Gelified reverse micellar dispersions as percutaneous formulations

Journal of Drug Delivery Science and Technology xxx (2015) 1e13

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Gelified reverse micellar dispersions as percutaneous formulations Elisabetta Esposito a, Laura Ravani a, Paolo Mariani b, Carmelo Puglia c, Stefania Mazzitelli a, Nicolas Huang d, Rita Cortesi a, *, Claudio Nastruzzi a, * a

Department of Life Sciences and Biotechnology, University of Ferrara, I-44121 Ferrara, Italy Department of Life and Environmental Sciences and CNISM, Polytechnic University of Marche, I-60100 Ancona, Italy c Department of Drug Science, University of Catania, I-95125 Catania, Italy d Institut Galien Paris Sud, UMR CNRS 8612, School of Pharmacy, University Paris-Sud, 92296 Chatenay-Malabry Cedex, France b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 February 2015 Received in revised form 8 June 2015 Accepted 9 June 2015 Available online xxx

This study describes the use of lecithin organogels (LO) as versatile vehicles for percutaneous administration of methyl nicotinate (MN), fenretinide (4HPR) and curcumin (CUR). LO are able to increase the solubility of poor water-soluble molecules probably due to the formation of a short-range hexagonal organization of the lecithin cylindrical reverse micelles. After six months from production, no phase separation and an almost absence of aggregates was observed. LO exhibited similar viscosities and rheological behaviour both in the absence and in the presence of drug. The in vitro drug diffusion from LO studied by Franz cell showed that higher viscous vehicles result in lower diffusion coefficient. The microscopic investigation of human skin displayed no significant alterations after treatment with LO and with PBS. In vivo topical activity on erythema after cutaneous application of LO showed that LO-MN induce a strong erythema, while LO-CUR inhibit skin erythema due to CUR anti-inflammatory activity. Tape-stripping experiments performed on skin after topical administration of LO showed a decrease in the amount of CUR in the stratum corneum after 1 h from the occlusion. Shelf life stability studies demonstrated the higher stability of LO-HPR as compared to the others drugs. © 2015 Elsevier B.V. All rights reserved.

Keywords: Lecithin organogel Methyl nicotinate Fenretinide Curcumin Franz cell Tape stripping

1. Introduction (Trans)dermal administration of drugs has great diffusion since it is characterized by many advantages including ease of administration and delivery benefits. Among the vehicles for cutaneous or percutaneous absorption many systems and strategies have been developed [1e8]. Particularly, the use of excipients based on biofriendly molecules such as lipids and phospholipids, has obtained large interest; for instance, supramolecular structures [9e11], such as lecithin organogels (LO), appear particularly promising. LO are thermodynamically stable, clear, viscoelastic, biocompatible and isotropic gels, often defined also as gel-like reverse micellar system or w/o microemulsion; LO are relatively simple in composition since they are constituted of highly pure lecithin (i.e. more than 90% content in phosphatidylcholine), an appropriate

Abbreviations: LO, lecithin organogels; MN, methyl nicotinate; 4HPR, fenretinide; CUR, curcumin; AUC, area under the curve; PIE, percentage of induced erythema. * Corresponding authors. E-mail addresses: [email protected] (R. Cortesi), [email protected] (C. Nastruzzi).

organic solvent and small (critical) amount of water [10,12,13]. The jelly-like phase of LO is characterized by a threedimensional network of entangled reverse cylindrical (polymerlike) micelles, which immobilizes the continuous external organic phase [14] (see Fig. 1). The formation of cylindrical worm like micelles is due to the unidimensional growth of the spherical micelle present at low water concentration, after the addition of a critical amount of water. The elongated tubular micelles subsequently entangle forming a three-dimensional network, resembling uncrosslinked polymer, in solution and are often called polymer-like micelles, living or equilibrium polymers, wormlike, or threadlike micelles [14e20]. LO being biocompatible non-aqueous semisolid systems [10] are able to assure controlled delivery of actives, minimizing at the same time toxic effects appears particularly appropriate for the treatment of cutaneous pathologies. Lecithin, the major constituent of LO, can be indeed considered one of the most promising and natural excipient able to increase the skin permeation, acting as penetration enhancer. In reason of these properties, lecithin is widely used in nutritional products, medicines and cosmetics. Synthetic lecithin containing residues of

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Fig. 1. Schematic representation of the preparation procedure for LO. Upon addition of water to PC in organic solvent (A) (typically fatty acids, cyclic hydrocarbons or linear hydrocarbons) the organization of PC molecules into reverse micelles occurs (B), when the critical amount of water is added, micellar unidimensional growth and entanglement takes place, resulting in the formation of a gelled structure (C). Panels D and E show a solution of PC in IPP, before (D) and after (E) the addition of the critical amount of water.

saturated fatty acids or hydrogenated soybean lecithin failed to form organogel [9] thus indicating the importance of using natural, unsaturated lecithin. It has been established that unsaturation in phospholipid molecules is a desired property for the formation of lecithin organogels. The unsaturation contributes to the volume factor of the nonpolar region of phospholipid molecules, altering the packing parameter, therefore promoting the formation of reverse micellar structures. The purity of lecithin is another parameter involved in the organogel formation. It has been demonstrated that poorly purified lecithin does not possess gel-forming properties. Indeed to obtain organogels, lecithin should contain about 90% of phosphatidylcholine [10,12,13].

Besides lecithin, the organic solvent exerts an important role [10,13]; the organic solvents able to form gel include linear, branched and cyclic alkanes, ethers and esters, fatty acids and amines (see Table 1). For topical applications the use of fatty acid esters, such as isopropyl palmitate, is of great interest due to their skin penetration enhancing property and to their biocompatible and biodegradability [21e23]. The third component necessary for the formation of the organogel, acting as a structure forming is a polar solvent. It has been established that gel-forming solvents possess high surface tension, relative permittivity (dielectric constant), solvent polarity (polarity index), and a strong tendency toward hydrogen bonding. The most commonly employed polar agent, is typified by water although some other polar solvents such

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E. Esposito et al. / Journal of Drug Delivery Science and Technology xxx (2015) 1e13 Table 1 Critical amount of water for different organic solvents used for preparation of LO. Solvent

Cosolvent (%, v/v)

w0a

Isopropyl palmitate

/ Ethyl acetate (10) Ethyl acetate (20) Propyl acetate (10) Propyl acetate (20) Hexyl acetate (10) Hexyl acetate (20) / 10% ethyl acetate 20% ethyl acetate 10% propyl acetate 20% propyl acetate 10% butyl acetate 20% butyl acetate 10% hexyl acetate 20% hexyl acetate 10% dodecane 20% dodecane / / / / / /

3 6 9 6 8 5 7 1.5 4 7 4 7 3 5 2 4 1 1 3 4 7 5 3 3

Cetearyl octanoate

Isoparaffin Ethyl laurate Butyl laurate Ethyl miristate Isopropyl miristate Isostearyl isostrarate a

Value of added water at which the maximum viscosity is reached.

