 9 and 12 presented presented(>(>50%) (( lowerFigure lower cell 1) ). viability. cell TheThe viabilitylowerlower than cellcell the than viabilityviability negative the negativeofof control compoundcompound (DMSO), control 6 might except (DMSO be compounds due), e xceptto the presencecompounds of a bromine9 and 12 atom(>50%) that (Figure is described 1). The lower as potentially cell viability cytotoxic of compound, causing 6 amight decrease be due in to cell the compounds9presenceandpresence12 ( > of of50%)9 a and a bromine bromine (Figure 12 (>50%) 1 atom ). atom The(Figure that that lower is 1 is ) described . described cellThe viabilitylower as ascell potentially ofpotentially viability compound cytotoxicof cytotoxic compound6 might, ,causing causing be 6 might due a a to decrease decreasebe the due presence to in in the cell cell presencepresence of of a brominea bromine atom atom that that is is described described as as potentially potentially cytotoxic cytotoxic, causing, causing a decreasea decrease in in cell cell viabilityviability [43][43]. . Compound Compound 8 8 showedshowed the the lowest lowest cell cell viability viability among among all all compounds. compounds. The The only only presenceofviability a bromine of[43][43] a atom..bromine Compound Compound that isatom described 8 thatshowedshowed is as described potentially the the lowest lowest as cytotoxic,potentially cell cell viability viability causing cytotoxic among among a decrease, causing all all compounds. compounds. in a cell decrease viability The The in [ onlycell only43]. differenceviability with [43] .compound Compound 9 was8 showed the presence the lowestof an ester cell group viability which among improved all compounds. the cytotoxicity. The only viabilityCompounddifferencedifference [43] with with8. showed Compound compound compound the 8lowest9 9wasshowed was the cellthe presence thepresence viability lowest of of among an cellan ester ester viability all group group compounds. which among which improved improved all The compounds. only the the di cytotoxicity. ffcytotoxicity.erence The onlywith differencedifference with with compound compound 9 was9 was the the presence presence of ofan an ester ester group group which which improved improved the the cytotoxicity. cytotoxicity. differencecompound with9 was compound the presence 9 was of anthe esterpresence group of whichan ester improved group which the cytotoxicity. improved the cytotoxicity. 140 140140 140140 140120120 120 100120 120100100 100100 1008080 80 6080 806060 6060 604040 40 2040 402020 2020

Cell viability Cell (%)viability HaCat 200

Cell viability Cell (%)viability HaCat 0

Cell viability Cell (%)viability HaCat 0

Cell viability Cell (%)viability HaCat 0 Cell viability Cell (%)viability HaCat 0 ER143 6 8 9 12 Negative Positive

Cell viability Cell (%)viability HaCat 0 ER143 6 8 9 12 Negative Positive ER143 6 8 9 12 Negative Positive (1)ER143 6 8 9 12 ControlNegativeControlPositive ER143(1)(1) 6 8 9 12 NegativeControlControlPositiveControlControl (1)(1) ControlControlControlControl (1) Control Control FigureFigure 1. 1. Cell Cell viability viability assay assay at at 50 50 µM µM concentration concentration of of compounds compounds ER143 ER143 (1) (1), 6, ,6 8, ,8 9, 9and and 12 12 in in HaCaT HaCaT Figure 1. Cell viability assay at 50 µM concentration of compounds ER143 (1),, 6,, 8,, 9 and 12 inin HaCaT cellsFigure for 24 1. h Cell of exposition viability assay time. at 50 µM concentration of compounds ER143 (1), 6, 8, 9 and 12 in HaCaT FigureFigurecellscells for 1.for 1. Cell24Cell 24 h hviabilityof viability of exposition exposition assay assay time. attime. at 50 50 µM µM concentration concentration of of compounds compounds ER143 (1) (1),, 66,, 88,, 99 andand 1212 inin HaCaT HaCaT cellscells for for 24 24h of h ofexposition exposition time. time. cellscells for for 24 24 h h of of exposition exposition time. time. Due to optimal inhibition activity against HNE and good cell viability of compounds 9 and 12, DueDue to to optimal optimal inhibition inhibition activity activity against against HNE HNE and and good good cell cell viability viability of of compounds compounds 9 9and and 12 12, , DueDue to tooptimal optimal inhibition inhibition activity activity against against HNE HNE and and good good cell cell viability viability of ofcompounds compounds 9 and9 and 12 12, , fluorescentfluorescentDue to optimalderivatives derivatives inhibition were were also activityalso synthesized synthesized against HNE(compounds (compounds and good 1 71 7celland and viability 1 61,6 ,respectively) respectively) of compounds for for the the 9 posterior andposterior 12, fluorescentfluorescent derivativesderivatives werewere alsoalso synthesizedsynthesized (compounds(compounds 17 and 16,, respectively)respectively) forfor thethe posterior drugfluorescent assay. derivatives were also synthesized (compounds 17 and 16, respectively) for the posterior fluorescentdrugdrug assay. assay. derivatives were also synthesized (compounds 17 and 16, respectively) for the posterior drugdrug assay. assay. drug assay.

