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The difficulties for a photolabile drug in topical formulations: The case of diclofenac
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International Journal of Pharmaceutics xxx (2014) xxx–xxx
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Pharmaceutical Nanotechnology
The difficulties for a photolabile drug in topical formulations: The case of diclofenac Giuseppina Ioele ∗ , Michele De Luca, Lorena Tavano, Gaetano Ragno Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Rende, CS, Italy
a r t i c l e
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Article history: Received 31 December 2013 Received in revised form 23 January 2014 Accepted 25 January 2014 Available online xxx Keywords: Diclofenac Photodegradation Photostabilization Gel formulation Light-absorbers agents Cyclodextrin
a b s t r a c t Topical commercial formulations containing diclofenac (DC) were submitted to photostability tests, according to the international rules, showing a clear degradation of the drug. The degradation process was monitored by applying the multivariate curve resolution technique to the UV spectral data from samples exposed to stressing irradiation. This method was able to estimate the number of components evolved as well as to draw their spectra and concentration profiles. Three photoproducts (PhPs) were resolved by the analysis of photodegradation kinetics, according to two consecutive reactions with a mechanism postulated as DC > PhP1 > PhP2 and PhP3 . Photodegradation rate of DC in gel was found to be very fast, with a residual content of 90% only after 3.90 min under a radiant exposure of 450 W m−2 . Because of a very slow skin uptake of DC, a prolonged time of exposure to light could lead to a significant decrease of drug available or the uptake of undesired photoproducts. New gel formulations were designed to increase the photostability of DC by incorporating chemical light-absorbers or entrapping the drug into cyclodextrin. Drug photostability resulted increased significantly in comparison with that of the commercial formulations. The gel containing the light-absorbers such as octisilate, octyl methoxycinnamate and a combination thereof showed a residual DC of 90% up to 12.22 min, 13.75 min and 15.71 min, respectively, under the same irradiation power. The best results were obtained by incorporating the drug in -cyclodextrin with a degradation of 10% after 25.01 min of light exposure. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Diclofenac, (2-[(2,6-dichlorophenyl)amino] phenylacetate) (DC), is a non-steroidal anti-inflammatory drug (NSAID), commonly used for the treatment of arthritis, soft tissue injuries (Fei et al., 2006; Todd and Sorkin, 1988), dysmenorrhoea, and menorrhagia (Dawood, 1993). On the other hand, DC treatment may have some adverse effects, such as gastrointestinal damage, platelet dysfunction, and convulsion. These effects are likely to be associated with the ability of this compound to compete with arachidonic acid for binding to cyclo-oxygenase, resulting in decreased prostaglandin formation (Small, 1989; Vane, 1996). Clinical evidence suggests that the topical use of NSAIDs is safer and at least as efficacious as oral administration in the treatment of rheumatic diseases (Boinpally et al., 2003; Heyneman et al., 2000). Unfortunately, DC is not easily absorbed on transdermal application (Nishihata et al., 1987), and many strategies have been suggested in order to overcome the low permeability of drugs through the skin (Barry, 1983; Nokhodchi et al., 2002).
∗ Corresponding author. Tel.: +39 0984 493268; fax: +39 0984 493201. E-mail address: [email protected] (G. Ioele).
DC has been reported to be not stable and undergo degradation processes. Stability studies of this compound showed several potential impurities (Gaudiano et al., 2003; Hartmann et al., 2008; Hofmann et al., 2007). The main impurity, 1(2,6-dichlorophenyl)indolin-2-one, may occur in pharmaceutical formulations after long-term storage (Hájková et al., 2002). One of the most studied degradation processes relating to DC is its photodegradation in the aquatic environment. Zhang et al. (2011) have studied the kinetic model for DC photodegradation in water and the variation of the degradation profile in the presence of different forms of nitrogen in the aquatic environment under simulated sunlight. Newly, Salgado et al. (2013) have investigated the degradation kinetics of DC by UV radiation used for wastewater disinfection. The identified photoproducts presented a quinone imine structure, probably obtained by decarboxylation and oxidation followed by dehalogenation and cyclization. Monitoring of drug degradation in dosage forms can be more difficult by the fact that its mechanism may vary depending on the formulation. Moreover, degradation may be influenced by temperature, humidity, light, container and often from the combination of these factors (Galmier et al., 2005). No studies have been reported up today about the photodegradation of DC in the semi-solid commercial specialties. Being the global consumption of DC estimated to be 940 tons per year (Vieno et al., 2007; Wiegel et al., 2004) and
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in consideration of the slow absorption of the drug through the skin, a detailed photostability study of DC and the design of novel light-stable semi-solid formulations seemed interesting. In this work, an in depth study about the photodegradation of DC in solution and semi-solid formulations is reported and attempts for the photostabilization of gel formulations containing DC are proposed through addition of chemical UV-absorbers or incorporating the drug into cyclodextrin supramolecular matrices. The protecting action of the UV-absorber compounds is based on the property of absorbing, partially or wholly, visible and/or UV sun radiations, showing absorption spectra overlapping that of the photosensitive drugs (Aloui et al., 2007; Atarashi et al., 2012; Gaspar and Maia Campos, 2006, 2007; Kockler et al., 2012; Mahltig et al., 2005; Venditti et al., 2008). However, the choice of these additives is limited as a result of the pharmaceutical, toxicological and economic requirements. Derivatives of 2-hydroxybenzophenone are extensively employed as UV absorbers (Negreira et al., 2009). In the European Union (EU), 2-hydroxy-4-methoxybenzophenone5-sulphonic acid (BP-4) and 2-hydroxy-4-methoxybenzophenone (BP-3) have been approved to be used as UV filters in sunscreens at maximum concentrations of 5% and 10%, respectively. In this work, UV absorbers were added in gel formulations of DC, at concentrations recommended by law (Parlamento Europeo, 2009), in order to increase the photoprotection of the drug but without altering the transparency of the gel. Mixtures of UV-absorbers were also tested to study possible synergic effects. Photostabilization of DC was besides approached by incorporation of the drug in cyclodextrins. Cyclodextrins are hydrophilic water-soluble oligosaccharides that form hydrophobic cavities in which water-insoluble drugs can be entrapped thus leading to a significant increase in the concentration of the drug in solution and, at the same time, to a physical shield from light. Actually, supramolecular systems have been proposed in the last years for enhancing the stability of drugs to light and many studies reporting significant successes in this field have been published. In particular, cyclodextrins have shown the most promising results (Garnero and Longhi, 2010; Wang et al., 2013) for the improvement of the chemical stability. The photodegradation kinetics of the new proposed formulations was defined and compared with that of the DC commercial gel specialties. The quantitative determination of the drug was performed by applying a chemometric approach to the analysis of the spectral data avoiding so any previous separation of the analytes. The algorithm Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) was selected for its ability in well describing the kinetics of drug photodegradation, offering a fast and powerful tool for the mathematical resolution of unknown mixtures (de Juan et al., 2009; Gemperline, 1999; Rajko and Istvan, 2005; Wentzell et al., 1998) with the estimation of the pure spectra and the concentration (time) profiles of the photoproducts (de Juan et al., 2000, 2009; De Luca et al., 2010, 2013; Mas et al., 2008). The results about the DC photodegradation process were compared with the data reported in the literature (Zhang et al., 2011).
