Ex vivo permeation of carprofen from nanoparticles: A comprehensive study through human, porcine and bovine skin as anti-inflammatory agent

Ex vivo permeation of carprofen from nanoparticles: A comprehensive study through human, porcine and bovine skin as anti-inflammatory agent

Accepted Manuscript Title: Ex vivo permeation of carprofen from nanoparticles: A compressive study through human, porcine and bovine skin as anti-infl...

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Accepted Manuscript Title: Ex vivo permeation of carprofen from nanoparticles: A compressive study through human, porcine and bovine skin as anti-inflammatory agent Author: Alexander Parra Beatriz Clares Ana Rossell´o Mar´ıa L. Gardu˜no-Ram´ırez Guadalupe Abrego Mar´ıa L. Garc´ıa Ana C. Calpena PII: DOI: Reference:

S0378-5173(16)30056-4 http://dx.doi.org/doi:10.1016/j.ijpharm.2016.01.056 IJP 15522

To appear in:

International Journal of Pharmaceutics

Received date: Revised date: Accepted date:

1-10-2015 21-1-2016 22-1-2016

Please cite this article as: Parra, Alexander, Clares, Beatriz, Rossell´o, Ana, Gardu˜noRam´irez, Mar´ia L., Abrego, Guadalupe, Garc´ia, Mar´ia L., Calpena, Ana C., Ex vivo permeation of carprofen from nanoparticles: A compressive study through human, porcine and bovine skin as anti-inflammatory agent.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2016.01.056 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Ex vivo permeation of carprofen from nanoparticles: A compressive study through human, porcine and bovine skin as anti-inflammatory agent Alexander Parraa,b, Beatriz Claresc* [email protected], Ana Rossellób, María L. GarduñoRamírezd, Guadalupe Abregod, María L. Garcíab, Ana C. Calpenaa a

Department of Pharmacy and Pharmaceutical Technology, Biopharmaceutics and Pharmacokinetics Unit, Faculty of Pharmacy, University of Barcelona, Joan XXIII Avenue, 08028 Barcelona, Spain b

Department of Physical Chemistry, Faculty of Pharmacy, University of Barcelona, Joan XXIII Avenue, 08028 Barcelona, Spain c

Department of Pharmacy and Pharmaceutical Technology, University of Granada, Campus de la Cartuja Street, 18071 Granada, Spain d

Centro de Investigaciones Químicas, Universidad Autónoma del Estado de Morelos, Av. Universidad No. 1001, Col Chamilpa, Cuernavaca, Morelos, México *

Corresponding author at: Department of Pharmacy and Pharmaceutical Technology, University of Granada, Campus de la Cartuja Street, 18071 Granada, Spain. Tel.: +34 958 243900; Fax: +34 958 248958.

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Graphical Abstract

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Abstract The purpose of this study was the development of poly(D,L-lactide-co-glycolide) acid (PLGA) nanoparticles (NPs) for the dermal delivery of carprofen (CP). The developed nanovehicle was then lyophilized using hydroxypropyl-β-cyclodextrin (HPβCD) as cryoprotectant. The ex vivo permeation profiles were evaluated using Franz diffusion cells using three different types of skin membranes: human, porcine and bovine. Furthermore, biomechanical properties of skin (trans-epidermal water loss and skin hydration) were tested. Finally, the in vivo skin irritation and the anti-inflammatory efficacy were also assayed. Results demonstrated the achievement of NPs 187.32 nm sized with homogeneous distribution, negatively charged surface (‒23.39 mV) and high CP entrapment efficiency (75.38%). Permeation studies showed similar diffusion values between human and porcine skins and higher for bovine. No signs of skin irritation were observed in rabbits. Topically applied NPs significantly decreased in vivo inflammation compared to the reference drug in a TPA-induced mouse ear edema model. Thus, it was concluded that NPs containing CP may be a useful tool for the dermal treatment of local inflammation.

Keywords: Carprofen; polymeric nanoparticles; freeze-drying; anti-inflammatory efficacy.

