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In vitro studies on release and skin permeation of nonivamide from novel oil-in-oil-emulsions
European Journal of Pharmaceutics and Biopharmaceutics xxx (2013) xxx–xxx
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Research paper
In vitro studies on release and skin permeation of nonivamide from novel oil-in-oil-emulsions Michael Rottke, Dominique Jasmin Lunter, Rolf Daniels ⇑ Department of Pharmaceutical Technology, Eberhard Karls University, Tuebingen, Germany
a r t i c l e
i n f o
Article history: Received 10 July 2013 Accepted in revised form 26 September 2013 Available online xxxx Keywords: Nonivamide Topical delivery Permeation Pig ear skin Silicone oil Oil-in-oil-emulsion
a b s t r a c t The purpose of this study was to develop oil-in-oil-emulsions that facilitate long-term treatment for chronic pruritus with capsaicinoids. To this end, oil-in-oil-emulsions, which comprised polydimethyl siloxanes, silicone surfactant and castor oil, were examined. We used nonivamide, a synthetic analogue of capsaicin as the active pharmaceutical ingredient. It was incorporated into castor oil that formed the dispersed phase of the emulsion. We evaluated the influence of formulation variables (nonivamide content, phase volume ratio and viscosity of the silicone oil) on the in vitro release and the permeation of nonivamide. Permeation was found to be controlled by the nonivamide concentration in the dispersed phase and the phase volume ratio. Oil-in-oil-emulsions were found to produce constant permeation rates over a period of 10 h. They are thus superior to conventional semisolid formulations as application intervals may be extended. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction The skin is the largest organ in the human body. It protects us not only from dehydration but also from intrusion of xenobiotics, parasites and microorganisms. Moreover, exogenous noxae may activate special nerves in the skin which physiologically results in itching and scratching. By scratching, potentially harmful substances are removed from the skin. But itch may also be a symptom that accompanies skin diseases [1]. Depending on the pathogenesis of the itch, different mediators and receptors may be involved in the development of pathologic itches. This makes therapy difficult and often insufficient [2]. One option in the therapy of chronic itch is the topical treatment with capsaicinoids. Although capsaicinoids may initially induce pain and itch when applied to the skin, longterm topical administration leads to effective relief. The mechanism of action is presumed to involve TPRV 1 (transient receptor potential type 1), a nonselective cation channel expressed on the peripheral endings of Ad- and C-fibres in the viable epidermis. The binding of capsaicinoids to TRPV 1 results in release of substance P, CGRP and other vasodilatative substances that induce neurogenic inflammation and oedema. If application of capsaicinoids is continued, the persistent influx of cations, mainly calcium, ⇑ Corresponding author. Department of Pharmaceutical Technology, Auf der Morgenstelle 8, 72076 Tuebingen, Germany. Tel.: +49 (0) 7071 2972462; fax: +49 (0) 7071 295531. E-mail addresses: [email protected] (M. Rottke), dominique. [email protected] (D.J. Lunter), [email protected] (R. Daniels).
leads to defunctionalisation of neurons that result in long lasting relief of pain and pruritus [3]. To date, no product that contains capsaicinoids is authorised with chronic pruritus as an indication although several semisolid capsaicin formulations for the temporary relief of muscle and joint pain are on the market. On the other hand, a magistral formula that is intended for the treatment of pain and chronic pruritus and that contains capsaicinoids is used [4]. In this cream, capsaicinoids are incorporated into a low dose (0.025– 0.1%) in order to avoid adverse effects such as pain or burning sensations. The fact that semisolids that contain capsaicinoids are used only sparsely results from the need to apply them several times a day. The reason for this high application frequency is the low substantivity of semisolids. In fact, up to 90% of the applied dose is withdrawn from the skin by clothing or contact to other surfaces. This means that up to 90% of the active cannot be delivered to the skin and cannot develop the desired pharmacological effect in the skin. Furthermore, application has to be done very carefully every time to avoid any unintended contact with the drug, which can induce an intense burning sensation. This makes therapy inconvenient and negatively affects patient’s compliance, which is mandatory for effective treatment [3]. The rationale of this work was therefore to develop a formulation that exhibits higher substantivity on the skin. High substantivity is closely associated with a prolonged delivery of the active pharmaceutical ingredient (API) to the skin as not only the formulation has to remain intact on the skin but also, more importantly, the API has to be delivered to the skin during the entire application interval [5]. We therefore aimed
0939-6411/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejpb.2013.09.018
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to develop a formulation with increased substantivity from which nonivamide permeates with a flux that is appropriate for effectively suppressing pain and itching. Thereby, the formulations will enable the extension of therapeutic intervals and reduction in the number of applications per day. This may lead to an increased compliance and a (more) successful treatment for chronic itching. Semisolid formulation principles that have been shown to exhibit high substantivity and/or to control release of an API to the skin are for example solid lipid nanoparticles [6] and film forming emulsions [7,8]. The latter concept for the formulation of semisolids with high substantivity was drawn from water-resistant sun screen products. These formulations employ adhesive polymers in o/w-type formulations. The polymers form a film on the skin which encapsulates the sun-screen agents and in the process ensures high substantivity. Another formulation principle that has already been successfully employed in sun-screen formulations is w/o-emulsions which have intrinsically a higher substantivity than o/w-systems. However, this simple approach is not suitable for the application of capsaicinoids because they are highly lipophilic and will thus be located in the outer phase of these dispersed systems. The capsaicinoids could therefore easily come into contact with the patients’ surrounding. Any contact to the contaminated surface could then cause a burning sensation. Alternatively, oil-in-silicon oil systems might be used as they may also lead to a higher substantivity and may show slow release of pharmaceutical drugs [9]. Such oil-in-oil emulsions contain polydimethyl siloxanes as their continuous phase and another oil as the dispersed phase and the solvent for the API. Polydimethyl siloxanes are chemically indifferent, non-occlusive and give the skin a soft and satiny touch [10]. Their high water-repellence makes them ideal as barrier creams. Polydimethyl siloxanes usually exhibit only very low solubility for organic compounds [11]. Therefore, another oil, for example castor oil, has to be used to incorporate the API. Castor oil shows high solubility for lipophilic APIs and is not miscible with polydimethyl siloxanes [12]. Hence, oil-in-oil emulsions may be formed that contain the active in the dispersed phase and the highly substantive polydimethyl siloxane in the continuous phase. We used nonivamide, a synthetic analogue of capsaicin as API instead of capsaicinoids as nonivamide exhibits equivalent pharmacodynamics and is, in contrast to capsaicinoids, a highly purified, chemically defined substance and may therefore be analysed with higher precision [13].
2.2. Preparation of emulsions Oil-in-oil emulsions were prepared by a ‘syringe to syringe technique’ (Fig. 1). To this end, polydimethyl siloxane, the dispersion of the silicone surfactant and castor oil that contained nonivamide were weighed precisely into a syringe. This syringe was connected to a second syringe by an adapter. Homogenisation was achieved by transferring the ingredients from one syringe to the other 70 times. The different formulations that were tested are listed in Table 1. 2.3. Preparation of ‘Hydrophilic Nonivamide Cream 0.1% and 1%’ ‘Hydrophilic Nonivamide Cream’ was prepared according to the monograph of ‘Hydrophile Capsaicinoid Creme NRF 11.125’ [4]. Capsaicinoids were replaced by nonivamide. In brief, ‘Cremor Basalis DAC’ was made by melting white soft paraffin, cetyl alcohol and glycerol monostearate, and then dissolving polyoxyethylene-20monostearate and propylene glycol in hot water and incorporating the aqueous phase into the oily phase to give an amphiphilic cream. A 10% or 1% (w/w) solution of nonivamide in ethanol 90% (v/v) was added to a solution of propylene glycol in water and this mixture was given to ‘Cremor Basalis’ in aliquots to form ‘Hydrophilic Nonivamide Cream 1% or 0.1%’. 2.4. Characterisation of oil-in-oil-emulsions Droplet sizes were determined by optical back reflection measurements using Sequip ADAS equipped with an IP68 sensor (Sequip S + E GmbH, D-Duesseldorf). Droplet sizes were determined by the MCSA software, d10, d50 and d90 values of droplet sizes were calculated. The experiments were performed in triplicate.
2. Materials and methods 2.1. Materials Nonivamide (Sigma–Aldrich Chemie GmbH, D-Steinheim) Castor oil (Caesar & Loretz GmbH, D-Hilden, Germany), Macrogol 600 (Pluriol E 600) (BASF SE, D-Ludwigshafen), medium chain triglycerides (MCT) (MiglyolÒ 818; MCT; Sasol GmbH, DWitten), glycerol monostearate (GMS) (Cutina GMSÒ, Cognis BASF SE, D-Ludwigshafen), cetyl alcohol (Henkel AG & Co. KG aA D-Duesseldorf), macrogol-20-glycerol monostearate (PEG-20GMS) (Tagat S2Ò, Evonik Goldschmidt GmbH, D-Essen), propylene glycol (PG) (BASF SE, D-Ludwigshafen), and polytetrafluorethylene (PTFE) filter membranes with a pore size of 5 lm (Sartorius Stedim Biotech GmbH, D-Goettingen). The 1:1-dispersion of silicone surfactant (BY 11-030) and polydimethyl siloxanes (Q7-9120 20 mPa s and 1000 mPa s) was kindly donated by (Dow Corning GmbH, B-Seneffe). Methanol and ethanol were HPLC gradient grade. Disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate and phosphoric acid were Ph. Eur. grade.
Fig. 1. Illustration of the syringe to syringe technique.