2. Materials and methods 2.1. Chemicals Diclofenac (DC), propylene glycol, microcrystalline cellulose, 2-hydroxy-4-methoxybenzophenone (BP-3), octocrylene (OC), octisilate (OS), octyl methoxycinnamate (OMC), ␣-cyclodextrin (␣C), -cyclodextrin (C), methyl-- cyclodextrin (mC), ␥cyclodextrin (␥C) were purchased from Sigma–Aldrich (Germany); ethanol, methanol and acetonitrile were from J.T. Baker (Holland).
All other reagents were of the highest purity commercially available. Pharmaceutical specialties Voltaren Emulgel® 1% and Voltaren Emulgel® 2% (Novartis Farma SpA, Italy) were obtained commercially.
2.2. Instruments UV spectra of the samples in a 10 mm quartz cell were registered on a Perkin-Elmer Lambda 40P Spectrophotometer at the following conditions: range 200–450 nm, scan rate 1 nm s−1 ; time response 1 s; spectral band 1 nm. The software UV Winlab 2.79.01 (Perkin-Elmer) was used for spectral acquisition and elaboration. All chemometric procedures were performed under Matlab® computer environment (Mathwork Inc., version 7). MCR routine computer methods were implemented as MATLAB functions. Photodegradation experiments were performed according to the ICH Guideline for photostability testing (International Conference on Harmonization, 2003) by using a light cabinet Suntest CPS+ (Heraeus, Milan, Italy), equipped with a Xenon lamp and an electronic device for measuring and controlling both irradiation and temperature inside the box. The system is able to closely simulate sunlight and to select spectral regions by interposition of filters. In the present study, the samples were irradiated in a range between 300 and 800 nm, by means of a glass filter, according to the ID65 standard of ICH rules. The spin-dryer was a Multispeed centrifuge PK 121 (ALC International s.r.l., Italy).
2.3. Standard solutions Two sets of DC standard solutions were prepared in the range of 5.0–50.0 mg ml−1 . The spectrophotometric analyses were performed after appropriate dilution with methanol for the first solutions set and Britton–Robinson buffer for the second ones. These solutions were used also to prepare validation samples.
2.4. Preparation of DC gel Gel formulation (20 g) was prepared as reported in the Pharmacopoeia (Council of Europe, 2010). 0.20 g of DC (1%, w/w) was dispersed in propylene glycol 2 g under continuous stirring for 15 min and so obtaining a complete emulsification. 0.60 g of microcrystalline cellulose (gelling agent) and 17.2 g of water were then added and the final emulsion was stirred for 50 min getting a homogeneous white gel.
2.5. Preparation of DC–UV absorbers gel DC gel with the addition of UV-absorbers was prepared in a manner similar to that of DC gel, where the UV-absorbers were added in propylene glycol together the DC powder. The optimal concentration of any compound was tested by varying the added percentage in the range 0.25–5.0%, according to the amount permitted in pharmaceutical and cosmetic formulations (Parlamento Europeo, 2009). Anyhow, the concentration of absorber was controlled that did not adversely affect the rheological properties of the gel. The eventual additive or synergistic effect from a combination of UV absorbers was also tested by preparing further formulations containing OS and OMC at concentrations between 0.25% and 0.50% were also tested.
Please cite this article in press as: Ioele, G., et al., The difficulties for a photolabile drug in topical formulations: The case of diclofenac. Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.01.030
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2.6. Preparation of DC–cyclodextrin gel 245 mg of DC were dissolved in 10 ml of cyclodextrin solution (1.32 g of cyclodextrin in 100 ml ethanol, 10 mM). 10.0 ml of Britton–Robinson buffer at pH 6.57 (0.04 M phosphoric acid; 0.04 M acetic acid; 0.04 M boric acid; 0.2 M NaOH) was added under stirring for 20 h at 37 ◦ C. A control drug-free solution was prepared under the same conditions. Four series of samples (4 × 5) were prepared by using ␣C, C, mC, and ␥C. All the samples were stored for 4 days at 4 ◦ C and then filtered through a 0.45 m membrane. The gel formulation containing the complex cyclodextrin–DC was prepared by adding 17.2 g of the complex solution to propylene glycol 2 g and microcrystalline cellulose 0.60 g under continuous stirring for 50 min. The concentration of DC in this preparation was measured to be about 1%. For the solubility tests, 245 mg of DC was added to each cyclodextrin with concentrations of 1, 5, 7.5 and 10 mM in 10 ml buffer. The suspensions were shaken at 25 ◦ C for 48 h. The sample for UV analysis was prepared by filtering an aliquot through a 0.45 m filter. The quantity of DC was measured by using the standard buffer solutions as reference. 2.7. Preparation of the analytical samples Gel 0.5 g was uniformly stratified on 5 glass plates (10 cm × 5 cm) to form a layer thickness of 0.25 mm and then exposed to forced irradiation. After each irradiation dose, the glass was plunged in acetonitrile 25 ml and sonicated for 10 min at room temperature. 10 ml of the suspension obtained was then centrifuged at 5000 rpm for 10 min. The supernatant was analyzed after 1:10 dilution with methanol. The concentration of drug residue was calculated by MCR applied on the UV spectrum of the methanol extract. The extraction procedure was optimized to maximize the drug recovery by evaluating the volume of acetonitrile, sonication time and centrifugation time. The final procedure was validated on a series of twenty reference gel samples with drug content in the range of 150–300 mg obtaining a percentage recovery between 91% and 108%. 2.8. Photodegradation test To minimize DC photodegradation, all laboratory experiments were carried out in a dark room under the illumination of a red lamp (60 W), kept at a distance of about 2 m. The stressed tests, according to the standard ID65 of ICH rules, were executed on DC in solution, in quartz cells perfectly stoppered, to prevent any evaporation of the solvent, and on gel. The samples were irradiated under an irradiance power of 450 W m−2 , corresponding to 27 kJ m−2 min, at a constant temperature of 25 ◦ C. The samples were assayed by UV spectrophotometry after appropriate preparation as described above. Analysis was performed just after preparation (t = 0 min) and at the following exposure times: 5–10–20–30–60 min. 2.9. Multivariate curve resolution-alternating least squares (MCR-ALS) The multivariate resolution methods provide to decompose an experimental data matrix from a chemical process into the pure contributions of the single components. The experimental data matrix, D, is decomposed into the product of two smaller factor matrices, C and ST : D = C · ST + E
Fig. 1. Absorbance spectra of DC methanol solution before ( ) and after irradiation intervals (··· ).