Chemical compounds studied in this article Carprofen (PubChem CID: 2581); Poly(D,L-lactide-co-glycolide) acid (PubChem CID: 23111554); Poloxamer 188 (PubChem CID: 24751) ; Hydroxypropyl-β-cyclodextrin (PubChem CID: 44134771)

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1. Introduction Carprofen (CP), 2-(6-chlorocarbazole) propionic acid, is a non-steroidal anti-inflammatory drug (NSAID). CP was licensed for systemic human use in several countries as an analgesic in the 1980s. It was withdrawn from the human market in the early 1990s for commercial reasons before reemerging for veterinary use (Kerr et al., 2008). The anti-inflammatory potency of CP, compared to other NSAIDs, is similar to indomethacin, piroxicam or diclofenac, and higher than ibuprofen or phenylbutazone (Baruth et al., 1986). Some phototoxic and photoallergic reactions were associated to oral administration. Thus its potential administration topically avoiding significant systemic concentrations of drug could be an important therapeutic tool as anti-inflammatory also for humans. The main effect of this drug is the inhibition of cyclooxygenase (COX). COX is an important enzyme in the arachidonic acid cascade. This process generates other mediators involved in some aspects of the inflammatory response, such as prostaglandins and thromboxanes ( Simmons et al., 2004). In veterinary CP is used in several treatments, for instance, osteoarthritis, with no recognized side effects (Sanderson et al., 2009; Slingsby et al., 2001), respiratory diseases and other pathologies in conjunction with antibiotics (Elitok and Elitok, 2004; Lockwood et al., 2003) or in bovine mastitis (Vangroenweghe et al., 2005). It has been administered as an analgesic in preoperative and postoperative patients (Bergmann et al., 2007; Sidler et al., 2013). The anti-inflammatory efficacy of CP when administrated parentally has been also reported (Karol, 1996, Sidler et al., 2013). However, the antiinflammatory efficacy of CP when administrated topically has been scarcely investigated. The use of NSAIDs in a topical formulation may reduce the adverse effects associated with systemic therapy (McPherson and Cimino, 2013). When treating a topic inflammation by directly applying the NSAID on the affected area, it should be ensured that drug reaches the site of action at determined concentrations and within effective therapeutic range. 4

Furthermore this therapeutic concentration should remain constant throughout the time required to reduce inflammation. The drug ability to penetrate through the skin is correlated to its physicochemical properties, as well as, the pharmaceutical dosage form (Patel et al., 2013; Zhang et al., 2009; Singh and Roberts, 1994). On the other hand, it is absolutely necessary the understanding of the processes, pathways and driving forces affecting the transdermal permeation of the drug. In this line, studies of new routes of administration for a particular drug the performance of in vivo studies is the most indicated. However, it is often not possible to conduct these types of studies in the initial stages because of regulations or ethical concerns. Furthermore, it is nearly impossible to assess the skin permeability addressing simply in vivo tests (Godin and Touitou, 2007). For this reason ex vivo studies are of crucial importance in the research of skin formulations for either topical or systemic drug actions (Flaten et al., 2015). Although variations in the setup of the experimental methodology could affect resulting data, it could be standardized (temperature, receptor media, diffusion area, etc.) to obtain a sufficient degree of representativity. The choice of an in vitro model with a specific skin model provides important information for the evaluation of a new therapeutic agent. Equally, the effectiveness and acceptability of skin formulations are directly related to the properties of the carrier/vehicle used (Bolzinger et al., 2012). The analysis of different type of skin for the same formulation is essential as most of the research on interspecies percutaneous absorption extrapolation is conducted to select animal model to predict absorption in humans (Magnusson et al., 2001). Thus the main goals of this work were the screening of different types of skin for the ex vivo permeation studies of CP, as well as, the evaluation of the antiinflammatory efficacy. For these purposes poly(D,L-lactide-co-glycolide) acid (PLGA) based nanoparticles (NPs) were designed and developed for the encapsulation of CP. 2. Materials and methods 5