Table 1 Composition of the oil-in-oil-emulsions and the experiments in which they were used (a: influence of NVA content; b: influence of phase volume ratio; c: influence of viscosity). 1:1Dispersion of silicone surfactant (g)
Dimethyl siloxane (g)
Castor oil (g)
Phase volume ratio
Nonivamide (g)
Experiment
4 4 4 4 4 4 4
47 47 47 86.2 66.6 47 86.2
48 48.5 48.9 8.8 28.4 48 8.8
0.5 0.5 0.5 0.1 0.3 0.5 0.1
1 0.5 0.1 1 1 1 1
a, b, c a a b, c b c c
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2.5. In vitro release experiments
2.8. Permeation through pig ear skin
In vitro release tests were conducted using modified Franz diffusion cells (Gauer Glas, D-Püttlingen) with a receptor-volume of 12 mL. Phosphate buffered PEG (PBP; pH 7.4 phosphate buffer containing 20% (V/V) PEG 600) was used as receptor fluid. Degassed, prewarmed (32 °C) receptor medium was filled into Franz diffusion cells. Thereafter, Franz diffusion cells were equipped with PTFE filters. Subsequently, 0.4 g of the emulsion was spread onto the filters. Cells were capped with Parafilm. In vitro release experiments were performed at 32 °C; the stirring speed was 500 rpm. Nonivamide was allowed to diffuse over a period of 24 h. Aliquots of 100 lL were withdrawn at 6 time points and the sample volume was replaced by fresh, prewarmed receptor medium. Samples were analysed by HPLC. Release rates were calculated by linear regression; release coefficients were calculated by dividing release rates by initial drug concentration in the vehicle according to the following equation:
In vitro permeation tests were conducted using modified Franz diffusion cells (Gauer Glas, D-Püttlingen) with a receptor-volume of 12 mL. Phosphate buffered ethanol (PBE; pH 7.4 phosphate buffer containing 50% (V/V) ethanol) was used as the receptor fluid. Degassed, prewarmed (32 °C) receptor medium was filled into Franz diffusion cells. Thereafter, Franz diffusion cells were equipped with dermatomed pig ear skin (thickness: 1 mm, diameter: 25 mm) and donor compartments. Subsequently, 0.4 g of the formulation was applied to the skin surface. Cells were capped with Parafilm to prevent evaporation of water. In vitro permeation experiments were performed at 32 °C; the stirring speed was 500 rpm. Nonivamide was allowed to diffuse across the skin over a period of 28 h. Aliquots of 0.6 mL were withdrawn at 5 time points and the sample volume was replaced by fresh, prewarmed receptor medium. Samples were analysed by HPLC. Cumulative permeated amounts were plotted against time. Permeation rates were calculated by linear regression; permeation coefficients were calculated by dividing permeation rates by initial drug concentration in the vehicle according to the following equation:
K r ¼ J ss =c0
ð1Þ
where Kr is the release coefficient (mg/cm2 h), Jss is the steady state flux through PTFE filter membranes (lg/cm2 h) and c0 is the initial nonivamide concentration in the formulation (lg/mg). The experiments were performed in quintuplicate.
K p ¼ J ss =c0
ð2Þ
where Kp is the permeation coefficient (mg/cm2 h), Jss is the steady state flux through skin (lg/cm2 h) and c0 is the initial nonivamide concentration in the formulation (lg/mg). The experiments were performed in quintuplicate.
2.6. Diffusion of nonivamide through silicone oil In vitro diffusion tests were conducted using modified Franz diffusion cells (Gauer Glas, D-Püttlingen) with a receptor-volume of 12 mL. Phosphate buffered PEG (PBP; pH 7.4 phosphate buffer containing 20% (V/V) PEG 600) was used as receptor fluid. PTFE filters were siliconised according to the following procedure. Silicone oil (Q7-9120 20 mPa s or 1000 mPa s) was diluted 1:10 with petroleum ether. PTFE filters were agitated in this diluted solution for 2 min. They were withdrawn from the solution and allowed to dry. Thereafter, the Franz diffusion cells were equipped with the filters. Subsequently, three aliquots of 50 lL of the 1:10 dilution of silicone oil in petroleum ether were added to the filters and allowed to dry. This corresponds to 15 lL of silicone oil, a quantity that was found to be sufficient to fill the pores of the filters and does not produce an excessive quantity of silicone oil on top of the filters. Degassed, prewarmed (32 °C) receptor medium was filled into the cells. Subsequently, 300 lL of a solution of nonivamide in castor oil was added to the donor compartments. Cells were capped with Parafilm. In vitro diffusion experiments were performed at 32 °C; the stirring speed was 500 rpm. Nonivamide was allowed to diffuse over a period of 24 h. Aliquots of 100 lL were withdrawn at 6 time points and the sample volume was replaced by fresh, prewarmed receptor medium. Samples were analysed by HPLC. Cumulative diffused amounts were plotted against time. The experiments were performed in quintuplicate.
2.9. Nonivamide quantification Nonivamide was quantified using the ‘LC-20A prominence’ HPLC system (Shimadzu, D-Duisburg). The HPLC-column ‘Nucleosil 100-5C 8 CC 125/4’ (Macherey–Nagel, D-Dueren) was used in combination with the HPLC-precolumn ‘Nucleosil 100-5 C8CC 8/3’ (Macherey–Nagel, D-Dueren). Column-oven temperature was set to 50 °C. For in vitro release and diffusion experiments the eluent consisted of 59.5% methanol and 40.5% phosphoric acid pH 3.0. The flow was set to 1.125 mL/min. Then 20 lL per sample was injected, and the UV-absorbance was measured at 230 nm. Nonivamide was eluted after approximately 3.7 min. For in vitro permeation experiments the eluent consisted of 50% methanol and 50% phosphoric acid pH 3.0. The flow was set to 1.15 mL/min. Then 200 lL per sample was injected, and the UVabsorbance was measured at 230 nm. Nonivamide was eluted after approximately 9.7 min. 2.10. Statistical analysis Data were obtained from repeated measurements (n P 3). Diagrams show mean ± standard deviation. Data were analysed by an unpaired two-sample t-test or one-sided, one factorial analysis of variance (ANOVA) followed by the Student–Newman–Keuls test. Data that are significantly different are marked with an asterisk (). 3. Results and discussion
2.7. Preparation of dermatomed pig ear skin Fresh pig ears were washed with isotonic saline. Postauricular skin was excised. Skin samples were cleaned from blood with isotonic saline and cotton swabs, patted dry with tissue, wrapped in aluminium foil and stored at 30 °C. On the day of the experiment, the skin was thawed at room temperature, cut into strips of approximately 3 cm width and fixed to a block of Styrofoam with pins. The skin was dermatomed to a thickness of 1 mm (Dermatom GA 630, Aesculap AG & Co. KG, D-Melsungen).
Pharmaceutically used emulsions usually consist of an aqueous and an oily phase. Depending on the hydrophilicity/lipophilicity (hydrophilic–lipophilic-balance; HLB-value) of the surfactant, oilin-water or water-in-oil emulsions are formed. In this work, we used oil-in-oil emulsions prepared from castor oil and silicone oil, which were stabilized by a silicone surfactant. Like water and oil, castor oil and silicone oil are not miscible and one phase may be dispersed within the other by mixing. Similar to emulsions made of oil and water, the phase distribution depends on the
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nature of the emulsifier. However, there is no characteristic figure, such as the HLB-value from which the preferred phase distribution may be derived. For each oil–silicone oil–silicone surfactant-combination, the phase distribution needs to be evaluated experimentally [14]. In our case, preliminary experiments showed, that stable castor oil-in-silicone oil-emulsions may be formed by the addition of 2% (m/m) of the silicone surfactant BY 11-030 [12]. The study’s objective was to investigate the factors that influence the release and permeation of an API which is dissolved in the inner oil phase of such oil-in-oil-emulsions. To this end, emulsions that contained different quantities of active, silicone oils of different viscosities, and emulsions that exhibited different phase volume ratios were prepared. The influence of these factors on in vitro release and permeation of the API was investigated. Table 1 shows the compositions of the emulsions that were used in in vitro release and permeation experiments. As the droplet size of the dispersed phase might also have an impact on the results, we attempted to keep this variable almost constant. Droplet sizes of all emulsions were found to be within a comparable range with d50 between 1.1 and 1.8 lm. Therefore, we concluded that all observed effects on in vitro release of the active from the emulsions and permeation through pig ear skin are solely related to the influence of varied composition and are not additionally affected by differences in the droplet size distribution. 3.1. In vitro release of nonivamide Fig. 2 illustrates the release of nonivamide from an oil-in-oilemulsion. It can be seen that release of the API from the inner oil phase is a sequence of diffusion and partitioning processes. In order to diffuse to the membrane/skin nonivamide has first to partition into the silicone oil and second to diffuse through the silicone oil. The partition coefficient of nonivamide (castor oil/silicone oil) is >1300. Therefore, nonivamide is almost exclusively located in the castor oil droplets. Diffusion may be described by Fick’s 1st law of diffusion [15]. According to that, the velocity of diffusion depends on (a) the concentration gradient of the diffusing substance within the diffusion medium, (b) the viscosity of the diffusion medium and (c) the distance that has travelled [15,16]. Nonivamide concentration is highest within the droplets as it is distributed between castor oil and silicone oil according to its partition coefficient. Alterations of the nonivamide concentration in the oil droplets lead to variations in the concentration gradient and should therefore affect the diffusion rate. It is thus reasonable to assume that not only the concentration in the formulation but also a variation in the concentration in the oil droplets (at a constant concentration in the whole formulation) may influence the release and permeation rates. This condition may be achieved by the use of a smaller amount of castor oil that contains a larger quantity of nonivamide. Whilst the viscosity of the diffusion medium depends
on the nature of the silicone oil, the distance that needs to be travelled may be altered by variation in the phase volume ratio. From this the following three factors that may affect the release of nonivamide are derived: (a) nonivamide content, (b) viscosity of the continuous phase and (c) phase volume ratio. 3.1.1. Influence of nonivamide content on release Table 2 shows the results of in vitro release of nonivamide from oil-in-oil-emulsions that contained different quantities of nonivamide. It can be seen that the release from all formulations is linear with time. As expected, the release rates increase with increasing nonivamide content. The release coefficients of all formulations are within the same range (0.35–0.37 lg/cm2 h). This can be explained by the thermodynamic activity of the API in the vehicle, which increases with increasing drug concentration in the vehicle. As thermodynamic activity is the driving force behind the release, higher concentrations increase drug release [17]. The release coefficients are similar for all formulations. This finding indicates a linear relationship between release rates and nonivamide concentration [17] and it can therefore be concluded that an increased API concentration is the reason for the increased release rates. 3.1.2. Influence of phase volume ratio and nonivamide content in the dispersed phase on release The influence of phase volume ratio on in vitro release of nonivamide was investigated using emulsions with a phase volume ratio between 0.1 and 0.5. Nonivamide concentrations in the castor oil were adjusted in order to maintain a constant nonivamide content of 1% (m/m) within the whole formulation. In order to achieve this, emulsions with a low phase volume ratio were prepared with higher concentrations of nonivamide in castor oil. Table 2 shows the results of the in vitro release experiments. Similar to the formulations previously tested, the release of nonivamide from all formulations is linear with time. Interestingly, the release rate and release coefficient are highest at a phase volume ratio of 0.1 and decrease with increasing phase volume ratio. As the distance that needs to be travelled decreases with increasing phase volume ratio the release rates should have decreased according to Fick’s 1st law. Evidently, release rates of the formulations do not follow this rule. This means that release rates do not primarily depend on the distance that has to be travelled. Instead, we observed an inverse relationship between distance and release rate. Therefore, the differing nonivamide concentrations in the dispersed phase must be the rate-determining factor. To evaluate this assumption, we plotted release rates vs. the concentration of nonivamide in the dispersed phase and calculated the coefficient of determination. We obtained a value of 0.95, which indicates an almost linear relationship between release rate and concentration of the dispersed phase. This proves that the nonivamide Table 2 Release rates and release coefficients of oil-in-oil-emulsions with varying nonivamide concentrations and phase volume ratios. Release rate (lg/cm2 h) ± standard deviation
Release coefficient (mg/cm2 h) ± standard deviation
Nonivamide concentration 0.1% 2 0.5% 10 1.0% 21
0.37 ± 0.13* 1.79 ± 0.09* 3.49 ± 0.57*
0.37 ± 0.13* 0.36 ± 0.02* 0.35 ± 0.06*
Phase volume ratio 0.1 114 0.3 35 0.5 21
7.20 ± 1.51* 4.75 ± 0.41* 3.39 ± 0.72*
0.72 ± 0.15* 0.47 ± 0.04* 0.34 ± 0.07*
Nonivamide concentration in inner oil phase (mg/g)
*
Fig. 2. Illustration of the release of nonivamide from an oil-in-oil-emulsion.
