http://informahealthcare.com/mnc ISSN: 0265-2048 (print), 1464-5246 (electronic) J Microencapsul, 2014; 31(7): 716–724 ! 2014 Informa UK Ltd. DOI: 10.3109/02652048.2014.918667
Ethosomes-based topical delivery system of antihistaminic drug for treatment of skin allergies Shishu Goindi, Bhavnita Dhatt, and Amanpreet Kaur
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Department of Pharmaceutics, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India
Abstract
Keywords
Cetirizine is indicated for the treatment of allergic conditions such as insect bites and stings, atopic and contact dermatitis, eczema, urticaria. This investigation deals with development of a novel ethosome-based topical formulation of cetirizine dihydrochloride for effective delivery. The optimised formulation consisting of drug, phospholipon 90 Gä and ethanol was characterised for drug content, entrapment efficiency, pH, vesicular size, spreadability and rheological behaviour. The ex vivo permeation studies through mice skin showed highest permeation flux (16.300 ± 0.300 mg/h/cm2) and skin retention (20.686 ± 0.517 mg/cm2) for cetirizine-loaded ethosomal vesicles as compared to conventional formulations. The in vivo pharmacodynamic evaluation of optimised formulation was assessed against oxazoloneinduced atopic dermatitis (AD) in mice. The parameters evaluated were reduction in scratching score, erythema score, skin hyperplasia and dermal eosinophil count. Our results suggest that ethosomes are effective carriers for dermal delivery of antihistaminic drug, cetirizine, for the treatment of AD.
Cetirizine, ethosomes, hapten, levocetirizine, skin allergy
Introduction Histamine is a biogenic amine present as a tissue hormone especially in the skin and in the lungs of humans. In the human body, it is stored in the basophilic granulocytes and the mastocytes. It is also present in bee poison and in the salivary secretion of biting/stinging insects. When allergic skin reactions occur, excess histamine is released and is one of the factors responsible for itching and the formation of skin wheals and flares. Currently, antihistamines are primarily administered orally to treat these effects of histamine releases (Walch and Baden, 2004). Cetirizine is a potent second-generation antihistaminic with marked affinity for peripheral H1 receptors and is used for the treatment of various allergies like urticaria, pollinosis and atopic dermatitis (AD). It inhibits the release of histamine and cytotoxic mediators from platelets as well as eosinophil chemotaxis during the secondary phase of allergic response. Studies have shown cetirizine to be effective in the treatment of skin inflammatory conditions by reducing the release of histamine, bradykinin and allergen-induced wheal and flare reactions; decreasing monocyte and T-lymphocyte chemotaxis; reducing eosinophil responses; and decreasing intercellular adhesion molecule-1 expression on epithelial cells (Campoli-Richards et al., 1990; Curran et al., 2004). The recommended oral dose for cetirizine is 10 mg once per day for adults and 2.5–5 mg once per day for children. In general, cetirizine is used to treat hay fever, allergic rhinitis, chronic urticaria and asthma. Recently, it has been recommended for treatment of pruritus associated with AD
Address for correspondence: Prof. Shishu Goindi, Professor in Pharmaceutics, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh – 160014, India. Tel: 0172-2541142, 0172-2534281 (O). E-mail: [email protected]
History Received 6 October 2013 Revised 16 April 2014 Accepted 21 April 2014 Published online 23 June 2014
(dry eczema). The oral administration of cetirizine is associated with setbacks like sedation, corneal and conjunctival staining due to ocular dryness, dry mouth and diarrhoea (Ousler et al., 2004). Therefore, to overcome the systemic side effects associated with oral therapy and for quick onset of action and targeted drug delivery to inflamed skin, a topical delivery using novel drug carrier may be more beneficial. No topical formulation of cetirizine is available in the market till date. Some literature reports suggest use of gels (Walch and Baden, 2004) and conventional liposomes (Elzainy et al., 2004) as topical carriers for cetirizine. Earlier, we have reported the feasibility of TransfersomesÕ (elastic vesicles) for the topical delivery of cetirizine. In the next step of our research, we aim to develop ethosomal carriers of cetirizine in context of obtaining better penetration as compared to conventional liposomes and investigating the in vivo pharmacodynamic activity against hapten, the oxazolone-induced AD murine model. Ethosomal carriers are innovative delivery systems containing vesicles composed mainly of phospholipid (PL; Phospholipon 90G), ethanol and water (Touitou et al., 2000; Dubey et al., 2007). The ethosomes being soft and malleable vesicles efficiently penetrate the skin and allow enhanced delivery of various compounds. In fact, ethosomes have emerged as nascent carriers for the delivery of both hydrophilic and lipophilic molecules (Dayan and Touitou, 2000; Mishraa et al., 2008). This work thus focuses on developing a novel ethosomal carrier-based topical delivery system of cetirizine to overcome the side effects associated with conventional oral therapy and to provide targeted therapy with enhanced skin delivery. The in vivo pharmacodynamic evaluation was done using hapten (oxazolone)induced AD in mice, and the activity of the developed formulation was compared with cetirizine oral solution, cetirizine cream and levocetirizine ethosomal gel.
Ethosomes-based topical delivery system
DOI: 10.3109/02652048.2014.918667
Materials and methods Materials Phospholipon 90G was a gift sample from Phospholipids GmbH (Nattermannallee, Koln, Germany). Cetirizine dihydrochloride and levocetirizine dihydrochloride were obtained as generous gift samples from Indswift Ltd (Mohali, India). Carbopol 980 NF was received as a gift sample from Lubrizol Advanced Materials India Private Limited, Mumbai, India. All other reagents and chemicals were of analytical grade.
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Animals Female mice (BALB/c strain, 25–27 g) were obtained from Central Animal House, Punjab University, and used for carrying out ex vivo permeation, histopathology and pharmacodynamic studies. Ethical approval to perform these studies was obtained from Institutional Animal Ethics Committee, Punjab University, India, and their guidelines were followed throughout the studies. Preparation of different topical formulations of cetirizine Ethosomal vesicles were prepared by method developed by Touitou et al., (2000). Accurately weighed amount of Phospholipon 90 G was dissolved in ethanol at 30 C in a specially designed closed beaker (Table 1). Cetirizine (0.1%) was dissolved in phosphate buffer saline (PBS) pH 6.4 under similar conditions. The aqueous phase was added slowly to ethanol in a fine stream with constant stirring at 2000–3000 rpm in wellclosed specially designed vessel to avoid loss of ethanol. Mixing was continued for an additional 5 min. The prepared vesicles were stored at 4–8 C in a refrigerator overnight and then subjected to further characterisation and evaluation studies. Furthermore, the optimised ethosomal dispersion was gelled using 1.25% carbopol to make it suitable for topical application. Liposomes were prepared by thin film hydration method using phosphatidylcholine and cholesterol (molar ratio 7:3) as previously described by Aggarwal and Goindi (2012). An oil/water cream containing 0.1% cetirizine was prepared by separately heating both oil phase (light liquid paraffin 10% w/w, isopropyl myristate 12% w/w, cetyl alcohol 5% w/w, stearic acid 6% w/w, butylated hydroxytoluene 0.2% w/w and glyceryl monostearate 5% w/w) and aqueous phase (drug 0.1% w/w, triethanolamine 1.6% w/w, propylene glycol 5% w/w, sodium metabisulfite 0.5% w/w and water q.s. 100% w/w) at 65 C. The aqueous
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phase was added to oil phase under continuous stirring. The resulting emulsion was allowed to cool down gradually under constant stirring to obtain a cream. Furthermore, the aqueous solution was prepared by dissolving 0.1% (w/v) of cetirizine in distilled water. Vesicle morphology, size analysis and zeta potential The ethosomal vesicles were visualised using transmission electron microscopy (TEM; Philips CM12 electron microscope, Eindhoven, The Netherlands) to study the morphology and structure of vesicles. The samples were negatively stained with a 1% aqueous solution of phosphotungstic acid, vesicles were dried on a microscopic carbon-coated grid and viewed under the microscope at a suitable magnification. Photo-micrographs of the vesicles were taken at suitable magnification. The vesicle size and size distribution were determined by dynamic light scattering (DLS) technique using a computerised Malvern ZetasizerÔ (Malvern Instruments Ltd., Worcestershire, UK). For determination of zeta potential and polydispersity index (PDI) of the selected formulation, Delsa Nano (Beckman Coulter, Brea, CA) was used. Zeta potential measurements were done at 25 C and the electric field strength was 23.2 V/cm. Entrapment efficiency and transmittance/turbidity determination For determination of entrapment efficiency, the vesicular formulation was kept overnight at 4 C followed by ultracentrifugation at 35 000 rpm for 3 h at 4 C. After removing the supernatant, the drug quantity was determined in both the sediment (after disrupting vesicles using methanol) and supernatant. The entrapment efficiency was calculated using the formula: ðT C Þ 100 T where T is the total amount of drug that is detected both in supernatant and sediment, and C is the amount of drug detected only in the supernatant (Touitou et al., 2000). Transmittance of different ethosomal formulations was determined at a wavelength of 500 nm by diluting the suspension (0.1 mL) to 10 mL with distilled water and setting it as blank at 100% transmittance (Jain et al., 2008). Rheological behaviour of the developed system
Table 1. Optimisation of ethosomal formulations for phospholipid, drug and ethanol concentration (n ¼ 3). Ethanol No. of % % entrapment Formulation Ratio of content vesicles/ code lipid:drug (%) mm3 103 transmittance efficiency E-1 E-2 E-3 E-4 E-5 E-6 E-7 E-8 E-9 E-10 E-11 E-12
20:1 25:1 30:1 35:1 40:1 45:1 50:1 35:1 35:1 35:1 35:1 35:1
30 30 30 30 30 30 30 25 35 40 45 50
265.0 347.5 375.0 415.0 405.0 412.5 380.0 225.0 447.5 500.0 312.5 212.5
60.2 53.0 44.1 34.7 38.9 37.8 36.0 55.6 36.8 33.8 52.7 58.7
45.11 ± 2.07 52.10 ± 1.05 61.57 ± 0.53 66.96 ± 0.57 63.66 ± 0.50 61.96 ± 2.19 62.06 ± 1.51 50.13 ± 0.42 68.85 ± 1.39 72.85 ± 1.57 53.35 ± 0.90 47.45 ± 2.21
E-4 is the optimized formulation on the basis of lipid:drug ratio. E-10 is the final optimized formulation on the basis of ethanol content and selected for further characterization studies.