2.1. Materials CP was obtained from Capot Chemical (Hangzhou, P.R. China). PLGA (75:25 ResomerRG753S, Mw 36510 Da) was from Boehringer Ingelheim (Ingelheim, Germany). Hydroxypropyl-β-cyclodextrin (HPβCD) was from Sigma-Aldrich (St Louis, MO, USA). Poloxamer 188 (LutrolF68; P188) was obtained from BASF (Barcelona, Spain). Double distilled water was obtained from a MilliQ® Plus System lab supplied. All other chemicals were of analytical grade and used without further purification. 2.2. Preparation of NPs A three-factor, five-level central rotatable composite design 23 + star was applied, by using the Statgraphics® Plus 5.1 software (Statpoint Technologies, Inc. Warrenton, VA, USA), to obtain the best formulation of NPs by testing the main effects and interactions of the amount of CP (cCP), amount of poloxamer 188 (cP188) and amount of PLGA (cPLGA) on the physicochemical properties such as average particle size (Z-ave), polydispersity index (PI), zeta potential (ZP), and entrapment efficiency (EE). Once selected, NPs were produced by the solvent displacement technique (Ribeiro et al., 2008). Briefly, an organic solution of polymer PLGA (1.98–7.02 mg/mL) in 10 mL of acetone containing CP (0.08–0.92 mg/mL) was poured while stirring moderately into 20 mL of an aqueous solution of P188 (5.80–14.20 mg/mL) and adjusted to 3.5 pH. Acetone was then evaporated and the NPs dispersion was concentrated to 20 mL under reduced pressure (Buchi B-480, Flawil, Switzerland). The free drug was separated from CP-NPs suspension by subsequent cycles of centrifugation at 3000 rpm for 30 min (Sigma 301K centrifuge, Osterode am Harz, Germany) and re-dispersion in water. Blank NPs were also prepared for comparison studies. 2.3. Freeze-drying

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The optimized CP-NPs (3 mL samples) were freeze-dried (L-CP-NPs) to maintain the stability using HPβCD (5%; w/v) as cryoprotectant. pH of samples were also adjusted to 7.4. The freeze-drying process consisted in a stabilization phase at +10 ºC for 1 h, a freezing phase at 55 ºC for 4 h, a primary drying I phase at 25 ºC for 37 h, a primary drying II phase at +20 ºC for 5 h and secondary drying phase at +20 ºC for 6 h using a Telstar LyoQuest freeze dryer (Telstar, Barcelona, Spain), equipped with Pirani and capacitance vacuum gauges at 5.2×10-2 mbar. The collapse temperature (Tc) of N90s was determined by a FDCS 196 freezedrying microscope (Linkam Scientific Instruments, Epsom, UK) equipped with camera (Olympus BX51) and a liquid nitrogen cooling system. The freeze-dried cake was rehydrated by slowly injecting 3 mL purified water onto the inside wall of the vial and stabilized for 5 min to ensure proper wetting of the cake. Finally, the vial was gently shaken for another 2 min to ensure complete disintegration and dissolution of the cake. 2.4. Physicochemical characterization The Z-ave, PI, and ZP were determined in triplicate by dynamic light scattering (DLS) with a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) at 25 ºC. The morphology characteristics were analyzed by transmission electron microscopy (TEM) with a JEOL 1010 instrument (Akishima, Japan) using 40.000 to 60.000 × magnification. The samples preparation was conducted following the negative staining technique with uranyl acetate solution 2%. The EE (%) of CP in NPs was determined by indirect determination of the concentration of non-entrapped drug in the dispersion medium by a reversed phase high-performance liquid chromatography (RP-HPLC) methodology validated according to international guidelines. Briefly, each sample was diluted MilliQ® water (1:20). The non-encapsulated CP was separated by filtration/centrifugation using an Ultracel YM-100 (Amicon® Millipore Corporation, Bedford, MA, USA) centrifugal filter at 3000 rpm for 30 min (Sigma 301K 7

centrifuge, Osterode am Harz, Germany). The EE was calculated using the following equation:

EE (%) 

W 0 W 1 100 W0

(1)

Where W0 and W1 are the total amount of CP and free drug in the filtrate, respectively. The RP-HPLC system consisted of a Waters 1525 pump (Waters, Milford, USA) with a UV– Vis 2487 detector (Waters, Milford, USA) (λ= 235 nm) and Hypersil Elite C-18, 150 x 4.6 mm, 5µm column (BC Aplicaciones Analíticas S.A., Spain) at flow rate of 1 mL/min. The mobile phase was methanol/potassium dihydrogen phosphate (75/25 v/v) with a final pH about 3.0. Validation of the developed methodology was performed in accordance of international guidelines (EMEA, 2011), including the evaluation of linearity, sensitivity, accuracy and precision. 2.5. Permeation studies 2.5.1. Skin membranes As permeation membranes three different skins were used: human, porcine and bovine. Human skin was obtained from abdominal plastic surgery of healthy patients. The experimental protocol was approved by the Bioethics Committee of the Barcelona SCIAS Hospital (Spain) with number 0012016, and written informed consent forms were provided by volunteers. The porcine skin from the flank area was obtained immediately after the animals (YorkshireLandrace) had been sacrificed for other purposes in the Animal Facility at Bellvitge Campus (University of Barcelona, Spain). Udder skin samples from healthy Holstein Frisian cows which were slaughtered according to legal requirements were obtained freshly from a local slaughterhouse (Barcelona, Spain).