Pharmaceutics 2020, 12, 358 11 of 19

Due to optimal inhibition activity against HNE and good cell viability of compounds 9 and 12, fluorescent derivatives were also synthesized (compounds 17 and 16, respectively) for the posterior Pharmaceutics 2020, 12, x FOR PEER REVIEW 11 of 20 drugPharmaceutics assay. 2020, 12, x FOR PEER REVIEW 11 of 20 Pharmaceutics 2020, 12, x FOR PEER REVIEW 11 of 20 PharmaceuticsThese ne new2020w, compounds1 2, x FOR PEER alsoREVIEW showed enzymatic activity against HNE at the sub sub-nanomolar-nanomolar11 of 20 range (TableThese3). It ne isw also compounds important also to showed notice thatenzymatic compound activity17 againstshowed HNE a at higher the sub activity-nanomolar than range the parent (Table These3). It neis walso compounds important also to showednotice thatenzymatic compound activity 17 against showed HNE a higher at the sub activity-nanomolar than rangethe parent (TableThese 3). It ne isw also compounds important also to showednotice that enzymatic compound activity 17 showed against HNEa higher at the activity sub-nanomolar than the parent range compound(Table 3). It9. is also important to notice that compound 17 showed a higher activity than the parent (Tablecompound 3). It 9 is. also important to notice that compound 17 showed a higher activity than the parent compoundSince these 9. new derivatives presented very similar activities, further studies of specificity specificity were compoundSince these 9. new derivatives presented very similar activities, further studies of specificity were performedSince tothese select new the derivatives best compound. presented In very this studysimilar (Table activities,3), di furtherfferent studies of specificity were used, were such as: performedperformedSince tothese to selectselect new the derivatives bestbest compound. compound. presented In In this verythis study studysimilar (Table (Table activities, 3), different3), different further enzymes studies enzymes were of specificity were used, used, such were as:such as: trypsin,performed chymotrypsin, to select the urokinasebest compound. and kallikrein. In this study (Table 3), different enzymes were used, such as: trypsin,performedtrypsin, chymotrypsin, chymotrypsin, to select the besturokinaseurokinase compound. and and kallikrein kallikreinIn this study. . (Table 3), different enzymes were used, such as: trypsin,All the chymotrypsin, enzymes tested urokinase belong and tokallikrein the serine. protease group, thus allowing evaluation if the trypsin,AllAll thechymotrypsin, the enzymes enzymes tested urokinase belong belong and to kallikrein to the the serine serine. protease protease group, group, thus thus allowing allowing evaluati evaluation if on the if the compoundsAll the act enzymes only on tested HNE belongor other to enzymes the serine of protease the same group, group. thus The all resultsowing showed evaluati thaton if the the tested compoundscompoundsAll the act act enzymes onlyonly on tested HNEHNE or belongor other other toenzymes enzymes the serine of ofthe protease the same same group. group, group. The thus The results all resultsowing showed showed evaluati that the thaton testedif the the tested compoundscompounds were act only specific on HNE for or enzyme other enzymes inhibition of the at a same higher group. range The like results the control showed ER143that the (1) tested. In other compoundscompounds wereactwere only specific on HNE forfor enzymeor enzyme other inhibitionenzymes inhibition of at the ata higher asame higher group.range range like The like the results controlthe showedcontrol ER143 thatER143 (1) the. In (1)tested other. In other compounds were specific for enzyme inhibition at a higher range like the control ER143 (1). In other words,compoundswords, duedue to towere thethe specific high potency potency for enzyme of of these these inhibition compounds, compounds, at a higher only only range a small a small like amount the amount control was was ER143required required (1) .to In reveal other to reveal words, due to the high potency of these compounds, only a small amount was required to reveal activitywords,activity against dueagainst to the HNEHNE high (Table potency 3) which of these was was compounds,not not sufficient su fficient only to toact a act onsmall onother otheramount enzymes enzymes was of required the of same the sameto class. reveal class. activityactivity against against HNE HNE (Table(Table 3)) whichwhich was was not not sufficient sufficient to toact act on onother other enzymes enzymes of the of samethe same class. class. activity against HNE (Table 3) which was not sufficient to act on other enzymes of the same class. TableTable 3. 3.Results Results of e enzymaticnzymatic inhibition inhibition and and specificity specificity assays assays for forcompounds compounds 16 and16 17and. Compound17. Compound TableTable 3. 3. Results Results ofof eenzymaticnzymatic inhibitioninhibition and and specificity specificity assays assays for forcompounds compounds 16 and 16 and17. Compound 17. Compound ER143TableER143 (1)3.(1) Results waswas used of e asnzymatic as reference reference. inhibition. and specificity assays for compounds 16 and 17. Compound ER143ER143 (1) (1) was was used used asas reference. . ER143 (1) was used as reference. ICIC ± SDSD 5050 ±± IC ± SD (µM) ICIC50±50 S DSD IC50 50 SD (µM) Compounds Structure IC(nM)± SD IC IC± ±SD± SD(µM) (µM) Compounds Structure (50nM) 50 ± 50 CompoundsCompounds StructureStructure (nM)(nM) IC50 SD (µM) Compounds Structure HNE(HNEnM) Trypsin Chymotrypsin Urokinase Urokinase Kalli Kallikreinkrein HNE Trypsin Chymotrypsin Urokinase Kalli krein HNEHNE TrypsinTrypsin ChymotrypsinChymotrypsin Urokinase Urokinase KallikKallirein krein ER143ER143 (1) (1) 0.42 ± 0.090.09 >>11 >>88 >>5050 >8 >8 ER143 (1) 0.42± ± 0.09 >1 >8 >50 >8 ER143 (1) 0.42 ± 0.09 >1 >8 >50 >8 ER143 (1) 0.42 ± 0.09 >1 >8 >50 >8

16 0.85 ± 0.10 >1 >1 >2 >5 1616 0.850.85 ±0.10 0.10 >>11 >1>1 >2 >5> 5 1616 0.850.85± ± 0.10± 0.10 >1 >1 >1 >1 >2 >2 >5 >5

17 0.64 ± 0.03 >1 >0.5 >2 >3 17 0.64 ± 0.03 >1 >0.5 >2 >3 1717 0.64 ± 0.030.03 >>11 >>0.50.5 >>22 >3 >3 17 0.64± ± 0.03 >1 >0.5 >2 >3

Compound 17 was more cytotoxic (38% of cell viability) than compound 9 (80% of cell viability), Compound 17 was more cytotoxic (38% of cell viability) than compound 9 (80% of cell viability), and Compound both had higher 17 was cell more viability cytotoxic than (38% compound of cell viability) ER143 than (1) (15%compound of cell 9 (80% viability) of cell (Figure viability), 2). andCompound both had higher17 was cell more viability cytotoxic than (38% compound of cell viability) ER143 (1) than (15% compound of cell viability) 9 (80% of (Figure cell viability), 2). andNevertheless,Compound both had compound higher17 was cell 16 more viability showed cytotoxic a than cell viability compound (38% (%) of ER143 cellcomparable viability) (1) (15% to the than of nega cell compoundtive viability) control. (Figure 9Thus,(80% this 2). of cell andNevertheless, both had compound higher cell 16 viability showed a thancell viability compound (%) comparable ER143 (1) to (15% the nega of tive cell control. viability) Thus, (Figure this 2). viability),Nevertheless,compound and was compound bothselected had to 16 be higher showed incorporated cell a cell viability viability into the than(%) topical comparable compound formulations. to theER143 nega (1)tive (15%control. of Thus, cell this viability) compound was selected to be incorporated into the topical formulations. (FigureNevertheless,compound2). Nevertheless,was compound selected to 16 compoundbe showed incorporated a cell16 intoviabilityshowed the topical (%) a cell comparable formulations. viability to (%) the comparable negative control. to the Thus, negative this compound was selected to be incorporated into the topical formulations. control. Thus, this compound140 was selected to be incorporated into the topical formulations. 140 140120 120 140120100 100 12010080 80 1008060 60 6040 8040 4020 6020 20400

Cell viability Cell (%)viability HaCat 0 Cell viability Cell (%)viability HaCat 200 ER143 (1) 16 17 Negative Positive Cell viability Cell (%)viability HaCat ER143 (1) 16 17 Negative Positive ER143 (1) 16 17 NegativeControl PositiveControl 0 Control Control Cell viability Cell (%)viability HaCat Control Control Figure 2. Cell viability assayER143 at 50 (1) µM concentration16 of fluorescent17 compoundsNegative ER143Positive (1), 16 and 17 Figure 2. Cell viability assay at 50 µM concentration of fluorescent compounds ER143 (1), 16 and 17 Figurein HaCaT 2. Cell cells viability for 24 h assayof exposition at 50 µM time. concentration of fluorescent compoundsControl ER143Control (1), 16 and 17 in HaCaT cells for 24 h of exposition time. Figurein HaCaT 2. Cell cells viability for 24 h of assay exposition at 50 µ Mtime. concentration of fluorescent compounds ER143 (1), 16 and 17 in Figure 2. Cell viability assay at 50 µM concentration of fluorescent compounds ER143 (1), 16 and 17 HaCaT cells for 24 h of exposition time. in HaCaT cells for 24 h of exposition time.

