Biopharmaceutical Study of Triamcinolone Acetonide Semisolid Formulations for Sublingual and Buccal Administration †

pharmaceutics

Article Biopharmaceutical Study of Triamcinolone Acetonide Semisolid Formulations for Sublingual and Buccal Administration †

Marta Márquez Valls 1, Alejandra Martínez Labrador 1, Lyda Halbaut Bellowa 1 , Doménica Bravo Torres 1, Paulo C. Granda 1 , Montserrat Miñarro Carmona 1, David Limón 2 and Ana C. Calpena Campmany 1,*

1 Department of Pharmacy and Pharmaceutical Technology and Physical Chemistry, Faculty of Pharmacy and Food Science, University of Barcelona, Av. Joan XXIII 29-31, 08028 Barcelona, Spain; [email protected] (M.M.V.); [email protected] (A.M.L.); [email protected] (L.H.B.); [email protected] (D.B.T.); [email protected] (P.C.G.); [email protected] (M.M.C.) 2 Department of Pharmacology, Toxicology and Therapeutic Chemistry, Faculty of Pharmacy and Food Science, University of Barcelona, Av. Joan XXIII 29-31, 08028 Barcelona, Spain; [email protected] * Correspondence: [email protected] † This paper is an extended versión of paper published in the 1st International Electronic Conference on Pharmaceutics, 1–15 December 2020.

Abstract: The mouth can be affected by important inflammatory processes resulting from localized or systemic diseases such as diabetes, AIDS and leukemia, among others, and are manifested in Citation: Márquez Valls, M.; various types of buccal sores typically presenting pain. This work focuses on the design, formulation, Martínez Labrador, A.; Halbaut and characterization of four semisolid formulations for oral mucosa in order to symptomatically treat Bellowa, L.; Bravo Torres, D.; Granda, these painful processes. The formulations have two active pharmaceutical ingredients, triamcinolone P.C.; Miñarro Carmona, M.; Limón, acetonide (TA) and lidocaine hydrochloride (LIDO). The formula also contains, as an excipient, D.; Calpena Campmany, A.C. ® Biopharmaceutical Study of Orabase , which is a protective, hydrophobic, and anhydrous adhesive vehicle, used to retain or Triamcinolone Acetonide Semisolid facilitate the application of active pharmaceutical ingredients to the oral mucosa. After designing Formulations for Sublingual and the formulations, an analytical method for TA was validated using HPLC so as to achieve reliable Buccal Administration. Pharmaceutics analytical results. Franz-type diffusion cells were used to perform drug release studies using synthetic 2021, 13, 1080. https://doi.org/ membrane, and permeation studies using buccal mucosa, estimating the amount and rate of TA 10.3390/pharmaceutics13071080 permeated across the tissue. Additionally, sublingual permeation studies were carried out to evaluate a scenario of a continuous contact of the tongue with the applied formulation. Permeation fluxes Academic Editors: Andrea Erxleben and the amount of TA retained within sublingual mucosa were similar to those in buccal mucosa, and Elisabetta Gavini also implying anti-inflammatory activity in the part of the tongue that is in direct contact with the formulation. In addition, the dynamic conditions of the mouth were recreated in terms of the Received: 21 April 2021 presence of phosphate buffered saline, constant movement of the tongue, pH, and temperature, using Accepted: 6 July 2021 Published: 15 July 2021 dissolution equipment. The amount of TA released into the phosphate buffered saline in dynamic conditions (subject to being ingested) is well below the normal oral doses of TA, for which the

Publisher’s Note: MDPI stays neutral formulation can be considered safe. The formulations applied to buccal or sublingual mucosas under with regard to jurisdictional claims in dynamic conditions permit the successful retention of TA within either tissue, where it exerts anti- published maps and institutional affil- inflammatory activity. The four formulations studied show a pseudoplastic and thixotropic behavior, iations. ideal for topical application. These results evidence the potential of these topical formulations in the treatment of inflammatory processes in the buccal mucosa.

Keywords: triamcinolone acetonide; buccal administration; semisolid formulations; thixotropic

Copyright: © 2021 by the authors. behaviour; lidocaine hydrochloride; Franz-type diffusion cells Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons 1. Introduction Attribution (CC BY) license (https:// The mouth can be affected by important inﬂammatory processes resulting from local- creativecommons.org/licenses/by/ ized or systemic diseases such as diabetes, AIDS and leukemia, among others, which can 4.0/).

Pharmaceutics 2021, 13, 1080. https://doi.org/10.3390/pharmaceutics13071080 https://www.mdpi.com/journal/pharmaceutics Pharmaceutics 2021, 13, 1080 2 of 15

manifest in various types of buccal sores, such as canker sores or lichen planus, conditions that typically present inflammation and pain [1]. Additionally, although the oral cavity has its own bacterial flora, a qualitative and quantitative imbalance of this ecosystem leads to infections that also cause inflammatory reactions. The treatment of oral lesions including inflammation may include lipophilic glu- cocorticosteroids with immunosuppressive and anti-inflammatory activity [2], such as triamcinolone acetonide (TA), at concentrations ranging from 0.05% to 0.5%, or fluoci- nolone acetonide at concentrations 0.025–0.05%, either in aqueous solutions or in Orabase®. Antiseptic agents such as chlorhexidine gluconate can be included as a mouthwash, and an- tibiotics such as doxycycline hyclate for lesions that involve infections, or topical anesthetics such as benzocaine hydrochloride for pain management [3]. For patients with recurrent lesions, such as AIDS patients, intralesional administration of TA has been used [4,5], which involves discomfort for the patient during treatment. In western European countries, semisolid formulations including TA can be found, including Aldoderma, Cemalyt, Positon, and Interderm, but none of them include local anesthetics such as benzocaine or lidocaine hydrochloride salts. A semisolid formulation including TA and lidocaine hydrochloride is marketed in the United States, but there is an overall lack of products containing both active ingredients around the world. Buccal and sublingual administration are routes intended for the administration of drugs through the (buccal or sublingual) mucosa for achieving a local or systemic effect. Buccal and sublingual administration may involve higher bioavailability than oral ingestion, as they lack the enzymatic degradation and presystemic elimination within the gastrointestinal tract, as well as the first pass hepatic metabolism involved [6,7]. Buccal and sublingual administration shows inherent challenges according to the local environment, with saliva being the most significant influence. As opposed to the gastrointestinal environment upon oral ingestion (high volume of water and pH 1–3), the limited amount of saliva (0.5–2 mL) constantly present, as well as the controlled pH (5.5–7), limits the amount of drug that can be dissolved. In this respect, hydrophilic drugs could dissolve in saliva and be further ingested orally, whereas lipophilic drugs (such as TA) cannot be easily dissolved in the saliva, but will most likely diffuse towards the mucosa. Additionally, the composition of the sublingual and buccal tissues importantly influences the permeability of drugs. Whereas the mucosas of the gingiva and hard palate are keratinized and contain ceramides and acylceramides that act as a barrier, soft buccal and sublingual mucosas contain only small amounts of polar lipids (cholesterol sulfate and glucosyl ceramides), making them more permeable to water. Therefore, lipophilic drugs might be retained close to the site of application to perform a local anti-inflammatory activity, rather than being systemically absorbed, which could involve systemic toxicity [3]. This work is an extension of the work presented at the 1st International Electronic Conference on Pharmaceutics, 1–15 December 2020 [8], and shows the design and development of four semisolid formulations for administration in the buccal mucosa with the aim of locally treating inflammatory processes and pain in this cavity. These formulations have one or two Active Pharmaceutical Ingredients (API): triamcinolone acetonide (TA) and lidocaine hydrochloride (LIDO). TA is a synthetic lipophilic glucocorticosteroid with immunosuppressive and anti-inflammatory activity [2], whereas lidocaine hydrochloride (LIDO) is a hydrophilic local anesthetic that blocks sodium ion channels. The formulations contain, as an excipient, Orabase®, which is a protective adhesive vehicle, hydrophobic and anhydrous, used to retain or facilitate the application of API in buccal mucosa. It has poor solubility and contains gelling agents that allow the adherence to the mucosa for periods of between 15 min and 2 h [9]. The aim of this research was the evaluation of the mechanical and biopharmaceutical properties of the semisolid formulations and to determine the influence of the concentration of TA or the presence and influence of LIDO on these properties. The suitability for topical application was therefore evaluated by performing rheology studies, while the amount and rate of TA that can be released from the formulation was determined. Additionally, Pharmaceutics 2021, 13, 1080 3 of 15