as glycerol, ethylene glycol, and formamide have been demonstrated to be able to transform an initial non-viscous lecithin solution into a jelly-like state or organogel [20]. LO are particularly interesting due to their ability to solubilize substances with different physico-chemical properties, their thermodynamic stability and their biocompatibility [24]. For instance, a wide variety of guest molecules such as vitamins, hormones, NSAIDS, peptides, amino acids, local anaesthetics and antifungal agents can be efficiently carried by lecithin LO [9,25]. For the penetration enhancer property of lecithin LO can be used as transdermal vehicle, able to promote skin permeation and partitioning of the drug into the skin layers. The transdermal effect is due to fluidization of membrane lipids as well as to a hydration mechanism [26e30]. In addition, the occlusive nature of LO provides smooth feel after dermal application [9,25,26]. Taking into account these considerations and due the simple and spontaneous production method along with a pronounced solubilising power, LO are ideal vehicle especially for thermolabile and/or lipophilic drugs. In the present study three different drugs were considered, namely methyl nicotinate (MN), fenretinide (4HPR) and curcumin (CUR) (see Table 2). MN is an urticant molecule causing vasodilation through the production of prostaglandin D2. It is used for the relief of aches and pains in muscles, tendons and joints and in cosmetics anti-cellulitis creams to improve blood

3

circulations. After in vivo topical application, MN is able to rapidly cross the skin and elicit a distinctive erythema which intensity can be monitored by means of non-invasive instrumental techniques, such as reflectance spectroscopy [31,32]. Fenretinide (4 hydroxypropyl phenyl retinamide, 4HPR) is a synthetic amide derivative of all-trans-retinoic acid [33,34] applied in the chemoprevention and in the treatment of many types of malignancies including neuroblastoma, breast, prostate and pancreas cancers. Moreover 4HPR has been proposed for the treatment of many skin tumours (e.g. basal cell carcinoma and squamous cell carcinoma) and diseases (e.g. actinic keratosis) [35e38]. Despite its pharmacologic advantages, 4HPR is difficult to administer because of its low solubility in aqueous medium. Curcumin (CUR) is a potential candidate for the treatment of head and neck squamous cell carcinoma (HNSCC), the sixth most common cancer worldwide. It has been recently demonstrated that CUR can be employed as a single agent in the treatment of HNSCC and also as an adjuvant agent in combination with standard platinum-based chemotherapy [39,40]. Unfortunately, CUR is characterized by scarce water solubility that prevents its administration [41]. In the first part of the present study the production and characterization of the LO is described. Particularly, rheological measurements and the analysis of the inner structure of LO by X-ray scattering techniques have been reported. The second part concerns the in vitro and in vivo comparison of release modalities of the considered model drug after the administration on the skin. In particular, MN and CUR delivery into the skin was studied determining their in vivo topical activity on erythema after cutaneous application of LO. The induced erythema was chosen as inflammatory model on healthy human volunteers and was monitored by reflectance visible spectrophotometry. Finally, tape-stripping experiments have been performed on skin after topical administration of LO to quantify CUR presence in the stratum corneum. 2. Materials and methods 2.1. Materials Isopropylpalmitate (IPP) and curcumin (CUR), (1E,6E)-1,7-bis(4Hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione were purchased from Sigma Chemical Company (St Louis, MO, USA). The soybean lecithin (90% phosphatidyl choline) Epikuron 200 was from Lucas Meyer (Hamburg, Germany). 4 N-hydroxypropyl phenyl retinamide, (4HPR) was from Cilag A.G. (Schaffhausen, Switzerland). All other materials and solvents of the high purity grade were from Sigma Chemical Co. All chemicals were used as received. Solvents were of HPLC grade and all other chemicals were of analytical grade.

Table 2 Structure and characteristics of drugs incorporated in lecithin organogels. Drug

Chemical structure

IUPAC name

MW

Solubility in water (mM)

Log P

Ref.

Methyl nicotinate (MN)

Methyl pyridine-3-carboxylate

137.14

347

0.80

[31,32]

Fenretinide (4HPR)

15-[(4-hydroxyphenyl) amino]retinal

391.55

0.003

6.31

[33e38]

Curcumin (CUR)

(1E,6E)-1,7-Bis(4-hydroxy-3-methoxyphenyl)-1, 6-heptadiene-3,5-dione

368.38

0.27

3.28

[39e41,45,48]

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E. Esposito et al. / Journal of Drug Delivery Science and Technology xxx (2015) 1e13

2.2. Production of lecithin organogels LO were prepared by dissolving lecithin (200 mM) in IPP followed by addition of water, under magnetic stirring. The amount of added water is expressed as w0 that is the molar water to lecithin ratio (w0 ¼ [H2O]/[lecithin]) [10]. The highest amount of water that can be incorporated into the lecithin solution with no phase separation (W0max) was determined as elsewhere described [10,24e26]. To produce drug containing LO the drug, namely MN (0.5% w/w), 4HPR (0.05, 0.1 or 0.15% w/w) or CUR (0.015% w/w), was alternatively added after three hours of stirring, to the obtained formulations. Afterwards, LO were maintained under stirring for 12 h. Solubility of MN, 4HPR and CUR in LO was determined by saturating the LO with an excess of drug. The obtained mixtures were maintained under stirring at room temperature protected from exposure to light for 72 h. Drug content in the LO was evaluated by extraction of drug with centrifugation cycles (15 min at 6000 rpm) followed by RP-HPLC chromatographic analysis. 2.3. X-ray diffraction measurements X-ray diffraction experiments were performed using a 3.5 kW Philips PW 1830 X-ray generator equipped with a Guinier-type focusing camera operating in vacuum with a bent quartz crystal monochromator (l ¼ 1.54 Å). Diffraction patterns were recorded on GNR Analytical Instruments Imaging Plate system. Samples were held in a tight vacuum cylindrical cell provided with thin mylar windows. Diffraction data were collected at three different temperatures 25, 37 and 45  C. In each experiment, a number of Bragg peaks were detected in the low-angle region and the peak indexing was performed considering the different symmetries commonly observed in lipid phases [42]. Once derived the lattice symmetry, the unit cell dimension, a, was calculated from the averaged spacing of the observed peaks. The disordered nature of the shortrange lipid conformation was confirmed by analysing the highangle X-ray diffraction profiles [43]. 2.4. Rheological measurements Rheological measurements (except for CUR) were carried out using a controlled strain RMS800 rheometer (Rheometrics, Possum Town, NY, USA) and concentric cylinder cuvette with 2 mm gap. Viscosity was measured at 100 s1 and 25  C. Oscillatory measurements were carried out at low amplitude (within the linear viscoelastic region) with an angular velocity (u) of between 0.1 and 100 rad/s both at 25  C and 35  C. In the case of CUR, rheological measurements were performed with an AR-G2 rotational rheometer (TA Instruments, New Castle, DE, USA). The geometry used was an aluminium cone-plate with a diameter of 40 mm and an angle of 1. Flow curve were obtained in varying the shear rate from 0.01 s1 to 5000 s1 with 5 points per decade, and each point was maintained for 180 s. Measurements were conducted at 25  C and 35  C. After setting the temperature was controlled with a Peltier plate. A solvent trap was used to prevent water evaporation. Measurements were performed in triplicate for each sample, to ensure reproducibility. The statistical analysis was performed calculating the standard deviation from the three measurements made for each sample. 2.5. Stability studies Physical stability of LO maintained at room temperature and at 40  C was routinely evaluated for a period of 12 months, namely day 1, 7, 15, 30, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330, 360. Stable systems were identified as those free of any physical change