Statistically different data (p 0.05).
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concentration in the dispersed phase predominantly controls release from the oil-in-oil-emulsions.
3.1.3. Influence of viscosity on release The influence of the viscosity on the in vitro release of nonivamide was investigated using emulsions that contained a silicone oil of low viscosity (20 mPa s) or high viscosity (1000 mPa s) and a phase volume ratio of 0.1 or 0.5. Nonivamide concentrations in the castor oil were adjusted in order to maintain a constant nonivamide content of 1% (m/m) within the formulation. Fig. 3 and Table 3 show the results of in vitro release experiments. As expected, release is linear with time. Comparing the release rates from emulsions with a phase volume ratio of 0.1 elucidates that nonivamide is released faster from emulsions that contain the low-viscous silicone oil. Although the release patterns seem to be the same for emulsions with a phase volume ratio of 0.5 (indicated by overlapping error bars), statistical analysis revealed a significant difference between release rates of nonivamide from the two emulsions at a phase volume ratio of 0.5. In accordance with the previous experiments, nonivamide is released faster from emulsions with a phase volume ratio of 0.1. The increase in phase volume ratio from 0.1 to 0.5 leads to a reduction in release rate by 61% (20 mPa s) and 56% (1000 mPa s), whilst increasing the viscosity reduces the release rate by only 48%. The fact that the release from the emulsion with the low-viscous silicone oil is faster than the release from the emulsion with the high-viscous silicone oil may be explained by a higher diffusivity in the low-viscous medium. However, the effect is not as pronounced as expected because according to Fick’s 1st law of diffusion a 50-fold increase in viscosity should reduce the flux by a factor of 50. To further evaluate the influence of the viscosity of the silicone oil on release of nonivamide from the emulsions, we performed an in vitro diffusion experiment in which we eliminated all other formulation variables that might possibly affect the release rate. For this, we used PTFE filter membranes which we loaded with the two silicone oils under investigation. The quantity of silicone oil used was adjusted to fill the pores of the filter but not to leave an excess silicone oil film on the filters. On top of the siliconised filters we added nonivamide solutions in castor oil that exhibited the same concentrations as the nonivamide solutions in emulsions with a phase volume ratio of 0.1 (114 mg/g) and 0.5 (21 mg/g), respectively. Apart from that, in vitro diffusion experiments were performed in a manner similar to in vitro release experiments. The results are shown in Fig. 4 and Table 3. The data clearly show that the diffusion rate is influenced by the viscosity of the silicone oil and the nonivamide concentration in
Fig. 3. Comparison of the in vitro release of nonivamide from oil-in-oil-emulsions with different phase volume ratios that contain silicone oils of different viscosities (d) 20 mPa s 0.1, (j) 20 mPa s 0.5, () 1000 mPa s 0.1, () 1000 mPa s 0.5 (n = 5; error bars = standard deviation).
Table 3 Release and diffusion characteristics of nonivamide from castor oil through silicone oils of different viscosities. Release or diffusion rate (lg/cm2 h) ± standard deviation
Release coefficient (mg/cm2 h) ± standard deviation
Release experiments 20 cst, 0.1 114 1000 cst, 0.1 21 20 cst, 0.5 114 1000 cst, 0.5 21
8.12 ± 1.06* 4.22 ± 0.75 3.14 ± 0.50 1.86 ± 0.27*
0.81 ± 0.11* 0.42 ± 0.07* 0.31 ± 0.05* 0.19 ± 0.02*
Diffusion experiments 20 cst 114 1000 cst 114 20 cst 21 1000 cst 21
8.71 ± 0.46* 6.04 ± 0.62* 2.53 ± 0.23* 1.37 ± 0.27*
Viscosity, phase volume ratio
*
Nonivamide concentration in inner oil phase (mg/g)
Statistically different data (p 0.05).
the castor oil. As the flux is directly related to the concentration gradient a 5.7-fold increase would have to be expected when the concentration is increased from 21 mg/g to 114 mg/g. Experimental data reveal factors of 3.44 (20 mPa s) and 4.41 (1000 mPa s) which closely match our expectations. On the other hand, flux is inversely related to the viscosity of the diffusion medium. Thus a 50-fold decrease in the release rate should result when the viscosity of the diffusion medium is increased from 20 mPa s to 1000 mPa s. However, only a marginal reduction by a factor of 1.44 (114 mg/g) and 1.84 (21 mg/g) was observed in the respective experiments. It is known from other studies, that the diffusion rate through silicone oils does not depend on the macro-viscosity of the silicone oil but on its micro-viscosity [18]. Obviously, the microviscosity of both silicon oils used is similar although they clearly differ in their macro-viscosity. Thus, this formulation variable cannot be used to effectively control the release rate. On the other hand, this provides the opportunity to freely choose the silicone oil that exhibits the most convenient application properties. 3.3. In vitro permeation through pig ear skin 3.3.1. Influence of nonivamide content on release In vitro release experiments showed a strong impact of nonivamide content on release rate. This was explained by a higher thermodynamic activity in formulations that contain higher nonivamide concentrations. As this situation does not change when formulations are applied to the skin, we expected permeation rates to depend on nonivamide concentration as well. Table 4
Fig. 4. Comparison of the in vitro diffusion of nonivamide from differently concentrated nonivamide solutions in castor oil through silicone oils of different viscosities (d) 20 mPa s, 114 mg/g, (j) 20 mPa s, 21 mg/g, () 1000 mPa s, 114 mg/ g, () 1000 mPa s, 21 mg/g (n = 5; error bars = standard deviation).
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shows the results of in vitro permeation experiments with formulations that contained 1.0%, 2.0% or 3.0% (m/m) nonivamide. Nonivamide permeates the skin after a lag time of approximately 10 h. The cumulative permeated amounts are linear with time in all formulations. The permeation rates increase with increasing nonivamide content. The permeation coefficients are independent of nonivamide concentration. The fact that the cumulative permeated amounts are linear with time is in accordance with Fick’s 1st law of diffusion [15] and indicates that permeation of nonivamide through skin is a diffusion limited process. As described above, higher nonivamide concentrations in the formulations lead to higher thermodynamic activity. As thermodynamic activity is the driving force behind permeation, higher thermodynamic activity leads to increased permeation rates. The fact that permeation rates increase linearly with nonivamide content and permeation coefficients remain constant confirms that the permeation rates depend directly on the nonivamide content. 3.3.2. Influence of phase volume ratio on in vitro permeation In vitro release experiments showed that release rates decrease with increasing phase volume ratio. We therefore evaluated the impact of phase volume ratio on permeation. The results of these experiments are shown in Fig. 5 and Table 4. The data show that the formulation with a phase volume ratio of 0.1 exhibits the highest permeation rate. The permeation rates of nonivamide from the formulations with a phase volume ratio of 0.3 and 0.5 are much lower than the permeation rate of nonivamide from the formulation with a phase volume ration of 0.1, and not significantly different from each other. These results agree with the results of release experiments, which showed that release rates clearly depend on phase volume ratio. We concluded that this is attributable to lower nonivamide concentrations in the dispersed phase of emulsions with a high phase volume ratio as we found a linear relationship between nonivamide concentration in the dispersed phase and release rates. Evidently, the situation is more complex in permeation. Between a phase volume ratio of 0.1 and 0.3, the nonivamide concentration in the dispersed phase clearly affects the permeation rate. The effect, however, seems to have levelled out at a phase volume ratio of 0.3. Obviously, diffusion through the emulsion is rate controlling in emulsions with a phase volume ratio of 0.1 whereas at higher phase volume ratios diffusion through skin determines the permeation rate. 3.3.3. Influence of viscosity on in vitro permeation The in vitro release experiments showed that viscosity may influence release rates in formulations with a low phase volume Table 4 Impact of the concentration of nonivamide in castor oil and the viscosity of the silicone oil on the permeation of nonivamide. Permeation rate (lg/cm2 h) ± standard deviation
Permeation coefficient (mg/cm2 h) ± standard deviation
Nonivamide concentration 1% 21 2% 2 3% 10
0.58 ± 0.18 1.16 ± 0.32 2.02 ± 0.80*
0.06 ± 0.02 0.06 ± 0.02 0.07 ± 0.03
Phase volume ratio 0.1 114 0.3 35 0.5 21
1.10 ± 0.16 0.38 ± 0.13 0.46 ± 0.16
0.11 ± 0.02 0.04 ± 0.01 0.05 ± 0.02
Viscosity 20 cst 1000 cst
1.10 ± 0.16* 2.15 ± 0.97*
0.11 ± 0.02* 0.21 ± 0.10*
Nonivamide concentration in inner oil phase (mg/g)
*
114 114
Statistically different data (p 0.05).