Pharmaceutics 2020, 12, 358 12 of 19 Pharmaceutics 2020, 12, x FOR PEER REVIEW 12 of 20

3.3. HNE HNE Inhibitor Inhibitor Loaded Loaded Emulsion Emulsion and and HNE HNE Inhibitor Inhibitor Loaded Microemulsion

3.3.1. Solubility Solubility Studies Studies According toto previousprevious results, results, compound compound16 16(AAN-16) (AAN-16) was was selected selected to beto incorporatedbe incorporated in an in O /anW O/Wemulsion emulsion (AAN-16 (AAN E)-16 and E) in and a microemulsion in a microemulsion (AAN-16 (AAN ME).-16 ME). The results showed that 0.1 mg/gmg/g of compound 16 was the maximum drug concentration solubilized in both both oil oil blends, blends, composed composed by by caprylic/capric caprylic/capric triglycerides, C15 C15-19-19 alkane and C21 C21-C28-C28 alkane oils for AAN-16AAN-16 MEME andand capryliccaprylic/capric/capric triglycerides triglycerides and and isopropyl isopropyl myristate myristate for for AANN-16 AANN-16 E. ETherefore,. Therefore, these these formulations formulations were were prepared prepared at the at samethe same concentration concentration of active of active compound compound (0.1 mg (0.1/g mg/gof formulation). of formulation). These final final formulations were fully characterized as described below.

3.3.3.3.2.2. Characterization Characterization Studies Studies of of AAN AAN-16-16 Loaded Emuls Emulsionion and AAN AAN-16-16 Loaded Microemulsion AANAAN-16-16 E revealed a yellow and opaque appearance with some consistency unlike the AAN AAN-16-16 ME correspond correspondinging to a clear and homogenous formulation with liquid appearance.appearance. AANAAN-16-16 E and AANAAN-16-16 ME presented a pH of 6.7 and and 7.5, 7.5, respectively. respectively. These values were appropriate for topical topical application application,, respecting respecting the the physiological physiological pH pH according according to to the the literature literature [44,45] [44,45].. Comparing with the pH obtained in placebos, there were no significantsignificant changes.changes. The rheological rheological characterization characterization was was performed performed by continuous by continuous shear and shear oscillation and experiments. oscillation experimentsAAN-16 E had. AAN a much-16 E higher had a viscositymuch higher (182.5 viscosity Pa.s) than (182.5 AAN-16 Pa.s) ME than (0.05 AAN Pa.s)-16 atME the (0.05 same Pa.s) shear at ratethe 1 same(at 0.1 shear s− ), asrate supposed (at 0.1 s−1 for), as these supposed systems. for Thesethese sy valuesstems. remained These values constant remained without constant any significant without anydifferences, significant when differences compared, when with compared placebos for with both placebos formulations. for both formulations. InIn addition, AAN-16AAN-16 ME ME was was a Newtoniana Newtonian fluid fluid in whichin which viscosity viscosity does does not changenot change when when a shear a shearrate is rateapplied, is applied, whereas whereas AAN-16 AAN E was-16 a shear-thinning E was a shear fluid-thinning characterized fluid characterized by a viscosity by decrease a viscosity with decreaseincreasing with shear increasing rate owing shear to therate orientation owing to the of orientation the molecules of the in themolecules structure in [the46, 47structure]. These [46,47] results. Thesewere in results agreement were in with agreement placebo studieswith placebo that others studies had tha reportedt others [had29,34 reported,48–51]. [29,34,48–51]. Figure 33 representsrepresents thethe oscillatoryoscillatory frequencyfrequency study,study, inin whichwhich thethe systemsystem responseresponse isis measuredmeasured as a function of frequency with constant shear strain. From From these these results, results, it it is is possible to obtain the storage modulus (G’)(G’) (elastic(elastic component) component) and and the the loss loss modulus modulus (G”) (G’’ (viscous) (viscous component) component) in whichin which the theenergy energy lost lost is reflected is reflected [52]. [52].

2500 6 AAN-16 E G' AAN-16 ME G' AAN-16 E G'' 2000 5 AAN-16 ME G'' 4 1500 3 1000

2 G' and G'' and G'' G' (Pa) G' and G'' and G'' G' (Pa) 500 1

0 0 0.01 0.1 1 10 0.01 0.1 1 10 Frequency (Hz) Frequency (Hz) (a) (b) Figure 3. OscillatoryOscillatory results results of of ( (aa)) AAN AAN-16-16 E E and and (b)) AAN AAN-16-16 ME. In this case, the values of the elastic modulus of AAN-16 E overcame the values of the viscosity In this case, the values of the elastic modulus of AAN-16 E overcame the values of the viscosity modulus (G’ > G”) and the loss tangent values were lower than 1 [52], indicating a solid-like behavior modulus (G’ > G’’) and the loss tangent values were lower than 1 [52], indicating a solid-like behavior with a strong network. In this structure type, the molecules are more bound (e.g., by Van der Walls with a strong network. In this structure type, the molecules are more bound (e.g., by Van der Walls interactions) and organized than in liquids, but less than in solids. On the contrary, the elastic interactions) and organized than in liquids, but less than in solids. On the contrary, the elastic modulus of AAN-16 ME was lower than the viscous modulus (G’ < G”) and the loss tangent values modulus of AAN-16 ME was lower than the viscous modulus (G’ < G’’) and the loss tangent values were lower than 1, thus presenting a liquid-like structure with a weak and easily disturbed network. were lower than 1, thus presenting a liquid-like structure with a weak and easily disturbed network. As a consequence, these formulations presented a suitable spreadability. As a consequence, these formulations presented a suitable spreadability. The d(50) for AAN-16 E was 23.52 µm with a span of 5.028, while AAN-16 ME presented a d(50) of 9.92 nm with a Pdi of 0.834. The droplet size obtained here was also concordant with the literature

Pharmaceutics 2020, 12, x FOR PEER REVIEW 13 of 20 Pharmaceutics 2020, 12, 358 13 of 19 related to these pharmaceutical forms [29,34,51]. Although AAN-16 ME had droplets in the nano range,The the d(50) Pdi for value AAN-16 was high, E was indicating 23.52 µm heterogeneity with a span of of droplet 5.028, while size within AAN-16 AAN ME-16 presented ME, or, aprobably d(50) of a9.92 bicontinuous nm with a Pdi system. of 0.834. Microemulsions The droplet typicallysize obtained exist here in the was form also concordant of micellar with and bicontinuous structures. However, it is very complex and difficult to characterize these systems at the literaturePharmaceutics related 2020, 1 to2, x theseFOR PEER pharmaceutical REVIEW forms [29,34,51]. Although AAN-16 ME had13 of 20 droplets the molecular level. in the nano range, the Pdi value was high, indicating heterogeneity of droplet size within AAN-16 Fluorescencerelated to these microscopy pharmaceutical was usedforms to[29,34,51] visualize. Although the internal AAN- 16structure ME had ofdroplets these formulationsin the nano with ME, or,range, probably the Pdi a bicontinuous value was high, system. indicating Microemulsions heterogeneity of typically droplet size exist within in the AAN form-16of ME, micellar or, and compound 16 (Figure 4). bicontinuousprobably structures. a bicontinuous However, system. it is Microemulsions very complex andtypically diffi cult exist to in characterize the form of these micellar systems and at the molecularbicontinuous level. structures. However, it is very complex and difficult to characterize these systems at Fluorescencethe molecular microscopy level. was used to visualize the internal structure of these formulations with Fluorescence microscopy was used to visualize the internal structure of these formulations with compound 16 (Figure4). compound 16 (Figure 4).