under visual inspection such as, turbidity, phase separation or macroscopic viscosity changes. The chemical stability of LO by time was studied on formulations stored in aluminium tubes, in N2 inert atmosphere at 40  C for three months [44]. At regular time intervals (30 days) for three months, 200 mg of each formulation were solubilized in 20 ml of methanol and then analysed for drug content by chromatographic HPLC analysis as reported in paragraph 2.9. Shelf life values were calculated as below reported. Log (drug residual content, %) was plotted against time and the slopes (m) were calculated by linear regression. The slopes (m) were then substituted into Equation (1) for the determination of k values.

k ¼ m  2:303

(1)

Shelf life values (the time for 10% LO, t90) were then calculated by Equation (2).

t90 ¼ 0:105=k

(2)

as reported by Wells [45]. 2.6. In vitro experiments The analyses were carried on using Franz type glass diffusion cell assembled with different membranes as reported in Table 3. The cell body was filled with an opportune receptor phase (see Table 3). 1 g of each organogel was alternatively placed into the donor cell compartment and tamped down on the membrane, previously moistened by immersion for 12 h with the receptor phase. Samples of receptor phase were withdrawn after predetermined time intervals, namely 0.5 h, 1 h, 1.5 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h and the drug concentration was measured by using an HPLC analytical procedure below reported. Each removed sample was replaced with an equal volume of simple receptor phase. To calculate the diffusion coefficients, the amount of drug that diffuse through the membrane(s) per unit area was plotted against time and the slopes were calculated by linear regression. The calculated regression coefficients were never less than 0.97. The slopes were then substituted into the equation Jn ¼ Jo/C, where Jn is the diffusion coefficient (normalized flux), Jo is the observed flux and C is the drug concentration (mg/ml). All the obtained diffusion rates were determined six times in independent experiments and the mean values ± standard deviations were calculated. In the case of CUR, samples of dried stratum corneum epidermis membranes (SCE) were rehydrated by immersion in distilled water at room temperature for 1 h before being mounted in Franz-type diffusion cells supplied by LGA (Berkeley, CA) as previously reported [46]. The exposed skin surface area was 0.78 cm2 area (1 cm diameter orifice). The receptor compartment contained 5 ml of a mixture of phosphate buffer 60 mM pH 7.4 (PBS) and ethanol (50:50, v/v) to allow the establishment of the sink conditions and to sustain permanent solubilization [45]. This solution was magnetically stirred at 500 rpm and maintained at 32 ± 1  C during all the experiments [47]. After Franz cell experiments, skin morphology was evaluated by optical and electron microscopy. In the first case, samples were treated with reducing agents and fixed with glutaraldehyde 2.5% (w/v) in phosphate buffer 0.1 M pH 7.4, post-osmicated with osmium tetroxide 2% in the same buffer, dehydrated with increasing quantities of acetone and included in Araldite Durcupan ACM (Fluka). Semifine sections were made on ultramicrotome Reichert Ultracut S, stained with an aqueous solution of toluidine blue 1% (w/v) and studied by light microscope Nikon Eclipse E800. Dried skin samples were also analyzed at 15e20 kV by scanning electron microscopy (SEM) (Zeiss EVO 40, Carl Zeiss AG) working

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Table 3 Experimental conditions for in vitro permeation experiments. Organogel

Membrane type

Membrane thickness (mm)/pore size (mm)

Receptor buffer

LO-MN LO-HPR LO-CUR

SCS systema nylon SCEb

Silastic®: 250 cellulose mixed esters: 150/0.45 120/0.2 80/0.08

Phosphate buffer (60 mM, pH 7.4) 0.9% (w/v) NaCl Phosphate buffer (0.1 M, pH 7.4) or methanol/water 20:80 v/v Phosphate buffer (0.1 M, pH 7.4) or methanol/water 20:80 v/v

Data are the mean of five independent experiments ± S.D. Experiments were performed by using Franz type glass diffusion cell as described in the Materials and Methods Section par. 2.6. a SCS system: Silastic®/cellulose/Silastic® sandwich obtained by hydrophilic cellulose esters membrane sandwiched between two lipophilic silicone membranes. b SCE: stratum corneum epidermis.

in high vacuum and extended Variable Pressure (XVP®). 2.7. In vivo studies In vivo experiments were designed according to the declaration of Helsinki. Particularly, experiments were performed on two groups of ten volunteers: group A enrolled for the in vivo evaluation of erythema and group B enrolled for tape-stripping. Volunteers of both sexes and in the age ranging between 25 and 55 years were recruited after medical screening followed by physical examination of the application sites. After they were fully informed on the nature of the study and on the procedures involved, they gave their written consent. Prior to the experiments the volunteers rested for 15 min in the experimental room set at 22 ± 2  C and 40e50% relative humidity. 2.7.1. In vivo evaluation of erythema Erythema was monitored by using a reflectance visible spectrophotometer X-Rite model 968 (XRite Inc. Grandville, MI, USA), calibrated and controlled as previously reported [32,48]. Reflectance spectra were obtained over the wavelength range 400e700 nm using illuminant C and 2 standard observer. From the spectral data obtained, the erythema index (EI) was calculated using equation (3):

   1 1 1 E:I: ¼100 log þ 1:5 log þ log R560 R540 R580   1 1 þ log  2 log R510 R610 where 1/R is the inverse reflectance at a specific wavelength (560, 540, 580, 510 and 610). In the case of MN, for each volunteer, four sites on the ventral surface of each forearm were defined using a circular template (1 cm2) and demarcated with permanent ink. One of the sites of each forearm was used as control and three sites were treated with 100 mg of LO-MN. After application, the sites were occluded for 30 min using Hill Top chambers (Hill Top Research, Cincinnati, USA) without cotton pad. Then, to remove the formulations tested, the chambers were removed, the skin surface washed and left to dry for 15 min. The induced erythema was monitored for 10 h. In the case of CUR, the skin erythema was induced by UVB irradiation using a UVM-57 ultraviolet lamp (UVP, San Gabriel, CA, USA) whose specific parameters are reported elsewhere [33]. The minimal erythema dose (MED) was preliminarily determined, and an irradiation dose corresponding to twice the value of MED was used throughout the study. For each subject, four sites on the ventral surface of each forearm were defined using a circular template (1 cm2) and demarcated with permanent ink. One of the sites of each forearm was used as control and three sites were treated with 300 mg of LO-CUR. The preparations were spread uniformly by means of a solid glass rod and then the sites were occluded for 6 h using Hill Top Chambers (Hill Top Research,

Cincinnati, USA). After the occlusion period, the chambers were removed and the skin surfaces were gently washed to remove the gel and allowed to dry for 15 min. Each pre-treated site was exposed to UV-B irradiation 1, 3 and 6 h (t ¼ 1, t ¼ 3 and t ¼ 6, respectively) after LO-CUR removal and the induced erythema was monitored for 52 h. Erythema index (EI) baseline values were taken at each site before application of gel formulation and they were subtracted from the EI values obtained after MN application or after UV-B irradiation at each time point to obtain DEI values. For each site, the AUC was computed using the trapezoidal rule. To better outline the results obtained, the percentage of induced erythema (PIE) was calculated from the AUC values using equation (4).