where D is the data matrix obtained from the experimental spectral measurements and contains as many rows as absorption spectra recorded along the chemical process (time, reaction conditions, etc.), C is the concentration matrix of n components involved in the process, ST is the spectral matrix of the pure components and E contains the unexplained data variance (Skoog and West, 2004). In a photodegradation study, the pathway of the process is often unknown and many products can be generated. The number of species involved is so difficult to determine and chemical rank analysis can give a lower number of components than the real number of absorbing species giving so a rank-deficiency (Izquierdo-Ridorsa et al., 1997). The rank deficiency problems could be removed by the simultaneous analysis of multiple experiments, under different conditions and/or together with simpler subsystems, via columnwise data matrix augmentation. In this way, the estimation of the correct number of components and the resolution of the whole system are achieved (Norgaard and Ridder, 1994). In this work, UV spectrophotometric data matrices from photodegradation experiments on different DC gel were simultaneously analyzed with the UV data matrices built from the calibration curves of DC solutions at five concentration levels. The spectral data of the absorber agents and cyclodextrin were added to the calibration matrices in modeling MCR. No interference was observed from cyclodextrins for the absence of overlap of the UV spectra. On the contrary, the UV spectra of the absorber agents overlapped the DC spectrum. Therefore, their spectral data were introduced in building the MCR method. Validation was made by removing one sample at a time from the calibration step and performing the calibration with all other samples. The concentration of the sample removed was then predicted with the obtained model. This step was in turn repeated for each sample considered. 3. Results and discussion 3.1. Photodegradation of DC solution A methanol solution of DC 10.0 mg ml−1 was subdued to forced photodegradation, under the standard conditions above reported. Degradation was monitored by spectrophotometry along irradiation after appropriate dilution of sample. The spectra, depicted in Fig. 1, were recorded just after the preparation and at several exposure times. The spectral data, as an average of six experiments, collected during the photodegradation tests, were used to construct the data matrix to be analyzed by MCR-ALS. Data elaboration showed the formation of one major photoproduct (PhP1 ) and traces of other
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Fig. 3. Concentration profiles of DC and photoproducts in the specialty Voltaren Emulgel® 1% from MCR elaboration.
3.2. Photodegradation of the commercial specialties
Fig. 2. Concentration profiles of DC (20.43 g ml−1 ) and photoproducts (a) and relative absorbance spectra (b) from MCR elaboration.
two by-products (PhP2 and PhP3 ). The spectra of these compounds were in accordance with those reported in the HPLC study by Zhang et al. (2011). Fig. 2 shows the concentration profiles (Fig. 2a) of DC and photoproducts and their absorbance spectra (Fig. 2b). The relative standard deviation values of all the points resulted ranging in the interval of 1.92–6.01%. The profile of photodegradation kinetics was hypothesized according to two consecutive reactions, with a mechanism postulated as DC > PhP1 > PhP2 . A third photoproduct PhP3 was also revealed in the first 10 min of irradiation but its content decreased until to a complete disappearance after about 40 min. Salgado et al. (2013) reported PhP1 corresponds to the decarboxylated product (E)-6-[2,6-dichlorophenyl)-imino]-3oxocyclohexa-1,4-dienecarbaldehyde. A full degradation of DC was observed after about 44.01 min. The degradation process proceeded via first-order kinetics, described by the following equation:
The fast photodegradation of DC in solution suggested to extend the study to the semi-solid specialties containing this drug. Two commercial formulations with DC concentration 1% and 2%, respectively, were exposed to forced degradation and then analyzed at various times as above described. A clear degradation of the drug was showed and degradation kinetics of first-order was confirmed. In particular, the values of t0.1 resulted 4.38 and 6.22 min for DC formulations at 1% and 2%, respectively. These experiments confirmed the high rate of degradation for both gel and its increase when the initial concentration of DC decreased, in accordance with Zhang et al. (2011). The specialty at 2% also contained butylated hydroxytoluene (BHT) as an excipient, usually used in pharmaceuticals for its antioxidant properties (Hocman, 1988). The better results obtained in photostability of this formulation was probably due also to the presence of this component. The complete disappearance of the drug was observed at 182.7 and 264.7 min for 1% and 2% formulations. MCR application to these data confirmed the formation of PhP1 as the main photoproduct. On the contrary, PhP2 was formed in minor amount while PhP3 was revealed only in traces, as shown in Fig. 3. As the photodegradation of the drug may be affected by the mode and the amount of the spread formulation, the influence of the thickness of the gel layer was also tested. For this aim, the 1% gel was spread uniformly over four glass plates to form layers of 0.25–0.50–0.75 and 1.0 mm thickness. The thickness of the gel was selected by using a purpose-built device. Two sheets of aluminum with a known thickness 0.25 mm were placed on a glass surface at a distance of 2 cm (Fig. 4). The gel was then layered on the glass by sliding a steel spatula on the aluminum sheets. Higher thicknesses were made by overlapping the aluminum sheets to each other up to a maximum thickness of 1 mm.
log[%DC] = −k1 · t + 2 where %DC is the percentage of residual drug, k the photodegradation rate constant, t the time (min), and 2 the logarithm of the starting concentration (100%). The parameter t0.1 (time to cause 10% degradation) was chosen as a criterion to compare the degradation behavior of the tested samples. This parameter is conventionally adopted because a drug can no longer be used when its purity falls below 90%. The value of k1 was 0.0681 and t0.1 resulted to be 2.52 min, corresponding to a radiant exposure of 68 kJ m−2 .
Fig. 4. Device for gel layering.
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Fig. 5. Influence of the gel layer thickness on DC photodegradation after 60 min of light irradiation. Fig. 7. Phase-solubility diagram of the DC–cyclodextrin complexes.
These samples were submitted to forced irradiation and analyzed up to 60 min. As shown in Fig. 5, the degradation decreased with increasing layer thickness, most likely for a simple physical effect of shielding the light. The layer thickness of 0.25 mm was so adopted for further photodegradation experiments in order to maximize the amount of drug exposed to light. 3.3. Photodegradation of DC–UV absorber gel Photodegradation of DC could involve not only the reduction of the therapeutic dose but also the formation of secondary products with unknown activity for health. For this purpose, new semi-solid preparations containing DC were designed to reduce drug photodegradation. In the recent years, the employment of UV filters added to topical formulations is one of the efficacious strategies proposed to prevent contact photosensitivity and drug photodegradation (Atarashi et al., 2012; Gaspar and Maia Campos, 2007). Among the various UV absorbers proposed in the literature, BP, OC, OS and OMC were selected to be added to the DC gel because of the overlap of their absorption spectra with that of DC. These formulations were prepared as described in the experimental part, with the filter content in the range 0.25–5%. As a control sample, a standard DC gel was prepared with DC at 1%. All gel formulations were then exposed to forced degradation up to 60 min and the residual drug analyses was then carried out. The standard gel presented a DC residual of 19.50%, whereas photostability resulted influenced in different manner for the other samples. Fig. 6 shows the drug percentage residue in the samples containing the absorbers at the various concentrations.
The OS and OMC samples showed a clear increase of the photostability for the absorber concentration 0.5% with a residual DC concentration of 64% and 68%, respectively. However, these values remained constant for the samples with higher content of these absorbers. Unsuccessful results were on the contrary carried out for the other absorbers BP and OC that showed no significant improvement of the drug stability. This behavior has been reported as a consequence of photochemical reactions of these molecules, such as trans-cis isomerization or keto-enol tautomerism (Kockler et al., 2012) or, moreover, production of by-products after light irradiation (Shaath, 2010). The degradation process of the DC control sample was confirmed to follow the first-order kinetics. The samples containing the absorbers instead showed a different behavior. By plotting the drug percentage concentration against degradation time, a second order kinetics was observed with straight lines according to the equation: 1 = k2 · t + 0.01 DC% where DC% is the percentage of the residual drug concentration, k2 the photodegradation rate constant, t the time (min) and 0.01 is the reciprocal value of the starting concentration percentage (100%). MCR-ALS analysis predicted the formation of PhP1 and only traces of PhP2 and PhP3 . The rate constants and the t0.1 values of the 0.5% absorber formulations were carried out. Successful t0.1 values of 12.22 min and 13.75 min for OS and OMC, respectively, were calculated showing a notable increase in comparison with a value of 3.90 min measured on the control gel. As more than one UV-filter is nowadays used in cosmetic preparations (Chatelain and Gabard, 2001; Gaspar and Campos, 2006; Lhiaubet-Vallet et al., 2010), the combination of OS and OMC was also verified in affecting photoprotection. Three formulations with OS/OMC ratio between 0.5 and 2 were tested, but the results did not show significant additive or synergistic effects. The best t0.1 value of 15.71 min was measured on the OS/OMC formulation containing both 0.5%. 3.4. Photodegradation of DC–cyclodextrin gel
Fig. 6. Influence of UV absorber concentration on DC photodegradation after 60 min of light irradiation.