The gelled system was evaluated for its rheological behaviour using rotational viscometer (Paar Physica MC 1, Brookfield DVII, UK) which is a cup and bob type viscometer (Martin et al., 1991) equipped with Z 4 sensor probe. The viscosity was determined at various shear rates by subjecting the gel to different torque values. The temperature was kept at 30 C, and spindle no. 29 was employed for viscosity determination. The rheograms thus generated provided useful insight into the flow properties of the formulations. Degree of deformability Vesicle extruder (Eastern Sci. Inc., Rockville, MD) was used to determine the degree of deformability. The deformability index of ethosomal vesicles was determined using mini filtration technique and compared with liposomal formulation. In brief, the vesicular dispersion was passed through polycarbonate filter of 50 nm. The vesicle size and size distribution measurement was monitored before and after filtration by DLS measurements using Malvern
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ZetasizerÔ 2000. Percentage deformability was calculated using the following formula: Vesicle size before extrusion vesicle size after extrusion % deformability ¼ 100 Vesicle size before extrusion Spreadability The spreadability of the developed vesicular gel was evaluated using Texture AnalyserÔ, equipped with a 5-kg load cell to determine different rheological properties of prepared vesicular gel such as work of shear, force of gel extrusion, stickiness and firmness (Chuang and Yeh, 2006).
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Skin permeation and deposition studies Franz diffusion cell assembly was used to study the permeation of cetirizine from different formulations through hairless dorsal skin of mice, with receptor compartment volume of 30 mL and effective diffusion area of 3.14 cm2. PBS pH 6.4 was selected as diffusion medium. The receptor cells containing diffusion medium were constantly stirred with a magnetic stirrer and equilibrated at 37 ± 1 C with the help of thermo-regulated outer water jacket. The different formulations of cetirizine equivalent to 0.5 mg were applied onto the preshaved mice skin in the donor compartment. Aliquots of 2 mL were withdrawn through the sampling port at suitable time intervals and analysed for drug concentration employing UV spectrophotometer at 232 nm after appropriate dilutions (Wankhede et al., 2012). Equal volume of diffusion medium was replaced into the receptor cells to maintain proper sink conditions. At the end of the permeation experiments, the skin surface in the donor compartment was rinsed with ethanol to remove excess drug from the surface. The receptor media was then replaced with 50% (v/v) ethanol (El Maghraby et al., 2001; Jain et al., 2003). The contents were allowed to stir for next 24 h followed by spectrophotometric determination at max 232 nm for amount of drug retained in skin. Similar permeation and skin retention studies were performed using blank formulations, i.e. without drug and absorbance values, were subtracted from the test formulations to account for the effect of excipients. The cumulative amount permeated per unit area (mg/cm2), flux (mg/h/cm2) and skin retention (mg/cm2) were calculated. All the experiments were performed in triplicate. All data were statistically analysed by one way analysis of variance followed by Dunnett’s method. Results were quoted as statistically significant where p50.05. Skin-sensitivity studies and histopathological examination Ethosomal gel was applied uniformly on the preshaved dorsal region of mice skin and kept in contact for 4 h. After that, the animals were sacrificed by cervical dislocation method and exposed dorsal surface was cut. Then, each specimen was fixed in 10% buffered formalin; embedded in paraffin and microtomed. The sections were stained with hematoxylin and eosin. Finally, the specimens were observed under a high-power light microscope and were evaluated for their integrity. In vivo pharmacodynamic evaluation Female BALB/c mice, 6–7 weeks old, were used to induce AD by topical treatment with hapten, the oxazolone. Animals were divided into six groups (A, B, C, D, E and F) containing four animals in each group (Table 2). The animals of group B, C, D, E
J Microencapsul, 2014; 31(7): 716–724
Table 2. Different treatments given to various animal groups during in vivo evaluations using oxazolone-induced atopic dermatitis in mice (n ¼ 4). Group A B C D E F
Treatment (formulation) Control/plain gel (without drug) Oxazolone Oxazolone + oral cetirizine aqueous solution Oxazolone + cetirizine liposomal gel Oxazolone + cetirizine ethosomal gel Oxazolone + levocetirizine ethosomal gel
and F were sensitised through topical application on to their shaved back skin of 10 mL of 5% oxazolone in ethanol daily for one week. After one week, the same animals were challenged with 60 mL of 0.1% oxazolone once every alternate day for an additional two weeks (Man et al., 2008). After half an hour of oxazolone application, cetirizine oral aqueous solution, cetirizine liposomal gel, cetirizine ethosomal gel and levocetirizine ethosomal gel each containing 1.0 mg drug was applied daily on the back of animals of group C, D, E and F, respectively. The animals were evaluated every week for change in erythema score and number of scratchings. After completion of three weeks, animals were sacrificed and skin was removed. Excised skin specimens were fixed in 10% neutral formalin and embedded in paraffin. Sections, 5-mm thick, were prepared and stained with hematoxylin and eosin. The number of eosinophils and mast cells were counted under light microscope (400 magnification) in 100 mm by 100 mm area (50 fields) and compared (Oshio et al., 2009).
Results and discussion Preparation of ethosomes Cetirizine-loaded ethosomes were prepared by cold method. The various ratios of PL to drug in the presence of fixed amount of ethanol were investigated (Table 1). For optimising the composition of vesicular formulations, various parameters like the magnitude of drug entrapment, number of vesicles per cubic mm and percent transmittance were studied. It was observed that the drug entrapment increases upon increasing the amount of PL due to the increased availability of the lipid phase for cetirizine and its subsequent incorporation in the lipid bilayers (Aggarwal and Goindi, 2013). Further increase in the amount of lipid decreases the entrapment efficiency because of decreased number of vesicular structures due to incomplete hydration of lipid or partly due to lower partitioning of hydrosoluble drug in PL. However, a very slight increase in entrapment efficiency was observed probably due to an increase in size of the decreased number of vesicles formed at such high PL ratios (50:1). Similar results were observed in case of number of vesicles per cubic mm and percent transmittance. Lower the transmittance, higher the turbidity, therefore, higher the number of vesicles. Thus, percent transmittance was found to decrease to a minimum of 34.7%. Of all the formulation batches containing constant ethanol content (30%), batch E-4 showed maximum entrapment efficiency of 66.96 ± 0.57%, maximum number of vesicles per cubic mm (415.0) and least % transmittance (34.7%). Thus, the above batch, E-4 with lipid–drug ratio 35:1, was finally selected for optimising the concentration of ethanol. The increase in concentration of ethanol from 20% to 50% increased the entrapment efficiency owing to increase in fluidity of membranes leading to higher drug loading on vesicles. The formulation batch, E-10 containing 40% of ethanol, was found to show maximum entrapment (72.85 ± 1.57%). However, further
Ethosomes-based topical delivery system
DOI: 10.3109/02652048.2014.918667
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increase in ethanol percentage probably made the vesicle membrane leaky due to greater disturbance of the lipid-bilayer organisation of vesicles by increased solubilisation of PL in ethanol causing breakdown of vesicular structures, thus leading to decrease in entrapment efficiency. The influence of increasing percentage of ethanol on number of vesicles per cubic mm, percent transmittance and their relative abundance was in coherence with the results of entrapment efficiency. Thus, the formulation batch E-10 showed highest entrapment efficiency of 72.85 ± 1.57%, maximum number of vesicles per cubic mm (500) and least percent transmittance (33.8%). On the basis of all the studies performed, the formulation batch E-10 containing PL:drug (35:1) and ethanol (40%) was selected as the optimised formulation, as it showed minimum transmittance, maximum number of vesicles and highest entrapment efficiency. Morphological evaluation of vesicles Morphological characterisation of the ethosomes as well as existence of their vesicular structure was confirmed by TEM. The prepared ethosomal vesicles were unilamellar and near spherical in shape as depicted in Figure 1(A).