the ability to permit the permeation of TA across either buccal or sublingual mucosa was studied using Franz cells and the amount of TA retained within the buccal mucosa, where the drug performs its anti-inﬂammatory activity, was calculated. In addition, the dynamic conditions of the mouth were recreated in terms of the presence of water, constant movement, pH, and temperature, and mucosas with the formulations applied were immersed in dynamic conditions such that it was possible to assess the amount of TA that can be released to the phosphate buffered saline and thus estimate possible systemic effects after oral ingestion. The amount of TA retained within these tissues under dynamic conditions was also analyzed to assess the inﬂuence of these parameters on the amount of TA retained within the tissue. To obtain fully reliable results from release, permeation, and retention studies, we designed and validated an analytical method using High-Performance Liquid Chromatog- raphy (HPLC).

2. Experiments 2.1. Materials Triamcinolone acetonide (TA), Lidocaine hydrochloride (LIDO) and Liquid parafﬁn were purchased in Fagron (Barcelona, Spain). Orabase® (Pectin 1–5%, Silica 5–10%, Sodium carboxymethylcellulose 5–10%, Parafﬁnum liquidum 75–100%) was purchased in Acofarma (Barcelona, Spain). Transcutol P was purchased from Gattefossé (Saint-Priest, France). Acetonitrile (MeCN) was purchased from Fisher Chemical (Barcelona, Spain). Ammonium acetate (≥98%) was purchased from Panreac (Barcelona, Spain).

2.2. Preparation and Composition of Formulations Four semisolid formulations were prepared using the bioadhesive platform Orabase® instead of water. For this task, TA, LIDO and liquid parafﬁn or mixtures were weighed and mixed in a mortar with a pestle. Then, pre-weighed Orabase® (ad 100%) was added to the formulation in increasing amounts by mixing up. The semisolid formulations obtained were further mixed and homogenized by an Ultra-Turrax T10 basic (IKA, Staufen, Germany) and placed in glass containers (Table1).

Table 1. Composition of the four different formulations.

Composition 0.05% TA 0.05% TA + LIDO 0.1% TA 0.1% TA + LIDO TA 0.05% 0.05% 0.1% 0.1% LIDO - 2% - 2% Liquid parafﬁn 5% 5% 5% 5% Orabase® ad 100% ad 100% ad 100% ad 100%

2.3. Rheological Properties The rheological characterization of the formulas was performed in duplicate at 25 ◦C, using a Thermo Scientific Haake Rheostress 1 rheometer (Thermo Fischer Scientific, Kalsruhe, Germany) equipped with a cone-plate geometry (C60/2◦ Ti), connected to a temperature control device (Thermo Haake Phoenix II + Haake C25P; Thermo Fischer Scientific, Kalsruhe, Germany) and operated using Haake Rheowin® Job Manager v. 3.3 software. The viscosity and flow curves were obtained in rotational mode by performing an ascendant shear rate ramp from 0 to 100 s−1 during 3 min, followed by 1 min at a constant rate of 100 s−1, and from 100 s−1 to 0 s−1 during 3 min. The data obtained for each formulation were adjusted to different mathematical models: Newton, Bingham, Casson, Ostwald, Herschel-Bulkley and Cross.

2.4. Analytical Method Validation The validation of the analytical method of TA using High Performance Liquid Chro- matography (HPLC) was carried out in a Waters HPLC system equipped with a Waters Pharmaceutics 2021, 13, x FOR PEER REVIEW 4 of 16

an ascendant shear rate ramp from 0 to 100 s−1 during 3 min, followed by 1 min at a constant rate of 100 s−1, and from 100 s−1 to 0 s−1 during 3 min. The data obtained for each formulation were adjusted to different mathematical models: Newton, Bingham, Casson, Ostwald, Herschel-Bulkley and Cross.

2.4. Analytical Method Validation Pharmaceutics 2021, 13, 1080 4 of 15 The validation of the analytical method of TA using High Performance Liquid Chromatography (HPLC) was carried out in a Waters HPLC system equipped with a Waters pump 1525, a UV-vis 2487 detector (Waters, Milford, EE. UU.) and a Supercosil LCpump-ABZ 1525,(15 cm; a UV-vis4.6 mm 2487 and detector5 µm) column. (Waters, The Milford, data were EE. collected UU.) and and a Supercosil processed LC-ABZ using the(15 Empower cm; 4.6 mm Pro and software 5 µm) (Waters, column. TheMilford, data CT, were USA). collected The andmobile processed phase consisted using the Em-of 50:50power (v/v Pro) water software/methanol. (Waters, Samples Milford, of CT,10 µL USA). were The injected mobile and phase TA consisted was detected of 50:50 at 232 (v/ v) nmwater/methanol. according to a validated Samples ofmethod 10 µL werefor a injecteddifferent and route TA of was administration detected at 232 [10,11,12] nm according. TA wasto a initially validated dissolved method for in a Transcutol different route P, of and administration further diluted [10– 12 using]. TA was a mixture initially of dis- MeCN:Ammoniumsolved in Transcutol acetate P, and buffer further pH diluted 4.7 (10:90) using [13] a. mixtureSix different of MeCN:Ammonium calibration curves acetatewere madebuffer by pHpreparing 4.7 (10:90) stock [13 solutions]. Six different of 205 μg calibration/mL TA, and curves further were dilutions made by of preparing 102.5 μg/mL, stock 68.3solutions μg/mL, of 41 205 μgµ/mL,g/mL 20.5 TA, μg and/mL further and 10.25 dilutions μg/mL. of 102.5Linearity,µg/mL, accuracy, 68.3 µ g/mL,precision, 41 µ limitg/mL, of20.5 detectionµg/mL (LOD and) 10.25 and limitµg/mL. of quantification Linearity, accuracy, (LOQ) precision, were estimated limit of as detection follows. (LOD) and limit of quantification (LOQ) were estimated as follows. 2.4.1. Linearity and Range 2.4.1. Linearity and Range Linearity of the method in the defined range of concentrations was evaluated by Linearity of the method in the defined range of concentrations was evaluated by per- performing a least squares regression on the experimental data and evaluating the forming a least squares regression on the experimental data and evaluating the correlation correlation coefficient (r) based on Equation (1): coefficient (r) based on Equation (1): y = SB · x + a (1) y = SB · x + a (1) where x is the concentration, y is the chromatographic area, SB is the value of the slope andwhere a is thex is y the-intercept concentration, [14]. y is the chromatographic area, SB is the value of the slope and a isLinearity the y-intercept is the ability [14]. within a defined range to obtain results directly proportional to the Linearity concentrations is the ability (amount) within of thea defined analyte range in the to obtain sample. results The directly range is proportional the interval to definedthe concentrations by the upper (amount) and lower of theconcentrations analyte in the of sample.the tested The drug range for is which the interval it has defined been provedby the that upper the andmethod lower achieves concentrations a suitable of level the testedof accuracy, drug forprecision which and it has linearity been proved [15]. Figurethat the 1 shows method a typical achieves chromatogram a suitable level obtained of accuracy, in the precision analysis and of linearitysamples [containing15]. Figure 1 TA.shows a typical chromatogram obtained in the analysis of samples containing TA.