The entrapment of DC in cyclodextrin was afterwards investigated to minimize light sensitivity of the drug and, at the same time, to enhance drug solubility in water. First, the influence of ␣C, C, mC, and ␥C on solubility of DC was tested as described by Higuchi and Connors (1965). The behavior of DC in the different cyclodextrins is shown in the phase-solubility diagram reported in Fig. 7.
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The photodegradation profile of the complex followed secondorder kinetics and was compared in Fig. 8 to that of 1% DC control sample and 1% commercial specialty. Table 1 summarizes the degradation rate constants and the values of t0.1 for all the studied matrices. All results were averages of six experiments. The relative standard deviation values of all the points resulted ranging in the interval 2.03–4.99%. 4. Conclusions
Fig. 8. Photodegradation profile of DC in gel control, Voltaren Emulgel® 1% and C-DC gel.
The most effective cyclodextrin in increasing the solubility of DC was C. A linear relationship between solubility and C concentration was observed with R2 equal to 0.997. This result was in agreement with an almost 1:1 inclusion complex (Higuchi and Connors, 1965). The binding constant (Kb ) of the complexes was calculated according to the following equation: Kb =
slope × (1 − slope) SDC
where SDC is the solubility in water of DC at 25 ◦ C. The values of Kb , expressed as M−1 , followed the order C-DC (1361.32), mC-DC (484.76), ␣C-DC (33.51), and ␥C-DC (21.83). The higher solubility of C-DC and mC-DC complexes can be explained by a better fitting of the drug molecule in the cavity of these cyclodextrins, whereas ␣C seems to have a cavity too small and ␥C, conversely, too large. C was finally selected to incorporate DC in the preparation of the gel to be submitted to the photostability test. The C-DC gel, with DC concentration at 1%, was prepared as above described and then submitted to the stressing test. DC residue along the experiments was each time assayed after appropriate dilution of 100 l of C-DC to 10 ml with methanol, in such a way to assure the drug release. The results from the stress testing showed a clear decrease of DC degradation, with a very successful t0.1 value of 25.01 min. The very good performance of this formulation in terms of photostabilization could be attributed to a dual action of the cyclodextrin complex: a real physical protection of the entrapped drug by means of a molecular shield (Bayomi et al., 2002; Mielcarek, 1997) aided by the increase of the drug solubility in this matrix.
Table 1 Degradation kinetics parameters of DC in different matrices. Matrix
k1
R2
t0.1
Methanol solution Voltaren Emulgel® 1% Voltaren Emulgel® 2% Standard gel
0.0681 0.0105 0.0074 0.0118
0.978 0.968 0.998 0.979
2.52 4.38 6.22 3.90
BP-DC gel OS-DC gel OC-DC gel OMC-DC gel OS 0.25/OMC 0.50 gel OS 0.50/OMC 0.50 gel OS 0.50/OMC 0.25 gel C-DC gel
k2 0.0006 0.00009 0.0004 0.00008 0.00006 0.00007 0.00006 0.00004
R2 0.992 0.950 0.961 0.951 0.977 0.967 0.952 0.938
t0.1 1.83 12.22 2.75 13.75 13.92 15.71 13.87 25.01
t is expressed as min.
In this work, the anti-inflammatory drug diclofenac in commercial gel formulations was demonstrated to undergo photodegradation. The study was performed by adopting photostability tests defined by international rules. In particular, under an irradiance power of 450 W m−2 , corresponding to 27 kJ m−2 min and at a constant temperature of 25 ◦ C, the drug resulted degraded of 10% in 4.38 min and 6.22 min in 1% and 2% formulations, respectively. The design of photoprotective pharmaceutical matrices for topical application is particularly important for the greater likelihood of exposure to light with the result of reducing the available drug and at the same time increasing the risk of formation of toxic photoproducts. Two different approaches were adopted to reduce drug photodegradation by incorporation in gel of UV-absorbers and by entrapping the drug in cyclodextrins. The use of the octisilate or octyl methoxycinnamate as excipients gave good results by increasing the drug photostability of 1% gel to 12.22 and 13.75 min. Better results were carried out by incorporating the drug in cyclodextrin, increasing this value up to 25.01 min, corresponding to a stability increase of almost six times. The systems proposed here appear to be of certain interest for the development of new pharmaceutical formulations for topical use of diclofenac, able to minimize the photodegradation of the drug. Acknowledgment This research was supported by grants from M.U.R.S.T. (Italy). References Aloui, F., Ahajji, A., Irmouli, Y., George, B., Charrier, B., Merlin, A., 2007. Inorganic UV absorbers for the photostabilisation of wood-clearcoating systems: comparison with organic UV absorbers. Appl. Surf. Sci. 253, 3737–3745. Atarashi, K., Takano, M., Kato, S., Kuma, H., Nakanishi, M., Tokura, Y., 2012. Addition of UVA-absorber butyl methoxy dibenzoylmethane to topical ketoprofen formulation reduces ketoprofen photoallergic reaction. J. Photochem. Photobiol. B 113, 56–62. Barry, B.W., 1983. Dermatological Formulations: Percutaneous Absorption. Marcel Dekker, New York. Bayomi, M.A., Abanumay, K.A., Al-Angary, A.A., 2002. Effect of inclusion complexation with cyclodextrins on photostability of nifedipine in solid state. Int. J. Pharm. 243, 107–117. Boinpally, R.R., Zhou, S.L., Poondru, S., Devraj, G., Jasti, B.R., 2003. Lecithin vesicles for topical delivery of diclofenac. Eur. J. Pharm. Biopharm. 56, 389–392. Chatelain, E., Gabard, B., 2001. Photostabilization of butyl methoxydibenzoylmethane (Avobenzone) and ethylhexyl methoxycinnamate by bisethylhexyloxyphenol methoxyphenyl triazine (Tinosorb S), a new UV broadband filter. Photochem. Photobiol. 74, 401–406. Council of Europe, 2010. European Pharmacopoeia, seventh ed. Council of Europe, Strasbourg. Dawood, M.Y., 1993. Nonsteroidal antiinflammatory drugs and reproduction. Am. J. Obstet. Gynecol. 169, 1255–1265. de Juan, A., Casassas, E., Tauler, R., 2000. Encyclopedia of Analytical Chemistry: Instrumentation and Applications, Soft-Modeling of Analytical Data, vol. 11. Wiley, New York. de Juan, A., Rutan, S.C., Maeder, M., Tauler, R., 2009. Comprehensive Chemometrics, vol. 2. Elsevier, Amsterdam. De Luca, M., Mas, S., Ioele, G., Oliverio, F., Ragno, G., Tauler, R., 2010. Kinetic studies of nitrofurazone photodegradation by multivariate curve resolution applied to UV-spectral data. Int. J. Pharm. 386, 99–107. De Luca, M., Tauler, R., Ioele, G., Ragno, G., 2013. Study of photodegradation kinetics of melatonin by multivariate curve resolution (MCR) with estimation of feasible band boundaries. Drug Test. Anal. 5, 96–102.