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stress is increased, the normally disarranged molecules begin to align their long axis in the direction of flow. This orientation reduces the internal resistance of the material and allows a greater rate of shear at each successive shearing stress (Martin et al., 1991). In addition, some of the solvent associated with the molecules may be released resulting in an effective lowering of the concentration and the size of the dispersed molecules. This results in lowering of apparent viscosity. Degree of deformability The results indicated that vesicle size of ethosomes before and after extrusion was 180.1 nm and 152.2 nm, whereas that of liposomes was 220.2 nm and 112.1 nm, respectively. Ethosomes got deformed by 15.490%, while liposomes got deformed by 49.09%, which showed that ethosomes were almost three times more flexible than liposomes. This was because liposomes were rigid due to the presence of cholesterol in their structure (Lo´pez-Pinto et al., 2005), while ethosomes were flexible due to the interdigitation induced by ethanol in the lipid bilayer. Ethanol also provided elasticity to vesicle membrane by reducing interfacial tension of the vesicle membrane. Similar results were reported in published literature (Vanden Bergh et al., 2001).
Vesicle size, size distribution and zeta potential The average vesicular size of prepared ethosomal formulation was 180.1 nm along with PDI of 0.278. The log size distribution curve showed normal distribution, and the low value of PDI indicates that the dispersion was homogeneous (Figure 1B). Zeta potential is the electric potential of the vesicle, including its ionic atmosphere (stern layer). The zeta-potential of the optimised formulation was 15.7 mV, i.e. negative value. This was due to the presence of ethanol, which confers a negative charge to the surface of ethosomes. Rheological studies The results of rheological profile of ethosomal dispersions gelled with 1.25% carbopol 980F depicted the non-Newtonian behaviour of flow. The characteristic bent of the rheogram towards the shear stress axis and the decrease in viscosity with increasing rate of shear indicated that the developed formulation exhibited pseudoplastic flow. This pseudoplasticity results from a shearing action on long chain molecules of the gelling polymer. As the shearing
Spreadability The non-sticky nature of optimised formulation was supported by various parameters such as firmness (2.297 kg), work of shear (1.020 kgsec), work of adhesion (0.271 kgsec) and stickiness (2.835 kg). The optimised formulation showed acceptable spreadability and was free from severe adhesion as it required less force and work of adhesion. Skin permeation and deposition studies The mean cumulative amount permeated per unit area from various cetirizine formulations (coded as CET 1 to CET 6) is shown in Figure 2(A). The mean cumulative amount permeated per unit area in 8 h was maximum from ethosomal dispersion (CET 5) was 119 ± 0.001 mg/cm2) followed by ethosomal gel (CET 6; 108 ± 0.079 mg/cm2), liposomal dispersion (CET 3; 60 ± 0.001 mg/cm2), liposomal gel (CET 4; 55 ± 0.001 mg/cm2), conventional cream (CET 2; 53 ± 0.001 mg/cm2), aqueous solution of cetirizine (CET 1; 52 ± 0.001 mg/cm2) in 8 h. This is attributed
Figure 1. (A) Transmission electron microscopy (magnification 40 000) and (B) vesicle size distribution of the prepared cetirizine ethosomal dispersion.
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Figure 2. Comparison of (A) ex-vivo permeation profiles, (B) permeation flux and skin retention of cetirizine from various formulations through mice skin (n ¼ 3).
Figure 3. Histopathological photographs showing the absence of any histological and pathological changes. (A) Control untreated skin and (B) skin treated with ethosomal gel (magnification, 100).
to the flexible membrane packing characteristics of the prepared ethosomal systems over conventional liposomal systems, which helps in better penetration and permeation. The permeation of drug into the deep layers of the skin and its transdermal absorption could be due to the fusion of ethosomes with skin lipids and drug release at various points along the penetration pathway (Touitou et al., 2000). The synergistic mechanism between ethanol, vesicles and skin lipids was responsible for maximal permeation from ethosomal vesicles. The dense packing of stratum corneum lipid multi-layers at physiological temperature helped it to possess high conformational order. The intercalation of ethanol into the polar head group environment can result in an increase in the membrane permeability (Berner and Liu, 1995). The rate of permeation/flux was significantly higher (16.300 ± 0.300 mg/h/cm2) for ethosomal dispersion (CET 5) as compared to liposomal dispersion (CET 3; 8.671 ± 0.570 mg/h/cm2), cream base (CET 2; 5.851 ± 0.148 mg/ h/cm2) and aqueous solution of cetirizine (CET 1; 5.776 ± 0.300 mg/h/cm2) as depicted in Figure 2(B). The ethosomal dispersion showed 1.88, 2.78 and 2.82 times enhancement in permeation flux values as compared to liposomal dispersion, conventional cream and aqueous solution, respectively. Skin retention studies revealed that the ethosomal dispersion was better and showed higher skin retention (CET 5; 20.428 ± 0.932%) as compared to aqueous solution (CET 1;
1.861 ± 0.148%), cream base (CET 2; 3.669 ± 0.226%) and liposomal dispersion (CET 3; 6.199 ± 0.352%). A significantly greater retention achieved with ethosomes suggests them as better delivery systems for topical use. The enhancement in skin retention was 3.29, 5.56 and 10.97 times as compared to liposomes, conventional cream and aqueous solution, respectively (Figure 2B). This may be attributed to the depot forming characteristic of the vesicular systems. Furthermore, the higher retention of drug in the skin with ethosome vis-a`-vis liposomal systems may be accounted for the elastic nature of ethosomes as compared to liposomes that not only help in the better penetration of drug across the skin but also form micro-depots within the skin layers. Thus, it can be inferred that the prepared ethosomal formulations could effectively make the drug molecules accessible within skin layers, retaining them within close vicinity of the target site. Skin sensitivity studies and histopathological examination No change was observed in the anatomical structure, and no pathological changes were found on the skin of treated mice as depicted in Figure 3. Thus, the results established the dermal safety of prepared formulations on mice skin.
DOI: 10.3109/02652048.2014.918667
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Figure 4. Effect of different formulations of antihistaminic drugs on scratching score in mice (n ¼ 4).
Figure 5. Visual evaluation of erythema. (A) Control/plain gel, (B) oxazolone, (C) oral cetirizine aqueous solution, (D) cetirizine liposomal gel, (E) cetirizine ethosomal gel and (F) levocetirizine ethosomal gel-treated mice.
In vivo pharmacodynamic evaluation AD with signs of acute erythema and pruritus was produced by use of hapten, the oxazolone that is thought to evoke primarily a Th1dominated response. However, it has been recently reported that multiple challenges with oxazolone to hairless mice skin over an extended period causes the skin inflammation to shift from a typical Th1-dominated delayed type hypersensitivity response to a chronic Th2-dominated inflammatory response that is similar to human AD (Matsumoto et al., 2004; Man et al., 2008). Indeed, 9–10 challenges with oxazolone to hairless mice produced a chronic Th2-like skin inflammation. The inflammation was characterised by dermal infiltration of Th2 lymphocytes that express the PGD2 receptor CRTH, mast cells and eosinophils, increased expression of IL-4 in the dermis and highly elevated IgE levels. Repeated challenge with oxazolone led to increased epidermal hyperplasia and decreased expression of the skin differentiation proteins filaggrin, loricrin and involucrin. A skin barrier abnormality became evident and was associated with decreased stratum corneum ceramide content, decreased
stratum corneum hydration, transepidermal water loss and impaired lamellar body secretion, resulting in reduced lamellar membranes, as observed in AD patients (Jin et al., 2009). Both cetirizine and levocetirizine counteracted the effect of permeated chemical and caused marked reduction in erythema and scratching. Scratching score The scratching behaviour shown by mice was decreased upon application of drug-loaded ethosomal gel formulation. There was a statistically significant reduction in the mean number of scratchings/20 min in mice treated with cetirizine ethosomal gel (4.000 ± 0.816) as compared to the mice treated with oxazolone control group (65.000 ± 4.690), cetirizine oral aqueous solution (11.250 ± 0.957) and cetirizine liposomal gel (10.750 ± 0.957) as depicted in Figure 4. Thus, it is vividly apparent from the results that ethosomal system is much more effective in treating the scratching behaviour in mice than oral aqueous solution and liposomes. This may be due to the rigid nature and slow
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Figure 6. Effect of different formulations of antihistaminic drugs on quantitative evaluation of erythema score [0 ¼ no erythema, 1 ¼ slight erythema, 2 ¼ moderate erythema, 3 ¼ moderate to severe erythema and 4 ¼ severe erythema] (n ¼ 4).