Figure 1. Chromatogram of the TA standard solution. Figure 1. Chromatogram of the TA standard solution. 2.4.2. Accuracy and Precision 2.4.2. AccuracyAccuracy and at eachPrecision concentration was expressed as the mean percentage deviation or relativeAccuracy error at (RE each, %) concentration calculated using was Equation expressed (2): as the mean percentage deviation or relative error (RE, %) calculated using Equation (2): %RE = [(Cobs − Cnom)/Cnom] · 100 (2) %RE = [(Cobs − Cnom)/Cnom] · 100 (2) where Cobs is the observed concentration and Cnom is the nominal concentration of each standard solution. Precision was calculated and expressed as the relative standard deviation (RSD, %) of each replicate series, using Equation (3):

%RSD = (SD/Cobs) · 100 (3)

where SD is the standard deviation and Cobs is the nominal concentration. Pharmaceutics 2021, 13, 1080 5 of 15

2.4.3. Determination of Limits LOD and LOQ were calculated based on the standard deviation of the response and the slope of the calibration curve using the following equation:

LOD or LOQ = K · SDsa/Sb (4)

where K is a factor related to the level of conﬁdence (3 for LOD and 10 for LOQ). SDSa is the standard deviation of the intercept (a) and Sb is the slope of the calibration line [16]. The results of the analytical method validation show that the 6 calibration lines are linear from 6.26 to 100.20 µg/mL, showing a correlation coefﬁcient (r2) in the range of 0.9993–0.9998 for each line. The method is accurate and precise in the range of 6.26 µg/mL to 100.20 µg/mL, with an accuracy of 92.49% and precision of 98.23% (at 6.26 µg/mL). Finally, the LOD of the method was 2.63 ± 1.19 µg/mL and the LOQ calculated was 7.97 ± 3.60 µg/mL.

2.5. Release Studies Static conditions: To assess the release of TA from the 4 different types of formulations, drug release experiments were performed in triplicate using Franz-type diffusion cells (FDC 400, Crown Glass, Somerville, NY, USA), with the donor and receptor chambers being separated by nylon synthetic membranes (Type NY1 µm). The receptor chambers were filled with a mixture of MeCN: Ammonium acetate buffer pH 4.7 (10:90), complying with sink conditions. The Franz-type diffusion cells were connected with a temperature- controlled circulating bath at 37 ◦C. The dose applied in the donor compartment was 1.5 g of formulation in a diffusion area of 2.54 cm2. Samples of 300 µL were collected from the receptor compartment and replaced with the same volume of fresh receptor fluid. Dynamic conditions: To assess the release of TA under recreated conditions of the mouth, a Hanson Research SR8 SRII Flask Dissolution Test Station was filled with phosphate buffered saline adjusted to a pH 6.5 as receptor fluid and the experimental conditions were adjusted to 37 ◦C. The sublingual (Figure2A) and buccal (Figure2B) mucosas ( n = 5) with ≈2 g of the formulation applied into each were immersed in the dissolution station under constant stirring for a period of 6 to 8 h, and samples (300 µL) were collected at known intervals with the micropipette MODEL 5000 (Gilson, Middleton, WI, USA), and stored frozen in HPLC vials until analyzed. The application surface of the formulations was approximately 10 and 12 cm2 for the sublingual and buccal mucosas, respectively.

2.6. Permeation and Retention Studies Static conditions: Ex vivo permeation and retention studies were conducted in Franz- type diffusion cells with a setup that is similar to that of release studies, but replacing the membrane for either sublingual (Figure2C) or buccal (Figure2D) porcine mucosa. The receptor ﬂuid consisted of Transcutol® P, the surface was 0.64 cm2. The amount of formulation applied into the donor compartment was 100 mg. The mucosa samples were frozen at −20 ◦C and longitudinally cut into 700 µm slabs with a dermatome GA 630 (Figure2B). Mucous membrane samples were placed between the receptor and donor compartments with the proximal side in contact with the receptor medium and the mucous side in contact with the donor chamber [17]. The ﬂux values of TA (µg/h) across mucous membranes were estimated through the slope of the cumulative amount of TA permeated versus time for each formulation. Moreover, the retention of TA was estimated in the mucous membranes after the permeation experiment. Pharmaceutics 2021, 13, 1080 6 of 15 Pharmaceutics 2021, 13, x FOR PEER REVIEW 6 of 18

Figure 2. Dissolution equipment showing porcine sublingual ( A) and buccal ( B) mucosas for release and retention of TA studies in recreated dynamicdynamic conditionsconditions ofof thethe mouth. (C) Porcine buccal and (D) sublingual mucosas used for permeation experiments in static conditions. experiments in static conditions.

2.6. PermeationAt the end and of Retention the permeation Studies study, the amount of drug retained in the mucosa (µg/cmStatic2) wasconditions: extracted Ex vivo by ultrasound-assisted permeation and retention extraction. studies The were biological conducted samples in Franz- were typecleaned diffusion and washed cells with with agauze setup soakedthat is simila in a 0.05%r to that solution of release of dodecyl studies, sulfate but replacing and distilled the water.membrane The for mucosa either epitheliumsublingual was(Figure removed 2C) or and buccal immersed (Figure in2D) 4 andporcine 2 mL mucosa. of acetoni- The receptortrile:transcutol fluid consisted (50:50 v:v )of for Transcutol the sublingual® P, the and surface buccal was mucosa, 0.64 cm respectively,2. The amount and of sonicated formulation(100 KHz) applied for 30into min. the Thedonor samples compartment were filtered was 100 and mg. quantified by HPLC. TheDynamic mucosa conditions: samplesThe were amount frozen of at TA −20 retained °C and within longitudinally buccal and cut sublingual into 700 µm mucosas slabs withafter a 8 dermatome h application GA in 630 dynamic (Figure conditions 2B). Mucous was membrane obtained with samples a similar were procedure placed between to that theof static receptor conditions. and donor The compartments receptor fluid with consisted the proximal of Phosphate side in buffered contact salinewith the adjusted receptor to 2 2 mediumpH 6.5, the and surface the mucous was 10 side cm inand contact 12 cm withfor sublingualthe donor chamber and buccal [17]. mucosa, The flux respectively. values of TAThe (µg/h) amount across of formulation mucous membranes applied into were mucosas estimated was through 2 g. the slope of the cumulative amount of TA permeated versus time for each formulation. Moreover, the retention of TA 2.7. Statistical Analysis was estimated in the mucous membranes after the permeation experiment. AtNon-parametric the end of the Student’s permeationt-tests study, and ANOVAthe amount test of were drug performed retained usingin the amucosa Graph- (µg/cmPad Prism2) was 3 (GraphPad extracted by Software ultrasound-assisted Inc., San Diego, ex CA,traction. USA) The for biological comparing samples the different were cleanedformulations. and washed with gauze soaked in a 0.05% solution of dodecyl sulfate and distilled water. The mucosa epithelium was removed and immersed in 4 and 2 mL of acetoni- 2.8. Histological Observations trile:transcutol (50:50 v:v) for the sublingual and buccal mucosa, respectively, and soni- catedThe (100 integrity KHz) for of 30 the min. sublingual The sample ands were buccal filtered mucosas and wasquantified studied by under HPLC. brightfield microscopy.Dynamic Eightconditions: hours The after amount application of TA retained of 0.05% within TA + LIDObuccal formulation, and sublingual tissues muco- in sascontact after with8 h application the formulation in dynamic were frozenconditions and was kept obtained until analyzed. with a similar Tissues procedure were fixed to overnight by immersion in 4% paraformaldehyde in phosphate buffer 20 mM pH 7.4, and