Inhibition ð%Þ ¼

AUCðCÞ  AUCðTÞ  100 AUCðCÞ

(4)

where AUC(C) is the area under the response/time curve of the vehicle-treated site (control) and AUC(T) is the area under the response/time curve of the drug-treated site. 2.7.2. Tape stripping For each subject of group B, four sites (2 cm2) were defined on the ventral surface of each forearm, and 200 mg of LO-CUR was applied on these cutaneous sites. The preparations were spread uniformly on the site by means of a solid glass rod and were then occluded for 6 h. After the occlusion period, the residual formulations were removed by gently wiping with cotton balls (different for each pretreated site). Ten individual 2 cm2 squares of adhesive tape (Scotch Book Tape 845, 3 M) were utilized to sequentially tapestrip the stratum corneum on the application sites. To obtain a realistic comparison between the results of this experimentation and the ones obtained in the previous in vivo study, the removal of stratum corneum in each pretreated site was effected at 1 h (t ¼ 1), 3 h (t ¼ 3) and 6 h (t ¼ 6) after gel removal [33]. Each adhesive square, before and after skin tape stripping, was weighed on a Sartorius balance (model ME415S, sensitivity 1 mg) to quantify the weight of stratum corneum removed. After each stripping, the tapes were put in the same vial containing 2 ml of the HPLC mobile phase (methanol, 2% acetic acid and acetonitrile, 5:30:65 v/v) and subjected to whirling stirring over 30 s. The extracted CUR was then quantified by HPLC. The validation of recovery of CUR was obtained by spiking tape-stripped samples of untreated stratum corneum with 2 ml of a mobile phase containing CUR 10 mg/ml. The extraction efficiency of CUR was 96.8 ± 0.9% (n ¼ 3). 2.8. Statistical analysis Statistical differences of in vivo data were determined using repeated-measures analysis of variance (ANOVA) followed by the BonferronieDunn post hoc pairwise comparison procedure. The employed software was Prism 5.0, Graph Pad Software Inc. (La Jolla, CA, USA). A probability of less than 0.05 is considered significant.

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2.9. HPLC procedure RP-HPLC determinations were performed using an HPLC apparatus (1200 series, Agilent Technologies, Santa Clara, USA) equipped with a two-plungers alternative pump, an UV-detector and a 7125 Rheodyne injection valve with a 100-ml loop. Sample pretreatment was not required. In all cases, 30 ml of receptor phase were injected into the liquid chromatograph and quantitated by comparison with a drug standard of known concentration. In the case of MN, analyses were performed on a Vydac C18 column (25  0.46 cm) stainless steel packed with 5 mm particles was eluted at room temperature with a mobile phase consisting of acetonitrile/0.1 M ammonium acetate buffer (pH 7.4) (20:80 v/v), the flow rate being 1.0 ml/min and a 265 nm lmax. Under these conditions MN showed a retention time of 7.5 min. In the case of 4HPR, analyses were performed on a C18 Hypersil BDS column (15  0.46 cm) using a mobile phase constituted of methanol/sodium acetate buffer 0.1 M (pH 3.5) 95:5 v/v at a 0.8 ml/ min flux and a 362 nm lmax. In these conditions 4HPR retention time was 3.9 min. In the case of CUR samples were loaded on a stainless steel C-18 reverse-phase column (15  0.46 cm) packed with 5 mm particles (Grace® e Alltima, Alltech, USA). Elution was performed with a mobile phase containing methanol, 2% acetic acid and acetonitrile 5:30:65 v/v at a flow rate of 0.5 ml/min and a 425 nm lmax. Retention time of CUR was 7.0 min. 3. Results 3.1. Preparation of lecithin organogels Solutions in the range of 50e250 mM phosphatidylcholine in organic solvent, such as isopropyl-palmitate (IPP) can be transformed into transparent gels by addition of critical and welldefined amounts of water [10]. The addition of water, as schematized in Fig. 1, leads to a strong change in viscosity, which is generally function of the amount of added water (expressed as w0) [10,12]. For the majority of LO for (trans)dermal applications, w0 is equal to 3 since, it results in formulations with a suitable viscosity for topical application. The produced LO are transparent (or slightly translucent), macroscopically monophasic and isotropic under polarized light. In Fig. 1 (panels D and E) are shown, as an example, the images of vials containing plain w03 lecithin organogel. Table 4 reports the model drugs content within the produced LO. It is to be underlined the high solubilizing power of LO. For instance, in the case of CUR, lecithin organogel is able to solubilize 5.5 mg/ml, a value 5.5 fold higher than the CUR solubility in ethanol (1 mg/ml) and dramatically higher than its solubility in water (11 ng/ml). 3.2. Characterization of LO X-ray diffraction was used to investigate the inner structural

organization of LO. Experiments were performed at 37  C, both in the presence and in the absence of CUR, taken as model drug. In order to derive structural information, the SAXS curves have been analysed using a phenomenological model previously employed to describe the clustering effects in macromolecular systems [49e52]. The function used to fit the curves is based on two terms, the first one defining the Porod scattering from large size clusters (related here to composition fluctuations in the entanglement of rodshaped tubules), while the second one describing the scattering from the flexible cylindrical reverse micelles [50]:

IðQ Þ ¼ A=Q n þ C




1 þ ðQ xÞm þ B

(5)