Figure 7. (A) Histological analysis of skin: (a) Control/plain gel, (b) oxazolone, (c) oral cetirizine aqueous solution, (d) cetirizine liposomal gel, (e) cetirizine ethosomal gel, (f) levocetirizine ethosomal gel treated mice (arrows shows the presence of eosinophils) and (B) Effect of different formulations of antihistaminic drugs on dermal mast and eosinophil count (n ¼ 4).
penetration of liposomes, while ethosomes being flexible, penetrate easily across the skin and result in rapid therapeutic effect. The reduction in mean number of scratchings/20 min in mice treated with cetirizine ethosomal gel (4.000 ± 0.816) was significantly better than in mice treated with levocetirizine ethosomal gel (7.250 ± 1.893). These results suggest that cetirizine, the racemic mixture is more effective as compared to levo isomer, i.e. levocetirizine, in reducing the scratching score after induction of the disease with oxazolone for three weeks (Garg and Thami, 2007). Skin hyperplasia Single oxazolone challenge produced mild hyperplasia; this feature became more prominent with successive challenges.
The decrease in hyperplasia was observed in all the treated groups and was more prominent in the group treated with ethosomal system. Erythema score Development of erythema in different treatment groups was evaluated on the basis of erythema score. Erythema score, observed at the end of each week after drug application, was found to decrease subsequently as depicted in Figure 5. The erythema score was maximum (score 3) for the oxazolone treated group, while no erythema (score 0) was seen in the control (blank ethosomal gel) group (Figure 6). Both cetirizine oral aqueous solution-treated group and liposomal gel-treated group showed a significant decrease in the erythema score from 3 to 1
Ethosomes-based topical delivery system
DOI: 10.3109/02652048.2014.918667
after 6-week treatment. However, the decrease in erythema was more prominent in cetirizine ethosomal gel-treated group (score decreased from 3 to 0 after 6-week treatment) than that in liposomal gel and oral aqueous solution treated group, indicating the effectiveness of ethosomal gel over conventional systems. The reduction in erythema score in levocetirizine ethosomal gel treated group was similar as that of cetirizine ethosomal gel-treated group.
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Histological analysis of mast cells and dermal eosinophil count The number of mast cells and eosinophils increased in oxazolone treated mice as compared to control (Figure 7A). Upon application of cetirizine containing formulations, the eosinophil count decreased, but no significant inhibitory action on mast cell count was observed (Campoli-Richards et al., 1990). Cetirizine and levocetirizine act on H1 histaminic receptors, which are absent on mast cells thus having no inhibitory effect on mast cell production. Thus, both drugs showed no effect on mast cell count. The effect of cetirizine-containing formulations and levocetirizine formulation on eosinophil count was notable. The eosinophil count in oxazolone-treated group (92.00 ± 1.73) was found to be five times as compared to control (blank ethosomal gel) group (18.33 ± 3.51). Application of various cetirizine containing formulations resulted in a significant decrease in the number of eosinophils as compared to oxazolone-treated group (Figure 7B). This is due to the inhibitory effect of cetirizine on eotaxin, IL-4, RANTES, etc., which are responsible for eosinophil recruitment in dermal tissue. Eotaxin acts as a chemoattractant for eosinophils and plays an important role in eosinophil dominant allergic inflammation by upregulating the expression of adhesion molecules on endothelial cells (Bae et al., 2002). Cetirizine ethosomal gel-treated group showed a fourfold decrease in number of eosinophils (21.67 ± 1.53) as compared to oxazolone-treated group. The eosinophil count decreased significantly in ethosomal-treated group than that in conventional liposomes (36.00 ± 3.00) and oral aqueous solution (41.00 ± 6.00), which may be attributed to the flexible and pliable nature of ethosomes, allowing them to penetrate effectively and efficiently. However, there was no significant difference in eosinophil count between oral aqueous solution- and liposomal gel-treated groups. Cetirizine ethosomal gel was statistically more effective in decreasing the eosinophil count as compared to levocetirizine ethosomal gel (30.67 ± 3.06).
Conclusion Cetirizine is highly selective H1 antagonist used for the treatment of various allergies like urticaria, pollinosis and AD. This investigation was an attempt to develop a therapeutically effective dermal delivery system of cetirizine. The enhanced penetration and retention of drug within the skin layers using ethosomal delivery system obtained during ex vivo permeation studies are the direct reflections of improved delivery. And the latter was translated into improved therapeutic performance in the in vivo pharmacodynamic studies using oxazolone induced AD model. Furthermore, the topical route may help in reducing the somnolence on oral intake and provide a targeted delivery of cetirizine for a quick onset of action, generating high local tissue levels with minimal side effects, thus leading to enhanced therapeutic efficacy in addition to patient compliance. These highly promising results strongly suggest further clinical investigations of the novel ethosomal topical formulation of cetirizine for the treatment of AD, contact dermatitis, insect bites/stings, nettle rash, sunburn, neurodermatitis, urticaria and eczema. Furthermore, the topical ethosomal formulation of cetirizine
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appear to be a better alternative to oral route for the treatment of various skin allergy conditions.
Acknowledgements The authors gratefully acknowledge gift samples of levocetirizine dihydrochloride and cetirizine dihydrochloride supplied by IndSwift Ltd., India; PhospholiponÕ 90G, provided by Phospholipid GmbH, Nattermannallee, Koln, Germany; and Carbopol 980 NF from Lubrizol Advanced Materials India Private Limited, Mumbai, India.
Declaration of interest The authors declare no conflict of interest.
References Aggarwal N, Goindi S. Preparation and evaluation of antifungal efficacy of griseofulvin loaded deformable membrane vesicles in optimized guinea pig model of Microsporum canis-Dermatophytosis. Int J Pharm, 2012;437:277–87. Aggarwal N, Goindi S. Dermatopharmacokinetic and pharmacodynamic evaluation of ethosomes of griseofulvin designed for dermal delivery. J Nanopart Res, 2013;15:1983. doi 10.1007/s11051-013-1983-9. Bae S, Tanaka Y, Ogawa F, Takenaka M, Hamazaki Y, Shimizu K, Katayama I. Inhibitory effect of cetirizine on histamine-induced eotaxin production in normal human fibroblasts. Curr Ther Res, 2002;63:128–40. Berner B, Liu P. 1995. Alcohol. In: Smith EW, Maibach HI, eds. Percutaneous enhancer. Boca Raton, FL: CRC Press, pp. 45–60. Campoli-Richards DMB, Micaela M-T, Fitton A. Cetirizine: A review of its pharmacological properties and clinical potential in allergic rhinitis, pollen-induced asthma, and chronic urticaria. Drugs, 1990; 40:762–81. Chuang GCC, Yeh A-I. Rheological characteristics and texture attributes of glutinous rice cakes (mochi). J Food Engineering, 2006;74: 314–23. Curran MP, Scott LJ, Perry CM. Cetirizine: A review of its use in allergic disorders. Drugs, 2004;64:523–61. Dayan N, Touitou E. Carriers for skin delivery of trihexyphenidyl HCl: Ethosomes vs. liposomes. Biomaterials, 2000;21:1879–85. Dubey V, Mishra D, Dutta T, Nahar M, Saraf DK, Jain NK. Dermal and transdermal delivery of an anti-psoriatic agent via ethanolic liposomes. J Control Release, 2007;123:148–54. Elzainy AAW, Gu X, Simons FER, Simons KJ. Cetirizine from topical phosphatidylcholine-hydrogenated liposomes: Evaluation of peripheral antihistaminic activity and systemic absorption in a rabbit model. AAPS J, 2004;6(3):7–12. El Maghraby GMM, Williams AC, Barry BW. Skin delivery of 5-fluorouracil from ultra deformable and standard liposomes in vitro. J Pharm Pharmacol, 2001;53:1069–77. Garg G, Thami GP. Comparative efficacy of cetirizine and levocetirizine in chronic idiopathic urticaria. J Dermatol Treat, 2007;18:23–4. Jain S, Jain P, Umamahashwari RB, Jain NK. Transfersomes – A novel vesicular carrier for enhanced transdermal delivery: Development, characterization, and performance evaluation. Drug Dev Ind Pharm, 2003;29:1013–26. Jain SK, Gupta Y, Jain A, Rai K. Enhanced transdermal delivery of acyclovir sodium via elastic liposomes. Drug Deliv, 2008;15:141–7. Jin H, He R, Oyoshi M, Geha R. Animal models of atopic dermatitis. J Invest Dermatol, 2009;129(1):31–40. Lo´pez-Pinto JM, Gonza´lez-Rodrı´guez ML, Rabasco AM. Effect of cholesterol and ethanol on dermal delivery from DPPC liposomes. Int J Pharm, 2005;298:1–12. Man M, Hatano Y, Lee SH, Man M, Chang S, Feingold KR, Leung DYM, Holleran W, Uchida Y, Elias PM. Characterization of a hapteninduced, murine model with multiple features of atopic dermatitis: Structural, immunologic, and biochemical changes following single versus multiple oxazolone challenges. J Invest Dermatol, 2008;128: 79–86. Martin A, Swarbrick J, Cammarata A. 1991. Rheology, physical pharmacy. Bombay: Varghese Publishing house, pp. 525–6. Matsumoto K, Mizukoshi K, Oyobikawa M, Ohshima H, Tagami H. Establishment of an atopic dermatitis-like skin model in a hairless mouse by repeated elicitation of contact hypersensitivity that enables to
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conduct functional analyses of the stratum corneum with various noninvasive biophysical instruments. Skin Res Technol, 2004;10:122–9. Mishraa D, Mishrab PK, Dubeya V, Nahara M, Dabadghaob S, Jain NK. Systemic and mucosal immune response induced by transcutaneous immunization using Hepatitis B surface antigen-loaded modified liposomes. Eur J Pharm Sci, 2008;33:424–33. Oshio T, Sasaki Y, Funakoshi-Tago M, Aizu-Yokota E, Sonoda Y, Matsuoka H, Kasahara T. Dermatophagoides farinae extract induces severe atopic dermatitis in NC/Nga mice, which is effectively suppressed by the administration of tacrolimus ointment. Int Immunopharmacol, 2009;9:403–11. Ousler GW, Wilcox KA, Gupta G, Abelson MB. An evaluation of the ocular drying effects of 2 systemic antihistamines: Loratadine and cetirizine hydrochloride. Ann Allergy Asthma Immunol, 2004;93: 460–4.