Fig. 5. Comparison of the in vitro permeation of nonivamide from oil-in-oilemulsions with different phase volume ratios (d) 0.1, (N) 0.3, (j) 0.5 (n = 5; error bars = standard deviation).
ratio. The effect of viscosity on permeation was evaluated with two formulations that contained either a low-viscous silicone oil (20 mPa s) or a high-viscous silicone oil (1000 mPa s) and showed a phase volume ratio of 0.1. The results are shown in Fig. 6 and Table 4. Interestingly, permeation of nonivamide is faster from the formulation that contains the high-viscous silicone oil. However, the standard deviations of the cumulative permeated amounts overlap and are substantially higher when nonivamide permeates from the formulation that contains the high-viscous silicone oil. Despite the overlapping standard deviations, the statistical analysis proves a significant difference between the permeation rates of the two formulations. Therefore, it must be concluded that viscosity of the coherent phase does influence permeation of nonivamide through pig ear skin. However, the difference is rather small and has no practical relevance. 3.3.4. Comparison to permeation from hydrophilic nonivamide cream Oil-in-oil-emulsions that contain nonivamide are intended to provide an alternative to semisolid formulations like Hydrophilic Nonivamide Cream. The latter is used in the therapy of chronic pruritus, but compliance with the treatment is low as it has to be applied 2–5 times per day. We therefore aimed to develop more substantial formulations in order to enable a reduction in the application frequency. The quantities of nonivamide needed to achieve relief of itch differ strongly from patient to patient. To be able to investigate whether the permeation rate of nonivamide from oil-in-oil-emulsions is within a suitable range for therapy we decided to compare permeation rates of nonivamide from oil-in-oil-emulsions to permeation rates from Hydrophilic Nonivamide Cream 0.1% (HNC 0.1%). This formulation contains nonivamide in a
Fig. 6. Comparison of the in vitro permeation of nonivamide from oil-in-oilemulsions that contain silicone oils of different viscosities (d) 20 mPa s, () 1000 mPa s (n = 5; error bars = standard deviation).
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almost independent of the macro-viscosity of the continuous phase and thus allows adjustment the consistency of the formulation in a wide range. Constant permeation rates can be maintained for a period of 10 h. This is a definite advantage of oil-in-oil-emulsions as application intervals can be extended. This results in enhanced compliance and ensures therapeutic success. The novel concept of oil-in-oil-emulsions may be used for a multitude of lipophilic substances utilising oil-in-oil-emulsions as a drug delivery system which may be tailored to specific needs of patients and thereby allow sustained dermal therapy of various diseases. Acknowledgements Fig. 7. Comparison of the in vitro permeation of nonivamide from (j) oil-in-oilemulsion that contains 1% nonivamide to () Hydrophilic Nonivamide Cream 1% and (e) Hydrophilic Nonivamide Cream 0.1% (n = 5; error bars = standard deviation).
concentration that is used in the therapy of chronic pruritus. Permeation rates of nonivamide from oil-in-oil-emulsions should be in the same range as permeation rates from HNC 0.1% in order to be suitable for therapy. To be able to extend application intervals, we needed to achieve prolonged release of the active from the formulation. This means that the formulation has to control release of the active. To investigate this, we compared release from an oil-in-oil-emulsion that contained 1% nonivamide to permeation from HNC 1%. If oil-inoil-emulsions control release/permeation of nonivamide permeation rates and coefficients of oil-in-oil-emulsions should be significantly lower than those obtained from HNC 1%. The results of permeation experiments (Fig. 7) show that permeation is linear with time for oil-in-oil-emulsions and HNC. Permeation is highest for HNC 1% (9.66 ± 0.83 lg/cm2 h). Permeation from HNC 0.1% (0.98 ± 0.10 lg/cm2 h) is 10 times less than permeation from HNC 1%. The permeation coefficients of nonivamide from HNC 0.1% (0.98 ± 0.10 mg/cm2 h) and HNC 1% (0.98 ± 0.08 mg/cm2 h) are not significantly different. This means that the higher permeation rate of nonivamide from HNC 1% is due to the higher nonivamide content. The permeation rate of nonivamide from oil-in-oil-emulsions (0.69 ± 0.30 lg/cm2 h) is similar to that of nonivamide from HNC 0.1%. This indicates that the permeation rate of the oil-in-oil-emulsion is within a suitable range for therapy. The fact that permeation rate (0.69 ± 0.30 lg/cm2 h) and coefficient (0.07 ± 0.03 mg/cm2 h) of nonivamide from the oil-in-oil-emulsion are substantially lower than the respective values for HNC 1% proves that the oil-in-oil-emulsion controls the release and permeation of nonivamide. Sustained release has therefore been achieved. 4. Conclusion The results of this work elucidate the possibility of formulating stable emulsions that consist of a silicone oil as the continuous phase and a drug solution in castor oil as the dispersed phase. It is shown that the permeation rate of the active substance from its oily solution can be controlled by the drug content in the dispersed phase and the phase volume ratio. The permeation rate is
The authors would like to thank Martin Schenk and his team from the Department of Experimental Medicine at the University of Tuebingen for the supply of pig ears and Dow Corning GmbH for the kind donation of silicone oils and surfactants. References [1] M. Metz, S. Ständer, Chronic pruritus – pathogenesis, clinical aspects and treatment, J. Eur. Acad. Dermatol. Venereol. 24 (2010) 1249–1260. [2] D. Siepmann, C. Weishaupt, T.A. Luger, S. Ständer, Evaluation of the German guideline for chronic pruritus: results of a retrospective study on 385 patients, Dermatology 223 (2011) 374–380. [3] P. Anand, K. Bley, Topical capsaicin for pain management: therapeutic potential and mechanisms of action of the new high-concentration capsaicin 8% patch, Br. J. Anaesth. 107 (2011) 490–502. [4] Bundesvereinigung Deutscher Apotheker, Hydrophile Capsaicinoid Creme 0.025%/0.05%/0.1% (NRF 11.125), Neues Rezeptur Formularium, D-Eschborn, 2010. [5] H. Kählig, A. Hasanovic, B. Biruss, S. Höller, J. Grimm, C. Valenta, Chitosan– glycolic acid: a possible matrix for progesterone delivery into skin, Drug Dev. Ind. Pharm. 35 (2009) 997–1002. [6] A.A. Attama, C. Weber, C.C. Müller-Goymann, Assessment of Drug Permeation from Lipid Nanoparticles formulated with a Novel Structured Lipid Matrix through Artificial Skin Construct Bio-engineered from HDF and HaCaT Cell Lines, Editions de sant, Paris, France, 2008. [7] D.J. Lunter, R. Daniels, New film forming emulsions containing EudragitÒ NE and/or RS 30D for sustained dermal delivery of nonivamide, Eur. J. Pharm. Biopharm. 82 (2012) 291–298. [8] D.J. Lunter, R. Daniels, In vitro skin permeation and penetration of nonivamide from novel film forming emulsions, Skin Pharmacol. Physiol. 26 (2012) 139– 146. [9] V. Jaitely, T. Sakthivel, G. Magee, A.T. Florence, Formulation of Oil in Oil Emulsions: Potential Drug Reservoirs for Slow Release, Editions de sant, Paris, FRANCE, 2004. [10] D. Floyd, Silicone surfactants: applications in the personal care industry, in: R.M. Hill (Ed.), Silicone Surfactants, Marcel Dekker Inc., New York, 1999, pp. 181–209. [11] B. Grüning, A. Bungard, Silicone surfactants: emulsification, in: R.M. Hill (Ed.), Silicone Surfactants, Marcel Dekker Inc., New York, 1999. [12] M. Rottke, Entwicklung und Charakterisierung nonivamidhaltiger Rizinussölin-Silikonöl-Emulsionen zur dermalen Anwendung, 2012. [13] M.d.L. Reyes-Escogido, E.G. Gonzalez-Mondragon, E. Vazquez-Tzompantzi, Chemical and pharmacological aspects of capsaicin, Molecules 16 (2011) 1253–1270. [14] O. Suitthimeathegorn, V. Jaitely, A.T. Florence, Novel anhydrous emulsions: formulation as controlled release vehicles, Int. J. Pharm. 298 (2005) 367–371. [15] A. Fick, Ueber diffusion, Ann. Phys. 170 (1855) 59–86. [16] A. Einstein, Über die von der molekularkinetischen Theorie der Wärme geforderte Bewegung von in ruhenden Flüssigkeiten suspendierten Teilchen, Ann. Phys. 322 (1905) 549–560. [17] J. Hadgraft, Skin, the final frontier, Int. J. Pharm. 224 (2001) 1–18. [18] T. Seki, M. Okamoto, O. Hosoya, D. Aiba, K. Morimoto, K. Juni, Effect of chondroitin sulfate on the diffusion coefficients of drugs in aqueous, Solutions 13 (2003) 215–218.