In the equation, the two multiplicative factors A and C, the incoherent background B, the parameter x and the two exponents n and m are fitting parameters. In particular, x is the correlation length for the micellar cylinders. Fitting curves are superposed to experimental data in Fig. 2. At ambient temperature it is found n ¼ 3.7 ± 0.3, x ¼ 280 ± 40 Å and m ¼ 0.3 ± 0.1 for empty organogels (LO-E) and n ¼ 4.3 ± 0.5, x ¼ 350 ± 30 Å and m ¼ 2.0 ± 0.1 for LOCUR. As main results, the clustering strength, defined by A/Qn, where Q ¼ 0.004 Å1 (a low enough Q value), is very similar in all samples and appears scarcely dependent on temperature, while the correlation length x as well as the coefficient C in the increase after the addition of the drug. Accordingly, the entanglement length, which is a measure of the distance along the micelle separating neighbouring entanglements [53] and which can be approximate by x5/3l2/3 where lp is the persistence length (the minimum disp tance over which the micelle begins to bend significantly), increases from about 420 to 620 Å (such values have been calculated using lp ¼ 150 Å), as detected in most of the studied reverse micelles. Wide-angle diffraction profiles (WAXS) shown in Fig. 2B evidence a broad band at Q ¼ 1.35 Å1 corresponding to a repeat distance of 4.65 Å. Taken together, the SAXS profile suggests that the presence of CUR induces the formation of a short-range, local 2D hexagonal organization of the lecithin cylindrical reverse micelles, while the broad band in WAXS profile confirms the fluid conformation of the hydrocarbon chains, independently on the presence of the drug. 3.3. Rheological analyses The flow properties of LO were investigated by viscosity measurements performed at room temperature (25  C) and with a shear rate of 100 s1. The obtained results are reported in Table 5. As expected, it was found that the viscosity of organogel increases by increasing the amount of water (data not shown), due to the increased networking among the tubular micelles [14e20]. Moreover, LO's viscosity is influenced either by the presence, the concentration and the type of the added drug. For instance, the viscosity of LO-E was 808 ± 78 mPa s while those of LO-MN and LOHPR (at the same drug concentration) were 910 ± 87 mPa s and 857 ± 92 mPa s, respectively. On the other hand, LO-CUR viscosity at a shear rate value of 100 s1 was 822 ± 88 mPa s, It was also observed that, in the case of 4HPR, the increase of drug

Table 4 Drug content and composition of lecithin organogels. Batch

Drug

Drug content (%, w/v)

PC type

LO-E LO-MN LO-HPR/0.05 LO-HPR/0.10 LO-HPR/0.15 LO-CUR

/ MN 4HPR 4HPR 4HPR CUR

/ 0.50 0.05 0.10 0.15 0.015

Soybean Soybean Soybean Soybean Soybean Soybean

lecithin lecithin lecithin lecithin lecithin lecithin

(90% (90% (90% (90% (90% (90%

PC) PC) PC) PC) PC) PC)

PC (mM)

Organic solvent

wo [H2O]/[lec]

200 200 200 200 200 200

IPP IPP IPP IPP IPP IPP

3 3 3 3 3 3

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7

In panel A, in the whole frequency range, G0 is higher than G00 at both temperatures, indicating that the elastic behaviour dominates the viscous one. The sweep test performed at 35  C resulted in an analogue behaviour, One can observe that G0 at 35  C shows an oscillatory behaviour at low frequencies before the intersection point, indicating multiple relaxation processes and an intermediate plateau. This rheological trend is typical of lecithin organogel as mathematically described by a Maxwell model. That is a blend of micellar aggregates possessing various molecular weights [54]. LO-CUR, at 25  C or 35  C, exhibit a Newtonian plateau at low shear rate, and a shear thinning behaviour at higher shear rate. Particularly, at 25  C, for both LO-E and LO-CUR the Newtonian plateau extends from 0.01 s1 to 1 s1, and the viscosity decreases significantly above 10 s1. At 35  C, the same behaviour is observed one decade higher in shear rate: the Newtonian plateau extends from 0.01 s1 to 10 s1, and the viscosity decreases significantly above 100 s1. The shear is high enough to break significantly the structure of the gel (shear thinning behaviour) and to conceal the effect of temperature. It should be noted that the second Newtonian plateau, normally observed at very high shear rate, was not reached at 10,000 s1. However, for the LO-CUR at 25  C, the slight decrease of the viscosity slope at 10,000 s1 may be the sign of the very beginning of this plateau. Concerning these results, it seems that the structure of the gels is quite unchanged for a wide range of shear rate (0.01e1 s1 at 25  C, and 0.01 to 10 at 35  C). Above these shear rates the structure is broken by the shear rate. Flow curves of LO-E and LO-CUR are superimposable, thus LO's viscosity is not affected by the presence of the drug. 3.4. Drug stability in LO

Fig. 2. SAXS (A) and WAXS (B) profiles of LO-E (a) and LO-CUR (b). Curves have been displaced along the intensity axis for clarity. Samples were measured at 37  C.

concentration from 0.5% to 0.15% leads to a 22.5% increase of viscosity, passing from 857 ± 92 mPa s to 1050 ± 120 mPa s. Thus it can be hypothesized that the interaction of this drug with the supramolecular structure of the organogels could be involved in the formation of the three-dimensional micellar network. Fig. 3A and B shows the oscillatory measurements performed on empty lecithin organogels (LO-E), respectively at 25  C and 35  C to mimic storage condition and cutaneous administration. G0 and G00 values are reported as a function of frequency. Moreover, the viscosity vs. shear rate for LO-E (Fig. 3C) and LO-CUR (Fig. 3D), taken as an example, performed at 25  C and 35  C, is reported. From the oscillatory measurements test it is possible to extrapolate information about the experimental dependence of the elastic modulus G0 and the loss modulus G00 on the frequency of applied mechanical oscillation.

The content of the two lipophilic compounds 4HPR and CUR within LO was calculated as a function of time and expressed as percentage of the total amount of drug used for LO preparation. Shelf life stability was calculated plotting Log of the drug residual content (% with respect to drug content at time 0) against time, obtaining first order kinetics. From the slopes (m) obtained by linear regression, the time at which the drug concentration has lost 10% (shelf life t90) was calculated and reported in Table 6. All data were statistically significant (p < 0.0001). It was found that LO-HPR are the more stable formulations as compared to the others LO. In fact LO-HPR could maintain 90% of 4HPR stability for almost 4 month, whilst in the case of LO-CUR, t90 is around 18 days. Where t90 is the time at which the drug concentration is reduced of 10% with respect to the initial drug content evaluated at accelerated conditions or at 40  C. However, the efficacy in controlling CUR stability is startling since CUR in phosphate buffer 0.1 M rapidly decomposes (t1/2 9.4 min) [49]. The macroscopic aspect of the produced LO did not change by time. Indeed, they did not show phase separation phenomena, maintaining the almost absence of aggregates after six months from production.

Table 5 Viscosity and drug content of produced lecithin organogels. Organogel

Organic solvent

wo [H2O]/[PC]

Drug content (% w/v)

Viscosity at 100 s1 (m Pa s)

LO-E LO-MN LO-HPR/0.05 LO-HPR/0.10 LO-HPR/0.15 LO-CUR

IPP IPP IPP IPP IPP IPP

3 3 3 3 3 3

0.000 0.500 0.050 0.100 0.150 0.015

808 910 857 1023 1050 822

± ± ± ± ± ±

78 87 92 116 116 88

Measurements were conducted at 25  C. Data represent the mean of three measurements ± S.D.

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Fig. 3. Effect of temperature and drug on the rheological behaviour of LO. Measurements were performed at 25  C (A, C) and 35  C (B, D). A, B: Oscillatory measurements of LO-E. The storage modulus G0 (open symbols) and loss modulus G00 (closed symbols) were measured as functions of angular frequency. C, D: Viscosity's behaviour of LO-E (open symbols) and LO-CUR (closed symbols) at different temperatures. Data represent the mean of three measurements ± S.D.