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Touitou E, Dayan N, Bergelson L, Godin B, Eliaz M. Ethosomes-novel vesicular carriers for enhanced delivery: Characterization and skin penetration properties. J Control Release, 2000;65:403–18. Vanden Bergh BAI, Wertz PW, Junginger HE, Bouwstra JA. Elasticity of vesicles assessed by electron spin resonance, electron microscopy and extrusion measurement. Int J Pharm, 2001;217:13–24. Walch H, Baden DE. 2004. Topical application of cetirizine and loratadine. United State Oramon Arzneimittel GmbH (Laupheim, DE). US Patent No. 6,790,847 B2. Available at: http://www.freepatentsonline.com/6790847.html. Accessed 12 May 2013. Wankhede SB, Lad KA, Chitlange SS. Development and validation of UV-spectrophotometric methods for simultaneous sstimation of cetirizine hydrochloride and phenylephrine hydrochloride in tablets. Int J Pharm Sci Drug Res, 2012;4:222–6.
Ethosomes-based topical delivery system of antihistaminic drug for treatment of skin allergies Shishu Goindi, Bhavnita Dhatt, and Amanpreet Kaur
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Department of Pharmaceutics, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India
Abstract
Keywords
Cetirizine is indicated for the treatment of allergic conditions such as insect bites and stings, atopic and contact dermatitis, eczema, urticaria. This investigation deals with development of a novel ethosome-based topical formulation of cetirizine dihydrochloride for effective delivery. The optimised formulation consisting of drug, phospholipon 90 Gä and ethanol was characterised for drug content, entrapment efficiency, pH, vesicular size, spreadability and rheological behaviour. The ex vivo permeation studies through mice skin showed highest permeation flux (16.300 ± 0.300 mg/h/cm2) and skin retention (20.686 ± 0.517 mg/cm2) for cetirizine-loaded ethosomal vesicles as compared to conventional formulations. The in vivo pharmacodynamic evaluation of optimised formulation was assessed against oxazoloneinduced atopic dermatitis (AD) in mice. The parameters evaluated were reduction in scratching score, erythema score, skin hyperplasia and dermal eosinophil count. Our results suggest that ethosomes are effective carriers for dermal delivery of antihistaminic drug, cetirizine, for the treatment of AD.
Cetirizine, ethosomes, hapten, levocetirizine, skin allergy
Introduction Histamine is a biogenic amine present as a tissue hormone especially in the skin and in the lungs of humans. In the human body, it is stored in the basophilic granulocytes and the mastocytes. It is also present in bee poison and in the salivary secretion of biting/stinging insects. When allergic skin reactions occur, excess histamine is released and is one of the factors responsible for itching and the formation of skin wheals and flares. Currently, antihistamines are primarily administered orally to treat these effects of histamine releases (Walch and Baden, 2004). Cetirizine is a potent second-generation antihistaminic with marked affinity for peripheral H1 receptors and is used for the treatment of various allergies like urticaria, pollinosis and atopic dermatitis (AD). It inhibits the release of histamine and cytotoxic mediators from platelets as well as eosinophil chemotaxis during the secondary phase of allergic response. Studies have shown cetirizine to be effective in the treatment of skin inflammatory conditions by reducing the release of histamine, bradykinin and allergen-induced wheal and flare reactions; decreasing monocyte and T-lymphocyte chemotaxis; reducing eosinophil responses; and decreasing intercellular adhesion molecule-1 expression on epithelial cells (Campoli-Richards et al., 1990; Curran et al., 2004). The recommended oral dose for cetirizine is 10 mg once per day for adults and 2.5–5 mg once per day for children. In general, cetirizine is used to treat hay fever, allergic rhinitis, chronic urticaria and asthma. Recently, it has been recommended for treatment of pruritus associated with AD
Address for correspondence: Prof. Shishu Goindi, Professor in Pharmaceutics, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh – 160014, India. Tel: 0172-2541142, 0172-2534281 (O). E-mail: [email protected]
History Received 6 October 2013 Revised 16 April 2014 Accepted 21 April 2014 Published online 23 June 2014
(dry eczema). The oral administration of cetirizine is associated with setbacks like sedation, corneal and conjunctival staining due to ocular dryness, dry mouth and diarrhoea (Ousler et al., 2004). Therefore, to overcome the systemic side effects associated with oral therapy and for quick onset of action and targeted drug delivery to inflamed skin, a topical delivery using novel drug carrier may be more beneficial. No topical formulation of cetirizine is available in the market till date. Some literature reports suggest use of gels (Walch and Baden, 2004) and conventional liposomes (Elzainy et al., 2004) as topical carriers for cetirizine. Earlier, we have reported the feasibility of TransfersomesÕ (elastic vesicles) for the topical delivery of cetirizine. In the next step of our research, we aim to develop ethosomal carriers of cetirizine in context of obtaining better penetration as compared to conventional liposomes and investigating the in vivo pharmacodynamic activity against hapten, the oxazolone-induced AD murine model. Ethosomal carriers are innovative delivery systems containing vesicles composed mainly of phospholipid (PL; Phospholipon 90G), ethanol and water (Touitou et al., 2000; Dubey et al., 2007). The ethosomes being soft and malleable vesicles efficiently penetrate the skin and allow enhanced delivery of various compounds. In fact, ethosomes have emerged as nascent carriers for the delivery of both hydrophilic and lipophilic molecules (Dayan and Touitou, 2000; Mishraa et al., 2008). This work thus focuses on developing a novel ethosomal carrier-based topical delivery system of cetirizine to overcome the side effects associated with conventional oral therapy and to provide targeted therapy with enhanced skin delivery. The in vivo pharmacodynamic evaluation was done using hapten (oxazolone)induced AD in mice, and the activity of the developed formulation was compared with cetirizine oral solution, cetirizine cream and levocetirizine ethosomal gel.
Ethosomes-based topical delivery system
DOI: 10.3109/02652048.2014.918667
Materials and methods Materials Phospholipon 90G was a gift sample from Phospholipids GmbH (Nattermannallee, Koln, Germany). Cetirizine dihydrochloride and levocetirizine dihydrochloride were obtained as generous gift samples from Indswift Ltd (Mohali, India). Carbopol 980 NF was received as a gift sample from Lubrizol Advanced Materials India Private Limited, Mumbai, India. All other reagents and chemicals were of analytical grade.