Figure 4. The inside structure of AAN-16 E (a) and emulsions’ placebo (b) using a fluorescence microscope. Figure 4. TheFigure inside 4. The structure inside structure of AAN-16 of AAN E (a-)16 and E (a emulsions’) and emulsions’ placebo placebo (b) using (b) using a fluorescence a fluorescence microscope. microscope. ItIt waswas onlyonly possiblepossible to to observe observe the the emulsion emulsion (E) (E) droplets droplets due due to to the the respective respective size. size In. I then the AAN-16 AAN- E,16 itE was, it was possibleIt possible was only to observe topossible observe to the observe the drug drug the both emulsionboth dispersed dispersed (E) droplets and and dissolved due dissolved to the inrespective thein the formulation, formulation,size. In the AAN as shownas- shown in Figurein Figure416. Thus, E4., itT hus,was the possible the formulation formulation to observe droplets thedroplets drug were both were fluorescent dispersed fluorescent and owing dissolved owing to this toin thisthe labeled formulation, labeled compound. compound. as shown in Figure 4. Thus, the formulation droplets were fluorescent owing to this labeled compound. 3.3.3. In Vitro Release Studies 3.3.3. In3.3. Vitro3. In ReleaseVitro Release Studies Studies InIn vitrovitroInAAN-16 AANvitro AAN-16 release release-16 release studies studies studies were were were performed performedperformed in in bothin both both final final final formulationsformulations formulations using using usingFranz Franz CellsFranz Cells Cells with ® awith synthetic a syntheticwith membranea synthetic membrane membrane (Tuffryn (Tuffryn (Tuffryn) (Figure® )® (Figure) (Figure5). 5).

10 10 AAN-16 E AAN-16 E AAN-16 ME 8 AAN-16 ME 8

6 (µg)

62 (µg)

2 4 percm 4

2 percm

Amount of compound 16 releasedof 16 compound Amount 2 0 0 2 4 6 Time (h) Amount of compound 16 releasedof 16 compound Amount 0 ® Figure 5. Drug release studies0 of AAN-16 E and2 AAN-16 ME using4 a synthetic membrane6 (Tuffryn ). Results expressed as average ± SD, n = 6. Time (h)

®® FigureFigure 5.After5. Drug 6 h release, the amount studies of ofthe AAN-16AAN compound-16 E and 16 released AAN-16AAN-16 was MEME different, using a synthetic i.e., 7.2 ± 2.1 membrane μg/cm2 and (Tu(Tuffryn ff3.3ryn ± )).. ResultsResults expressedexpressed2 asas averageaverage ± SD, n = 66.. 1.2 μg/cm for AAN-16 E and± AAN-16 ME, respectively. The emulsion released a higher amount of drug than the microemulsion, despite the higher bulk viscosity of the AAN-16 E. In fact, the structure 2 AfterAfterof vehicles 6 h h,, the the plays amount amount an important of of the the compoundrole compound in the release 1616 released ofreleased the active was wascompound different, different, [53] i.e.,. i.e., 7.2 7.2± 2.1 μg/cm2.1 µg2/ cmand 3.3and ± 2 ± 3.3 1.2 µ2g/cm for AAN-16 E and AAN-16 ME, respectively. The emulsion released a higher amount 1.2 ±μg/cm for AAN-16 E and AAN-16 ME, respectively. The emulsion released a higher amount of ofdrug drug than than the the microemulsion, microemulsion, despite despite the the higher higher bulk bulk viscosity viscosity of of the the AAN AAN-16-16 E. E. In fact, the structure ofof vehiclesvehicles playsplays anan importantimportant rolerole inin thethe releaserelease ofof thethe activeactive compoundcompound [[53]53]..

Pharmaceutics 2020, 12, 358 14 of 19

Regarding drug properties, compound 16 is mainly lipophilic (log P (predicted) = 4.68), uncharged 1 and withPharmaceutics a high 20 molecular20, 12, x FOR PEER weight REVIEW (M = 643.68 g.mol− ), thus its diffusion into the receptor14 of aqueous 20 phase could be compromised. AAN-16Regarding E and drug AAN-16 properties, ME present compound a structure 16 is type mainly with lipophilic aqueous (log and P occlusive (predicted) lipidic = 4.68), phases. −1 It wasuncharged further noted and with that a AAN-16high molecular was dissolvedweight (M in= 643.68 the oily g.mol phase), thus of the its diffusion microemulsion, into the receptor while it was aqueous phase could be compromised. both dispersed and dissolved in the emulsion. AAN-16 E and AAN-16 ME present a structure type with aqueous and occlusive lipidic phases. AAN-16 E is an O/W emulsion, where the drug is incorporated in the internal phase of the It was further noted that AAN-16 was dissolved in the oily phase of the microemulsion, while it was formulation.both dispersed To reach and the dissolved membrane in the and emulsion. the receptor phase, the compound needs to pass through the structureAAN of the-16 external E is an aqueous O/W emulsion, phase. where the drug is incorporated in the internal phase of the Theformulation. diffusivity To reach of compound the membrane16 in and the the formulations receptor phase, was the an compound inverse functionneeds to pass of the through surfactant concentration.the structure In of addition, the external several aqueous studies phase [54. –56] indicate that drug flow is inversely proportional to the amountThe of diffusivity surfactant. of compound 16 in the formulations was an inverse function of the surfactant concentration. In addition, several studies [54–56] indicate that drug flow is inversely proportional2 Diffusion was the drug release mechanism for both formulations, where the Higuchi (R ajusted to the amount of surfactant. 2 = 0.934 0.029) and First order models (R ajusted = 0.784 0.069) could be applied for AAN-16 E ±Diffusion was the drug release mechanism for both formulations,± where the Higuchi (R2ajusted = and AAN-16 ME, respectively. Finally, no drug was detected in the receptor phase when a silicone 0.934 ± 0.029) and First order models (R2ajusted = 0.784 ± 0.069) could be applied for AAN-16 E and membrane was used. AAN-16 ME, respectively. Finally, no drug was detected in the receptor phase when a silicone Themembrane use of was synthetic used. membranes is useful in quality assurance, but it does not represent how a formulationThe use will of behavesynthetic once membranes applied is on useful skin. in Manyquality drugs assurance, developed but it does for thenot topicalrepresent treatment how a of skin diseaseformulation are poorlywill behave water-soluble once applied and ondi skin.fficult Many to formulate.drugs develo Thus,ped for certain the topical strategies treatment can be of used. For example,skin disease AAN are 16poorly was water composed-soluble by and permeation difficult to enhancers formulate. (isopropylThus, certain myristate, strategies propylenecan be used. glycol and oleicFor example, acid) and AAN 6% emulsifiers 16 was composed (hydrogenated by permeation lecithin enhancers (and) C12-16 (isopropyl alcohols myristate, (and) propyle palmiticne acid), whichglycol may and aff ectoleic the acid) solubility and 6% emulsifiers and/or interact (hydrogenated with the lecithin membrane (and) C12 used-16 alcohols [34,53,57 (and)]. Consequently, palmitic AAN-16acid) E, exhibitedwhich may higher affect drug the release solubility over and/or time when interact compared with the to membrane AAN-16 ME used formulated [34,53,57]. with Consequently, AAN-16 E exhibited higher drug release over time when compared to AAN-16 ME 40% of a non-ionic surfactant (PEG-20 glyceryl triisostearate). formulated with 40% of a non-ionic surfactant (PEG-20 glyceryl triisostearate). 3.3.4. In Vivo Antipsoriatic Activity 3.3.4. In Vivo Antipsoriatic Activity TheThe imiquimod imiquimod (IMQ)-induced (IMQ)-inducedpsoriasis-like psoriasis-like inflammation inflammation model model was was used used in this in this study study to to evaluateevaluate the antipsoriaticthe antipsoriatic activity activity of of AAN-16 AAN-16 EE and AAN AAN-16-16 ME ME topically topically applied. applied. In this In model, this model, stratumstratum corneum corneumdisturbance disturbance by imiquimodby imiquimod dramatically dramatically promotes promotes the the cutaneous cutaneous drugdrug absorptionabsorption [58]. After[58] treatment. After tre withatment test with compounds test compounds and commercial and commercial corticosteroid corticosteroid control, control, several several parameters parameters were assessedwere as assessed erythema, as erythema scaling,, scaling, and skin and thickness skin thickness (Figure (Figure6). 6).