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Research paper
In vitro studies on release and skin permeation of nonivamide from novel oil-in-oil-emulsions Michael Rottke, Dominique Jasmin Lunter, Rolf Daniels ⇑ Department of Pharmaceutical Technology, Eberhard Karls University, Tuebingen, Germany
a r t i c l e
i n f o
Article history: Received 10 July 2013 Accepted in revised form 26 September 2013 Available online xxxx Keywords: Nonivamide Topical delivery Permeation Pig ear skin Silicone oil Oil-in-oil-emulsion
a b s t r a c t The purpose of this study was to develop oil-in-oil-emulsions that facilitate long-term treatment for chronic pruritus with capsaicinoids. To this end, oil-in-oil-emulsions, which comprised polydimethyl siloxanes, silicone surfactant and castor oil, were examined. We used nonivamide, a synthetic analogue of capsaicin as the active pharmaceutical ingredient. It was incorporated into castor oil that formed the dispersed phase of the emulsion. We evaluated the influence of formulation variables (nonivamide content, phase volume ratio and viscosity of the silicone oil) on the in vitro release and the permeation of nonivamide. Permeation was found to be controlled by the nonivamide concentration in the dispersed phase and the phase volume ratio. Oil-in-oil-emulsions were found to produce constant permeation rates over a period of 10 h. They are thus superior to conventional semisolid formulations as application intervals may be extended. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction The skin is the largest organ in the human body. It protects us not only from dehydration but also from intrusion of xenobiotics, parasites and microorganisms. Moreover, exogenous noxae may activate special nerves in the skin which physiologically results in itching and scratching. By scratching, potentially harmful substances are removed from the skin. But itch may also be a symptom that accompanies skin diseases [1]. Depending on the pathogenesis of the itch, different mediators and receptors may be involved in the development of pathologic itches. This makes therapy difficult and often insufficient [2]. One option in the therapy of chronic itch is the topical treatment with capsaicinoids. Although capsaicinoids may initially induce pain and itch when applied to the skin, longterm topical administration leads to effective relief. The mechanism of action is presumed to involve TPRV 1 (transient receptor potential type 1), a nonselective cation channel expressed on the peripheral endings of Ad- and C-fibres in the viable epidermis. The binding of capsaicinoids to TRPV 1 results in release of substance P, CGRP and other vasodilatative substances that induce neurogenic inflammation and oedema. If application of capsaicinoids is continued, the persistent influx of cations, mainly calcium, ⇑ Corresponding author. Department of Pharmaceutical Technology, Auf der Morgenstelle 8, 72076 Tuebingen, Germany. Tel.: +49 (0) 7071 2972462; fax: +49 (0) 7071 295531. E-mail addresses: [email protected] (M. Rottke), dominique. [email protected] (D.J. Lunter), [email protected] (R. Daniels).
leads to defunctionalisation of neurons that result in long lasting relief of pain and pruritus [3]. To date, no product that contains capsaicinoids is authorised with chronic pruritus as an indication although several semisolid capsaicin formulations for the temporary relief of muscle and joint pain are on the market. On the other hand, a magistral formula that is intended for the treatment of pain and chronic pruritus and that contains capsaicinoids is used [4]. In this cream, capsaicinoids are incorporated into a low dose (0.025– 0.1%) in order to avoid adverse effects such as pain or burning sensations. The fact that semisolids that contain capsaicinoids are used only sparsely results from the need to apply them several times a day. The reason for this high application frequency is the low substantivity of semisolids. In fact, up to 90% of the applied dose is withdrawn from the skin by clothing or contact to other surfaces. This means that up to 90% of the active cannot be delivered to the skin and cannot develop the desired pharmacological effect in the skin. Furthermore, application has to be done very carefully every time to avoid any unintended contact with the drug, which can induce an intense burning sensation. This makes therapy inconvenient and negatively affects patient’s compliance, which is mandatory for effective treatment [3]. The rationale of this work was therefore to develop a formulation that exhibits higher substantivity on the skin. High substantivity is closely associated with a prolonged delivery of the active pharmaceutical ingredient (API) to the skin as not only the formulation has to remain intact on the skin but also, more importantly, the API has to be delivered to the skin during the entire application interval [5]. We therefore aimed
0939-6411/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejpb.2013.09.018
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to develop a formulation with increased substantivity from which nonivamide permeates with a flux that is appropriate for effectively suppressing pain and itching. Thereby, the formulations will enable the extension of therapeutic intervals and reduction in the number of applications per day. This may lead to an increased compliance and a (more) successful treatment for chronic itching. Semisolid formulation principles that have been shown to exhibit high substantivity and/or to control release of an API to the skin are for example solid lipid nanoparticles [6] and film forming emulsions [7,8]. The latter concept for the formulation of semisolids with high substantivity was drawn from water-resistant sun screen products. These formulations employ adhesive polymers in o/w-type formulations. The polymers form a film on the skin which encapsulates the sun-screen agents and in the process ensures high substantivity. Another formulation principle that has already been successfully employed in sun-screen formulations is w/o-emulsions which have intrinsically a higher substantivity than o/w-systems. However, this simple approach is not suitable for the application of capsaicinoids because they are highly lipophilic and will thus be located in the outer phase of these dispersed systems. The capsaicinoids could therefore easily come into contact with the patients’ surrounding. Any contact to the contaminated surface could then cause a burning sensation. Alternatively, oil-in-silicon oil systems might be used as they may also lead to a higher substantivity and may show slow release of pharmaceutical drugs [9]. Such oil-in-oil emulsions contain polydimethyl siloxanes as their continuous phase and another oil as the dispersed phase and the solvent for the API. Polydimethyl siloxanes are chemically indifferent, non-occlusive and give the skin a soft and satiny touch [10]. Their high water-repellence makes them ideal as barrier creams. Polydimethyl siloxanes usually exhibit only very low solubility for organic compounds [11]. Therefore, another oil, for example castor oil, has to be used to incorporate the API. Castor oil shows high solubility for lipophilic APIs and is not miscible with polydimethyl siloxanes [12]. Hence, oil-in-oil emulsions may be formed that contain the active in the dispersed phase and the highly substantive polydimethyl siloxane in the continuous phase. We used nonivamide, a synthetic analogue of capsaicin as API instead of capsaicinoids as nonivamide exhibits equivalent pharmacodynamics and is, in contrast to capsaicinoids, a highly purified, chemically defined substance and may therefore be analysed with higher precision [13].
2.2. Preparation of emulsions Oil-in-oil emulsions were prepared by a ‘syringe to syringe technique’ (Fig. 1). To this end, polydimethyl siloxane, the dispersion of the silicone surfactant and castor oil that contained nonivamide were weighed precisely into a syringe. This syringe was connected to a second syringe by an adapter. Homogenisation was achieved by transferring the ingredients from one syringe to the other 70 times. The different formulations that were tested are listed in Table 1. 2.3. Preparation of ‘Hydrophilic Nonivamide Cream 0.1% and 1%’ ‘Hydrophilic Nonivamide Cream’ was prepared according to the monograph of ‘Hydrophile Capsaicinoid Creme NRF 11.125’ [4]. Capsaicinoids were replaced by nonivamide. In brief, ‘Cremor Basalis DAC’ was made by melting white soft paraffin, cetyl alcohol and glycerol monostearate, and then dissolving polyoxyethylene-20monostearate and propylene glycol in hot water and incorporating the aqueous phase into the oily phase to give an amphiphilic cream. A 10% or 1% (w/w) solution of nonivamide in ethanol 90% (v/v) was added to a solution of propylene glycol in water and this mixture was given to ‘Cremor Basalis’ in aliquots to form ‘Hydrophilic Nonivamide Cream 1% or 0.1%’. 2.4. Characterisation of oil-in-oil-emulsions Droplet sizes were determined by optical back reflection measurements using Sequip ADAS equipped with an IP68 sensor (Sequip S + E GmbH, D-Duesseldorf). Droplet sizes were determined by the MCSA software, d10, d50 and d90 values of droplet sizes were calculated. The experiments were performed in triplicate.
2. Materials and methods 2.1. Materials Nonivamide (Sigma–Aldrich Chemie GmbH, D-Steinheim) Castor oil (Caesar & Loretz GmbH, D-Hilden, Germany), Macrogol 600 (Pluriol E 600) (BASF SE, D-Ludwigshafen), medium chain triglycerides (MCT) (MiglyolÒ 818; MCT; Sasol GmbH, DWitten), glycerol monostearate (GMS) (Cutina GMSÒ, Cognis BASF SE, D-Ludwigshafen), cetyl alcohol (Henkel AG & Co. KG aA D-Duesseldorf), macrogol-20-glycerol monostearate (PEG-20GMS) (Tagat S2Ò, Evonik Goldschmidt GmbH, D-Essen), propylene glycol (PG) (BASF SE, D-Ludwigshafen), and polytetrafluorethylene (PTFE) filter membranes with a pore size of 5 lm (Sartorius Stedim Biotech GmbH, D-Goettingen). The 1:1-dispersion of silicone surfactant (BY 11-030) and polydimethyl siloxanes (Q7-9120 20 mPa s and 1000 mPa s) was kindly donated by (Dow Corning GmbH, B-Seneffe). Methanol and ethanol were HPLC gradient grade. Disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate and phosphoric acid were Ph. Eur. grade.
Fig. 1. Illustration of the syringe to syringe technique.