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Animals Female mice (BALB/c strain, 25–27 g) were obtained from Central Animal House, Punjab University, and used for carrying out ex vivo permeation, histopathology and pharmacodynamic studies. Ethical approval to perform these studies was obtained from Institutional Animal Ethics Committee, Punjab University, India, and their guidelines were followed throughout the studies. Preparation of different topical formulations of cetirizine Ethosomal vesicles were prepared by method developed by Touitou et al., (2000). Accurately weighed amount of Phospholipon 90 G was dissolved in ethanol at 30 C in a specially designed closed beaker (Table 1). Cetirizine (0.1%) was dissolved in phosphate buffer saline (PBS) pH 6.4 under similar conditions. The aqueous phase was added slowly to ethanol in a fine stream with constant stirring at 2000–3000 rpm in wellclosed specially designed vessel to avoid loss of ethanol. Mixing was continued for an additional 5 min. The prepared vesicles were stored at 4–8 C in a refrigerator overnight and then subjected to further characterisation and evaluation studies. Furthermore, the optimised ethosomal dispersion was gelled using 1.25% carbopol to make it suitable for topical application. Liposomes were prepared by thin film hydration method using phosphatidylcholine and cholesterol (molar ratio 7:3) as previously described by Aggarwal and Goindi (2012). An oil/water cream containing 0.1% cetirizine was prepared by separately heating both oil phase (light liquid paraffin 10% w/w, isopropyl myristate 12% w/w, cetyl alcohol 5% w/w, stearic acid 6% w/w, butylated hydroxytoluene 0.2% w/w and glyceryl monostearate 5% w/w) and aqueous phase (drug 0.1% w/w, triethanolamine 1.6% w/w, propylene glycol 5% w/w, sodium metabisulfite 0.5% w/w and water q.s. 100% w/w) at 65 C. The aqueous
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phase was added to oil phase under continuous stirring. The resulting emulsion was allowed to cool down gradually under constant stirring to obtain a cream. Furthermore, the aqueous solution was prepared by dissolving 0.1% (w/v) of cetirizine in distilled water. Vesicle morphology, size analysis and zeta potential The ethosomal vesicles were visualised using transmission electron microscopy (TEM; Philips CM12 electron microscope, Eindhoven, The Netherlands) to study the morphology and structure of vesicles. The samples were negatively stained with a 1% aqueous solution of phosphotungstic acid, vesicles were dried on a microscopic carbon-coated grid and viewed under the microscope at a suitable magnification. Photo-micrographs of the vesicles were taken at suitable magnification. The vesicle size and size distribution were determined by dynamic light scattering (DLS) technique using a computerised Malvern ZetasizerÔ (Malvern Instruments Ltd., Worcestershire, UK). For determination of zeta potential and polydispersity index (PDI) of the selected formulation, Delsa Nano (Beckman Coulter, Brea, CA) was used. Zeta potential measurements were done at 25 C and the electric field strength was 23.2 V/cm. Entrapment efficiency and transmittance/turbidity determination For determination of entrapment efficiency, the vesicular formulation was kept overnight at 4 C followed by ultracentrifugation at 35 000 rpm for 3 h at 4 C. After removing the supernatant, the drug quantity was determined in both the sediment (after disrupting vesicles using methanol) and supernatant. The entrapment efficiency was calculated using the formula: ðT C Þ 100 T where T is the total amount of drug that is detected both in supernatant and sediment, and C is the amount of drug detected only in the supernatant (Touitou et al., 2000). Transmittance of different ethosomal formulations was determined at a wavelength of 500 nm by diluting the suspension (0.1 mL) to 10 mL with distilled water and setting it as blank at 100% transmittance (Jain et al., 2008). Rheological behaviour of the developed system
Table 1. Optimisation of ethosomal formulations for phospholipid, drug and ethanol concentration (n ¼ 3). Ethanol No. of % % entrapment Formulation Ratio of content vesicles/ code lipid:drug (%) mm3 103 transmittance efficiency E-1 E-2 E-3 E-4 E-5 E-6 E-7 E-8 E-9 E-10 E-11 E-12
20:1 25:1 30:1 35:1 40:1 45:1 50:1 35:1 35:1 35:1 35:1 35:1
30 30 30 30 30 30 30 25 35 40 45 50
265.0 347.5 375.0 415.0 405.0 412.5 380.0 225.0 447.5 500.0 312.5 212.5
60.2 53.0 44.1 34.7 38.9 37.8 36.0 55.6 36.8 33.8 52.7 58.7
45.11 ± 2.07 52.10 ± 1.05 61.57 ± 0.53 66.96 ± 0.57 63.66 ± 0.50 61.96 ± 2.19 62.06 ± 1.51 50.13 ± 0.42 68.85 ± 1.39 72.85 ± 1.57 53.35 ± 0.90 47.45 ± 2.21
E-4 is the optimized formulation on the basis of lipid:drug ratio. E-10 is the final optimized formulation on the basis of ethanol content and selected for further characterization studies.
The gelled system was evaluated for its rheological behaviour using rotational viscometer (Paar Physica MC 1, Brookfield DVII, UK) which is a cup and bob type viscometer (Martin et al., 1991) equipped with Z 4 sensor probe. The viscosity was determined at various shear rates by subjecting the gel to different torque values. The temperature was kept at 30 C, and spindle no. 29 was employed for viscosity determination. The rheograms thus generated provided useful insight into the flow properties of the formulations. Degree of deformability Vesicle extruder (Eastern Sci. Inc., Rockville, MD) was used to determine the degree of deformability. The deformability index of ethosomal vesicles was determined using mini filtration technique and compared with liposomal formulation. In brief, the vesicular dispersion was passed through polycarbonate filter of 50 nm. The vesicle size and size distribution measurement was monitored before and after filtration by DLS measurements using Malvern
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ZetasizerÔ 2000. Percentage deformability was calculated using the following formula: Vesicle size before extrusion vesicle size after extrusion % deformability ¼ 100 Vesicle size before extrusion Spreadability The spreadability of the developed vesicular gel was evaluated using Texture AnalyserÔ, equipped with a 5-kg load cell to determine different rheological properties of prepared vesicular gel such as work of shear, force of gel extrusion, stickiness and firmness (Chuang and Yeh, 2006).
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Skin permeation and deposition studies Franz diffusion cell assembly was used to study the permeation of cetirizine from different formulations through hairless dorsal skin of mice, with receptor compartment volume of 30 mL and effective diffusion area of 3.14 cm2. PBS pH 6.4 was selected as diffusion medium. The receptor cells containing diffusion medium were constantly stirred with a magnetic stirrer and equilibrated at 37 ± 1 C with the help of thermo-regulated outer water jacket. The different formulations of cetirizine equivalent to 0.5 mg were applied onto the preshaved mice skin in the donor compartment. Aliquots of 2 mL were withdrawn through the sampling port at suitable time intervals and analysed for drug concentration employing UV spectrophotometer at 232 nm after appropriate dilutions (Wankhede et al., 2012). Equal volume of diffusion medium was replaced into the receptor cells to maintain proper sink conditions. At the end of the permeation experiments, the skin surface in the donor compartment was rinsed with ethanol to remove excess drug from the surface. The receptor media was then replaced with 50% (v/v) ethanol (El Maghraby et al., 2001; Jain et al., 2003). The contents were allowed to stir for next 24 h followed by spectrophotometric determination at max 232 nm for amount of drug retained in skin. Similar permeation and skin retention studies were performed using blank formulations, i.e. without drug and absorbance values, were subtracted from the test formulations to account for the effect of excipients. The cumulative amount permeated per unit area (mg/cm2), flux (mg/h/cm2) and skin retention (mg/cm2) were calculated. All the experiments were performed in triplicate. All data were statistically analysed by one way analysis of variance followed by Dunnett’s method. Results were quoted as statistically significant where p50.05. Skin-sensitivity studies and histopathological examination Ethosomal gel was applied uniformly on the preshaved dorsal region of mice skin and kept in contact for 4 h. After that, the animals were sacrificed by cervical dislocation method and exposed dorsal surface was cut. Then, each specimen was fixed in 10% buffered formalin; embedded in paraffin and microtomed. The sections were stained with hematoxylin and eosin. Finally, the specimens were observed under a high-power light microscope and were evaluated for their integrity. In vivo pharmacodynamic evaluation Female BALB/c mice, 6–7 weeks old, were used to induce AD by topical treatment with hapten, the oxazolone. Animals were divided into six groups (A, B, C, D, E and F) containing four animals in each group (Table 2). The animals of group B, C, D, E
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Table 2. Different treatments given to various animal groups during in vivo evaluations using oxazolone-induced atopic dermatitis in mice (n ¼ 4). Group A B C D E F
Treatment (formulation) Control/plain gel (without drug) Oxazolone Oxazolone + oral cetirizine aqueous solution Oxazolone + cetirizine liposomal gel Oxazolone + cetirizine ethosomal gel Oxazolone + levocetirizine ethosomal gel
and F were sensitised through topical application on to their shaved back skin of 10 mL of 5% oxazolone in ethanol daily for one week. After one week, the same animals were challenged with 60 mL of 0.1% oxazolone once every alternate day for an additional two weeks (Man et al., 2008). After half an hour of oxazolone application, cetirizine oral aqueous solution, cetirizine liposomal gel, cetirizine ethosomal gel and levocetirizine ethosomal gel each containing 1.0 mg drug was applied daily on the back of animals of group C, D, E and F, respectively. The animals were evaluated every week for change in erythema score and number of scratchings. After completion of three weeks, animals were sacrificed and skin was removed. Excised skin specimens were fixed in 10% neutral formalin and embedded in paraffin. Sections, 5-mm thick, were prepared and stained with hematoxylin and eosin. The number of eosinophils and mast cells were counted under light microscope (400 magnification) in 100 mm by 100 mm area (50 fields) and compared (Oshio et al., 2009).