FigureFigure 6. Results 6. Results of theof thein in vivo vivoantipsoriatic antipsoriatic activity:activity: erythema, erythema, scaling scaling and and skin skin thickness thickness of mice of mice challengedchallenged with with imiquimod imiquimod (IMQ) (IMQ) and and treated treated withwith each formulation formulation (Dermovate (Dermovate® , AAN®, AAN-16-16 E and E and emulsionemulsion placebo). placebo Score). Score 0–4 0– (none4 (none to to marked). marked). Results expressed expressed as asaverage average ± SD. SD. ±

PharmaceuticsPharmaceutics 20202020,, 1212,, 358x FOR PEER REVIEW 1515 ofof 1920

Mice treated with AAN-16 E and placebo obtained similar and higher scores, for all parameters Mice treated with AAN-16 E and placebo obtained similar and higher scores, for all parameters in in opposition to the results of the positive control group (treated with Dermovate® containing opposition to the results of the positive control group (treated with Dermovate® containing clobetasol clobetasol propionate). Unexpectedly, mice exposed to AAN-16 ME had to be sacrificed within 5 days propionate). Unexpectedly, mice exposed to AAN-16 ME had to be sacrificed within 5 days of of application due to the presence of physiological and behavioral signs of distress (humane application due to the presence of physiological and behavioral signs of distress (humane endpoints). endpoints). In addition, it was not possible to verify any significant difference between In addition, it was not possible to verify any significant difference between microemulsions’ placebo microemulsions’ placebo and AAN-16 ME. and AAN-16 ME. IMQ-induced psoriasis-like model is a commonly used model to test antipsoriatic drugs. In IMQ-induced psoriasis-like model is a commonly used model to test antipsoriatic drugs. In BALB/c BALB/c mice, IMQ topical application induces psoriasis-like dermatitis mediated via the IL-23/IL-17 mice, IMQ topical application induces psoriasis-like dermatitis mediated via the IL-23/IL-17 axis [59]. axis [59]. However, in mice, this model causes not only plaque psoriasis, but also systemic disease However, in mice, this model causes not only plaque psoriasis, but also systemic disease comprising comprising more severe symptoms, like anorexia, malaise, and pain [60]. These severe systemic more severe symptoms, like anorexia, malaise, and pain [60]. These severe systemic features are usually features are usually only observed in non-treated animals in contrast to animals treated with only observed in non-treated animals in contrast to animals treated with antipsoriatic drugs, like antipsoriatic drugs, like clobetasol propionate cream (Dermovate® —Figure 6), which show clobetasol propionate cream (Dermovate®—Figure6), which show amelioration in the development amelioration in the development of IMQ-induced psoriasis-like signs. In this study, we treated mice of IMQ-induced psoriasis-like signs. In this study, we treated mice with ME-based formulations with ME-based formulations containing approximately 40% (w/w) of PEG-20 glyceryl triisostearate. containing approximately 40% (w/w) of PEG-20 glyceryl triisostearate. This compound is considered This compound is considered safe for cosmetic use, being favorably used as a penetration enhancer safe for cosmetic use, being favorably used as a penetration enhancer [61]. Surfactants have effects on the [61]. Surfactants have effects on the permeability of several biological membranes including skin permeability of several biological membranes including skin mainly due to their potential to solubilize mainly due to their potential to solubilize lipids within the stratum corneum. Some authors tested lipids within the stratum corneum. Some authors tested the cytotoxicity and inflammation potency the cytotoxicity and inflammation potency of several surfactants on reconstructed human epidermis of several surfactants on reconstructed human epidermis tissues, and concluded that PEG ethers tissues, and concluded that PEG ethers appeared to be more toxic than PEG esters. The results also appeared to be more toxic than PEG esters. The results also revealed the mildness of polyoxyethylene revealed the mildness of polyoxyethylene sorbitan esters independently of their alkyl chain length sorbitan esters independently of their alkyl chain length [62]. Frequently, the cleansing and leave-on [62]. Frequently, the cleansing and leave-on products contain up to 20% and 5% PEG-20 glyceryl products contain up to 20% and 5% PEG-20 glyceryl triisostearate, respectively [61]. Considering the triisostearate, respectively [61]. Considering the results obtained in this work, we are convinced that results obtained in this work, we are convinced that the exacerbated systemic effects observed in mice the exacerbated systemic effects observed in mice treated with ME-based formulations are related to treated with ME-based formulations are related to the enhanced IMQ transdermal absorption and not the enhanced IMQ transdermal absorption and not to the in vivo toxicity of ME-based formulation to the in vivo toxicity of ME-based formulation per se. per se. Figure7 shows the histopathology results of mice skin previously exposed to imiquimod, Figure 7 shows the histopathology results of mice skin previously exposed to imiquimod, and and thereafter, to Dermovate®, emulsion placebo and AAN-16 E. thereafter, to Dermovate® , emulsion placebo and AAN-16 E.