Pharmaceutics 2021, 13, x FOR PEER REVIEW 7 of 18

that of static conditions. The receptor fluid consisted of Phosphate buffered saline adjusted to pH 6.5, the surface was 10 cm2 and 12 cm2 for sublingual and buccal mucosa, respectively. The amount of formulation applied into mucosas was 2 g.

2.7. Statistical Analysis Non-parametric Student’s t-tests and ANOVA test were performed using a GraphPad Prism 3 (GraphPad Software Inc., San Diego, CA, USA) for comparing the different formulations.

2.8. Histological Observations The integrity of the sublingual and buccal mucosas was studied under brightfield microscopy. Eight hours after application of 0.05% TA + LIDO formulation, tissues in contact with the formulation were frozen and kept until analyzed. Tissues were fixed overnight by immersion in 4% paraformaldehyde in phosphate buffer 20 mM pH 7.4, and fur- Pharmaceutics 2021, 13, 1080 7 of 15 ther processed for paraffin embedding. Vertical histological sections were obtained, stained with hematoxylin and eosin and mounted under a cover slip. Samples were observed using a Leica DMD 108 optical microscope (Spain). further processed for paraffin embedding. Vertical histological sections were obtained, stained3. Results with and hematoxylin Discussion and eosin and mounted under a cover slip. Samples were observed using a Leica DMD 108 optical microscope (Spain). 3.1. Composition of the Formulations 3. ResultsFour anddifferent Discussion formulations (Table 1) containing TA for topical administration were 3.1.developed Composition so as of to the evaluate Formulations the influence of TA concentration and the presence or absence, and Fourinfluence different of LIDO formulations on the mechanical (Table1) containing and biopharmaceutical TA for topical administration properties wereof the formu- developedlation. For so instance, as to evaluate TA was the prepared influenceof at TA 0.05% concentration and 0.1%and (w/v the), and presence LIDO or was absence, tested at 2% and influence of LIDO on the mechanical and biopharmaceutical properties of the formula- concentration. Liquid paraffin and Orabase® were included as excipients for promoting tion. For instance, TA was prepared at 0.05% and 0.1% (w/v), and LIDO was tested at 2% concentration.the formation Liquid of a homogeneous paraffin and Orabase and consistent® were included hydrophobic as excipients film for upon promoting application the in or- formationder to maximize of a homogeneous the retention and consistentof the API hydrophobic in the area filmof application. upon application in order to maximize the retention of the API in the area of application. 3.2. Rheological Properties 3.2. Rheological Properties The rheological characteristics of the formulations play an important role in physical stability,The rheological and are a characteristicscrucial attribute of the for formulations the development play an of important topical drug role in products physical [18]. To stability, and are a crucial attribute for the development of topical drug products [18]. To find out the mechanical properties of the formulations, rheology studies were performed, find out the mechanical properties of the formulations, rheology studies were performed, and theythey revealed revealed a pseudoplastica pseudoplastic and and apparent apparent thixotropic thixotropic behavior behavior in all the in formula-all the formulations (Figure(Figure3), 3), both both being being desirable desirable characteristics characteristics for topical for topical application, application, allowing allowing the the formation of of a a consistent consistent film film covering covering the applicationthe application area that area facilitates that facilitates the diffusion the diffusion of of the drugdrug through through the the matrix matrix [19 –[19–22].22].

Figure 3.3. ViscosityViscosity curve curve (blue (blue line) line) and and ﬂow flow curve curve (red (red line) line) of the of 4the formulations 4 formulations under under study. study. (a) ◦ (0.05%a) 0.05% TA; TA; (b ()b 0.05%) 0.05% TA TA + LIDO; LIDO; ((cc)) 0.1% 0.1% TA; TA; (d ()d 0.1%) 0.1% TA TA + LIDO, + LIDO, at 25 at± 250.1 ± 0.1C. °C.

Table2 shows that the viscosity is similar in all formulations, except for 0.1% TA, in which it is slightly lower. On the basis of the precision of the rotational test for these kinds of highly viscous products, the lower viscosity of 0.1% TA is difﬁcult to explain, since it was expected to be in the same range. This is presumably due to the form of disturbance of the microstructure of the gel under the effect of shear. The viscosity of our formulations was approximately eight times higher than that of a reference formula [22], because the Transcutol used as a cosolvent in the reference was not incorporated in these formulations. This fact implies that it will not confer the same adhesive force, but we can explain or predict that, with the high viscosity values found, the difference in the formula 0.1% TA will hardly translate into less adherence. Pharmaceutics 2021, 13, 1080 8 of 15

Table 2. Rheological evaluation of the different formulations at 100 s−1. Values represent Means ± SD (n = 2).

Formulations Viscosity (mPa·s) at 100 s−1 0.05% TA 3890.0 ± 39.8 0.05% TA + LIDO 3833.0 ± 27.9 0.1% TA 3662.0 ± 42.3 0.1% TA + LIDO 3819.0 ± 39.8

Moreover, the four formulations are shear thinning systems with the same ﬂow behavior adjusted by Cross model (Equation (5)), for both the ascendant and descendant sections: . η∞ + ( η0 − η∞) Cross equation: τ = g· . n (5) 1 + g g0 . where τ is the shear stress (Pa); g is the shear rate (1/s); G0 is the zero-shear rate (1/s); η∞ is the inﬁnite shear rate viscosity; η0 is the zero-shear rate viscosity (Pa·s); n is a dimensionless rate constant.

3.3. Release Studies To ensure that the TA can be released from the matrix of the pharmaceutical form and can reach the biophase, drug release studies were performed using Franz-type diffusion cells (static conditions). For each formulation, the cumulative released amount of TA (µg) Pharmaceutics 2021, 13, x FOR PEER REVIEW 11 of 18 versus time (h) was obtained in triplicate (Figure4), all of them following a Boltzmann sigmoidal model in accordance with the coefﬁcients of determination (r2) ≥ 0.98.

1500 g) μ 1250

1000

750

500 0.05% TA 0.05% TA + LIDO 250 0.1% TA 0.1% TA + LIDO Cumulative TA released ( released TA Cumulative 0 0 20 40 60 80 100 time (h)

FigureFigure 4. 4. CumulativeCumulative amount amount (µg) (µg) of TATA releasedreleased versus versus time time (h) (h) from from the the four four different different formulations. formulations.Values Values represent represent Mean Mean± SD ± ( nSD= 3).(n = 3).