Table 6 Shelf life stability testing of lecithin organogels. Organogel

LO-MN LO-HPR LO-CUR

Drug content at 40  C after the indicated periods (% by weight of initial content) 1 month

2 months

3 months

n.d. 102 ± 1.8 85 ± 1.7

n.d. 97 ± 1.3 n.d.

n.d. 95 ± 0.7 83 ± 0.5

t90a (days)

Drug content after 6 months storage at room temperature (% by weight of initial content)

n.d. 118.4 18.4

n.d. 96 ± 0.9 89 ± 1.3

n.d.: not determined. Data represent the average of three independent experiments ± S.D. a t90: time at which the drug concentration is reduced of 10% with respect to the initial drug content after maintaining the samples at 40  C.

3.5. In vitro experiments A simple method to characterize the efficiency of topical semisolid forms is represented by the study of in vitro drug diffusion. The in vitro drug diffusion from LO was studied by Franz cell under the experimental conditions reported in Table 3. Table 7 In vitro diffusion coefficients of drugs incorporated in lecithin organogels. Formulation

Jn (mg/cm2/h)

Log Jn

LO-MN LO-HPR LO-CUR

42.70 11.24 0.82

1.630 1.050 0.086

Particularly, for MN the in vitro permeation studies were performed using a multi-membrane system consisting of a hydrophilic cellulose ester membrane sandwiched between two lipophilic Silastic® membranes (SCS) tentatively reproducing the human skin [55]. A synthetic nylon membrane was used for 4HPR, while for CUR the natural stratum corneum epidermis (SCE) was employed. Concerning receptor phase, in the case of lipophilic drugs, such as 4HPR and CUR, the use of physiological media as receptor phase led to negligible diffusion kinetics, because of their poor solubility in water. Hence a non-physiological receptor phase with 20% v/v of methanol was used [55]. The cumulative plots of the amount of drug permeated through the selected membranes as a function of time are reported in Fig. 4. From the obtained equation the drug steady state flux value (Jn) for

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the different vehicles was calculated. Table 7 summarizes the obtained results. The highest Jn was obtained for MN followed by 4HPR and CUR. The differences in Jn should be ascribed both to the partition coefficients of the three molecules and to the different membranes employed. Moreover, as a general rule, higher viscous vehicles result in lower Jn. In addition, other parameters that are known to affect the in vitro drug release should be considered, such as the partition of molecules in the matrix of LO as well as the interaction between the drug and the gel components such as IPP or lecithin [56e58]. The lower Jn was obtained for LO-CUR that resulted 0.82 mg/cm2. This behavior could be ascribed both to the use of SCE membrane and to the partition coefficient of CUR (see Table 2). In order to seek whether, and to what extent, LO are harmful to human skin [26], a microscopic investigation of human skin was carried out by mean of light and scanning electron microscopies. Skin samples treated for 8 h with LO-E or PBS (taken as control) were observed. As shown in Fig. 5, no significant alterations of the skin are evident between the skin samples treated with LO-E

9

(Fig. 5A and B) and those with PBS (Fig. 5C and D). In particular, the stratum corneum was still intact after treatment. 3.6. In vivo studies In the present study two different drugs with opposite effect of erythema have been considered, namely MN and CUR. Particularly, after cutaneous application MN induces a strong erythema, while CUR inhibits skin erythema due to its anti-inflammatory activity [50,56]. In order to study the effect of LO-MN on in vivo percutaneous absorption, the extent of MN induced erythema after its topical application was evaluated. The amount of MN penetrated into the skin was assessed indirectly by estimating the vasodilatory effect generated by MN, whose intensity and lasting depends on MN concentration present in the dermal vasculature. This effect was monitored non-invasively by skin reflectance spectrophotometry. LO-MN cause an erythema rapid and intense which declines to undetectable values within 2 or 3 h. As reported in the literature, MN erythema depends on both vehicle composition and application time [55]. Accordingly to Ryatt et al. [59] a contact time of 30 min was used to allow the opportunity to observe the influence of vehicle on the vasodilator response. Due to inter-subject variations, it would be preferable to calculate the area under responseetime curve (AUC) rather than other parameters, thus in our experimental conditions, AUC and subsequent percentage of induced erythema (PIE) were considered. The obtained values are reported in Fig. 6A. LO-CUR was studied in vivo to determine its ability to inhibit the UVB-induced skin erythema on healthy human volunteers. Skin reflectance spectrophotometry was used to determine the extent of the erythema and to assess the inhibition capacity of the formulations after their preventive application onto the skin [48]. The AUC was determined for each subject plotting DEI values versus time. Fig. 6B reports the PIE values. LO-CUR showed to be effective in inhibiting the induced erythema 1 h after its removal (P < 0.05). In the case of LO-CUR for quantifying drug depletion in the viable epidermis and the amount of CUR responsible for the antiinflammatory effect a tape-stripping experiment was also conducted [48]. From the analysis of the data, a decrease in the amount of CUR found in the stratum corneum was detected (Fig. 6B). Particularly, after application of LO-CUR the amount of CUR recovered in the stratum corneum decreased from 37 ng/cm2 after 1 h to 20 ng/cm2 after 6 h. The low CUR amount found in the stratum corneum after 1 h from the occlusion with LO-CUR could be related to the type of interaction between the formulation and the skin. It is known that lecithin exert a penetration enhancement effect in contact with skin and the whole LO-CUR is able to promote drug absorption through the skin [48,59]. In this view it is supposed that after one hour from the occlusion CUR has penetrated through the upper compartments of the epidermis and it is reaching the dermal zone displaying anti-inflammatory activity as evidenced by reflectance spectroscopy data. After 3 and 6 h from the occlusion period the amounts of CUR undergo a further decrease. 4. Discussion

Fig. 4. In vitro diffusion kinetics of MN (A), 4HPR (B) and CUR (C) from LO. Data represent the mean of six independent experiments ± S.D.

LO have a potential as matrixes able to dissolve and deliver active molecules in a controlled fashion, thereby improving their bioavailability and reducing side effects. Topical application of LO allows the treatment of cutaneous pathologies improving local delivery of the incorporated drugs or modulating drug diffusion, as a function of the lipid components. In the present study we produced LO based on a w/o lecithin microemulsion constituted of an entanglement of elongated

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Fig. 5. Effect of lecithin organogels (LO-E) on the morphological characteristics of dried stratum corneum epidermis membranes. The membranes treated for 8 h with LO-E were analysed by optical (A) or scanning electron microscopy (B). For comparison in panels C and D the micrographs of SCE membranes treated with PBS were also reported.