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The studies were conducted under a protocol approved by the Animal Experimentation Ethics Committee of the University of Barcelona (Spain) with number 7428 and the Committee of Animal Experimentation of the regional autonomous government of Catalonia (Spain). After being frozen to −20 ºC, the skins were cut with a dermatome GA 630 (Aesculap, Tuttlingen, Germany) at different thickness according to the type of skin, 400, 700 and 1000 µm for human, porcine and bovine udder skin, respectively (Brugués et al., 2015; Netzlaff et al., 2006; Seto et al., 2010). The integrity of the skin tissues was verified prior the experiment according to section 2.6. 2.5.2. Franz diffusion cells Ex vivo permeation study of NPs was performed in Franz diffusion cells with diffusion area of 2.54 cm2. Skin samples were placed between the receptor and donor compartments with the dermal side in contact with the receptor medium and the epidermis side in contact with the donor chamber. 300 µL samples were placed in the donor compartment (L-NPs-CP and NPsCP). A saturated solution of CP in phosphate buffered saline (PBS) was also assayed. As receptor medium PBS at pH 7.4 was used and kept at 32 ± 0.5 ºC under continuous stirring in accordance with sink conditions. Samples of 300 µL were withdrawn from the receptor compartment at pre-selected times for 24 h and replaced by an equivalent volume of fresh PBS at the same temperature. Samples were analyzed in triplicate by RP-HPLC. 2.5.3. Permeation parameters The cumulative amounts of CP (mg) that had penetrated per unit surface area of the skin membrane (cm2) were corrected for the sample removal and plotted versus time (h). The permeation profiles were analyzed on the basis of a diffusion model for an infinite dose condition. CP fluxes (Jss, μg/cm2/h) through the skin was calculated by plotting the cumulative amount of drug permeating the skin against time, determining the slope of the linear portion of the 9

curve by linear regression analysis using the Prism®, V. 3.00 software (GraphPad Software Inc., San Diego, CA, USA) and dividing by the diffusion area. In this plot the lag time (Tl, h) is the intercept with the X-axis (time). The permeability coefficients (Kpss, cm/h) were obtained by dividing Jss by the initial drug concentration (C0) in the donor compartment, and it assumed that under sink conditions the drug concentration in the receiver compartment is negligible compared to that in the donor compartment. The P1 (related with the partition coefficient, cm) and the P2 (related with the diffusion parameter, h‒1) parameters were also estimated from the following equations:

Kpss  P1  P2

(2)

Tl  1

(3)

6  P2

The predicted steady-state plasma concentration (Css) of drug, that would penetrate skin barrier after topical application, was obtained using the following equation: Css 

JA Clp

(4)

where Css is the plasma steady-state concentration, Jss the flux determined in this study, A the hypothetical area of application and Clp the plasmatic clearance. Calculations were addressed on the basis of a maximum application area of 1 cm2 and Clp values of 2.18 L/h ± 0.42 for human (Crevoisier, 1982) , this same value of 2.18 ± 0.42 L/h for porcine, and 0.17 ± 0.12 L/h for bovine (Delatour et al., 1996). Besides, the mean transit time (MTT, h) of the drug in the skin was also determined from the following equation: V1    1  MTT      2  P2  P 1  P 2  A E 

(5)

where V1 (mL) is the volume of the donor compartment and AE (cm2) is the area of experimental skin sample.