Results and discussion Preparation of ethosomes Cetirizine-loaded ethosomes were prepared by cold method. The various ratios of PL to drug in the presence of fixed amount of ethanol were investigated (Table 1). For optimising the composition of vesicular formulations, various parameters like the magnitude of drug entrapment, number of vesicles per cubic mm and percent transmittance were studied. It was observed that the drug entrapment increases upon increasing the amount of PL due to the increased availability of the lipid phase for cetirizine and its subsequent incorporation in the lipid bilayers (Aggarwal and Goindi, 2013). Further increase in the amount of lipid decreases the entrapment efficiency because of decreased number of vesicular structures due to incomplete hydration of lipid or partly due to lower partitioning of hydrosoluble drug in PL. However, a very slight increase in entrapment efficiency was observed probably due to an increase in size of the decreased number of vesicles formed at such high PL ratios (50:1). Similar results were observed in case of number of vesicles per cubic mm and percent transmittance. Lower the transmittance, higher the turbidity, therefore, higher the number of vesicles. Thus, percent transmittance was found to decrease to a minimum of 34.7%. Of all the formulation batches containing constant ethanol content (30%), batch E-4 showed maximum entrapment efficiency of 66.96 ± 0.57%, maximum number of vesicles per cubic mm (415.0) and least % transmittance (34.7%). Thus, the above batch, E-4 with lipid–drug ratio 35:1, was finally selected for optimising the concentration of ethanol. The increase in concentration of ethanol from 20% to 50% increased the entrapment efficiency owing to increase in fluidity of membranes leading to higher drug loading on vesicles. The formulation batch, E-10 containing 40% of ethanol, was found to show maximum entrapment (72.85 ± 1.57%). However, further
Ethosomes-based topical delivery system
DOI: 10.3109/02652048.2014.918667
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increase in ethanol percentage probably made the vesicle membrane leaky due to greater disturbance of the lipid-bilayer organisation of vesicles by increased solubilisation of PL in ethanol causing breakdown of vesicular structures, thus leading to decrease in entrapment efficiency. The influence of increasing percentage of ethanol on number of vesicles per cubic mm, percent transmittance and their relative abundance was in coherence with the results of entrapment efficiency. Thus, the formulation batch E-10 showed highest entrapment efficiency of 72.85 ± 1.57%, maximum number of vesicles per cubic mm (500) and least percent transmittance (33.8%). On the basis of all the studies performed, the formulation batch E-10 containing PL:drug (35:1) and ethanol (40%) was selected as the optimised formulation, as it showed minimum transmittance, maximum number of vesicles and highest entrapment efficiency. Morphological evaluation of vesicles Morphological characterisation of the ethosomes as well as existence of their vesicular structure was confirmed by TEM. The prepared ethosomal vesicles were unilamellar and near spherical in shape as depicted in Figure 1(A).
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stress is increased, the normally disarranged molecules begin to align their long axis in the direction of flow. This orientation reduces the internal resistance of the material and allows a greater rate of shear at each successive shearing stress (Martin et al., 1991). In addition, some of the solvent associated with the molecules may be released resulting in an effective lowering of the concentration and the size of the dispersed molecules. This results in lowering of apparent viscosity. Degree of deformability The results indicated that vesicle size of ethosomes before and after extrusion was 180.1 nm and 152.2 nm, whereas that of liposomes was 220.2 nm and 112.1 nm, respectively. Ethosomes got deformed by 15.490%, while liposomes got deformed by 49.09%, which showed that ethosomes were almost three times more flexible than liposomes. This was because liposomes were rigid due to the presence of cholesterol in their structure (Lo´pez-Pinto et al., 2005), while ethosomes were flexible due to the interdigitation induced by ethanol in the lipid bilayer. Ethanol also provided elasticity to vesicle membrane by reducing interfacial tension of the vesicle membrane. Similar results were reported in published literature (Vanden Bergh et al., 2001).
Vesicle size, size distribution and zeta potential The average vesicular size of prepared ethosomal formulation was 180.1 nm along with PDI of 0.278. The log size distribution curve showed normal distribution, and the low value of PDI indicates that the dispersion was homogeneous (Figure 1B). Zeta potential is the electric potential of the vesicle, including its ionic atmosphere (stern layer). The zeta-potential of the optimised formulation was 15.7 mV, i.e. negative value. This was due to the presence of ethanol, which confers a negative charge to the surface of ethosomes. Rheological studies The results of rheological profile of ethosomal dispersions gelled with 1.25% carbopol 980F depicted the non-Newtonian behaviour of flow. The characteristic bent of the rheogram towards the shear stress axis and the decrease in viscosity with increasing rate of shear indicated that the developed formulation exhibited pseudoplastic flow. This pseudoplasticity results from a shearing action on long chain molecules of the gelling polymer. As the shearing
Spreadability The non-sticky nature of optimised formulation was supported by various parameters such as firmness (2.297 kg), work of shear (1.020 kgsec), work of adhesion (0.271 kgsec) and stickiness (2.835 kg). The optimised formulation showed acceptable spreadability and was free from severe adhesion as it required less force and work of adhesion. Skin permeation and deposition studies The mean cumulative amount permeated per unit area from various cetirizine formulations (coded as CET 1 to CET 6) is shown in Figure 2(A). The mean cumulative amount permeated per unit area in 8 h was maximum from ethosomal dispersion (CET 5) was 119 ± 0.001 mg/cm2) followed by ethosomal gel (CET 6; 108 ± 0.079 mg/cm2), liposomal dispersion (CET 3; 60 ± 0.001 mg/cm2), liposomal gel (CET 4; 55 ± 0.001 mg/cm2), conventional cream (CET 2; 53 ± 0.001 mg/cm2), aqueous solution of cetirizine (CET 1; 52 ± 0.001 mg/cm2) in 8 h. This is attributed
Figure 1. (A) Transmission electron microscopy (magnification 40 000) and (B) vesicle size distribution of the prepared cetirizine ethosomal dispersion.
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Figure 2. Comparison of (A) ex-vivo permeation profiles, (B) permeation flux and skin retention of cetirizine from various formulations through mice skin (n ¼ 3).
Figure 3. Histopathological photographs showing the absence of any histological and pathological changes. (A) Control untreated skin and (B) skin treated with ethosomal gel (magnification, 100).
to the flexible membrane packing characteristics of the prepared ethosomal systems over conventional liposomal systems, which helps in better penetration and permeation. The permeation of drug into the deep layers of the skin and its transdermal absorption could be due to the fusion of ethosomes with skin lipids and drug release at various points along the penetration pathway (Touitou et al., 2000). The synergistic mechanism between ethanol, vesicles and skin lipids was responsible for maximal permeation from ethosomal vesicles. The dense packing of stratum corneum lipid multi-layers at physiological temperature helped it to possess high conformational order. The intercalation of ethanol into the polar head group environment can result in an increase in the membrane permeability (Berner and Liu, 1995). The rate of permeation/flux was significantly higher (16.300 ± 0.300 mg/h/cm2) for ethosomal dispersion (CET 5) as compared to liposomal dispersion (CET 3; 8.671 ± 0.570 mg/h/cm2), cream base (CET 2; 5.851 ± 0.148 mg/ h/cm2) and aqueous solution of cetirizine (CET 1; 5.776 ± 0.300 mg/h/cm2) as depicted in Figure 2(B). The ethosomal dispersion showed 1.88, 2.78 and 2.82 times enhancement in permeation flux values as compared to liposomal dispersion, conventional cream and aqueous solution, respectively. Skin retention studies revealed that the ethosomal dispersion was better and showed higher skin retention (CET 5; 20.428 ± 0.932%) as compared to aqueous solution (CET 1;
1.861 ± 0.148%), cream base (CET 2; 3.669 ± 0.226%) and liposomal dispersion (CET 3; 6.199 ± 0.352%). A significantly greater retention achieved with ethosomes suggests them as better delivery systems for topical use. The enhancement in skin retention was 3.29, 5.56 and 10.97 times as compared to liposomes, conventional cream and aqueous solution, respectively (Figure 2B). This may be attributed to the depot forming characteristic of the vesicular systems. Furthermore, the higher retention of drug in the skin with ethosome vis-a`-vis liposomal systems may be accounted for the elastic nature of ethosomes as compared to liposomes that not only help in the better penetration of drug across the skin but also form micro-depots within the skin layers. Thus, it can be inferred that the prepared ethosomal formulations could effectively make the drug molecules accessible within skin layers, retaining them within close vicinity of the target site. Skin sensitivity studies and histopathological examination No change was observed in the anatomical structure, and no pathological changes were found on the skin of treated mice as depicted in Figure 3. Thus, the results established the dermal safety of prepared formulations on mice skin.