Fig. 6. In vivo effect, after the indicate length of time, of methyl nicotinate (A) and curcumin (B) after topical application of LO-MN and LO-CUR. Closed bars represent the percentage of erythema index while open bars indicate the curcumin accumulated in the stratum corneum. Details on the experimental procedure used for in vivo tests are reported in the Materials and methods. Data represent the mean of six subjects ± SD.

reverse micelles with the aim to obtain new vehicles for cutaneous administration of drugs. The produced LO are characterized by a typical supramolecular organization described by other authors as a three-dimensional network of entangled interpenetrating polymer-like or worm-like micelles [11e17]. Upon addition of water, the micelles evolve resulting in a branching of aggregates from the initial linear growth and then disintegrate into a mixture of shorter and longer ones constituting a three-dimensional network stabilized by hydrogen bonds between lecithin and polar solvent molecules. The LO were prepared using of a lecithin solution in IPP a biocompatible oil suitable for transdermal delivery [24e26]. Lecithin concentration was fixed at 200 mM on the basis of results of literature, as this concentration was found effective for penetration enhancement. Particularly, at the beginning of nineties Willimann and Luisi [25] have studied LOs as matrix systems for transdermal delivery of scopolamine and broxaterol using LOs composed of 200 mM of lecithin in IPP and they observed a significantly higher transdermal flux of both drugs in lecithin-IPP gel as compared to that of each drug in aqueous solution. Subsequent studies have investigated the role of LOs, prepared employing soybean lecithin with IPP, in transskin permeation of drugs. It was noted that the solubility of various drugs (i.e. nifedipine, clonidine, scopolamine, broxaterol, aromatic tetra-amidines) increases in lecithin-IPP system as compared to the drug solubility in IPP alone, thus suggesting a solubility enhancing properties of the organogels [25e27,60]. Moreover, the trans-skin permeability across human cadaver skin of a poorly permeable and water-soluble drug (i.e. propranolol hydrochloride) incorporated in LO was studied [28]. It was observed that the employment of drug in 200 mM lecithin organogel system significantly enhances (~10 times higher) the permeability of the drug across the human skin as compared to that of pure drug in solution form. In addition, the transdermal delivery of various NSAIDs, such as indomethacin and diclofenac, formulated in LO (250 mM of soy lecithin in IPP) showed that the transdermal delivery of both the drugs was higher using LO as compared to IPP alone [27,29]. This enhanced permeation effect of the organogel was attributed to the vectoring

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properties of the reverse micelles. It was suggested that the micellar entities, being small in size and with hydrocarbon sheath, might be received by skin barrier as hydrophobic entities, allowing for closer interaction with skin barrier and leading to an enhanced permeation of the drug molecules. Furthermore, skin irritation tests performed with 200 mM of lecithin LO showed no significant irritation reaction during the period of application (1e5 days) [9,27]. From a rheological point of view, the produced LO exhibited similar viscosities and behaviour both in the absence and in the presence of drug (see Table 4). Indeed, as resulted by a near constant viscosity over shear rate, a Newtonian flow was found for over the low hear rate range [45,53] suggesting that the network structure formed by the worm-like micelles does not break in this range. On the other hand, at higher shear rates, the example of LOCUR displays a non-Newtonian flow as expressed by a decrease in viscosity with increasing shear rate. This effect could be ascribed to the collapse of the network structure due to disentanglement and/ or breakage of the worm-like micelles due to the increase of shear rate. It was also evidenced that, taken 4HPR as an example, the higher the drug content, the higher the viscosity of LO. Concerning drug solubility it was pointed out that LO are able to significantly the increase solubility of lipophilic drugs, such as 4HPR and CUR. This phenomenon may occur since, as found for other molecules, within the LO the drug is probably localized between the acyl chains of the lecithin molecules bound by hydrophobic interactions and partially dissolved in the external oil phase. In vitro studies were carried out using artificial or natural membrane depending on the physico-chemical characteristics of the chemical compounds used in the present study. In particular, three different membranes were used. Firstly the simpler, reproducible and low cost membrane typified by a nylon membrane, was employed. This membrane showed good results for the analysis of the lipophilic compound 4HPR, however in order to simulate more precisely in vivo conditions, the subsequent experiments testing CUR diffusion were performed using cadaver skin. Although the obtained results were acceptable, the variability of different types of skin together with the difficult in finding, addressed us to the choice of a synthetic multi-membrane system [55] the study of the more hydrophilic and small molecule MN. The Silastic®/cellulose/ Silastic® multi-membrane system consists of a hydrophilic cellulose ester membrane sandwiched between two lipophilic Silastic® membranes, which satisfactorily reproduce the lipophilicehydrophilic structure of human skin, thus mimicking the stratum corneum barrier properties. The system was found to be appropriate for simulating the dermal absorption of MN enabling differentiation of the physicochemical dependent properties of permeation from the permeation processes that depend on the biological properties of the skin, and providing a quality control tool to ascertain batch-to-batch uniformity. Indeed the physicochemical properties of drugs, such as their molecular weights and the lipophilic/hydrophilic, have been shown to affect permeability through membranes. These parameters that can be obtained from in vitro permeation experiments, depend on the characteristics as well as penetrant properties of the membrane. Notably, the highest Jn was obtained for MN followed by 4HPR and CUR. As above reported, the differences in Jn should be ascribed both to the partition coefficients (log P) of the three molecules and to the different membranes employed. In fact, notwithstanding the similarity between SCS system and natural SCE, the diffusion coefficient of CUR from LO-CUR (Jn) is almost 52 fold lower as compared to that of MN from LO-MN. The light microscopic investigation of human skin after in vitro diffusion experiments demonstrated that no significant alterations

11

of the skin were apparent for all the treated samples. Moreover, in vivo experiments gave precious information about the biodistribution of the drug within the skin after topical application by LO. Concerning MN, the rapid reduction of the erythema found for LO-MN indicates that, when applied with LO, MN induces an initial rapid and intense effect followed by a similarly rapid decline. The short persistence of the erythema induced by LO could suggest a rapid and intense initial penetration of MN. The high initial penetration could be justified by a strong interaction with the stratum corneum lipids and could result in high concentration of MN in the vascularized section of the skin, from which MN is rapidly removed by blood stream. This strong interaction could be due to: (a) the “solubilization” of stratum corneum lipid caused by IPP contained in LO and (b) the peculiar supramolecular aggregation structure of phospholipids in LO. Previous studies indicated that skin pretreatment by IPP provide greater AUC values compared to aqueous solution alone, but timeeresponse profiles of similar shape indicating that the rapid and total absorption of MN from LO can be attributed to the phospholipids supramolecular aggregation structure, probably causing strong interactions with the stratum corneum lipids. Similar behavior has been shown for CUR. Indeed, the high initial anti-inflammatory effect induced by LO-CUR followed by a rapid decrease suggests a rapid and intense initial penetration of drug justified by a strong interaction with the lipids of stratum corneum and could result in high concentration of CUR in the vascularized section of the skin, from which CUR is rapidly removed by the blood stream. The strong interaction is possibly due to the peculiar supramolecular aggregation structure of phospholipids in the LO-CUR promoting a CUR penetration enhancer effect. Tape stripping experiments helped to elucidate the in vivo behaviour of drug applied on the skin. 5. Conclusions This study has highlighted the performances of LO as topical delivery system. In the field of topical drug delivery, LO provide a new perspective for the topical delivery of many drugs and have emerged as one of the most potential carrier systems. In contrast to other lipid-based systems, such as vesicular systems, LO show to have an edge in terms of efficacy, stability, and technological feasibility exhibiting many desirable physico-chemical properties essential for topical vehicles. LO can dissolve both hydrophilic and lipophilic drugs and hence acts as effective vehicle to deliver wide variety of drugs across the skin. For instance, the topical delivery of biotech molecules in the protective nonpolar microenvironment of these systems may be helpful to protect these macromolecules from degradation during transport to the desired site. In addition, LO are easy to prepare and to handle, and they contain natural, biocompatible, safe and stable ingredients. The in vitro data here reported have demonstrated no skin alterations and intact stratum corneum after treatment with LO. Nevertheless, in vivo data showed that LO-MN and LO-CUR facilitate transcutaneous absorption and thus can be employed for transdermal administration. Particularly, LO-CUR, promoting CUR absorption through skin, appear to be a promising strategy to treat skin diseases such as scleroderma, psoriasis and skin cancer. Concerning lipophilic drugs such as 4HPR, the use of LO is important to increase its bioavailability since the drug is easily solubilized in the LO. Moreover, it was found that LO-HPR shelf life maintains the 90% of 4HPR stability for almost 4 months. The investigations on the structural and functional aspects of these systems and the influence of the organogel components, such