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2.5.4. Determination of the amount of drug remaining in the skin At the end of the permeation study, the skin was used to determine the amount of retained drug. Skin samples mounted in the Franz cells were carefully removed and cleaned with gauze soaked in a 0.05% solution of sodium lauryl sulphate, washed in distilled water and blotted dry with filter paper. The permeation area of the skin was then excised and weighed. Its CP content was extracted with methanol/buffer phosphate solution in an ultrasonic processor for 20 min. The resulting solutions were measured by RP-HPLC yielding the amount of CP retained in the skin (Qr, μg/gcm2 of skin). 2.6. Transepidermal water loss (TEWL) The measure of the quantity of water that passes through the epidermal layer of the skin to the surrounding atmosphere by diffusion and evaporation processes was performed by the TEWL assessment using a Tewameter® TM 300 (Courage & Khazaka Electronics GmbH, Cologne, Germany) (Amores et al., 2014). To measure TEWL, the probe, a small hollow cylinder, was held on the skin surface for 1 minute. Results were expressed as g/m2/h. TEWL values of the three types of skin were also assayed after L-CP-NPs application for 1 h. Values (g/m2h) are reported as the mean of 10 replications ± SD. 2.7. Stratum corneum hydration (SCH) The measurements of skin humidity were carried out by a Corneometer® 825, which was mounted on a Multi Probe Adapter® MPA5 (Courage & Khazaka Electronics GmbH, Cologne, Germany). The measurement was performed by the capacitance method that uses the relatively high dielectric constant of water compared to the ones of other substances of the skin. SCH values of the three types of skin were also assayed after L-CP-NPs application for 1 h. Values (arbitrary units, AU) are reported as the mean of 10 replications ± SD. 2.8. Skin irritation testing (Draize rabbit test)

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Skin irritation potential of L-CP-NPs was assessed by the Draize skin irritation test on albino rabbits. This study was approved by the animal research ethical committee of the University of Barcelona according to the regulations of the Spanish Government (Law 32/2007 of 7 November 2007, and Royal Decree 1201/2005 of 10 October) under veterinary supervision. New Zealand male albino rabbits (weighing 2.5 -3 Kg) were acclimatized for 7 days before the study. Backs were clipped free of hair 24 h before the assay without damaging the skin. Animals were divided into four groups (n=3): Group 1: 0.9% (w/v) NaCl solution (Control); Group 2: CP saturated solution; Group 3: blank NPs; and Group 4: L-NPs-CP. 0.5 mL of each formulation was applied on the hair-free skin of rabbits by uniform spreading within the area of 4 cm2 covering the site with gauze and a polyethylene film (parafilm®) and fixed with hypoallergenic sticking plaster. The animals were then returned to their cages and examined at 24, 48 and 72 h after the application of formulations. The exposed skin was scored for the formation of edema (graded 0-4), and erythema (graded 0-4). The sites were inspected for dermal reactions such as erythema and edema. The mean erythemal and edemal scores were recorded on the basis of degree of severity (from 0 to 4).Taking into account the primary irritation index value, formulations may be classified as "non-irritant" (< 0.5), "irritant" (2-5), or "highly irritant" (5 - 8) (Draize et al., 1944). 2.9. Anti-inflammatory testing TPA-induced mouse ear edema was carried out using groups of three male Wistar CD-1 mouse with a body weight ranging from 20 to 25 g following the protocol described elsewhere (Domínguez-Villegas et al., 2014). Edema was induced by topical application of 2.5 µg per ear of TPA (12Otetradecanoylphorbol13acetate) dissolved in 5 μL ethanol. CP in buffer solution (100 µL/ear 0.075%), blank NPs (100 µL/ear 0.075%) L-NPs-CP (100 µL/ear 0.075%), and the standard drug indomethacin (1 mg/ear) was used as reference. The TPA solution was applied to both sides of the ear (50 µL each side) simultaneously with the 12

test formulations, which were only applied in the right ear. Four hours after the application of the formulations, the animals were sacrificed and circular sections with 7 mm of diameter were cut from left and right ears and weighted to determine the anti-inflammatory activity. All experiments were performed in compliance with the Norma Oficial Mexicana NOM-062ZOO-1999 and with the approval of the Academic Committee of Ethics of the Vivarium of the Autonomous University of the Morelos State of Mexico with number 0122013. 2.10. Mouse ear skin retention studies Ex vivo retention study of L-NPs-CP formulation and a CP buffered solution (both with the same concentration) were performed in mouse ear skin. The aim of this experiment was to obtain the concentration of retained CP after 4 h and then could be related to the results of the TPA-induced mouse ear edema using mouse ear skin as permeation membrane following the protocol described in section 2.5.2. 2.11. Statistical analysis Statistical evaluation of data was performed using one-way analysis of variance (ANOVA) using the Prism®, v. 3.0 software (Graphpad Software Inc., California, USA). The KruskalWallis test was used to assess the significance of the differences among various groups in permeation studies, p value < 0.05 was accepted as significant. 3. Result and discussion 3.1. Elaboration, stabilization and physicochemical characterization of NPs After the application of the experimental design the polynomial equation generated to establish relationships among selected factors was EE (%) = 0.023 + 12.88cCP +1.59cP188 + 0.16(cCP)2 + 0.0025(cP188)2 + 0.023cCP. From the 16 experiments, the optimized NPs formulation selected was composed of CP 0.075%, PLGA 0.3% and P188 1.25% (Parra et al., 2015).