AnotherTA is released question to ais different that in order extent to depending more closely on the estimate formulation, the amount after 76.2 of TA h being that is re- releasedleased 1154.33 from theµg semisolid (0.08%) from formulation TA 0.1% +to LIDO, the saliva 609.11 uponµg (0.04%) application from onto TA 0.1%, either 546.33 buccalµg µ or(0.04%) sublingual from mucosa, TA 0.05% and + LIDO, theref andore 190.78estimateg the (0.01%) possible from systemic TA 0.05% effect formulation. due to oral There- in- fore, the presence of LIDO promotes a higher amount of TA released. These results might gestion of the corticoid, the conditions inside the mouth® were recreated in terms of solvent,suggest pH, that temperature, either the higherand constant (2%) amount movement of Orabase of the tonguein the formulations(dynamic conditions). without LIDO For might account for a higher retention of TA in the formulation, or that the ionic nature of this purpose, a Dissolution Test Station containing phosphate buffered saline at 37 °C pH LIDO, which can undergo a faster solvation and diffusion in the medium, could indirectly 6.5 as receptor fluid. The mucosas (n = 5) with each formulation applied onto it (≈2 g) were promote a faster release of TA. The overall low release in static conditions observed implies submerged into the phosphate buffered saline and they were kept under constant stirring that it prevents the active ingredient to be released to the saliva and be swallowed. Instead, for 6–8 h. Samples from the receptor fluid were taken at certain intervals, and the amount the main purpose of the formulation is to promote the penetration of TA in the tissue to act of TA released (µg) was plotted as a function of time (h) (Figure 5). For both sublingual in situ. and buccal mucosas, the mathematical model that best fits the TA released is the hyper- Another question is that in order to more closely estimate the amount of TA that is bola according to the coefficients of determination (r2) ≥ 0.99. released from the semisolid formulation to the saliva upon application onto either buccal or sublingual mucosa, and therefore estimate the possible systemic effect due to oral ingestion 150 ABSublingual mucosa40 Buccal mucosa

30 100

50 0.05% TA 10 0.05% TA + LIDO 0.1% TA 0 0 0.1% TA + LIDO

Cumulative TA (μg) released Cumulative TA 0 2 4 6 0 2 4 6 8

Time (h)

Figure 5. Cumulative amount of TA released (µg) to the phosphate buffered saline in dynamic conditions over time (h) when applied onto the (A) sublingual or (B) buccal mucosa. Values represent Mean ± SD (n = 5).

In sublingual mucosa, the amount of TA released after 6 h in dynamic conditions was 50.2 µg (0.003%) for 0.05% TA formulation, 102.5 µg (0.005%) for 0.05% TA + LIDO, 82.1 µg (0.004%) for 0.1% TA, and 123.4 µg (0.006%) for 0.1% TA + LIDO. In buccal mucosa, the amount of TA released after 6 h in dynamic conditions was 28.2 µg (0.001%) for 0.05% TA formulation, 22.5 µg (0.001%) for 0.05% TA + LIDO, 29.9 µg (0.001%) for 0.1% TA, and 26.2 µg (0.001%) for 0.1% TA + LIDO. In general, a lower amount of TA released is observed in dynamic conditions in comparison to static conditions, which could be explained by the low solubility of TA in aqueous media such as the saliva. Moreover, in both tissues a higher concentration of TA in the formulation promotes a higher release of TA.

Pharmaceutics 2021, 13, x FOR PEER REVIEW 11 of 18

1500 g) μ 1250

1000

750

500 0.05% TA 0.05% TA + LIDO 250 0.1% TA 0.1% TA + LIDO Cumulative TA released ( released TA Cumulative 0 0 20 40 60 80 100 time (h)

Figure 4. Cumulative amount (µg) of TA released versus time (h) from the four different formulations. Values represent Mean ± SD (n = 3).

Pharmaceutics 2021, 13, 1080 Another question is that in order to more closely estimate the amount of TA that9 of 15is released from the semisolid formulation to the saliva upon application onto either buccal or sublingual mucosa, and therefore estimate the possible systemic effect due to oral ingestion of the corticoid, the conditions inside the mouth were recreated in terms of sol- of the corticoid, the conditions inside the mouth were recreated in terms of solvent, pH, vent, pH, temperature, and constant movement of the tongue (dynamic conditions). For temperature, and constant movement of the tongue (dynamic conditions). For this purpose, this purpose, a Dissolution Test Station containing phosphate buffered saline at 37 °C pH a Dissolution Test Station containing phosphate buffered saline at 37 ◦C pH 6.5 as receptor 6.5fluid. as receptor The mucosas fluid. (Then = 5)mucosas with each (n =formulation 5) with each appliedformulation onto applied it (≈2 g) onto were it submerged(≈2 g) were submergedinto the phosphate into the phosphate buffered saline buffered and saline they were and they kept were under kept constant under stirringconstant for stirring 6–8 h. forSamples 6–8 h. fromSamples the from receptor the receptor fluid were fluid taken were at taken certain at certain intervals, intervals, and the and amount the amount of TA ofreleased TA released (µg) was (µg) plotted was plotted as a function as a function of time of (h)time (Figure (h) (Figure5). For 5). both For sublingualboth sublingual and andbuccal buccal mucosas, mucosas, the mathematicalthe mathematical model model that that best best fits fits the the TA TA released released is the is the hyperbola hyper- 2 bolaaccording according to the to coefficients the coefficients of determination of determination (r2) ≥ (r0.99.) ≥ 0.99.

150 ABSublingual mucosa40 Buccal mucosa

30 100

50 0.05% TA 10 0.05% TA + LIDO 0.1% TA 0 0 0.1% TA + LIDO

Cumulative TA (μg) released Cumulative TA 0 2 4 6 0 2 4 6 8

Time (h)

µ FigureFigure 5. 5. CumulativeCumulative amount amount of ofTA TA released released (µg) ( tog) the to thephosphate phosphate buffered buffered saline saline in dynamic in dynamic conditionsconditions over over time time (h) (h)when when applied applied onto onto the the (A ()A sublingual) sublingual or or (B (B) buccal) buccal mucosa. mucosa. Values Values represent represent MeanMean ±± SDSD (n (n == 5). 5).

InIn sublingual sublingual mucosa, mucosa, the the amount amount of of TA TA released released after 6 h h in in dynamic dynamic conditions conditions was was 50.250.2 µgµg (0.003%) forfor 0.05%0.05% TATA formulation, formulation, 102.5 102.5µ gµg (0.005%) (0.005%) for for 0.05% 0.05% TA TA + LIDO, + LIDO, 82.1 82.1µg µg(0.004%) (0.004%) for for 0.1% 0.1% TA, TA, and and 123.4 123.4µg (0.006%)µg (0.006%) for 0.1%for 0.1% TA +TA LIDO. + LIDO. In buccal In buccal mucosa, mucosa, the µ theamount amount of TA of TA released released after after 6 h 6 in h dynamic in dynamic conditions conditions was was 28.2 28.2g (0.001%)µg (0.001%) for for 0.05% 0.05% TA µ µ TAformulation, formulation, 22.5 22.5g µg (0.001%) (0.001%) for for 0.05% 0.05% TA TA + + LIDO, LIDO, 29.9 29.9 µgg(0.001%) (0.001%) forfor 0.1%0.1% TA, and µ 26.226.2 µgg (0.001%)(0.001%) forfor 0.1% 0.1% TA TA + LIDO.+ LIDO. In In general, general, a lower a lower amount amount of TA of releasedTA released is observed is ob- servedin dynamic in dynamic conditions conditions in comparison in comparison to static conditions, to static conditions, which could which be explained could be by ex- the low solubility of TA in aqueous media such as the saliva. Moreover, in both tissues a higher plained by the low solubility of TA in aqueous media such as the saliva. Moreover, in both concentration of TA in the formulation promotes a higher release of TA. Nonetheless, the tissues a higher concentration of TA in the formulation promotes a higher release of TA. presence of LIDO has a different inﬂuence depending on the tissue. In sublingual mucosa, the presence of LIDO promotes a signiﬁcantly higher TA release, similar to observations in release experiments under static conditions, whereas in buccal mucosa, the observed

differences are not signiﬁcant with the presence of LIDO. According to these results, the formulations that could imply a lower amount of TA released into the saliva and therefore ingested, for either sublingual or buccal application, are those with 0.05% TA. However, the amount of TA released to the receptor ﬂuid (<125 µg) does not in either case represent a dose that could induce systemic effects upon ingestion, as oral doses of triamcinolone in adults normally range from 4 to 50 mg per day [23,24].