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Pharmaceutical Nanotechnology
The difficulties for a photolabile drug in topical formulations: The case of diclofenac Giuseppina Ioele ∗ , Michele De Luca, Lorena Tavano, Gaetano Ragno Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Rende, CS, Italy
a r t i c l e
i n f o
Article history: Received 31 December 2013 Received in revised form 23 January 2014 Accepted 25 January 2014 Available online xxx Keywords: Diclofenac Photodegradation Photostabilization Gel formulation Light-absorbers agents Cyclodextrin
a b s t r a c t Topical commercial formulations containing diclofenac (DC) were submitted to photostability tests, according to the international rules, showing a clear degradation of the drug. The degradation process was monitored by applying the multivariate curve resolution technique to the UV spectral data from samples exposed to stressing irradiation. This method was able to estimate the number of components evolved as well as to draw their spectra and concentration profiles. Three photoproducts (PhPs) were resolved by the analysis of photodegradation kinetics, according to two consecutive reactions with a mechanism postulated as DC > PhP1 > PhP2 and PhP3 . Photodegradation rate of DC in gel was found to be very fast, with a residual content of 90% only after 3.90 min under a radiant exposure of 450 W m−2 . Because of a very slow skin uptake of DC, a prolonged time of exposure to light could lead to a significant decrease of drug available or the uptake of undesired photoproducts. New gel formulations were designed to increase the photostability of DC by incorporating chemical light-absorbers or entrapping the drug into cyclodextrin. Drug photostability resulted increased significantly in comparison with that of the commercial formulations. The gel containing the light-absorbers such as octisilate, octyl methoxycinnamate and a combination thereof showed a residual DC of 90% up to 12.22 min, 13.75 min and 15.71 min, respectively, under the same irradiation power. The best results were obtained by incorporating the drug in -cyclodextrin with a degradation of 10% after 25.01 min of light exposure. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Diclofenac, (2-[(2,6-dichlorophenyl)amino] phenylacetate) (DC), is a non-steroidal anti-inflammatory drug (NSAID), commonly used for the treatment of arthritis, soft tissue injuries (Fei et al., 2006; Todd and Sorkin, 1988), dysmenorrhoea, and menorrhagia (Dawood, 1993). On the other hand, DC treatment may have some adverse effects, such as gastrointestinal damage, platelet dysfunction, and convulsion. These effects are likely to be associated with the ability of this compound to compete with arachidonic acid for binding to cyclo-oxygenase, resulting in decreased prostaglandin formation (Small, 1989; Vane, 1996). Clinical evidence suggests that the topical use of NSAIDs is safer and at least as efficacious as oral administration in the treatment of rheumatic diseases (Boinpally et al., 2003; Heyneman et al., 2000). Unfortunately, DC is not easily absorbed on transdermal application (Nishihata et al., 1987), and many strategies have been suggested in order to overcome the low permeability of drugs through the skin (Barry, 1983; Nokhodchi et al., 2002).
∗ Corresponding author. Tel.: +39 0984 493268; fax: +39 0984 493201. E-mail address: [email protected] (G. Ioele).
DC has been reported to be not stable and undergo degradation processes. Stability studies of this compound showed several potential impurities (Gaudiano et al., 2003; Hartmann et al., 2008; Hofmann et al., 2007). The main impurity, 1(2,6-dichlorophenyl)indolin-2-one, may occur in pharmaceutical formulations after long-term storage (Hájková et al., 2002). One of the most studied degradation processes relating to DC is its photodegradation in the aquatic environment. Zhang et al. (2011) have studied the kinetic model for DC photodegradation in water and the variation of the degradation profile in the presence of different forms of nitrogen in the aquatic environment under simulated sunlight. Newly, Salgado et al. (2013) have investigated the degradation kinetics of DC by UV radiation used for wastewater disinfection. The identified photoproducts presented a quinone imine structure, probably obtained by decarboxylation and oxidation followed by dehalogenation and cyclization. Monitoring of drug degradation in dosage forms can be more difficult by the fact that its mechanism may vary depending on the formulation. Moreover, degradation may be influenced by temperature, humidity, light, container and often from the combination of these factors (Galmier et al., 2005). No studies have been reported up today about the photodegradation of DC in the semi-solid commercial specialties. Being the global consumption of DC estimated to be 940 tons per year (Vieno et al., 2007; Wiegel et al., 2004) and
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in consideration of the slow absorption of the drug through the skin, a detailed photostability study of DC and the design of novel light-stable semi-solid formulations seemed interesting. In this work, an in depth study about the photodegradation of DC in solution and semi-solid formulations is reported and attempts for the photostabilization of gel formulations containing DC are proposed through addition of chemical UV-absorbers or incorporating the drug into cyclodextrin supramolecular matrices. The protecting action of the UV-absorber compounds is based on the property of absorbing, partially or wholly, visible and/or UV sun radiations, showing absorption spectra overlapping that of the photosensitive drugs (Aloui et al., 2007; Atarashi et al., 2012; Gaspar and Maia Campos, 2006, 2007; Kockler et al., 2012; Mahltig et al., 2005; Venditti et al., 2008). However, the choice of these additives is limited as a result of the pharmaceutical, toxicological and economic requirements. Derivatives of 2-hydroxybenzophenone are extensively employed as UV absorbers (Negreira et al., 2009). In the European Union (EU), 2-hydroxy-4-methoxybenzophenone5-sulphonic acid (BP-4) and 2-hydroxy-4-methoxybenzophenone (BP-3) have been approved to be used as UV filters in sunscreens at maximum concentrations of 5% and 10%, respectively. In this work, UV absorbers were added in gel formulations of DC, at concentrations recommended by law (Parlamento Europeo, 2009), in order to increase the photoprotection of the drug but without altering the transparency of the gel. Mixtures of UV-absorbers were also tested to study possible synergic effects. Photostabilization of DC was besides approached by incorporation of the drug in cyclodextrins. Cyclodextrins are hydrophilic water-soluble oligosaccharides that form hydrophobic cavities in which water-insoluble drugs can be entrapped thus leading to a significant increase in the concentration of the drug in solution and, at the same time, to a physical shield from light. Actually, supramolecular systems have been proposed in the last years for enhancing the stability of drugs to light and many studies reporting significant successes in this field have been published. In particular, cyclodextrins have shown the most promising results (Garnero and Longhi, 2010; Wang et al., 2013) for the improvement of the chemical stability. The photodegradation kinetics of the new proposed formulations was defined and compared with that of the DC commercial gel specialties. The quantitative determination of the drug was performed by applying a chemometric approach to the analysis of the spectral data avoiding so any previous separation of the analytes. The algorithm Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) was selected for its ability in well describing the kinetics of drug photodegradation, offering a fast and powerful tool for the mathematical resolution of unknown mixtures (de Juan et al., 2009; Gemperline, 1999; Rajko and Istvan, 2005; Wentzell et al., 1998) with the estimation of the pure spectra and the concentration (time) profiles of the photoproducts (de Juan et al., 2000, 2009; De Luca et al., 2010, 2013; Mas et al., 2008). The results about the DC photodegradation process were compared with the data reported in the literature (Zhang et al., 2011).