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The freeze-drying process (lyophilization) is the most used method to handle and stabilize NPs, avoiding undesirable changes upon storage (Sameti et al., 2003; Vega et al., 2012). The incorporation of cyclodextrins such as HPβCD as a cryoprotectant helps to maintain the properties of NPs (Felton et al., 2002). Tc was determined to be ‒33.60 °C. These results are in accordance with those obtained by other authors (Murthy et al., 2004). The optimized, lyophilized and subsequently rehydrated NPs exhibited a Z-ave of 187.32±1.85 nm, with narrow distribution (PI=0.05±0.07). The negative value of ZP (‒23.39±0.72 mV) probably due to the ionization of the carboxyl end-groups of the PLGA chains in presence of water and represent particle stability, because the repulsive forces prevent aggregation with ageing due to the ionization of carboxylic end groups of surface polymer (Domínguez-Villegas et al., 2014). The EE of 75.38±1.07% assured an optimal drug loading to provide sufficient amount for the topical administration. The morphology of the L-NPs-CP was determinate by TEM. The images of the optimized LNPs-CP using HPβCD as cryoprotectant are depicted in Fig. 1. It can be observed the NPs with spherical shape and smooth surface. It can be also observed the uniform distribution. Sizes were smaller than 200 nm, suitable for skin application, confirming the DLS characterization results. It is well known that the smaller the particles the higher their adhesiveness to the surfaces such as tissues (Badihi et la., 2014). A closer contact between the drug-loaded particles and the biological membrane may enable more efficient drug permeation (Cho et al., 2014; Luengo et al., 2006). 3.2. Permeation studies The epidermal thickness is an important factor in transcutaneous drug flux and therefore different thickness were used according to data provided in specialized literature (Brugués et al., 2015; Netzlaff et al., 2006; Monteiro-Riviere et al., 1990; Seto et al., 2010).

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The CP saturated solution was assayed in order to study the intrinsic characteristics of CP, comparing the excipient effect of the CP permeation at maximum thermodynamic activity in order to find the intrinsic permeation parameters. Results of permeation studies are depicted in Table 1. A nonparametric analysis of the experimental data was performed in these studies because drug permeation through animal tissues follows more closely a log-normal than a normal (Gaussian) distribution, in accordance with what was reported by Williams et al. (1992). For this reason, the results are presented as median values, as the arithmetic mean as well as the dispersion of the experimental data, expressed in terms of standard deviation, tends to be artificially increased because of the nature and degree of a skewed distribution. The permeation of CP saturated solution through the three types of skin does not present statistical differences after 24 h among skin membranes. Equally, no statistically significant differences were observed for the CP-NPs or L-NPs-CP. However, some trends could be discussed. Tl for the bovine skin, below 2 hours, is to some extent lower than for the other two species, 4.54 and 5.48 h, human and porcine, respectively in CP-NPs, and 3.05 and 2.19 h, in L-NPs-CP. Tl indicates the time required to reach the steady state. Therefore, results suggested that the bovine skin had a high diffusion. Regarding the permeation behavior of LNPs-CP, the bovine skin was also the skin that retained the biggest amount of CP; 300 µg/cm2/g, when compared to 26.10 µg/cm2/g and 63.20 µg/cm2/g of human and porcine, respectively. This was also consistent with the higher permeability coefficient through bovine skin (6.92104 cm/h). An adequate explanation to this difference could be the fact that NPs penetrated through the hair's follicle route (Raber et al., 2014), since bovine has considerably more hair follicles, 759.3/cm2 versus 40-70/cm2 of human and 52/cm2 for porcine skin (Stahl et al., 2009). Regarding MTT, it can be observed the same trend. Finally, when compared permeation parameters of NPs-CP and L-NPs-CP no statistical differences were observed between them. However, L-NPs-CP provide the advantage that they are stable for a longer 15