FigureFigure 7. 7. Representative microphotographs of of skin (dorsum) of mice previously (a)) treatedtreated with with ® DermovateDermovate® ;;( b (b)) emulsion emulsion placebo; placebo; (c) AAN-16 (c) AAN E.- Changes16 E. Changes were characterized were characterized by epidermal by hyperplasia epidermal (blackhyperplasia arrowhead), (black arrowhead), and focally extensive and focally inflammation extensive inflammation expanding the expanding dermis (white the dermis arrowhead). (white Hematoxylin and eosin stain. Original magnification, 10 (bar, 250 µm). arrowhead). Hematoxylin and eosin stain. Original magnifica× tion, 10× (bar, 250 µm). Histopathological changes were evaluated by means of epidermal and dermal thickness. Histopathological changes were evaluated by means of epidermal and dermal thickness. Through the present study, it could be observed (Figure7b,c) that the skin changes were characterized Through the present study, it could be observed (Figure 7b,c) that the skin changes were by diffuse epidermal hyperplasia resulting in increased thickness (73.91 µm and 63.48 µm for AAN-16 characterized by diffuse epidermal hyperplasia resulting in increased thickness (73.91 µm and 63.48 E and emulsion placebo, respectively) of the Malpighian layer (highlighted with a black arrow, µm for AAN-16 E and emulsion placebo, respectively) of the Malpighian layer (highlighted with a Figure7c,d), which is composed of stratum basale and stratum spinosum. A focally extensive minimal black arrow, Figure 7c,d), which is composed of stratum basale and stratum spinosum. A focally to mild inflammation expanding the dermis (237.13 µm and 204.79 µm for AAN-16 E and emulsion extensive minimal to mild inflammation expanding the dermis (237.13 µm and 204.79 µm for AAN- placebo, respectively) was observed promoting a diffuse thickening (highlighted with a white arrow, 16 E and emulsion placebo, respectively) was observed promoting a diffuse thickening (highlighted Figure7). In this inflammation model, the infiltrates corresponded to a mixed cell population with with a white arrow, Figure 7). In this inflammation model, the infiltrates corresponded to a mixed

Pharmaceutics 2020, 12, 358 16 of 19 a marked mononuclear-cell component. It is important to notice that the edema was minimal, and no changes were found in the untreated animals (data not show). Therefore, the results are in accordance with the literature [42,59,63,64], where the application of IMQ causes epidermal hyperplasia resulting in increased thickness. Comparing the visual score of the emulsion placebo and AAN-16 E it is not possible to find a significant difference between these two groups. In other studies, using the same animal model, higher scores were obtained for the different parameters evaluated when placebo formulations were tested (negative control groups) [42,59,64,65]. In this study, even if it is not possible to distinguish between the AAN-16 E and the placebo results, both by visual score or by histology analysis, the results do not show high severity on skin lesions if compared to the data presented in the literature. Further studies are required to clarify the role of the placebo emulsion and its components on the inflammation control.

4. Conclusions From the results presented in this work, we can conclude that these new synthesized inhibitors showed high in vitro activity and selectivity against HNE, constituting promising scaffolds. According to biological assays, only two compounds were selected (9 and 12), to synthesize new ones (16 and 17) with fluorescence labeling. As compound 16 presented higher cell viability, it was chosen to be further incorporated into topical emulsion and microemulsion formulations (AAN-16 E and AAN-16 ME, respectively). The in vitro AAN-16 release studies showed a great dependence on the formulation type. In particular, a higher release rate was obtained for emulsion compared to microemulsion. Finally, according to in vivo antipsoriatic activity data, no significant differences between the placebo and AAN-16 E were observed. Moreover, results obtained for the tested formulations were not equivalent or superior to those of the reference formulation (Dermovate®), indicating that the therapeutic effect was not detected in this model. Consequently, future in vivo studies will provide a clarification of the mechanism of action of this compound among others within this scaffold.

Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4923/12/4/358/s1, the characterization of all compounds is presented in the Supplementary Materials. Author Contributions: A.N., J.M., R.F., L.M.G. and S.S. contributed to the conceptualization, methodology, validation, formal analysis, and investigation; A.N. contributed to the writing—original draft preparation; A.A., J.M., F.L. and H.M.R. contributed to supervision, writing—review and editing; F.L. and H.M.R. contributed to project administration, resources, and funding acquisition. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by Fundação para a Ciência e a Tecnologia, Portugal (UID/DTP/04138/2019 to iMedUlisboa and and PTDC/MEC-DER/30198/2017) and Labodidática, Equipamentos de Laboratório e Didácticos L.da. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

References

1. Hawkes, J.E.; Chan, T.C.; Krueger, J.G. Psoriasis pathogenesis and the development of novel targeted immune therapies. J. Allergy Clin. Immunol. 2017, 140, 645–653. [CrossRef][PubMed] 2. Parisi, R.; Symmons, D.P.M.; Griffiths, C.E.M.; Ashcroft, D.M. Global epidemiology of psoriasis: A systematic review of incidence and prevalence. J. Investig. Dermatol. 2013, 133, 377–385. [CrossRef][PubMed] 3. Noncommunicable Diseases and Their Risk Factors. Psoriasis. Available online: https://www.who.int/ncds/ management/psoriasis/en/ (accessed on 22 April 2019). 4. Deng, Y.; Chang, C.; Lu, Q. The Inflammatory Response in Psoriasis: A Comprehensive Review. Clin. Rev. Allergy Immunol. 2016, 50, 377–389. [CrossRef][PubMed] 5. Kim, J.; Krueger, J.G. The Immunopathogenesis of Psoriasis. Dermatol. Clin. 2015, 33, 13–23. [CrossRef] 6. Liang, Y.; Sarkar, M.K.; Tsoi, L.C.; Gudjonsson, J.E. Psoriasis: A mixed autoimmune and autoinflammatory disease. Curr. Opin. Immunol. 2017, 49, 1–8. [CrossRef] Pharmaceutics 2020, 12, 358 17 of 19