Table 1 Composition of the oil-in-oil-emulsions and the experiments in which they were used (a: influence of NVA content; b: influence of phase volume ratio; c: influence of viscosity). 1:1Dispersion of silicone surfactant (g)
Dimethyl siloxane (g)
Castor oil (g)
Phase volume ratio
Nonivamide (g)
Experiment
4 4 4 4 4 4 4
47 47 47 86.2 66.6 47 86.2
48 48.5 48.9 8.8 28.4 48 8.8
0.5 0.5 0.5 0.1 0.3 0.5 0.1
1 0.5 0.1 1 1 1 1
a, b, c a a b, c b c c
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2.5. In vitro release experiments
2.8. Permeation through pig ear skin
In vitro release tests were conducted using modified Franz diffusion cells (Gauer Glas, D-Püttlingen) with a receptor-volume of 12 mL. Phosphate buffered PEG (PBP; pH 7.4 phosphate buffer containing 20% (V/V) PEG 600) was used as receptor fluid. Degassed, prewarmed (32 °C) receptor medium was filled into Franz diffusion cells. Thereafter, Franz diffusion cells were equipped with PTFE filters. Subsequently, 0.4 g of the emulsion was spread onto the filters. Cells were capped with Parafilm. In vitro release experiments were performed at 32 °C; the stirring speed was 500 rpm. Nonivamide was allowed to diffuse over a period of 24 h. Aliquots of 100 lL were withdrawn at 6 time points and the sample volume was replaced by fresh, prewarmed receptor medium. Samples were analysed by HPLC. Release rates were calculated by linear regression; release coefficients were calculated by dividing release rates by initial drug concentration in the vehicle according to the following equation:
In vitro permeation tests were conducted using modified Franz diffusion cells (Gauer Glas, D-Püttlingen) with a receptor-volume of 12 mL. Phosphate buffered ethanol (PBE; pH 7.4 phosphate buffer containing 50% (V/V) ethanol) was used as the receptor fluid. Degassed, prewarmed (32 °C) receptor medium was filled into Franz diffusion cells. Thereafter, Franz diffusion cells were equipped with dermatomed pig ear skin (thickness: 1 mm, diameter: 25 mm) and donor compartments. Subsequently, 0.4 g of the formulation was applied to the skin surface. Cells were capped with Parafilm to prevent evaporation of water. In vitro permeation experiments were performed at 32 °C; the stirring speed was 500 rpm. Nonivamide was allowed to diffuse across the skin over a period of 28 h. Aliquots of 0.6 mL were withdrawn at 5 time points and the sample volume was replaced by fresh, prewarmed receptor medium. Samples were analysed by HPLC. Cumulative permeated amounts were plotted against time. Permeation rates were calculated by linear regression; permeation coefficients were calculated by dividing permeation rates by initial drug concentration in the vehicle according to the following equation:
K r ¼ J ss =c0
ð1Þ
where Kr is the release coefficient (mg/cm2 h), Jss is the steady state flux through PTFE filter membranes (lg/cm2 h) and c0 is the initial nonivamide concentration in the formulation (lg/mg). The experiments were performed in quintuplicate.
K p ¼ J ss =c0
ð2Þ
where Kp is the permeation coefficient (mg/cm2 h), Jss is the steady state flux through skin (lg/cm2 h) and c0 is the initial nonivamide concentration in the formulation (lg/mg). The experiments were performed in quintuplicate.
2.6. Diffusion of nonivamide through silicone oil In vitro diffusion tests were conducted using modified Franz diffusion cells (Gauer Glas, D-Püttlingen) with a receptor-volume of 12 mL. Phosphate buffered PEG (PBP; pH 7.4 phosphate buffer containing 20% (V/V) PEG 600) was used as receptor fluid. PTFE filters were siliconised according to the following procedure. Silicone oil (Q7-9120 20 mPa s or 1000 mPa s) was diluted 1:10 with petroleum ether. PTFE filters were agitated in this diluted solution for 2 min. They were withdrawn from the solution and allowed to dry. Thereafter, the Franz diffusion cells were equipped with the filters. Subsequently, three aliquots of 50 lL of the 1:10 dilution of silicone oil in petroleum ether were added to the filters and allowed to dry. This corresponds to 15 lL of silicone oil, a quantity that was found to be sufficient to fill the pores of the filters and does not produce an excessive quantity of silicone oil on top of the filters. Degassed, prewarmed (32 °C) receptor medium was filled into the cells. Subsequently, 300 lL of a solution of nonivamide in castor oil was added to the donor compartments. Cells were capped with Parafilm. In vitro diffusion experiments were performed at 32 °C; the stirring speed was 500 rpm. Nonivamide was allowed to diffuse over a period of 24 h. Aliquots of 100 lL were withdrawn at 6 time points and the sample volume was replaced by fresh, prewarmed receptor medium. Samples were analysed by HPLC. Cumulative diffused amounts were plotted against time. The experiments were performed in quintuplicate.
2.9. Nonivamide quantification Nonivamide was quantified using the ‘LC-20A prominence’ HPLC system (Shimadzu, D-Duisburg). The HPLC-column ‘Nucleosil 100-5C 8 CC 125/4’ (Macherey–Nagel, D-Dueren) was used in combination with the HPLC-precolumn ‘Nucleosil 100-5 C8CC 8/3’ (Macherey–Nagel, D-Dueren). Column-oven temperature was set to 50 °C. For in vitro release and diffusion experiments the eluent consisted of 59.5% methanol and 40.5% phosphoric acid pH 3.0. The flow was set to 1.125 mL/min. Then 20 lL per sample was injected, and the UV-absorbance was measured at 230 nm. Nonivamide was eluted after approximately 3.7 min. For in vitro permeation experiments the eluent consisted of 50% methanol and 50% phosphoric acid pH 3.0. The flow was set to 1.15 mL/min. Then 200 lL per sample was injected, and the UVabsorbance was measured at 230 nm. Nonivamide was eluted after approximately 9.7 min. 2.10. Statistical analysis Data were obtained from repeated measurements (n P 3). Diagrams show mean ± standard deviation. Data were analysed by an unpaired two-sample t-test or one-sided, one factorial analysis of variance (ANOVA) followed by the Student–Newman–Keuls test. Data that are significantly different are marked with an asterisk (). 3. Results and discussion
2.7. Preparation of dermatomed pig ear skin Fresh pig ears were washed with isotonic saline. Postauricular skin was excised. Skin samples were cleaned from blood with isotonic saline and cotton swabs, patted dry with tissue, wrapped in aluminium foil and stored at 30 °C. On the day of the experiment, the skin was thawed at room temperature, cut into strips of approximately 3 cm width and fixed to a block of Styrofoam with pins. The skin was dermatomed to a thickness of 1 mm (Dermatom GA 630, Aesculap AG & Co. KG, D-Melsungen).
Pharmaceutically used emulsions usually consist of an aqueous and an oily phase. Depending on the hydrophilicity/lipophilicity (hydrophilic–lipophilic-balance; HLB-value) of the surfactant, oilin-water or water-in-oil emulsions are formed. In this work, we used oil-in-oil emulsions prepared from castor oil and silicone oil, which were stabilized by a silicone surfactant. Like water and oil, castor oil and silicone oil are not miscible and one phase may be dispersed within the other by mixing. Similar to emulsions made of oil and water, the phase distribution depends on the
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nature of the emulsifier. However, there is no characteristic figure, such as the HLB-value from which the preferred phase distribution may be derived. For each oil–silicone oil–silicone surfactant-combination, the phase distribution needs to be evaluated experimentally [14]. In our case, preliminary experiments showed, that stable castor oil-in-silicone oil-emulsions may be formed by the addition of 2% (m/m) of the silicone surfactant BY 11-030 [12]. The study’s objective was to investigate the factors that influence the release and permeation of an API which is dissolved in the inner oil phase of such oil-in-oil-emulsions. To this end, emulsions that contained different quantities of active, silicone oils of different viscosities, and emulsions that exhibited different phase volume ratios were prepared. The influence of these factors on in vitro release and permeation of the API was investigated. Table 1 shows the compositions of the emulsions that were used in in vitro release and permeation experiments. As the droplet size of the dispersed phase might also have an impact on the results, we attempted to keep this variable almost constant. Droplet sizes of all emulsions were found to be within a comparable range with d50 between 1.1 and 1.8 lm. Therefore, we concluded that all observed effects on in vitro release of the active from the emulsions and permeation through pig ear skin are solely related to the influence of varied composition and are not additionally affected by differences in the droplet size distribution. 3.1. In vitro release of nonivamide Fig. 2 illustrates the release of nonivamide from an oil-in-oilemulsion. It can be seen that release of the API from the inner oil phase is a sequence of diffusion and partitioning processes. In order to diffuse to the membrane/skin nonivamide has first to partition into the silicone oil and second to diffuse through the silicone oil. The partition coefficient of nonivamide (castor oil/silicone oil) is >1300. Therefore, nonivamide is almost exclusively located in the castor oil droplets. Diffusion may be described by Fick’s 1st law of diffusion [15]. According to that, the velocity of diffusion depends on (a) the concentration gradient of the diffusing substance within the diffusion medium, (b) the viscosity of the diffusion medium and (c) the distance that has travelled [15,16]. Nonivamide concentration is highest within the droplets as it is distributed between castor oil and silicone oil according to its partition coefficient. Alterations of the nonivamide concentration in the oil droplets lead to variations in the concentration gradient and should therefore affect the diffusion rate. It is thus reasonable to assume that not only the concentration in the formulation but also a variation in the concentration in the oil droplets (at a constant concentration in the whole formulation) may influence the release and permeation rates. This condition may be achieved by the use of a smaller amount of castor oil that contains a larger quantity of nonivamide. Whilst the viscosity of the diffusion medium depends
on the nature of the silicone oil, the distance that needs to be travelled may be altered by variation in the phase volume ratio. From this the following three factors that may affect the release of nonivamide are derived: (a) nonivamide content, (b) viscosity of the continuous phase and (c) phase volume ratio. 3.1.1. Influence of nonivamide content on release Table 2 shows the results of in vitro release of nonivamide from oil-in-oil-emulsions that contained different quantities of nonivamide. It can be seen that the release from all formulations is linear with time. As expected, the release rates increase with increasing nonivamide content. The release coefficients of all formulations are within the same range (0.35–0.37 lg/cm2 h). This can be explained by the thermodynamic activity of the API in the vehicle, which increases with increasing drug concentration in the vehicle. As thermodynamic activity is the driving force behind the release, higher concentrations increase drug release [17]. The release coefficients are similar for all formulations. This finding indicates a linear relationship between release rates and nonivamide concentration [17] and it can therefore be concluded that an increased API concentration is the reason for the increased release rates. 3.1.2. Influence of phase volume ratio and nonivamide content in the dispersed phase on release The influence of phase volume ratio on in vitro release of nonivamide was investigated using emulsions with a phase volume ratio between 0.1 and 0.5. Nonivamide concentrations in the castor oil were adjusted in order to maintain a constant nonivamide content of 1% (m/m) within the whole formulation. In order to achieve this, emulsions with a low phase volume ratio were prepared with higher concentrations of nonivamide in castor oil. Table 2 shows the results of the in vitro release experiments. Similar to the formulations previously tested, the release of nonivamide from all formulations is linear with time. Interestingly, the release rate and release coefficient are highest at a phase volume ratio of 0.1 and decrease with increasing phase volume ratio. As the distance that needs to be travelled decreases with increasing phase volume ratio the release rates should have decreased according to Fick’s 1st law. Evidently, release rates of the formulations do not follow this rule. This means that release rates do not primarily depend on the distance that has to be travelled. Instead, we observed an inverse relationship between distance and release rate. Therefore, the differing nonivamide concentrations in the dispersed phase must be the rate-determining factor. To evaluate this assumption, we plotted release rates vs. the concentration of nonivamide in the dispersed phase and calculated the coefficient of determination. We obtained a value of 0.95, which indicates an almost linear relationship between release rate and concentration of the dispersed phase. This proves that the nonivamide Table 2 Release rates and release coefficients of oil-in-oil-emulsions with varying nonivamide concentrations and phase volume ratios. Release rate (lg/cm2 h) ± standard deviation
Release coefficient (mg/cm2 h) ± standard deviation
Nonivamide concentration 0.1% 2 0.5% 10 1.0% 21
0.37 ± 0.13* 1.79 ± 0.09* 3.49 ± 0.57*
0.37 ± 0.13* 0.36 ± 0.02* 0.35 ± 0.06*
Phase volume ratio 0.1 114 0.3 35 0.5 21
7.20 ± 1.51* 4.75 ± 0.41* 3.39 ± 0.72*
0.72 ± 0.15* 0.47 ± 0.04* 0.34 ± 0.07*
Nonivamide concentration in inner oil phase (mg/g)
*
Fig. 2. Illustration of the release of nonivamide from an oil-in-oil-emulsion.