DOI: 10.3109/02652048.2014.918667
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Figure 4. Effect of different formulations of antihistaminic drugs on scratching score in mice (n ¼ 4).
Figure 5. Visual evaluation of erythema. (A) Control/plain gel, (B) oxazolone, (C) oral cetirizine aqueous solution, (D) cetirizine liposomal gel, (E) cetirizine ethosomal gel and (F) levocetirizine ethosomal gel-treated mice.
In vivo pharmacodynamic evaluation AD with signs of acute erythema and pruritus was produced by use of hapten, the oxazolone that is thought to evoke primarily a Th1dominated response. However, it has been recently reported that multiple challenges with oxazolone to hairless mice skin over an extended period causes the skin inflammation to shift from a typical Th1-dominated delayed type hypersensitivity response to a chronic Th2-dominated inflammatory response that is similar to human AD (Matsumoto et al., 2004; Man et al., 2008). Indeed, 9–10 challenges with oxazolone to hairless mice produced a chronic Th2-like skin inflammation. The inflammation was characterised by dermal infiltration of Th2 lymphocytes that express the PGD2 receptor CRTH, mast cells and eosinophils, increased expression of IL-4 in the dermis and highly elevated IgE levels. Repeated challenge with oxazolone led to increased epidermal hyperplasia and decreased expression of the skin differentiation proteins filaggrin, loricrin and involucrin. A skin barrier abnormality became evident and was associated with decreased stratum corneum ceramide content, decreased
stratum corneum hydration, transepidermal water loss and impaired lamellar body secretion, resulting in reduced lamellar membranes, as observed in AD patients (Jin et al., 2009). Both cetirizine and levocetirizine counteracted the effect of permeated chemical and caused marked reduction in erythema and scratching. Scratching score The scratching behaviour shown by mice was decreased upon application of drug-loaded ethosomal gel formulation. There was a statistically significant reduction in the mean number of scratchings/20 min in mice treated with cetirizine ethosomal gel (4.000 ± 0.816) as compared to the mice treated with oxazolone control group (65.000 ± 4.690), cetirizine oral aqueous solution (11.250 ± 0.957) and cetirizine liposomal gel (10.750 ± 0.957) as depicted in Figure 4. Thus, it is vividly apparent from the results that ethosomal system is much more effective in treating the scratching behaviour in mice than oral aqueous solution and liposomes. This may be due to the rigid nature and slow
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Figure 6. Effect of different formulations of antihistaminic drugs on quantitative evaluation of erythema score [0 ¼ no erythema, 1 ¼ slight erythema, 2 ¼ moderate erythema, 3 ¼ moderate to severe erythema and 4 ¼ severe erythema] (n ¼ 4).
Figure 7. (A) Histological analysis of skin: (a) Control/plain gel, (b) oxazolone, (c) oral cetirizine aqueous solution, (d) cetirizine liposomal gel, (e) cetirizine ethosomal gel, (f) levocetirizine ethosomal gel treated mice (arrows shows the presence of eosinophils) and (B) Effect of different formulations of antihistaminic drugs on dermal mast and eosinophil count (n ¼ 4).
penetration of liposomes, while ethosomes being flexible, penetrate easily across the skin and result in rapid therapeutic effect. The reduction in mean number of scratchings/20 min in mice treated with cetirizine ethosomal gel (4.000 ± 0.816) was significantly better than in mice treated with levocetirizine ethosomal gel (7.250 ± 1.893). These results suggest that cetirizine, the racemic mixture is more effective as compared to levo isomer, i.e. levocetirizine, in reducing the scratching score after induction of the disease with oxazolone for three weeks (Garg and Thami, 2007). Skin hyperplasia Single oxazolone challenge produced mild hyperplasia; this feature became more prominent with successive challenges.
The decrease in hyperplasia was observed in all the treated groups and was more prominent in the group treated with ethosomal system. Erythema score Development of erythema in different treatment groups was evaluated on the basis of erythema score. Erythema score, observed at the end of each week after drug application, was found to decrease subsequently as depicted in Figure 5. The erythema score was maximum (score 3) for the oxazolone treated group, while no erythema (score 0) was seen in the control (blank ethosomal gel) group (Figure 6). Both cetirizine oral aqueous solution-treated group and liposomal gel-treated group showed a significant decrease in the erythema score from 3 to 1
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after 6-week treatment. However, the decrease in erythema was more prominent in cetirizine ethosomal gel-treated group (score decreased from 3 to 0 after 6-week treatment) than that in liposomal gel and oral aqueous solution treated group, indicating the effectiveness of ethosomal gel over conventional systems. The reduction in erythema score in levocetirizine ethosomal gel treated group was similar as that of cetirizine ethosomal gel-treated group.
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Histological analysis of mast cells and dermal eosinophil count The number of mast cells and eosinophils increased in oxazolone treated mice as compared to control (Figure 7A). Upon application of cetirizine containing formulations, the eosinophil count decreased, but no significant inhibitory action on mast cell count was observed (Campoli-Richards et al., 1990). Cetirizine and levocetirizine act on H1 histaminic receptors, which are absent on mast cells thus having no inhibitory effect on mast cell production. Thus, both drugs showed no effect on mast cell count. The effect of cetirizine-containing formulations and levocetirizine formulation on eosinophil count was notable. The eosinophil count in oxazolone-treated group (92.00 ± 1.73) was found to be five times as compared to control (blank ethosomal gel) group (18.33 ± 3.51). Application of various cetirizine containing formulations resulted in a significant decrease in the number of eosinophils as compared to oxazolone-treated group (Figure 7B). This is due to the inhibitory effect of cetirizine on eotaxin, IL-4, RANTES, etc., which are responsible for eosinophil recruitment in dermal tissue. Eotaxin acts as a chemoattractant for eosinophils and plays an important role in eosinophil dominant allergic inflammation by upregulating the expression of adhesion molecules on endothelial cells (Bae et al., 2002). Cetirizine ethosomal gel-treated group showed a fourfold decrease in number of eosinophils (21.67 ± 1.53) as compared to oxazolone-treated group. The eosinophil count decreased significantly in ethosomal-treated group than that in conventional liposomes (36.00 ± 3.00) and oral aqueous solution (41.00 ± 6.00), which may be attributed to the flexible and pliable nature of ethosomes, allowing them to penetrate effectively and efficiently. However, there was no significant difference in eosinophil count between oral aqueous solution- and liposomal gel-treated groups. Cetirizine ethosomal gel was statistically more effective in decreasing the eosinophil count as compared to levocetirizine ethosomal gel (30.67 ± 3.06).
Conclusion Cetirizine is highly selective H1 antagonist used for the treatment of various allergies like urticaria, pollinosis and AD. This investigation was an attempt to develop a therapeutically effective dermal delivery system of cetirizine. The enhanced penetration and retention of drug within the skin layers using ethosomal delivery system obtained during ex vivo permeation studies are the direct reflections of improved delivery. And the latter was translated into improved therapeutic performance in the in vivo pharmacodynamic studies using oxazolone induced AD model. Furthermore, the topical route may help in reducing the somnolence on oral intake and provide a targeted delivery of cetirizine for a quick onset of action, generating high local tissue levels with minimal side effects, thus leading to enhanced therapeutic efficacy in addition to patient compliance. These highly promising results strongly suggest further clinical investigations of the novel ethosomal topical formulation of cetirizine for the treatment of AD, contact dermatitis, insect bites/stings, nettle rash, sunburn, neurodermatitis, urticaria and eczema. Furthermore, the topical ethosomal formulation of cetirizine
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appear to be a better alternative to oral route for the treatment of various skin allergy conditions.
Acknowledgements The authors gratefully acknowledge gift samples of levocetirizine dihydrochloride and cetirizine dihydrochloride supplied by IndSwift Ltd., India; PhospholiponÕ 90G, provided by Phospholipid GmbH, Nattermannallee, Koln, Germany; and Carbopol 980 NF from Lubrizol Advanced Materials India Private Limited, Mumbai, India.
Declaration of interest The authors declare no conflict of interest.
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