7. Current and Potential New Therapies for the Treatment of Psoriasis. Available online: https://www.pharmaceutical-journal.com/news-and-analysis/news/current-and-potential-new-therapies- for-the-treatment-of-psoriasis/11013061.article?firstPass=false (accessed on 19 July 2019). 8. Hansen, G.; Gielen-haertwig, H.; Reinemer, P.; Schomburg, D.; Harrenga, A.; Niefind, K. Unexpected Active-Site Flexibility in the Structure of Human Neutrophil Elastase in Complex with a New Dihydropyrimidone Inhibitor. J. Mol. Biol. 2011, 409, 681–691. [CrossRef] 9. Shreder, K.R.; Cajica, J.; Du, L.; Fraser, A.; Hu, Y.; Kohno, Y.; Lin, E.C.; Liu, S.J.; Okerberg, E.; Pham, L.; et al. Synthesis and optimization of 2-pyridin-3-yl-benzo[d][1,3]oxazin-4-one based inhibitors of human neutrophil elastase. Bioorganic Med. Chem. Lett. 2009, 19, 4743–4746. [CrossRef] 10. Lucas, S.D.; Costa, E.; Guedes, R.C.; Moreira, R. Targeting COPD: Advances on Low-Molecular-Weight Inhibitors of Human Neutrophil Elastase. Med. Res. Rev. 2013, 33, E73–E101. [CrossRef] 11. Heutinck, K.M.; ten Berge, I.J.M.; Hack, C.E.; Hamann, J.; Rowshani, A.T. Serine proteases of the human in health and disease. Mol. Immunol. 2010, 47, 1943–1955. [CrossRef] 12. Crocetti, L.; Giovannoni, M.P.; Schepetkin, I.A.; Quinn, M.T.; Khlebnikov, A.I.; Cilibrizzi, A.; Dal Piaz, V.; Graziano, A.; Vergelli, C. Design, synthesis and evaluation of N-benzoylindazole derivatives and analogues as inhibitors of human neutrophil elastase. Bioorganic Med. Chem. 2011, 19, 4460–4472. [CrossRef] 13. Giovannoni, M.P.; Schepetkin, I.A.; Quinn, M.T.; Cantini, N.; Crocetti, L.; Guerrini, G.; Iacovone, A.; Paoli, P.; Rossi, P.; Bartolucci, G.; et al. Synthesis, biological evaluation, and molecular modelling studies of potent human neutrophil elastase (HNE) inhibitors. J. Enzyme Inhib. Med. Chem. 2018, 33, 1108–1124. [CrossRef] [PubMed] 14. Korkmaz, B.; Horwitz, M.S.; Jenne, D.E.; Gauthier, F. Neutrophil Elastase, , and G as Therapeutic Targets in Human Diseases. Pharmacol. Rev. 2010, 62, 726–759. [CrossRef][PubMed] 15. Santana, A.B.; Lucas, S.D.; Gonçalves, L.M.; Correia, H.F.; Cardote, T.A.F.; Guedes, R.C.; Iley, J.; Moreira, R. N-Acyl and N-sulfonyloxazolidine-2,4-diones are pseudo-irreversible inhibitors of serine proteases. Bioorg. Med. Chem. Lett. 2012, 22, 3993–3997. [CrossRef] 16. Yang, X.; Yan, H.; Zhai, Z.; Hao, F.; Ye, Q.; Zhong, B. Neutrophil elastase promotes proliferation of HaCaT cell line and transwell psoriasis organ culture model. Int. J. Dermatol. 2010, 49, 1068–1074. [CrossRef] 17. Zhong, J.; Groutas, W. Recent Developments in the Design of Mechanism-based and Alternate Substrate Inhibitors of Serine Proteases. Curr. Top. Med. Chem. 2005, 4, 1203–1216. [CrossRef] 18. Leung, D.; Abbenante, G.; Fairlie, D.P. Protease inhibitors: Current status and future prospects. J. Med.Chem. 2000, 43, 305–341. [CrossRef] 19. Guyot, N.; Butler, M.W.; McNally, P.; Weldon, S.; Greene, C.M.; Levine, R.L.; O’Neill, S.J.; Taggart, C.C.; McElvaney, N.G. Elafin, an elastase-specific inhibitor, is cleaved by its cognate enzyme neutrophil elastase in sputum from individuals with cystic fibrosis. J. Biol. Chem. 2008, 283, 32377–32385. [CrossRef] 20. Powers, J.C.; Asgian, J.L.; Ekici, O.D.; James, K.E. Irreversible inhibitors of serine, cysteine, and threonine proteases. Chem. Rev. 2002, 102, 4639–4750. [CrossRef] 21. Marto, J.; Ruivo, E.; Lucas, S.D.; Gonçalves, L.M.; Simões, S.; Gouveia, L.F.; Felix, R.; Moreira, R.; Ribeiro, H.M.; Almeida, A.J. Starch nanocapsules containing a novel neutrophil elastase inhibitor with improved pharmaceutical performance. Eur. J. Pharm. Biopharm. 2018, 127, 1–11. [CrossRef] 22. Maryanoff, B.E.; Costanzo, M.J. Inhibitors of proteases and amide that employ an α-ketoheterocycle as a key enabling functionality. Bioorg. Med. Chem. 2008, 16, 1562–1595. [CrossRef] 23. Groutas, W.C.; Dou, D.; Alliston, K.R. Neutrophil Elastase Inhibitors. Expert Opin. Ther. Pat. 2012, 21, 339–354. [CrossRef] 24. Mulchande, J.; Oliveira, R.; Carrasco, M.; Gouveia, L.; Guedes, R.C.; Iley, J.; Moreira, R. 4-Oxo-β-lactams (Azetidine-2, 4-diones) Are Potent and Selective Inhibitors of Human Leukocyte Elastase. J. Med. Chem. 2010, 53, 241–253. [CrossRef] 25. Mulchande, J.; Simões, S.I.; Gaspar, M.M.; Eleutério, C.V.; Oliveira, R.; Cruz, M.E.M.; Moreira, R.; Iley, J. Synthesis, stability, biochemical and pharmacokinetic properties of a new potent and selective 4-oxo-β-lactam inhibitor of human leukocyte elastase. J. Enzyme Inhib. Med. Chem. 2011, 26, 169–175. [CrossRef] 26. Mulchande, J.; Guedes, R.C.; Tsang, W.; Page, M.I.; Moreira, R.; Iley, J. Azetidine-2,4-diones (4-Oxo-β-lactams) as Scaffolds for Designing Elastase Inhibitors. J. Med. Chem. 2008, 51, 1783–1790. [CrossRef] Pharmaceutics 2020, 12, 358 18 of 19

27. Carneiro, R.; Salgado, A.; Raposo, S.; Marto, J.; Simões, S.; Urbano, M.; Ribeiro, H.M. Topical emulsions containing ceramides: Effects on the skin barrier function and anti-inflammatory properties. Eur. J. Lipid Sci. Technol. 2011, 113, 961–966. [CrossRef] 28. Raposo, S.C.; Simões, S.D.; Almeida, A.J.; Ribeiro, H.M. Advanced systems for glucocorticoids’ dermal delivery. Expert Opin. Drug Deliv. 2013, 10, 857–877. [CrossRef] 29. Simões, S.; Ribeiro, H.M.; Almeida, A.J. Microemulsões e nanoemulsões. In Novas Formas Farmacêuticas para Administração de Fármacos, 1st ed.; Souto, E.B., Lopes, C.M., Eds.; Edições Universidade Fernando Pessoa: Porto, Portugal, 2011; pp. 271–296. 30. Sakamoto, K.; Lochhead, R.Y.; Maibach, H.I.; Yamashita, Y. Cosmetic Science and Technology: Theoretical Principles and Applications, 1st ed.; Elseivier: Amsterdam, The Netherlands, 2017; pp. 1–835. [CrossRef] 31. Marto, J.; Ascenso, A.; Simões, S.; Almeida, A.; Ribeiro, H. Pickering emulsions: Challenges and opportunities in topical delivery. Expert Opin. Drug Deliv. 2016, 13, 1093–1107. [CrossRef] 32. Anton, N.; Vandamme, T.F. Nano-emulsions and micro-emulsions: Clarifications of the critical differences. Pharm. Res. 2011, 28, 978–985. [CrossRef] 33. Kogan, A.; Garti, N. Microemulsions as transdermal drug delivery vehicles. Adv. Colloid Interface Sci. 2006, 123, 369–385. [CrossRef] 34. Lopes, L.B. Overcoming the cutaneous barrier with microemulsions. Pharmaceutics 2014, 6, 52–77. [CrossRef] 35. Tadros, T.F. Microemulsions. In Applied Surfactants: Principles and Applications, 1st ed.; Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, Germany, 2005; pp. 309–334. [CrossRef] 36. Virtual Computational Chemistry Laboratory. Available online: http://www.virtuallaboratory.org/lab/alogps/ (accessed on 24 March 2020). 37. Lucas, S.D.; Gonçalves, L.M.; Cardote, T.A.F.; Correia, H.F.; Moreira, R.; Guedes, R.C. Structure Based Virtual Screening for Discovery of Novel Human Neutrophil Elastase Inhibitors. Med. Chem. Commun. 2012, 3, 1299–1304. [CrossRef] 38. Strober, W. Trypan Blue Exclusion Test of Cell Viability. Curr. Protoc. Immunol. 2015, 111.[CrossRef] 39. Zhang, Y.; Huo, M.; Zhou, J.; Zou, A.; Li, W.; Yao, C.; Xie, S. DDSolver: An add-in program for modeling and comparison of drug dissolution profiles. AAPS J. 2010, 12, 263–271. [CrossRef] 40. Mateus, D.; Marto, J.; Trindade, P.; Gonçalves, H.; Salgado, A.; Machado, P.; Melo-Gouveia, A.; Ribeiro, H.M.; Almeida, A.J. Improved morphine-loaded hydrogels for wound-related pain relief. Pharmaceutics 2019, 11, 76. [CrossRef] 41. Costa, P.; Lobo, J.M.S. Modeling and comparison of dissolution profiles. Eur. J. Pharm. Sci. 2001, 13, 123–133. [CrossRef] 42. Pivetta, T.P.; Simões, S.; Araújo, M.M.; Carvalho, T.; Arruda, C.; Marcato, P.D. Development of nanoparticles from natural lipids for topical delivery of thymol: Investigation of its anti-inflammatory properties. Colloids Surf. B Biointerfaces 2018, 164, 281–290. [CrossRef] 43. Centers for Disease Control and Prevention: Facts about Bromine. Available online: https://emergency.cdc. gov/agent/bromine/basics/facts.asp (accessed on 26 July 2019). 44. Baroni, A.; Buommino, E.; De Gregorio, V.; Ruocco, E.; Ruocco, V.; Wolf, R. Structure and function of the epidermis related to barrier properties. Clin. Dermatol. 2012, 30, 257–262. [CrossRef] 45. Lambers, H.; Piessens, S.; Bloem, A.; Pronk, H.; Finkel, P. Natural skin surface pH is on average below 5, which is beneficial for its resident flora. Int. J. Cosmet. Sci. 2006, 28, 359–370. [CrossRef] 46. Barnes, H.A.; Hutton, J.F.; Walters, K. An Introduction to Rheology, 1st ed.; Elsevier Science: Amsterdam, The Netherlands, 1989; pp. 1–200. 47. Schramm, G. A Practical Approach to Rheology and Rheometry, 2nd ed.; Thermo Electron: Waltham, MA, USA, 2004; pp. 1–259. 48. Gilbert, L.; Picard, C.; Savary, G.; Grisel, M. Rheological and textural characterization of cosmetic emulsions containing natural and synthetic polymers: Relationships between both data. Colloids Surf. A Physicochem. Eng. Asp. 2013, 421, 150–163. [CrossRef] 49. Eccleston, G.M. Emulsions and microemulsions. In Encyclopedia of Biomedical Polymers and Polymeric Biomaterials, 1st ed.; Mishra, M., Ed.; CRC Press: Boca Raton, FL, USA, 2015; Volume 11, pp. 3259–3276. [CrossRef] Pharmaceutics 2020, 12, 358 19 of 19