Statistically different data (p 0.05).
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concentration in the dispersed phase predominantly controls release from the oil-in-oil-emulsions.
3.1.3. Influence of viscosity on release The influence of the viscosity on the in vitro release of nonivamide was investigated using emulsions that contained a silicone oil of low viscosity (20 mPa s) or high viscosity (1000 mPa s) and a phase volume ratio of 0.1 or 0.5. Nonivamide concentrations in the castor oil were adjusted in order to maintain a constant nonivamide content of 1% (m/m) within the formulation. Fig. 3 and Table 3 show the results of in vitro release experiments. As expected, release is linear with time. Comparing the release rates from emulsions with a phase volume ratio of 0.1 elucidates that nonivamide is released faster from emulsions that contain the low-viscous silicone oil. Although the release patterns seem to be the same for emulsions with a phase volume ratio of 0.5 (indicated by overlapping error bars), statistical analysis revealed a significant difference between release rates of nonivamide from the two emulsions at a phase volume ratio of 0.5. In accordance with the previous experiments, nonivamide is released faster from emulsions with a phase volume ratio of 0.1. The increase in phase volume ratio from 0.1 to 0.5 leads to a reduction in release rate by 61% (20 mPa s) and 56% (1000 mPa s), whilst increasing the viscosity reduces the release rate by only 48%. The fact that the release from the emulsion with the low-viscous silicone oil is faster than the release from the emulsion with the high-viscous silicone oil may be explained by a higher diffusivity in the low-viscous medium. However, the effect is not as pronounced as expected because according to Fick’s 1st law of diffusion a 50-fold increase in viscosity should reduce the flux by a factor of 50. To further evaluate the influence of the viscosity of the silicone oil on release of nonivamide from the emulsions, we performed an in vitro diffusion experiment in which we eliminated all other formulation variables that might possibly affect the release rate. For this, we used PTFE filter membranes which we loaded with the two silicone oils under investigation. The quantity of silicone oil used was adjusted to fill the pores of the filter but not to leave an excess silicone oil film on the filters. On top of the siliconised filters we added nonivamide solutions in castor oil that exhibited the same concentrations as the nonivamide solutions in emulsions with a phase volume ratio of 0.1 (114 mg/g) and 0.5 (21 mg/g), respectively. Apart from that, in vitro diffusion experiments were performed in a manner similar to in vitro release experiments. The results are shown in Fig. 4 and Table 3. The data clearly show that the diffusion rate is influenced by the viscosity of the silicone oil and the nonivamide concentration in
Fig. 3. Comparison of the in vitro release of nonivamide from oil-in-oil-emulsions with different phase volume ratios that contain silicone oils of different viscosities (d) 20 mPa s 0.1, (j) 20 mPa s 0.5, () 1000 mPa s 0.1, () 1000 mPa s 0.5 (n = 5; error bars = standard deviation).
Table 3 Release and diffusion characteristics of nonivamide from castor oil through silicone oils of different viscosities. Release or diffusion rate (lg/cm2 h) ± standard deviation
Release coefficient (mg/cm2 h) ± standard deviation
Release experiments 20 cst, 0.1 114 1000 cst, 0.1 21 20 cst, 0.5 114 1000 cst, 0.5 21
8.12 ± 1.06* 4.22 ± 0.75 3.14 ± 0.50 1.86 ± 0.27*
0.81 ± 0.11* 0.42 ± 0.07* 0.31 ± 0.05* 0.19 ± 0.02*
Diffusion experiments 20 cst 114 1000 cst 114 20 cst 21 1000 cst 21
8.71 ± 0.46* 6.04 ± 0.62* 2.53 ± 0.23* 1.37 ± 0.27*
Viscosity, phase volume ratio
*
Nonivamide concentration in inner oil phase (mg/g)
Statistically different data (p 0.05).
the castor oil. As the flux is directly related to the concentration gradient a 5.7-fold increase would have to be expected when the concentration is increased from 21 mg/g to 114 mg/g. Experimental data reveal factors of 3.44 (20 mPa s) and 4.41 (1000 mPa s) which closely match our expectations. On the other hand, flux is inversely related to the viscosity of the diffusion medium. Thus a 50-fold decrease in the release rate should result when the viscosity of the diffusion medium is increased from 20 mPa s to 1000 mPa s. However, only a marginal reduction by a factor of 1.44 (114 mg/g) and 1.84 (21 mg/g) was observed in the respective experiments. It is known from other studies, that the diffusion rate through silicone oils does not depend on the macro-viscosity of the silicone oil but on its micro-viscosity [18]. Obviously, the microviscosity of both silicon oils used is similar although they clearly differ in their macro-viscosity. Thus, this formulation variable cannot be used to effectively control the release rate. On the other hand, this provides the opportunity to freely choose the silicone oil that exhibits the most convenient application properties. 3.3. In vitro permeation through pig ear skin 3.3.1. Influence of nonivamide content on release In vitro release experiments showed a strong impact of nonivamide content on release rate. This was explained by a higher thermodynamic activity in formulations that contain higher nonivamide concentrations. As this situation does not change when formulations are applied to the skin, we expected permeation rates to depend on nonivamide concentration as well. Table 4
Fig. 4. Comparison of the in vitro diffusion of nonivamide from differently concentrated nonivamide solutions in castor oil through silicone oils of different viscosities (d) 20 mPa s, 114 mg/g, (j) 20 mPa s, 21 mg/g, () 1000 mPa s, 114 mg/ g, () 1000 mPa s, 21 mg/g (n = 5; error bars = standard deviation).
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shows the results of in vitro permeation experiments with formulations that contained 1.0%, 2.0% or 3.0% (m/m) nonivamide. Nonivamide permeates the skin after a lag time of approximately 10 h. The cumulative permeated amounts are linear with time in all formulations. The permeation rates increase with increasing nonivamide content. The permeation coefficients are independent of nonivamide concentration. The fact that the cumulative permeated amounts are linear with time is in accordance with Fick’s 1st law of diffusion [15] and indicates that permeation of nonivamide through skin is a diffusion limited process. As described above, higher nonivamide concentrations in the formulations lead to higher thermodynamic activity. As thermodynamic activity is the driving force behind permeation, higher thermodynamic activity leads to increased permeation rates. The fact that permeation rates increase linearly with nonivamide content and permeation coefficients remain constant confirms that the permeation rates depend directly on the nonivamide content. 3.3.2. Influence of phase volume ratio on in vitro permeation In vitro release experiments showed that release rates decrease with increasing phase volume ratio. We therefore evaluated the impact of phase volume ratio on permeation. The results of these experiments are shown in Fig. 5 and Table 4. The data show that the formulation with a phase volume ratio of 0.1 exhibits the highest permeation rate. The permeation rates of nonivamide from the formulations with a phase volume ratio of 0.3 and 0.5 are much lower than the permeation rate of nonivamide from the formulation with a phase volume ration of 0.1, and not significantly different from each other. These results agree with the results of release experiments, which showed that release rates clearly depend on phase volume ratio. We concluded that this is attributable to lower nonivamide concentrations in the dispersed phase of emulsions with a high phase volume ratio as we found a linear relationship between nonivamide concentration in the dispersed phase and release rates. Evidently, the situation is more complex in permeation. Between a phase volume ratio of 0.1 and 0.3, the nonivamide concentration in the dispersed phase clearly affects the permeation rate. The effect, however, seems to have levelled out at a phase volume ratio of 0.3. Obviously, diffusion through the emulsion is rate controlling in emulsions with a phase volume ratio of 0.1 whereas at higher phase volume ratios diffusion through skin determines the permeation rate. 3.3.3. Influence of viscosity on in vitro permeation The in vitro release experiments showed that viscosity may influence release rates in formulations with a low phase volume Table 4 Impact of the concentration of nonivamide in castor oil and the viscosity of the silicone oil on the permeation of nonivamide. Permeation rate (lg/cm2 h) ± standard deviation
Permeation coefficient (mg/cm2 h) ± standard deviation
Nonivamide concentration 1% 21 2% 2 3% 10
0.58 ± 0.18 1.16 ± 0.32 2.02 ± 0.80*
0.06 ± 0.02 0.06 ± 0.02 0.07 ± 0.03
Phase volume ratio 0.1 114 0.3 35 0.5 21
1.10 ± 0.16 0.38 ± 0.13 0.46 ± 0.16
0.11 ± 0.02 0.04 ± 0.01 0.05 ± 0.02
Viscosity 20 cst 1000 cst
1.10 ± 0.16* 2.15 ± 0.97*
0.11 ± 0.02* 0.21 ± 0.10*
Nonivamide concentration in inner oil phase (mg/g)
*
114 114
Statistically different data (p 0.05).