2. Materials and methods 2.1. Chemicals Diclofenac (DC), propylene glycol, microcrystalline cellulose, 2-hydroxy-4-methoxybenzophenone (BP-3), octocrylene (OC), octisilate (OS), octyl methoxycinnamate (OMC), ␣-cyclodextrin (␣C), -cyclodextrin (C), methyl-- cyclodextrin (mC), ␥cyclodextrin (␥C) were purchased from Sigma–Aldrich (Germany); ethanol, methanol and acetonitrile were from J.T. Baker (Holland).
All other reagents were of the highest purity commercially available. Pharmaceutical specialties Voltaren Emulgel® 1% and Voltaren Emulgel® 2% (Novartis Farma SpA, Italy) were obtained commercially.
2.2. Instruments UV spectra of the samples in a 10 mm quartz cell were registered on a Perkin-Elmer Lambda 40P Spectrophotometer at the following conditions: range 200–450 nm, scan rate 1 nm s−1 ; time response 1 s; spectral band 1 nm. The software UV Winlab 2.79.01 (Perkin-Elmer) was used for spectral acquisition and elaboration. All chemometric procedures were performed under Matlab® computer environment (Mathwork Inc., version 7). MCR routine computer methods were implemented as MATLAB functions. Photodegradation experiments were performed according to the ICH Guideline for photostability testing (International Conference on Harmonization, 2003) by using a light cabinet Suntest CPS+ (Heraeus, Milan, Italy), equipped with a Xenon lamp and an electronic device for measuring and controlling both irradiation and temperature inside the box. The system is able to closely simulate sunlight and to select spectral regions by interposition of filters. In the present study, the samples were irradiated in a range between 300 and 800 nm, by means of a glass filter, according to the ID65 standard of ICH rules. The spin-dryer was a Multispeed centrifuge PK 121 (ALC International s.r.l., Italy).
2.3. Standard solutions Two sets of DC standard solutions were prepared in the range of 5.0–50.0 mg ml−1 . The spectrophotometric analyses were performed after appropriate dilution with methanol for the first solutions set and Britton–Robinson buffer for the second ones. These solutions were used also to prepare validation samples.
2.4. Preparation of DC gel Gel formulation (20 g) was prepared as reported in the Pharmacopoeia (Council of Europe, 2010). 0.20 g of DC (1%, w/w) was dispersed in propylene glycol 2 g under continuous stirring for 15 min and so obtaining a complete emulsification. 0.60 g of microcrystalline cellulose (gelling agent) and 17.2 g of water were then added and the final emulsion was stirred for 50 min getting a homogeneous white gel.
2.5. Preparation of DC–UV absorbers gel DC gel with the addition of UV-absorbers was prepared in a manner similar to that of DC gel, where the UV-absorbers were added in propylene glycol together the DC powder. The optimal concentration of any compound was tested by varying the added percentage in the range 0.25–5.0%, according to the amount permitted in pharmaceutical and cosmetic formulations (Parlamento Europeo, 2009). Anyhow, the concentration of absorber was controlled that did not adversely affect the rheological properties of the gel. The eventual additive or synergistic effect from a combination of UV absorbers was also tested by preparing further formulations containing OS and OMC at concentrations between 0.25% and 0.50% were also tested.
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2.6. Preparation of DC–cyclodextrin gel 245 mg of DC were dissolved in 10 ml of cyclodextrin solution (1.32 g of cyclodextrin in 100 ml ethanol, 10 mM). 10.0 ml of Britton–Robinson buffer at pH 6.57 (0.04 M phosphoric acid; 0.04 M acetic acid; 0.04 M boric acid; 0.2 M NaOH) was added under stirring for 20 h at 37 ◦ C. A control drug-free solution was prepared under the same conditions. Four series of samples (4 × 5) were prepared by using ␣C, C, mC, and ␥C. All the samples were stored for 4 days at 4 ◦ C and then filtered through a 0.45 m membrane. The gel formulation containing the complex cyclodextrin–DC was prepared by adding 17.2 g of the complex solution to propylene glycol 2 g and microcrystalline cellulose 0.60 g under continuous stirring for 50 min. The concentration of DC in this preparation was measured to be about 1%. For the solubility tests, 245 mg of DC was added to each cyclodextrin with concentrations of 1, 5, 7.5 and 10 mM in 10 ml buffer. The suspensions were shaken at 25 ◦ C for 48 h. The sample for UV analysis was prepared by filtering an aliquot through a 0.45 m filter. The quantity of DC was measured by using the standard buffer solutions as reference. 2.7. Preparation of the analytical samples Gel 0.5 g was uniformly stratified on 5 glass plates (10 cm × 5 cm) to form a layer thickness of 0.25 mm and then exposed to forced irradiation. After each irradiation dose, the glass was plunged in acetonitrile 25 ml and sonicated for 10 min at room temperature. 10 ml of the suspension obtained was then centrifuged at 5000 rpm for 10 min. The supernatant was analyzed after 1:10 dilution with methanol. The concentration of drug residue was calculated by MCR applied on the UV spectrum of the methanol extract. The extraction procedure was optimized to maximize the drug recovery by evaluating the volume of acetonitrile, sonication time and centrifugation time. The final procedure was validated on a series of twenty reference gel samples with drug content in the range of 150–300 mg obtaining a percentage recovery between 91% and 108%. 2.8. Photodegradation test To minimize DC photodegradation, all laboratory experiments were carried out in a dark room under the illumination of a red lamp (60 W), kept at a distance of about 2 m. The stressed tests, according to the standard ID65 of ICH rules, were executed on DC in solution, in quartz cells perfectly stoppered, to prevent any evaporation of the solvent, and on gel. The samples were irradiated under an irradiance power of 450 W m−2 , corresponding to 27 kJ m−2 min, at a constant temperature of 25 ◦ C. The samples were assayed by UV spectrophotometry after appropriate preparation as described above. Analysis was performed just after preparation (t = 0 min) and at the following exposure times: 5–10–20–30–60 min. 2.9. Multivariate curve resolution-alternating least squares (MCR-ALS) The multivariate resolution methods provide to decompose an experimental data matrix from a chemical process into the pure contributions of the single components. The experimental data matrix, D, is decomposed into the product of two smaller factor matrices, C and ST : D = C · ST + E
Fig. 1. Absorbance spectra of DC methanol solution before ( ) and after irradiation intervals (··· ).
where D is the data matrix obtained from the experimental spectral measurements and contains as many rows as absorption spectra recorded along the chemical process (time, reaction conditions, etc.), C is the concentration matrix of n components involved in the process, ST is the spectral matrix of the pure components and E contains the unexplained data variance (Skoog and West, 2004). In a photodegradation study, the pathway of the process is often unknown and many products can be generated. The number of species involved is so difficult to determine and chemical rank analysis can give a lower number of components than the real number of absorbing species giving so a rank-deficiency (Izquierdo-Ridorsa et al., 1997). The rank deficiency problems could be removed by the simultaneous analysis of multiple experiments, under different conditions and/or together with simpler subsystems, via columnwise data matrix augmentation. In this way, the estimation of the correct number of components and the resolution of the whole system are achieved (Norgaard and Ridder, 1994). In this work, UV spectrophotometric data matrices from photodegradation experiments on different DC gel were simultaneously analyzed with the UV data matrices built from the calibration curves of DC solutions at five concentration levels. The spectral data of the absorber agents and cyclodextrin were added to the calibration matrices in modeling MCR. No interference was observed from cyclodextrins for the absence of overlap of the UV spectra. On the contrary, the UV spectra of the absorber agents overlapped the DC spectrum. Therefore, their spectral data were introduced in building the MCR method. Validation was made by removing one sample at a time from the calibration step and performing the calibration with all other samples. The concentration of the sample removed was then predicted with the obtained model. This step was in turn repeated for each sample considered. 3. Results and discussion 3.1. Photodegradation of DC solution A methanol solution of DC 10.0 mg ml−1 was subdued to forced photodegradation, under the standard conditions above reported. Degradation was monitored by spectrophotometry along irradiation after appropriate dilution of sample. The spectra, depicted in Fig. 1, were recorded just after the preparation and at several exposure times. The spectral data, as an average of six experiments, collected during the photodegradation tests, were used to construct the data matrix to be analyzed by MCR-ALS. Data elaboration showed the formation of one major photoproduct (PhP1 ) and traces of other
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Fig. 3. Concentration profiles of DC and photoproducts in the specialty Voltaren Emulgel® 1% from MCR elaboration.