period of time. This implies that at industrial scale it can be stored until they need to be rehydrated for use, as an extemporaneous medication, guaranteeing its stability (Abdelwahed et al., 2006). Css of saturated CP solution were: 2.44±0.10 µg/L for human, 0.63±0.01 µg/L for porcine and 27.33±1.10 µg/L for bovine. This last higher theoretical concentration is due to the minor clearance rate in bovine. Statistical differences (p = 0.0273) among species were also observed in NPs-CP with values of 0.37±0.10, 0.08±0.01 and 0.24±0.01 µg/L for human, porcine and bovine, respectively. On the other hand, L-NPs-CP showed the following Css values: 0.06±0.01 µg/L for human and porcine, and 3.02±0.10 µg/L for bovine. This last formulation would reach statistically lower CP plasmatic concentrations in humans (p = 0.001) than NPs-CP, and contrary, higher CP plasmatic concentrations in bovine (p = 0.0001). Therefore, it could be assumed that L-NPs-CP would be more secure for its use in humans avoiding potential systemic toxicity. 3.2. TEWL and SCH Table 2 shows the results of TEWL and SCH measurements before and after the application of L-NPs-CP for 1 h. As can be observed the biomechanical properties (TEWL and SCH) of skins changed with this application. However, this change was barely appreciable for TEWL values in the case of human and porcine skins. As expected, SCH and TEWL values in nontreated (basal) skin were similar for human and porcine skins due to its anatomical similarity. The increase of TEWL values after application of L-CP-NPs for 1 h could be due to a destabilization of skin surface and follicular ducts causing a modification on the stratum corneum (Rancan et al., 2009). On the other hand, the effect of L-CP-NPs of SCH was more pronounced indicating their hydration power. Among three types of skins, bovine skin was the most hydrated. The SCH results may be correlated with the ex vivo drug retention results (Qr bovine > Qr porcine > Qr human; SCH bovine > SCH porcine > SCH human). In this 16

way, the hydration level is a function of the water concentration gradient between the dermis and the surface of the skin. An increase in skin water corresponds to an augmentation in permeability of topical applied compounds (Morganti et al., 2001). 3.3. Skin Draize Test The risk of causing cutaneous damage is a crucial factor to be considered during the development of topical formulations. The developed formulation should minimize the risk of irritant or sensitization reactions following application. The most common adverse effect associated with topical NSAIDs is local skin irritation or application site reactions (McPherson and Cimino, 2013). The obtained result from the in vivo irritancy test demonstrated that none of the assayed formulations showed signs of erythema, edema or swelling on intact rabbit skin at the end of the study. The primary irritancy index determined was 0 in all cases. 3.4. TPA-induced mouse ear edema This in vivo experiment was carried to evaluate the anti-inflammatory efficacy of the formulation. Results are represented in Fig. 2. It can be observed that lyophilized NPs-CP have a higher efficacy compared to the reference drug indomethacin and CP buffered solution as the inhibition of inflammation is significantly higher. Due to the experiment was conducted in mouse ear; the CP amount retained in ear mouse skin was also calculated. Fig. 3 shows the retained amount of CP and the reference antiinflammatory drug indomethacin. The lowest value was for the L-CP-NPs and the highest for indomethacin. However, as observed above, L-NPs-CP was the formulation with best antiinflammatory efficacy. It could be explained for the enhanced capacity of permeation by the effect of the excipients P188 and HPβCD (Lv et al., 2009; Maia et al., 2000) and (Felton et al., 2002; Lopez et al., 2000) allowing the CP to start inhibiting the inflammatory process earlier (Másson et al., 1999). The anti-inflammatory effect could therefore not be directly 17

related to the amount of CP retained, but related to the synergy factors or the sustained delivery of CP from the vehicle (Babu and Pandit, 2004; Loftsson and Masson, 2001). The results obtained with the CP in buffer solution are not as good as L-NPs-CP, as its structure is not optimal to interact with the skin composition and different intermediaries affect the therapeutic response (Jain et al., 2005). In addition the pH of both CP formulations is different, which could affect the drug penetration 3.5 and 7.4 for L-NPs-CP and CP-solution, respectively (Dominguez-Villegas et al., 2014; Bralley et al., 2008). 3. Conclusion By means of a skin permeation studies, it has been demonstrated that both formulations NPsCP and L-NPs-CP can provide sufficient amount of CP to reach anti-inflammatory effect through the three types of skin, human, bovine and porcine. Lyophilization contributes to greater stability of the NPs. These systems could be useful to deliver CP into the skin and the stratum corneum over a prolonged time period. A sustained drug release might reduce systemic drug absorption in local treatment of inflammation avoiding side effects associated to the oral administration of NSAIDs. However, further work is required to understand the interactions of NPs with the common structures of skin such as hair follicles, which are absolutely critical to the improvement of topical drug delivery. Acknowledgments The authors would like to acknowledge the financial support of the Spanish Ministry of Science and Innovation (grant MAT2011-26994 and MAT2014-59134R) and valuable collaboration of Dr. Saša Nikolić and Dr. Gladys Ramos from Reig Jofre, S.A., for their technical assistance in lyophilization assays, as well as, Dr. Alvaro Gimeno for his help in animal studies.