50. Ribeiro, H.M.; Morais, J.A.; Eccleston, G.M. Structure and rheology of semisolid o/w creams containing cetyl alcohol/non-ionic surfactant mixed emulsifier and different polymers. Int. J. Cosmet. Sci. 2004, 26, 47–59. [CrossRef] 51. Callender, S.P.; Mathews, J.A.; Kobernyk, K.; Wettig, S.D. Microemulsion utility in pharmaceuticals: Implications for multi-drug delivery. Int. J. Pharm. 2017, 526, 425–442. [CrossRef][PubMed] 52. Lippacher, A.; Müller, R.H.; Mäder, K. Liquid and semisolid SLNTM dispersions for topical application: Rheological characterization. Eur. J. Pharm. Biopharm. 2004, 58, 561–567. [CrossRef][PubMed] 53. Otto, A.; Du Plessis, J.; Wiechers, J.W. Formulation effects of topical emulsions on transdermal and dermal delivery. Int. J. Cosmet. Sci. 2009, 31, 1–19. [CrossRef][PubMed] 54. Dalvi, U.G.; Zatz, J.L. Effect of nonionic surfactants on penetration of dissolved benzocaine through hairless mouse skin. J. Soc. Cosmet. Chem. 1981, 32, 87–94. 55. Florence, A.T.; Gillan, J.M.N. Biological implications of the use of surfactants in medicines and the biphasic effects of surfactants in biological systems. Pestic. Sci. 1975, 6, 429–439. [CrossRef] 56. Sarpotdar, P.P.; Zatz, J.L. Evaluation of penetration enhancement of lidocaine by nonionic surfactants through hairless mouse skin in vitro. J. Pharm. Sci. 1986, 75, 176–181. [CrossRef] 57. Mohammed, D.; Hirata, K.; Hadgraft, J.; Lane, M.E. Influence of skin penetration enhancers on skin barrier function and skin protease activity. Eur. J. Pharm. Sci. 2014, 51, 118–122. [CrossRef] 58. Lin, Y.K.; Yang, S.H.; Chen, C.C.; Kao, H.C.; Fang, J.Y. Using imiquimod-induced psoriasis-like skin as a model to measure the skin penetration of anti-psoriatic drugs. PLoS ONE 2015, 10, e0137890. [CrossRef] 59. Van der Fits, L.; Mourits, S.; Voerman, J.S.A.; Kant, M.; Boon, L.; Laman, J.D.; Cornelissen, F.; Mus, A.M.; Florencia, E.; Prens, E.P.; et al. Imiquimod-Induced Psoriasis-Like Skin Inflammation in Mice Is Mediated via the IL-23/IL-17 Axis. J. Immunol. 2009, 182, 5836–5845. [CrossRef] 60. Alvarez, P.; Jensen, L.E. Imiquimod Treatment Causes Systemic Disease in Mice Resembling Generalized Pustular Psoriasis in an IL-1 and IL-36 Dependent Manner. Mediators Inflamm. 2016, 2016, 6756138. [CrossRef] 61. Jang, H.J.; Shin, C.Y.; Kim, K.B. Safety Evaluation of Polyethylene Glycol (PEG) Compounds for Cosmetic Use. Toxicol. Res. 2015, 31, 105–136. [CrossRef][PubMed] 62. Lémery, E.; Briançon, S.; Chevalier, Y.; Bordes, C.; Oddos, T.; Gohier, A.; Bolzinger, M.A. Skin toxicity of surfactants: Structure/toxicity relationships. Colloids Surf. A Physicochem. Eng. Asp. 2015, 469, 166–179. [CrossRef] 63. Sun, J.; Zhao, Y.; Hu, J. Curcumin Inhibits Imiquimod-Induced Psoriasis-Like Inflammation by Inhibiting IL-1beta and IL-6 Production in Mice. PLoS ONE 2013, 8, e67078. [CrossRef][PubMed] 64. Chen, Y.H.; Wu, C.S.; Chao, Y.H.; Lin, C.C.; Tsai, H.Y.; Li, Y.R.; Chen, Y.Z.; Tsai, W.H.; Chen, Y.K. Lactobacillus pentosus GMNL-77 inhibits skin lesions in imiquimod-induced psoriasis-like mice. J. Food Drug Anal. 2017, 25, 559–566. [CrossRef][PubMed] 65. Grine, L.; Dejager, L.; Libert, C.; Vandenbroucke, R.E. Dual Inhibition of TNFR1 and IFNAR1 in Imiquimod-Induced Psoriasiform Skin Inflammation in Mice. J. Immunol. 2015, 194, 5094–5102. [CrossRef] [PubMed]

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