3.2. Photodegradation of the commercial specialties
Fig. 2. Concentration profiles of DC (20.43 g ml−1 ) and photoproducts (a) and relative absorbance spectra (b) from MCR elaboration.
two by-products (PhP2 and PhP3 ). The spectra of these compounds were in accordance with those reported in the HPLC study by Zhang et al. (2011). Fig. 2 shows the concentration profiles (Fig. 2a) of DC and photoproducts and their absorbance spectra (Fig. 2b). The relative standard deviation values of all the points resulted ranging in the interval of 1.92–6.01%. The profile of photodegradation kinetics was hypothesized according to two consecutive reactions, with a mechanism postulated as DC > PhP1 > PhP2 . A third photoproduct PhP3 was also revealed in the first 10 min of irradiation but its content decreased until to a complete disappearance after about 40 min. Salgado et al. (2013) reported PhP1 corresponds to the decarboxylated product (E)-6-[2,6-dichlorophenyl)-imino]-3oxocyclohexa-1,4-dienecarbaldehyde. A full degradation of DC was observed after about 44.01 min. The degradation process proceeded via first-order kinetics, described by the following equation:
The fast photodegradation of DC in solution suggested to extend the study to the semi-solid specialties containing this drug. Two commercial formulations with DC concentration 1% and 2%, respectively, were exposed to forced degradation and then analyzed at various times as above described. A clear degradation of the drug was showed and degradation kinetics of first-order was confirmed. In particular, the values of t0.1 resulted 4.38 and 6.22 min for DC formulations at 1% and 2%, respectively. These experiments confirmed the high rate of degradation for both gel and its increase when the initial concentration of DC decreased, in accordance with Zhang et al. (2011). The specialty at 2% also contained butylated hydroxytoluene (BHT) as an excipient, usually used in pharmaceuticals for its antioxidant properties (Hocman, 1988). The better results obtained in photostability of this formulation was probably due also to the presence of this component. The complete disappearance of the drug was observed at 182.7 and 264.7 min for 1% and 2% formulations. MCR application to these data confirmed the formation of PhP1 as the main photoproduct. On the contrary, PhP2 was formed in minor amount while PhP3 was revealed only in traces, as shown in Fig. 3. As the photodegradation of the drug may be affected by the mode and the amount of the spread formulation, the influence of the thickness of the gel layer was also tested. For this aim, the 1% gel was spread uniformly over four glass plates to form layers of 0.25–0.50–0.75 and 1.0 mm thickness. The thickness of the gel was selected by using a purpose-built device. Two sheets of aluminum with a known thickness 0.25 mm were placed on a glass surface at a distance of 2 cm (Fig. 4). The gel was then layered on the glass by sliding a steel spatula on the aluminum sheets. Higher thicknesses were made by overlapping the aluminum sheets to each other up to a maximum thickness of 1 mm.
log[%DC] = −k1 · t + 2 where %DC is the percentage of residual drug, k the photodegradation rate constant, t the time (min), and 2 the logarithm of the starting concentration (100%). The parameter t0.1 (time to cause 10% degradation) was chosen as a criterion to compare the degradation behavior of the tested samples. This parameter is conventionally adopted because a drug can no longer be used when its purity falls below 90%. The value of k1 was 0.0681 and t0.1 resulted to be 2.52 min, corresponding to a radiant exposure of 68 kJ m−2 .
Fig. 4. Device for gel layering.
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Fig. 5. Influence of the gel layer thickness on DC photodegradation after 60 min of light irradiation. Fig. 7. Phase-solubility diagram of the DC–cyclodextrin complexes.
These samples were submitted to forced irradiation and analyzed up to 60 min. As shown in Fig. 5, the degradation decreased with increasing layer thickness, most likely for a simple physical effect of shielding the light. The layer thickness of 0.25 mm was so adopted for further photodegradation experiments in order to maximize the amount of drug exposed to light. 3.3. Photodegradation of DC–UV absorber gel Photodegradation of DC could involve not only the reduction of the therapeutic dose but also the formation of secondary products with unknown activity for health. For this purpose, new semi-solid preparations containing DC were designed to reduce drug photodegradation. In the recent years, the employment of UV filters added to topical formulations is one of the efficacious strategies proposed to prevent contact photosensitivity and drug photodegradation (Atarashi et al., 2012; Gaspar and Maia Campos, 2007). Among the various UV absorbers proposed in the literature, BP, OC, OS and OMC were selected to be added to the DC gel because of the overlap of their absorption spectra with that of DC. These formulations were prepared as described in the experimental part, with the filter content in the range 0.25–5%. As a control sample, a standard DC gel was prepared with DC at 1%. All gel formulations were then exposed to forced degradation up to 60 min and the residual drug analyses was then carried out. The standard gel presented a DC residual of 19.50%, whereas photostability resulted influenced in different manner for the other samples. Fig. 6 shows the drug percentage residue in the samples containing the absorbers at the various concentrations.
The OS and OMC samples showed a clear increase of the photostability for the absorber concentration 0.5% with a residual DC concentration of 64% and 68%, respectively. However, these values remained constant for the samples with higher content of these absorbers. Unsuccessful results were on the contrary carried out for the other absorbers BP and OC that showed no significant improvement of the drug stability. This behavior has been reported as a consequence of photochemical reactions of these molecules, such as trans-cis isomerization or keto-enol tautomerism (Kockler et al., 2012) or, moreover, production of by-products after light irradiation (Shaath, 2010). The degradation process of the DC control sample was confirmed to follow the first-order kinetics. The samples containing the absorbers instead showed a different behavior. By plotting the drug percentage concentration against degradation time, a second order kinetics was observed with straight lines according to the equation: 1 = k2 · t + 0.01 DC% where DC% is the percentage of the residual drug concentration, k2 the photodegradation rate constant, t the time (min) and 0.01 is the reciprocal value of the starting concentration percentage (100%). MCR-ALS analysis predicted the formation of PhP1 and only traces of PhP2 and PhP3 . The rate constants and the t0.1 values of the 0.5% absorber formulations were carried out. Successful t0.1 values of 12.22 min and 13.75 min for OS and OMC, respectively, were calculated showing a notable increase in comparison with a value of 3.90 min measured on the control gel. As more than one UV-filter is nowadays used in cosmetic preparations (Chatelain and Gabard, 2001; Gaspar and Campos, 2006; Lhiaubet-Vallet et al., 2010), the combination of OS and OMC was also verified in affecting photoprotection. Three formulations with OS/OMC ratio between 0.5 and 2 were tested, but the results did not show significant additive or synergistic effects. The best t0.1 value of 15.71 min was measured on the OS/OMC formulation containing both 0.5%. 3.4. Photodegradation of DC–cyclodextrin gel
Fig. 6. Influence of UV absorber concentration on DC photodegradation after 60 min of light irradiation.