3.4. Permeation and Retention Studies Ex vivo permeation studies of the four different formulations (n = 5) were carried out in static conditions to test the ability of TA to permeate the buccal mucosa and it being retained within the tissue upon application. The experiment setup was similar to the release studies in static conditions using Franz cells, but replacing the membrane for either the porcine sublingual or buccal mucosa. The amount of TA (µg) permeated across either mucous tissue was plotted versus time (h), and a linear least squares regression was performed (Figure6). Pharmaceutics 2021, 13, x FOR PEER REVIEW 12 of 18

Nonetheless, the presence of LIDO has a different influence depending on the tissue. In sublingual mucosa, the presence of LIDO promotes a significantly higher TA release, similar to observations in release experiments under static conditions, whereas in buccal mucosa, the observed differences are not significant with the presence of LIDO. According to these results, the formulations that could imply a lower amount of TA released into the saliva and therefore ingested, for either sublingual or buccal application, are those with 0.05% TA. However, the amount of TA released to the receptor fluid (<125 µg) does not in either case represent a dose that could induce systemic effects upon ingestion, as oral doses of triamcinolone in adults normally range from 4 to 50 mg per day [23,24].

3.4. Permeation and Retention Studies Ex vivo permeation studies of the four different formulations (n = 5) were carried out in static conditions to test the ability of TA to permeate the buccal mucosa and it being retained within the tissue upon application. The experiment setup was similar to the release studies in static conditions using Franz cells, but replacing the membrane for either the porcine sublingual or buccal mucosa. The amount of TA (µg) permeated across either mucous tissue was plotted versus time (h), and a linear least squares regression was performed (Figure 6). The results show that TA can permeate buccal mucosa at approximately 9.2 µg/h re- Pharmaceutics 2021, 13, 1080 10 of 15 gardless of the TA concentration or the presence or absence of LIDO (Table 3), as no significant differences were observed (>0.05) according to Student’s t-tests.

Figure 6.6. BuccalBuccal permeationpermeation kinetics kinetics of of TA TA for for the the different different formulations. formulations. (a) ( 0.05%a) 0.05% TA. TA. (b) 0.05%(b) 0.05% TA +TA LIDO. + LIDO. (c) 0.1% (c) 0.1% TA. TA. (d) 0.1% (d) 0.1% TA +LIDO. TA +LIDO. Values Values represent represent Means Means± SD ± (SDn = ( 5).n = 5).

TableThe 3. Amount results of show TA permeated that TA canin buccal permeate mucosa buccal per hour mucosa (flow). at Values approximately represent Means 9.2 µ g/h± SD regardless(n = 5). No significant of the TA differences concentration were observed or the presence (p < 0.05). or absence of LIDO (Table3), as no significant differences were observed (>0.05) according to Student’s t-tests. Formulations Flow (µg/h) Table 3. Amount of0.05% TA permeated TA in buccal mucosa per hour (flow). 9.24 Values ± 0.03 represent Means ± SD (n = 5). No significant0.05% differencesTA + LIDO were observed (p < 0.05). 9.19 ± 0.06 0.1% TA 9.24 ± 0.03 Formulations Flow (µg/h) 0.05% TA 9.24 ± 0.03 0.05% TA + LIDO 9.19 ± 0.06 0.1% TA 9.24 ± 0.03 0.1% TA + LIDO 9.22 ± 0.02

Considering a possible systemic effect after application of the formulations, Argenti D et al. [23] determined the multiple-dose pharmacokinetics, pharmacodynamics, and tolerability of a newly developed formulation of inhaled TA. They found that the maximum serum concentration (Cmax) at the steady state was 1.83 ng/mL. In addition, they found that TA treatment reduced the basal serum cortisol concentrations by 20% relative to the placebo treatment. For this reason, the concentrations at a steady state (Css) for each formulation were calculated according to the permeation parameters obtained and the reported pharmacoki- netic parameters of TA. For instance, upon treatment with these formulations, Css values would oscillate between 1.54 and 1.57 ng/mL, values that are 15% below those reported (1.83 ng/mL), with all formulations having similar systemic safety proﬁles. The amount of TA retained within the buccal mucosa was calculated by extracting the drug from the tissue after permeation experiments with the four different formulations (Figure7), ﬁnding that application of 0.05% TA leads to 9.2 ± 2.4 mg TA retained per gram and square centimeter of tissue, whereas application of 0.05% TA + LIDO leads to 14.8 ± 2.7 mg·g−1·cm−2, representing a 60% increase. Similarly, the application of 0.1% TA results in 8.0 ± 1.4 mg·g−1·cm−2 while 0.1% TA + LIDO results in 15.6 ± 2.2 mg·g−1·cm−2 Pharmaceutics 2021, 13, x FOR PEER REVIEW 13 of 18

0.1% TA + LIDO 9.22 ± 0.02

Considering a possible systemic effect after application of the formulations, Argenti D et al. [23] determined the multiple-dose pharmacokinetics, pharmacodynamics, and tolerability of a newly developed formulation of inhaled TA. They found that the maximum serum concentration (Cmax) at the steady state was 1.83 ng/mL. In addition, they found that TA treatment reduced the basal serum cortisol concentrations by 20% relative to the placebo treatment. For this reason, the concentrations at a steady state (Css) for each formulation were calculated according to the permeation parameters obtained and the reported pharmaco- kinetic parameters of TA. For instance, upon treatment with these formulations, Css values would oscillate between 1.54 and 1.57 ng/mL, values that are 15% below those reported (1.83 ng/mL), with all formulations having similar systemic safety profiles. The amount of TA retained within the buccal mucosa was calculated by extracting the drug from the tissue after permeation experiments with the four different formulations (Figure 7), finding that application of 0.05% TA leads to 9.2 ± 2.4 mg TA retained per gram and square centimeter of tissue, whereas application of 0.05% TA + LIDO leads to Pharmaceutics 2021, 13, 1080 11 of 15 14.8 ± 2.7 mg·g−1·cm−2, representing a 60% increase. Similarly, the application of 0.1% TA results in 8.0 ± 1.4 mg·g−1·cm−2 while 0.1% TA + LIDO results in 15.6 ± 2.2 mg·g−1·cm−2 representing a 95% increase in retained TA. Student t-tests confirmed that there is a signifi- cantrepresenting increase a(p 95% < 0.01) increase in the in amount retained of TA. TA Student that can t-tests be retained conﬁrmed in the that tissue there so is aas sig- to performniﬁcant increaseits therapeutic (p < 0.01) activity in the when amount the form of TAulations that can include be retained LIDO, suggesting in the tissue this so drug as to alsoperform behaves its therapeutic as a penetration activity enhancer. when the formulations include LIDO, suggesting this drug also behaves as a penetration enhancer.