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as cosurfactant, organic solvent, or other additives on the microstructures that get generated within the system as well as on the topical drug transport mechanism should be still investigated. However, LO are highly promising in realizing the drug delivery objectives and to play an increasingly important role in pharmacy as well as in cosmetics in the treatment of skin. Acknowledgements The authors are grateful to Paola Boldrini for microscopy characterization of skin samples. References [1] R.H. Guy, J. Hadgraft, Transdermal Drug Delivery, Marcel Dekker, New York, 2003. [2] A. Williams, Transdermal and Topical Drug Delivery, Pharmaceutical Press, London, 2003. [3] M.R. Prausnitz, S. Mitragotri, R. Langer, Current status and future potential of transdermal drug delivery, Nat. Rev. Drug Discov. 3 (2004) 115e124. [4] R.L. Bronaugh, H.I. Maibach, Percutaneous Absorption, fourth ed., Marcel Dekker, New York, 2005. [5] G. Cevc, Drug delivery across the skin, Expert Opin. Investig. Drugs 6 (1997) 1887e1937. [6] H.A.E. Benson, Transdermal drug delivery: penetration enhancement techniques, Curr. Drug Deliv. 2 (2005) 23e33. [7] M.R. Prausnitz, R. Langer, Transdermal drug delivery, Nat. Biotechnol. 26 (2008) 1261e1268. [8] K.A. Walters, R. Keith, Brain, in: K.A. Walters (Ed.), Dermatological and Transdermal Formulations, Informa Healthcare USA, Inc., New York, 2007, pp. 319e399. [9] R. Kumar, O.P. Katare, Lecithin organogels as a potential phospholipidStructured system for topical drug delivery: a review, AAPS PharmSciTech 6 (2) (2005). Article 40. [10] R. Scartazzini, P.L. Luisi, Organogels from lecithins, J. Phys. Chem. 92 (1988) 829e833. [11] L. Zarif, Elongated supramolecular assemblies in drug delivery, J. Control Release 81 (2002) 7e23. [12] P. Schurtenberger, R. Scartazzini, L.J. Magid, M.E. Leser, P.L. Luisi, Structural and dynamic properties of polymer-like reverse micelles, J. Phys. Chem. 94 (1990) 3695e3701. [13] Y.A. Shchipunov, Lecithin organogel: a micellar system with unique properties, Colloids Surf. A Physicochem. Eng. Asp. 185 (2001) 541e554. [14] D. Capitani, A.L. Segre, F. Dreher, P. Walde, P.L. Luisi, Multinuclear NMR investigation of phosphatidylcholine organogels, J. Phys. Chem. 100 (1996) 15211e15217. [15] P. Walde, A.M. Giuliani, C.A. Boicelli, P.L. Luisi, Phospholipid-based reverse micelles, Chem. Phys. Lipids 53 (1990) 265e288. [16] E.V. Shumilina, Y. Khromova, Y.A. Shchipunov, A study of the structure of lecithin organic gels by Fourier-transform IR spectroscopy, Zhurnal Fiz. Khimii 74 (2000) 1210e1219. [17] Y.A. Shchipunov, Self-organizing structures of lecithin, Usp. Khim 66 (1997) 328e352. [18] S.A. Mezzasalma, G.J.M. Koper, Y.A. Shchipunov, Lecithin organogel as a binary blend of monodisperse polymer-like micelles, Langmuir 16 (2000) 10564e10565. [19] Y.A. Shchipunov, Lecithin organogels: rheological properties of polymer-like micelles formed in the presence of water, Colloid J. 57 (1995) 556e560. [20] Y.A. Shchipunov, E.V. Shumilina, Lecithin bridging by hydrogen bonds in the organogel, Mater. Sci. Eng. C 3 (1995) 43e50. [21] J. Moore, Final report on the safety assessment of octyl palmitate, cetyl palmitate and isopropyl palmitate, J. Am. Coll. Toxicol. 1 (1982) 13e35. [22] A. Arellano, S. Santoyo, C. Martin, P. Ygartua, Influence of propylene glycol and isopropyl myristate on in vitro percutaneous penetration of diclofenac sodium from carbopol gel, Eur. J. Pharm. Sci. 7 (1999) 129e135. [23] S. Parsaee, M.N. Sarbolouki, M. Parnianpour, In vitro release of diclofenac diethylammonium from lipid-based formulations, Int. J. Pharm. 241 (2002) 185e190. [24] P. Walde, H. Willimann, C. Nastruzzi, R. Scartazzini, P. Schurtenberger, P.L. Luisi, Lecithin microemulsion gels as matrix for the transdermal delivery of drugs, Proc. Intern. Symp. Control Release Bioact. Mater. 17 (1990) 421e422. [25] H.L. Willimann, P.L. Luisi, Lecithin organogels as matrix for the transdermal transport of drugs, Biochem. Biophys. Res. Commun. 177 (1991) 897e900. [26] H. Willimann, P. Walde, P.L. Luisi, F. Stroppolo, Lecithin organogel as matrix for transdermal transport of drugs, J. Pharm. Sci. 81 (1992) 871. [27] F. Dreher, P. Walde, R. Walther, E. Wehrli, Interaction of a lecithin microemulsion gel with human stratum corneum and its effect on transdermal transport, J. Control Release 45 (1997) 131e140. [28] S. Bhatnagar, S.P. Vyas, Organogel-based systems for transdermal delivery of

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Please cite this article in press as: E. Esposito, et al., Gelified reverse micellar dispersions as percutaneous formulations, Journal of Drug Delivery Science and Technology (2015), http://dx.doi.org/10.1016/j.jddst.2015.06.007