The entrapment of DC in cyclodextrin was afterwards investigated to minimize light sensitivity of the drug and, at the same time, to enhance drug solubility in water. First, the influence of ␣C, C, mC, and ␥C on solubility of DC was tested as described by Higuchi and Connors (1965). The behavior of DC in the different cyclodextrins is shown in the phase-solubility diagram reported in Fig. 7.
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The photodegradation profile of the complex followed secondorder kinetics and was compared in Fig. 8 to that of 1% DC control sample and 1% commercial specialty. Table 1 summarizes the degradation rate constants and the values of t0.1 for all the studied matrices. All results were averages of six experiments. The relative standard deviation values of all the points resulted ranging in the interval 2.03–4.99%. 4. Conclusions
Fig. 8. Photodegradation profile of DC in gel control, Voltaren Emulgel® 1% and C-DC gel.
The most effective cyclodextrin in increasing the solubility of DC was C. A linear relationship between solubility and C concentration was observed with R2 equal to 0.997. This result was in agreement with an almost 1:1 inclusion complex (Higuchi and Connors, 1965). The binding constant (Kb ) of the complexes was calculated according to the following equation: Kb =
slope × (1 − slope) SDC
where SDC is the solubility in water of DC at 25 ◦ C. The values of Kb , expressed as M−1 , followed the order C-DC (1361.32), mC-DC (484.76), ␣C-DC (33.51), and ␥C-DC (21.83). The higher solubility of C-DC and mC-DC complexes can be explained by a better fitting of the drug molecule in the cavity of these cyclodextrins, whereas ␣C seems to have a cavity too small and ␥C, conversely, too large. C was finally selected to incorporate DC in the preparation of the gel to be submitted to the photostability test. The C-DC gel, with DC concentration at 1%, was prepared as above described and then submitted to the stressing test. DC residue along the experiments was each time assayed after appropriate dilution of 100 l of C-DC to 10 ml with methanol, in such a way to assure the drug release. The results from the stress testing showed a clear decrease of DC degradation, with a very successful t0.1 value of 25.01 min. The very good performance of this formulation in terms of photostabilization could be attributed to a dual action of the cyclodextrin complex: a real physical protection of the entrapped drug by means of a molecular shield (Bayomi et al., 2002; Mielcarek, 1997) aided by the increase of the drug solubility in this matrix.
Table 1 Degradation kinetics parameters of DC in different matrices. Matrix
k1
R2
t0.1
Methanol solution Voltaren Emulgel® 1% Voltaren Emulgel® 2% Standard gel
0.0681 0.0105 0.0074 0.0118
0.978 0.968 0.998 0.979
2.52 4.38 6.22 3.90
BP-DC gel OS-DC gel OC-DC gel OMC-DC gel OS 0.25/OMC 0.50 gel OS 0.50/OMC 0.50 gel OS 0.50/OMC 0.25 gel C-DC gel
k2 0.0006 0.00009 0.0004 0.00008 0.00006 0.00007 0.00006 0.00004
R2 0.992 0.950 0.961 0.951 0.977 0.967 0.952 0.938
t0.1 1.83 12.22 2.75 13.75 13.92 15.71 13.87 25.01
t is expressed as min.
In this work, the anti-inflammatory drug diclofenac in commercial gel formulations was demonstrated to undergo photodegradation. The study was performed by adopting photostability tests defined by international rules. In particular, under an irradiance power of 450 W m−2 , corresponding to 27 kJ m−2 min and at a constant temperature of 25 ◦ C, the drug resulted degraded of 10% in 4.38 min and 6.22 min in 1% and 2% formulations, respectively. The design of photoprotective pharmaceutical matrices for topical application is particularly important for the greater likelihood of exposure to light with the result of reducing the available drug and at the same time increasing the risk of formation of toxic photoproducts. Two different approaches were adopted to reduce drug photodegradation by incorporation in gel of UV-absorbers and by entrapping the drug in cyclodextrins. The use of the octisilate or octyl methoxycinnamate as excipients gave good results by increasing the drug photostability of 1% gel to 12.22 and 13.75 min. Better results were carried out by incorporating the drug in cyclodextrin, increasing this value up to 25.01 min, corresponding to a stability increase of almost six times. The systems proposed here appear to be of certain interest for the development of new pharmaceutical formulations for topical use of diclofenac, able to minimize the photodegradation of the drug. Acknowledgment This research was supported by grants from M.U.R.S.T. (Italy). References Aloui, F., Ahajji, A., Irmouli, Y., George, B., Charrier, B., Merlin, A., 2007. Inorganic UV absorbers for the photostabilisation of wood-clearcoating systems: comparison with organic UV absorbers. Appl. Surf. Sci. 253, 3737–3745. Atarashi, K., Takano, M., Kato, S., Kuma, H., Nakanishi, M., Tokura, Y., 2012. Addition of UVA-absorber butyl methoxy dibenzoylmethane to topical ketoprofen formulation reduces ketoprofen photoallergic reaction. J. Photochem. Photobiol. B 113, 56–62. Barry, B.W., 1983. Dermatological Formulations: Percutaneous Absorption. Marcel Dekker, New York. Bayomi, M.A., Abanumay, K.A., Al-Angary, A.A., 2002. Effect of inclusion complexation with cyclodextrins on photostability of nifedipine in solid state. Int. J. Pharm. 243, 107–117. Boinpally, R.R., Zhou, S.L., Poondru, S., Devraj, G., Jasti, B.R., 2003. Lecithin vesicles for topical delivery of diclofenac. Eur. J. Pharm. Biopharm. 56, 389–392. Chatelain, E., Gabard, B., 2001. Photostabilization of butyl methoxydibenzoylmethane (Avobenzone) and ethylhexyl methoxycinnamate by bisethylhexyloxyphenol methoxyphenyl triazine (Tinosorb S), a new UV broadband filter. Photochem. Photobiol. 74, 401–406. Council of Europe, 2010. European Pharmacopoeia, seventh ed. Council of Europe, Strasbourg. Dawood, M.Y., 1993. Nonsteroidal antiinflammatory drugs and reproduction. Am. J. Obstet. Gynecol. 169, 1255–1265. de Juan, A., Casassas, E., Tauler, R., 2000. Encyclopedia of Analytical Chemistry: Instrumentation and Applications, Soft-Modeling of Analytical Data, vol. 11. Wiley, New York. de Juan, A., Rutan, S.C., Maeder, M., Tauler, R., 2009. Comprehensive Chemometrics, vol. 2. Elsevier, Amsterdam. De Luca, M., Mas, S., Ioele, G., Oliverio, F., Ragno, G., Tauler, R., 2010. Kinetic studies of nitrofurazone photodegradation by multivariate curve resolution applied to UV-spectral data. Int. J. Pharm. 386, 99–107. De Luca, M., Tauler, R., Ioele, G., Ragno, G., 2013. Study of photodegradation kinetics of melatonin by multivariate curve resolution (MCR) with estimation of feasible band boundaries. Drug Test. Anal. 5, 96–102.
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