Fig. 5. Comparison of the in vitro permeation of nonivamide from oil-in-oilemulsions with different phase volume ratios (d) 0.1, (N) 0.3, (j) 0.5 (n = 5; error bars = standard deviation).
ratio. The effect of viscosity on permeation was evaluated with two formulations that contained either a low-viscous silicone oil (20 mPa s) or a high-viscous silicone oil (1000 mPa s) and showed a phase volume ratio of 0.1. The results are shown in Fig. 6 and Table 4. Interestingly, permeation of nonivamide is faster from the formulation that contains the high-viscous silicone oil. However, the standard deviations of the cumulative permeated amounts overlap and are substantially higher when nonivamide permeates from the formulation that contains the high-viscous silicone oil. Despite the overlapping standard deviations, the statistical analysis proves a significant difference between the permeation rates of the two formulations. Therefore, it must be concluded that viscosity of the coherent phase does influence permeation of nonivamide through pig ear skin. However, the difference is rather small and has no practical relevance. 3.3.4. Comparison to permeation from hydrophilic nonivamide cream Oil-in-oil-emulsions that contain nonivamide are intended to provide an alternative to semisolid formulations like Hydrophilic Nonivamide Cream. The latter is used in the therapy of chronic pruritus, but compliance with the treatment is low as it has to be applied 2–5 times per day. We therefore aimed to develop more substantial formulations in order to enable a reduction in the application frequency. The quantities of nonivamide needed to achieve relief of itch differ strongly from patient to patient. To be able to investigate whether the permeation rate of nonivamide from oil-in-oil-emulsions is within a suitable range for therapy we decided to compare permeation rates of nonivamide from oil-in-oil-emulsions to permeation rates from Hydrophilic Nonivamide Cream 0.1% (HNC 0.1%). This formulation contains nonivamide in a
Fig. 6. Comparison of the in vitro permeation of nonivamide from oil-in-oilemulsions that contain silicone oils of different viscosities (d) 20 mPa s, () 1000 mPa s (n = 5; error bars = standard deviation).
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almost independent of the macro-viscosity of the continuous phase and thus allows adjustment the consistency of the formulation in a wide range. Constant permeation rates can be maintained for a period of 10 h. This is a definite advantage of oil-in-oil-emulsions as application intervals can be extended. This results in enhanced compliance and ensures therapeutic success. The novel concept of oil-in-oil-emulsions may be used for a multitude of lipophilic substances utilising oil-in-oil-emulsions as a drug delivery system which may be tailored to specific needs of patients and thereby allow sustained dermal therapy of various diseases. Acknowledgements Fig. 7. Comparison of the in vitro permeation of nonivamide from (j) oil-in-oilemulsion that contains 1% nonivamide to () Hydrophilic Nonivamide Cream 1% and (e) Hydrophilic Nonivamide Cream 0.1% (n = 5; error bars = standard deviation).
concentration that is used in the therapy of chronic pruritus. Permeation rates of nonivamide from oil-in-oil-emulsions should be in the same range as permeation rates from HNC 0.1% in order to be suitable for therapy. To be able to extend application intervals, we needed to achieve prolonged release of the active from the formulation. This means that the formulation has to control release of the active. To investigate this, we compared release from an oil-in-oil-emulsion that contained 1% nonivamide to permeation from HNC 1%. If oil-inoil-emulsions control release/permeation of nonivamide permeation rates and coefficients of oil-in-oil-emulsions should be significantly lower than those obtained from HNC 1%. The results of permeation experiments (Fig. 7) show that permeation is linear with time for oil-in-oil-emulsions and HNC. Permeation is highest for HNC 1% (9.66 ± 0.83 lg/cm2 h). Permeation from HNC 0.1% (0.98 ± 0.10 lg/cm2 h) is 10 times less than permeation from HNC 1%. The permeation coefficients of nonivamide from HNC 0.1% (0.98 ± 0.10 mg/cm2 h) and HNC 1% (0.98 ± 0.08 mg/cm2 h) are not significantly different. This means that the higher permeation rate of nonivamide from HNC 1% is due to the higher nonivamide content. The permeation rate of nonivamide from oil-in-oil-emulsions (0.69 ± 0.30 lg/cm2 h) is similar to that of nonivamide from HNC 0.1%. This indicates that the permeation rate of the oil-in-oil-emulsion is within a suitable range for therapy. The fact that permeation rate (0.69 ± 0.30 lg/cm2 h) and coefficient (0.07 ± 0.03 mg/cm2 h) of nonivamide from the oil-in-oil-emulsion are substantially lower than the respective values for HNC 1% proves that the oil-in-oil-emulsion controls the release and permeation of nonivamide. Sustained release has therefore been achieved. 4. Conclusion The results of this work elucidate the possibility of formulating stable emulsions that consist of a silicone oil as the continuous phase and a drug solution in castor oil as the dispersed phase. It is shown that the permeation rate of the active substance from its oily solution can be controlled by the drug content in the dispersed phase and the phase volume ratio. The permeation rate is
The authors would like to thank Martin Schenk and his team from the Department of Experimental Medicine at the University of Tuebingen for the supply of pig ears and Dow Corning GmbH for the kind donation of silicone oils and surfactants. References [1] M. Metz, S. Ständer, Chronic pruritus – pathogenesis, clinical aspects and treatment, J. Eur. Acad. Dermatol. Venereol. 24 (2010) 1249–1260. [2] D. Siepmann, C. Weishaupt, T.A. Luger, S. Ständer, Evaluation of the German guideline for chronic pruritus: results of a retrospective study on 385 patients, Dermatology 223 (2011) 374–380. [3] P. Anand, K. Bley, Topical capsaicin for pain management: therapeutic potential and mechanisms of action of the new high-concentration capsaicin 8% patch, Br. J. Anaesth. 107 (2011) 490–502. [4] Bundesvereinigung Deutscher Apotheker, Hydrophile Capsaicinoid Creme 0.025%/0.05%/0.1% (NRF 11.125), Neues Rezeptur Formularium, D-Eschborn, 2010. [5] H. Kählig, A. Hasanovic, B. Biruss, S. Höller, J. Grimm, C. Valenta, Chitosan– glycolic acid: a possible matrix for progesterone delivery into skin, Drug Dev. Ind. Pharm. 35 (2009) 997–1002. [6] A.A. Attama, C. Weber, C.C. Müller-Goymann, Assessment of Drug Permeation from Lipid Nanoparticles formulated with a Novel Structured Lipid Matrix through Artificial Skin Construct Bio-engineered from HDF and HaCaT Cell Lines, Editions de sant, Paris, France, 2008. [7] D.J. Lunter, R. Daniels, New film forming emulsions containing EudragitÒ NE and/or RS 30D for sustained dermal delivery of nonivamide, Eur. J. Pharm. Biopharm. 82 (2012) 291–298. [8] D.J. Lunter, R. Daniels, In vitro skin permeation and penetration of nonivamide from novel film forming emulsions, Skin Pharmacol. Physiol. 26 (2012) 139– 146. [9] V. Jaitely, T. Sakthivel, G. Magee, A.T. Florence, Formulation of Oil in Oil Emulsions: Potential Drug Reservoirs for Slow Release, Editions de sant, Paris, FRANCE, 2004. [10] D. Floyd, Silicone surfactants: applications in the personal care industry, in: R.M. Hill (Ed.), Silicone Surfactants, Marcel Dekker Inc., New York, 1999, pp. 181–209. [11] B. Grüning, A. Bungard, Silicone surfactants: emulsification, in: R.M. Hill (Ed.), Silicone Surfactants, Marcel Dekker Inc., New York, 1999. [12] M. Rottke, Entwicklung und Charakterisierung nonivamidhaltiger Rizinussölin-Silikonöl-Emulsionen zur dermalen Anwendung, 2012. [13] M.d.L. Reyes-Escogido, E.G. Gonzalez-Mondragon, E. Vazquez-Tzompantzi, Chemical and pharmacological aspects of capsaicin, Molecules 16 (2011) 1253–1270. [14] O. Suitthimeathegorn, V. Jaitely, A.T. Florence, Novel anhydrous emulsions: formulation as controlled release vehicles, Int. J. Pharm. 298 (2005) 367–371. [15] A. Fick, Ueber diffusion, Ann. Phys. 170 (1855) 59–86. [16] A. Einstein, Über die von der molekularkinetischen Theorie der Wärme geforderte Bewegung von in ruhenden Flüssigkeiten suspendierten Teilchen, Ann. Phys. 322 (1905) 549–560. [17] J. Hadgraft, Skin, the final frontier, Int. J. Pharm. 224 (2001) 1–18. [18] T. Seki, M. Okamoto, O. Hosoya, D. Aiba, K. Morimoto, K. Juni, Effect of chondroitin sulfate on the diffusion coefficients of drugs in aqueous, Solutions 13 (2003) 215–218.
Please cite this article in press as: M. Rottke et al., In vitro studies on release and skin permeation of nonivamide from novel oil-in-oil-emulsions, Eur. J. Pharm. Biopharm. (2013), http://dx.doi.org/10.1016/j.ejpb.2013.09.018