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Figure Captions

Fig. 1. TEM image of the optimized L-NPs-CP using HPβCD as cryoprotectant.

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Fig. 2. In vivo anti-inflammatory efficacy after TPA-induced mouse ear edema. Mean ± SD (n=3).

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Fig. 3. Box-whisker plot of retained amounts of CP and the reference anti-inflammatory drug indomethacin in the mouse ear skin.

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Tables Table 1 Median (maximum and minimum) values of flux (Jss), lag time (Tl), P1, P2, permeability coefficient (Kpss), retained amount (Qr) and mean transit time (MTT) of CP at 24 h from the saturated solution of carprofen (CP), non-lyophilized CP loaded nanoparticles (NPs-CP) and lyophilized NPs-CP through different types of skin (human, porcine and bovine). Human

CP Saturated solution Porcine Bovine

5.10

Jss/Sup (µg/hcm2) (0.05-10.86) Tl (h) P2  102 (h-1) P1  102 (cm) Kpss 104 (cm/h) MTT  10-3 (h)

7.92 (2.39-12.00) 2.11 (1.39-6.96) 12.74 (0.03-22.47) 22.76 (0.21-48.46) 0.46 (0.31-18.39)

1.37 (0.31-4.35) 4.58 (1.47-8.85) 3.64 (1.88-11.33) 1.19 (0.24-5.34) 6.13 (1.39-19.40) 1.43 (0.37-2.84)

Human

NPs-CP Porcine

4.71 0.81 0.17 (1.73-8.34) (0.12-1.39) (0.02-0.62) 4.25 4.54 5.48 (3.02-5.44) (1.54-9.31) (4.09-13.51) 3.92 3.67 0.30 (3.06-5.52) (1.51-10.82) (0.12-4.07) 6.35 1.78 0.70 (1.73-9.41) (0.15-8.27) (0.06-3.28) 21.01 10.81 2.25 (7.71-37.21) (1.58-18.59) (0.25-8.24) 0.81 1.71 1.77 (0.11-2.17) (0.22-5.21) (0.50-16.05)

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Bovine 0.54 (0.04-2.33) 1.76 (0.61-5.86) 9.47 (2.85-27.32) 0.11 (0.01-5.97) 7.56 (0.50-31.06) 0.78 (0.17-23.66)

Lyophilized NPs-CP Human Porcine Bovine 0.12 (0.04-0.19) 3.05 (2.30-3.80) 5.82 (4.39-7.26) 0.32 (0.07-0.58) 1.54 (0.51-2.58) 4.64 (1.54-7.75)

0.13 (0.13-0.14) 2.19 (0.30-4.07) 7.61 (4.09-55.50) 0.44 (0.01-0.81) 3.48 (3.34-3.62) 2.36 (2.21-2.51)

0.52 (0.02-0.68) 1.60 (1.13-16.20) 10.42 (1.03-14.75) 0.52 (0.02-0.67) 6.92 (0.22-9.12) 0.57 (0.44-18.22)

Table 2 TEWL and SCH values of different types of skin before and after application of LCP-NPs for 1 h (mean ± SD; ANOVA Tukey's multiple comparison test, n = 10; p < 0.05). TEWL (g/hm2) basal L-CP-NPs SCH (UA) basal L-CP-NPs

human skin 10.7±1.5 13.1±1.9

Bovine skin 19.2± 2.1 a,b 26.1± 4.3 a,b

Porcine skin 13.1±1.3 a,b 15.4±1.9 a

45.2±2.1 56.7±2.2

8.76± 0.5 a,b 118.2± 6.4 a,b

47.67±1.8 a 105.5±5.4 a

Statistically significant differences regarding human skin (a) and porcine skin ( b).

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