) 20 -2 0.05% TA ** ***

cm 0.05% TA + LIDO -1 15 0.1% TA 0.1% TA + LIDO

5 Amount of TA retained in buccal mucosa (mg g (mg mucosa buccal 0 A A O O

Figure 7. Amount of TA retained per gram and square centimeter of buccal mucosa, 6 h after Figureapplication 7. Amount of each of formulationTA retained (0.05%per gram TA and or 0.05%square TA centimeter + LIDO, of or buccal 0.1% TA mucosa, or 0.1% 6 h TA after + LIDO).appli- cationValues of represent each formulation Mean ± SEM (0.05% (n =TA 5). or Statistical 0.05% TA differences + LIDO, or ** 0.1% (p < 0.01),TA or*** 0.1% (p

(µg)permeated TA 0.1% TA 0.1% TA + LIDO 70 70 Cummulative amount of amount Cummulative 60 60 50 50 40 40 0.05% TA 30 30 0.05% TA + LIDO 20 20 0.1% TA 10 10 0.1% TA + LIDO 0 0 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 Time (h)

Figure 8. SublingualSublingual permeation permeation kinetics kinetics of of TA TA for the different formulations: ( a)) 0.05% 0.05% TA, TA, ( b) 0.05% TA + LIDO, LIDO, ( (cc)) 0.1% 0.1% TA, (d)) 0.1%0.1% TATA +LIDO.+LIDO. Values representrepresent MeansMeans ±± SD ( n = 5). 5).

SublingualSublingual permeation permeation also also shows shows a a linear linear behavior, behavior, with fluxes fluxes slightly higher than thosethose observed observed in in buccal permeation, rangingranging between 10.1 µµg/hg/h and and 12.4 µg/h,µg/h, as as ob- ob- servedserved in in Table Table 44.. Student’sStudent’s tt-tests-tests were were performed in in order order to evaluate the influence influence of thethe presence presence of of lidocaine lidocaine hydrochloride hydrochloride in in the the formulations, formulations, revealing revealing significantly significantly higher higher fluxes both at 0.05% TA concentration (p < 0.001) and 0.1% TA concentrations (p < 0.05), and suggesting that LIDO behaves as a permeation enhancer in sublingual mucosa, through mechanisms of action that could include the reversible loss of integrity of the skin and mucosa barriers, the increase in the partitioning of the drug in the tissue, or the increase in the solubility of the drug [24,25]. The effects of a permeation enhancer may differ when combined with one or another drug [26].

Table 4. Amount of TA permeated in sublingual tissue per hour (flow). Values represent Mean ± SD (n = 5). Significant differences versus formulation with the same concentration of TA but without LIDO: * (p < 0.05), *** (p < 0.001).

Formulations Flow (µg/h) 0.05% TA 10.10 ± 0.12 0.05% TA + LIDO 12.40 ± 0.42 *** 0.1% TA 10.74 ± 0.20 0.1% TA + LIDO 11.04 ± 0.14 *

Pharmaceutics 2021, 13, 1080 12 of 15

ﬂuxes both at 0.05% TA concentration (p < 0.001) and 0.1% TA concentrations (p < 0.05), and suggesting that LIDO behaves as a permeation enhancer in sublingual mucosa, through mechanisms of action that could include the reversible loss of integrity of the skin and mucosa barriers, the increase in the partitioning of the drug in the tissue, or the increase in the solubility of the drug [24,25]. The effects of a permeation enhancer may differ when combined with one or another drug [26].

Table 4. Amount of TA permeated in sublingual tissue per hour (ﬂow). Values represent Mean ± SD (n = 5). Signiﬁcant differences versus formulation with the same concentration of TA but without LIDO: * (p < 0.05), *** (p < 0.001).

Formulations Flow (µg/h) 0.05% TA 10.10 ± 0.12 0.05% TA + LIDO 12.40 ± 0.42 *** 0.1% TA 10.74 ± 0.20 0.1% TA + LIDO 11.04 ± 0.14 *

For this reason, the concentrations at steady state (Css) for each formulation were also calculated and resulted in a range of 1.67–2.06 ng/mL, similar to the values reported (1.83 nm/mL) [23], and which could indicate some possible systemic effects in the semisolid formulations of TA. Corticosteroids can affect keratinocytes and prevent the secretion of collagen and hyaluronic acid by ﬁbroblasts in the dermis, interfering with cell proliferation and with long- term glucocorticoid usage. As a result, skin thinning ensues. Topical administration could produce local side effects, including skin atrophy, ecchymosis, erosions, striae, delayed wound healing, purpura, easy bruising, acne, hirsutism and hair loss [27]. Therefore, it is important to point out that these semisolid formulations should be used for short periods only, and in accordance with the medical prescription. The amount of TA retained in sublingual or buccal mucosa 8 h after application of the different formulations was also studied in dynamic conditions. In sublingual mucosa, both a higher TA concentration in the formulation and the presence of LIDO promoted a higher retention of TA within the tissue (Figure9A), a behavior also observed under static conditions. In contrast, in buccal mucosa (Figure9B), a higher concentration of TA promotes a higher amount of TA retained within the tissue, but the presence of LIDO decreases the amount of TA retained. Overall, a higher amount of TA is retained in the mucosas under static conditions (Franz cells experiments) than under dynamic conditions (dissolution equipment), which can be explained by the higher and prolonged adherence of the formulation to the mucosa in dry, static conditions, permitting the partitioning and further diffusion towards the tissue.

3.5. Histological Observations Histological observations were performed with sublingual and buccal mucosa tissues after treatment with 0.05% TA + LIDO formulation. Untreated tissues were processed in parallel as a control. Vertical histological sections were obtained and treated with hematoxylin (this was to stain the nucleus) and eosin (this was to stain the cytoplasm and extracellular matrix), and were observed under brightfield microscopy (Figure 10). For both buccal and sublingual mucosas, no important differences between the untreated samples and those with the formulations were observed. The stratified flat keratinized epithelium (outermost part of the tissue) is intact after being in contact with the formulation, similar to untreated tissues. Additionally, the thickness of the dermal papilla (between the flat keratinized epithelium and the basal layer) is similar in treated and untreated tissues and no alteration of cellular structures is observed. These observations indicate that the application of the formulation does not induce any local tissue damage. Pharmaceutics 2021, 13, x FOR PEER REVIEW 15 of 18

wound healing, purpura, easy bruising, acne, hirsutism and hair loss [27]. Therefore, it is important to point out that these semisolid formulations should be used for short periods only, and in accordance with the medical prescription. The amount of TA retained in sublingual or buccal mucosa 8 h after application of the different formulations was also studied in dynamic conditions. In sublingual mucosa, both a higher TA concentration in the formulation and the presence of LIDO promoted a higher retention of TA within the tissue (Figure 9A), a behavior also observed under static conditions. In contrast, in buccal mucosa (Figure 9B), a higher concentration of TA promotes a higher amount of TA retained within the tissue, but the presence of LIDO decreases the amount of TA retained. Overall, a higher amount of TA is retained in the mucosas under static conditions (Franz cells experiments) than under dynamic conditions (dissolution equipment), which can be explained by the higher and prolonged adherence Pharmaceutics 2021, 13, 1080 of the formulation to the mucosa in dry, static conditions, permitting the partitioning13 ofand 15 further diffusion towards the tissue.

ABSublingual mucosa Buccal mucosa

800 1000 *** *** ** *** *** 800 *** *** 600 *** ) -2 600 *** 400 *** 400 (μg cm *** 0.05% TA 200 0.05% TA + LIDO 200 0.1% TA Amount of TA retained of TA Amount 0.1% TA + LIDO Pharmaceutics 2021, 13, x FOR PEER0 REVIEW 0 16 of 18

A A FigureFigure 9.9. AmountAmount ofof TATA retainedretained inin ((AA)) sublingualsublingual oror ((BB)) buccalbuccal mucosamucosa 88 hh afterafter applicationapplication ofof thethe formulationformulation inin dynamicdynamic conditions.conditions. ValuesValues representrepresent Means Means± ± SD (n = 5). Significant Signiﬁcant differences: ** ( p < 0.01) 0.01) *** *** ( (pp << 0.001). 0.001).

3.5. Histological Observations Histological observations were performed with sublingual and buccal mucosa tissues after treatment with 0.05% TA + LIDO formulation. Untreated tissues were processed in parallel as a control. Vertical histological sections were obtained and treated with hematoxylin (this was to stain the nucleus) and eosin (this was to stain the cytoplasm and extracellular matrix), and were observed under brightfield microscopy (Figure 10). For both buccal and sublingual mucosas, no important differences between the untreated samples and those with the formulations were observed. The stratified flat keratinized epithelium (outermost part of the tissue) is intact after being in contact with the formulation, similar to untreated tissues. Additionally, the thickness of the dermal papilla (between the flat keratinized epithelium and the basal layer) is similar in treated and untreated tissues and no alteration of cellular structures is observed. These observations indicate that the application of the formulation does not induce any local tissue damage.

Figure 10. Histological observations of buccal (top) and sublingual (bottom) mucosas after appli- Figurecation 10. Histological of the formulation observations 0.05% of TA buccal + LIDO (top ()left and). sublingual Untreated tissues(bottom were) mucosas processed after inapplica- parallel as a tion of the formulation 0.05% TA + LIDO (left). Untreated tissues were processed in parallel as a control (right). (a) Stratified flat keratinized epithelium, (b) basal layer (c) collagen fibers. Scalebar control (right). (a) Stratified flat keratinized epithelium, (b) basal layer (c) collagen fibers. Scalebar represents 100 µm. represents 100 µm. 4. Conclusions 4. Conclusions Formulations containing 0.05% or 0.1% TA, and in the presence or absence of 2% FormulationsLIDO, were designedcontaining and 0.05% developed or 0.1% for TA, buccal and in application the presence as potential or absence treatments of 2% for LIDO,important were designed inflammatory and developed processes for in bucca the buccall application mucosa, as suchpotential as those treatments occurring for upon importantbuccal inflammatory cancer radiotherapy, processes lichen in the planus, buccal and mucosa, canker such sores, as amongthose occurring others. The upon effect of buccalTA cancer concentration radiotherapy, and the lichen presence planus, or and absence canker and sores, therefore among the others. influence The of effect LIDO of on the TA concentrationmechanical orand biopharmaceutical the presence or absence properties and of therefore the formulations the influence were of extensively LIDO on the studied. mechanicalThe four or biopharmaceutical different formulations properties showed of athe pseudoplastic formulations and were thixotropic extensively behavior, studied. ideal The fourfor topicaldifferent application. formulations In static showed conditions a pseudoplastic using Franz and cells, thixotropic TA can behavior, be released ideal from the for topicalformulations application. following In static a Boltz conditions Sigmoidal using behavior, Franz cells, and itTA was can found be released that that from the presencethe formulationsof LIDO following promotes a between Boltz Sigmoidal 107% and behavior, 212% more and TA it was being found released that afterthat 92the h pres- (p < 0.05), ence of LIDO promotes between 107% and 212% more TA being released after 92 h (p < 0.05), and that the formulation of 0.05% TA + LIDO showed the highest amount of TA released (1330 µg). Moreover, under the recreated dynamic conditions of the mouth (constant movement, presence of phosphate buffered saline, temperature), the formulations applied onto buccal or sublingual mucosa can release small (<150 µg) amounts of TA into the phosphate-buffered saline (subject to being ingested) due to the poor solubility of TA in aqueous media, which is much below the normal oral doses of TA (4–50 mg per day), indicating no risk of systemic effects due to oral ingestion. Additionally, permeation studies using Franz cells (static conditions) showed that TA can successfully permeate buccal mucosa at rates ranging between 9.19 and 9.24 µg/h, with no apparent influence from the concentration of TA or the presence of LIDO. After application, TA is successfully retained beneath the buccal mucosa and then performs its anti-inflammatory action, with it being found that the presence of LIDO can increase the

Pharmaceutics 2021, 13, 1080 14 of 15

and that the formulation of 0.05% TA + LIDO showed the highest amount of TA released (1330 µg). Moreover, under the recreated dynamic conditions of the mouth (constant movement, presence of phosphate buffered saline, temperature), the formulations applied onto buccal or sublingual mucosa can release small (<150 µg) amounts of TA into the phosphate- buffered saline (subject to being ingested) due to the poor solubility of TA in aqueous media, which is much below the normal oral doses of TA (4–50 mg per day), indicating no risk of systemic effects due to oral ingestion. Additionally, permeation studies using Franz cells (static conditions) showed that TA can successfully permeate buccal mucosa at rates ranging between 9.19 and 9.24 µg/h, with no apparent influence from the concentration of TA or the presence of LIDO. After application, TA is successfully retained beneath the buccal mucosa and then performs its anti-inflammatory action, with it being found that the presence of LIDO can increase the amount of TA in the tissue by 60% or 95% (p < 0.01). In sublingual mucosa, TA can permeate at fluxes ranging from 10.1 to 12.4 µg/h, and the presence of LIDO enhances the permeation through the tissue. In the recreated dynamic conditions of the mouth, TA can be successfully retained as well in the buccal mucosa (179 to 800 µg/cm2), observing that the presence of LIDO decreases the amount of TA retained. Provided there is a continuous contact of the tongue with the zone of application, TA could also penetrate into the sublingual mucosa, especially if the formulation contains LIDO, representing a possible anti-inflammatory and anesthetic effect both in the buccal mucosa as well as in the region of the tongue that is in contact with the formulation.

Author Contributions: Conception of the research: A.C.C.C.; Design of the experiments: A.C.C.C. and L.H.B.; Investigation: M.M.V., A.M.L., L.H.B. and D.B.T.; Data processing: M.M.V., A.M.L., L.H.B., P.C.G., D.B.T., D.L. and M.M.C.; Writing of first draft: M.M.V.; Revisions and final editing: D.L., L.H.B., P.C.G. and A.C.C.C.; Funding acquisition: A.C.C.C. and L.H.B. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: The study was carried out in accordance with the guidelines of the Declaration of Helsinki and was approved by Alvaro Gimeno, a member of the ethics committee of the University of Barcelona, approval code 10617 (14/06/2019). Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations The following abbreviations are used in this manuscript:

API Active Pharmaceutical Ingredient TA Triamcinolone acetonide LIDO Lidocaine hydrochloride HPLC High-Performance Liquid Chromatography MeCN Acetonitrile LOD Limit of detection LOQ Limit of quantiﬁcation SD Standard deviation Css Concentration at steady state Cmax Maximum concentration Pharmaceutics 2021, 13, 1080 15 of 15

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